Association of a rapidly selected 4.3kb transposon-containing structural variation with a P450-based resistance to pyrethroids in the African malaria vector Anopheles funestus.

Deciphering the evolutionary forces controlling insecticide resistance in malaria vectors remains a prerequisite to designing molecular tools to detect and assess resistance impact on control tools. Here, we demonstrate that a 4.3kb transposon-containing structural variation is associated with pyrethroid resistance in central/eastern African populations of the malaria vector Anopheles funestus. In this study, we analysed Pooled template sequencing data and direct sequencing to identify an insertion of 4.3kb containing a putative retro-transposon in the intergenic region of two P450s CYP6P5-CYP6P9b in mosquitoes of the malaria vector Anopheles funestus from Uganda. We then designed a PCR assay to track its spread temporally and regionally and decipher its role in insecticide resistance. The insertion originates in or near Uganda in East Africa, where it is fixed and has spread to high frequencies in the Central African nation of Cameroon but is still at low frequency in West Africa and absent in Southern Africa. A marked and rapid selection was observed with the 4.3kb-SV frequency increasing from 3% in 2014 to 98% in 2021 in Cameroon. A strong association was established between this SV and pyrethroid resistance in field populations and is reducing pyrethroid-only nets' efficacy. Genetic crosses and qRT-PCR revealed that this SV enhances the expression of CYP6P9a/b but not CYP6P5. Within this structural variant (SV), we identified putative binding sites for transcription factors associated with the regulation of detoxification genes. An inverse correlation was observed between the 4.3kb SV and malaria parasite infection, indicating that mosquitoes lacking the 4.3kb SV were more frequently infected compared to those possessing it. Our findings highlight the underexplored role and rapid spread of SVs in the evolution of insecticide resistance and provide additional tools for molecular surveillance of insecticide resistance.

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.

Overexpression and nonsynonymous mutations of UDP-glycosyltransferases are potentially associated with pyrethroid resistance in Anopheles funestus.

UDP-glycosyltransferases (UGTs) enzymes are pivotal in insecticide resistance by transforming hydrophobic substrates into more hydrophilic forms for efficient cell elimination. This study provides the first comprehensive investigation of Anopheles funestus UGT genes, their evolution, and their association with pyrethroid resistance. We employed a genome-wide association study using pooled sequencing (GWAS-PoolSeq) and transcriptomics on pyrethroid-resistant An. funestus, along with deep-targeted sequencing of UGTs in 80 mosquitoes Africa-wide. UGT310B2 was consistently overexpressed Africa-wide and significant gene-wise Fst differentiation was observed between resistant and susceptible populations: UGT301C2 and UGT302A3 in Malawi, and UGT306C2 in Uganda. Additionally, nonsynonymous mutations in UGT genes were identified. Gene-wise Tajima's D density curves provide insights into population structures within populations across these countries, supporting previous observations. These findings have important implications for current An. funestus control strategies facilitating the prediction of cross-resistance to other UGT-metabolised polar insecticides, thereby guiding more effective and targeted insecticide resistance management efforts.

Entomological indicators of Plasmodium species transmission in Goma Tsé-Tsé and Madibou districts, in the Republic of Congo.

BACKGROUND: Malaria remains a major public health problem in the Republic of Congo, with Plasmodium falciparum being the deadliest species of Plasmodium in humans. Vector transmission of malaria is poorly studied in the country and no previous report compared rural and urban data. This study aimed to determine the Anopheles fauna and the entomological indices of malaria transmission in the rural and urban areas in the south of Brazzaville, and beyond. METHODS: Indoor household mosquitoes capture using electric aspirator was performed in rural and urban areas during raining and dry seasons in 2021. The identification of Anopheles species was done using binocular magnifier and nested-PCR. TaqMan and nested-PCR were used to detect the Plasmodium species in the head/thorax and abdomens of Anopheles. Some entomological indices including the sporozoite infection rate, the entomological inoculation rate and the man biting rate were estimated. RESULTS: A total of 699 Anopheles mosquitoes were collected: Anopheles gambiae sensu lato (s.l.) (90.7%), Anopheles funestus s.l. (6.9%), and Anopheles moucheti (2.4%). Three species of An. gambiae s.l. were identified including Anopheles gambiae sensu stricto (78.9%), Anopheles coluzzii (15.4%) and Anopheles arabiensis (5.7%). The overall sporozoite infection rate was 22.3% with a predominance of Plasmodium falciparum, followed by Plasmodium malariae and Plasmodium ovale. Anopheles aggressiveness rate was higher in households from rural area (1.1 bites/night) compared to that from urban area (0.8 ib/p/n). The overall entomological inoculation rate was 0.13 ib/p/n. This index was 0.17 ib/p/n and 0.092 ib/p/n in rural and in urban area, respectively, and was similar during the dry (0.18 ib/p/n) and rainy (0.14 ib/p/n) seasons. CONCLUSION: These findings highlight that malaria transmission remains high in rural and urban area in the south of Republic of Congo despite the ongoing control efforts, thereby indicating the need for more robust interventions.

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

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

Indoor residual spraying of experimental huts in Cameroon highlights the potential of Fludora® Fusion to control wild pyrethroid-resistant malaria vectors.

Elevated resistance to pyrethroids in major malaria vectors has led to the introduction of novel insecticides including neonicotinoids. There is a fear that efficacy of these new insecticides could be impacted by cross-resistance mechanisms from metabolic resistance to pyrethroids. In this study, after evaluating the resistance to deltamethrin, clothianidin and mixture of clothianidin + deltamethrin in the lab using CDC bottle assays, the efficacy of the new IRS formulation Fludora® Fusion was tested in comparison to clothianidin and deltamethrin applied alone using experimental hut trials against wild free-flying pyrethroid-resistant Anopheles funestus from Elende and field An. gambiae collected from Nkolondom reared in the lab and released in the huts. Additionally, cone tests on the treated walls were performed each month for a period of twelve months to evaluate the residual efficacy of the sprayed products. Furthermore, the L1014F-kdr target-site mutation and the L119F-GSTe2 mediated metabolic resistance to pyrethroids were genotyped on a subset of mosquitoes from the EHT to assess the potential cross-resistance. All Anopheles species tested were fully susceptible to clothianidin and clothianidin + deltamethrin mixture in CDC bottle assay while resistance was noted to deltamethrin. Accordingly, Fludora® Fusion (62.83% vs 42.42%) and clothianidin (64.42% vs 42.42%) induced significantly higher mortality rates in EHT than deltamethrin (42.42%) against free flying An. funestus from Elende in month 1 (M1) and no significant difference in mortality was observed between the first (M1) and sixth (M6) months of the evaluation (P > 0.05). However, lower mortality rates were recorded against An. gambiae s.s from Nkolondom (mortality rates 50%, 45.56% and 26.68%). In-situ cone test on the wall showed a high residual efficacy of Fludora® Fusion and clothianidin on the susceptible strain KISUMU (> 12 months) and moderately on the highly pyrethroid-resistant An. gambiae strain from Nkolondom (6 months). Interestingly, no association was observed between the L119F-GSTe2 mutation and the ability of mosquitoes to survive exposure to Fludora® Fusion, whereas a trend was observed with the L1014F-kdr mutation. This study highlights that Fludora® Fusion, through its clothianidin component, has good potential of controlling pyrethroid-resistant mosquitoes with prolonged residual efficacy. This could be therefore an appropriate tool for vector control in several malaria endemic regions.

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.

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.

Functional Validation of Endogenous Redox Partner Cytochrome P450 Reductase Reveals the Key P450s CYP6P9a/-b as Broad Substrate Metabolizers Conferring Cross-Resistance to Different Insecticide Classes in Anopheles funestus.

The versatility of cytochrome P450 reductase (CPR) in transferring electrons to P450s from other closely related species has been extensively exploited, e.g., by using An. gambiae CPR (AgCPR), as a homologous surrogate, to validate the role of An. funestus P450s in insecticide resistance. However, genomic variation between the AgCPR and An. funestus CPR (AfCPR) suggests that the full metabolism spectrum of An. funestus P450s might be missed when using AgCPR. To test this hypothesis, we expressed AgCPR and AfCPR side-by-side with CYP6P9a and CYP6P9b and functionally validated their role in the detoxification of insecticides from five different classes. Major variations were observed within the FAD- and NADP-binding domains of AgCPR and AfCPR, e.g., the coordinates of the second FAD stacking residue AfCPR-Y456 differ from that of AgCPR-His456. While no significant differences were observed in the cytochrome c reductase activities, when co-expressed with their endogenous AfCPR, the P450s significantly metabolized higher amounts of permethrin and deltamethrin, with CYP6P9b-AfCPR membrane metabolizing α-cypermethrin as well. Only the CYP6P9a-AfCPR membrane significantly metabolized DDT (producing dicofol), bendiocarb, clothianidin, and chlorfenapyr (bioactivation into tralopyril). This demonstrates the broad substrate specificity of An. funestus CYP6P9a/-b, capturing their role in conferring cross-resistance towards unrelated insecticide classes, which can complicate resistance management.

Geographical emergence of sulfadoxine-pyrimethamine drug resistance-associated P. falciparum and P. malariae alleles in co-existing Anopheles mosquito and asymptomatic human populations across Cameroon.

Malaria molecular surveillance remains critical in detecting and tracking emerging parasite resistance to anti-malarial drugs. The current study employed molecular techniques to determine Plasmodium species prevalence and characterize the genetic diversity of Plasmodium falciparum and Plasmodium malariae molecular markers of sulfadoxine-pyrimethamine resistance in humans and wild Anopheles mosquito populations in Cameroon. Anopheles mosquito collections and parasitological survey were conducted in villages to determine Plasmodium species infection, and genomic phenotyping of anti-folate resistance was accomplished by sequencing the dihydrofolate-reductase (dhfr) and dihydropteroate-synthase (dhps) genes of naturally circulating P. falciparum and P. malariae isolates. The malaria prevalence in Elende was 73.5% with the 5-15 years age group harboring significant P. falciparum (27%) and P. falciparum + P. malariae (19%) infections. The polymorphism breadth of the pyrimethamine-associated Pfdhfr marker revealed a near fixation (94%) of the triple-mutant -A16I51R59N108I164. The Pfdhps backbone mediating sulfadoxine resistance reveals a high frequency of the V431A436G437K540A581A613 alleles (20.8%). Similarly, the Pmdhfr N50K55L57R58S59S114F168I170 haplotype (78.4%) was predominantly detected in the asexual blood stage. In contrast, the Pmdhps- S436A437occured at 37.2% frequency. The combined quadruple N50K55L57R58S59S114F168I170_ S436G437K540A581A613 (31.9%) was the major circulating haplotype with similar frequency in humans and mosquitoes. This study highlights the increasing frequency of the P. malariae parasite mostly common in asymptomatic individuals with apparent P. falciparum infection. Interventions directed at reducing malaria transmission such as the scaling-up of SP are favoring the emergence and spread of multiple drug-resistant alleles between the human and mosquito host systems.

Impact of insecticide resistance on malaria vector competence: a literature review.

Since its first report in Anopheles mosquitoes in 1950s, insecticide resistance has spread very fast to most sub-Saharan African malaria-endemic countries, where it is predicted to seriously jeopardize the success of vector control efforts, leading to rebound of disease cases. Supported mainly by four mechanisms (metabolic resistance, target site resistance, cuticular resistance, and behavioural resistance), this phenomenon is associated with intrinsic changes in the resistant insect vectors that could influence development of invading Plasmodium parasites. A literature review was undertaken using Pubmed database to collect articles evaluating directly or indiretly the impact of insecticide resistance and the associated mechanisms on key determinants of malaria vector competence including sialome composition, anti-Plasmodium immunity, intestinal commensal microbiota, and mosquito longevity. Globally, the evidence gathered is contradictory even though the insecticide resistant vectors seem to be more permissive to Plasmodium infections. The actual body of knowledge on key factors to vectorial competence, such as the immunity and microbiota communities of the insecticide resistant vector is still very insufficient to definitively infer on the epidemiological importance of these vectors against the susceptible counterparts. More studies are needed to fill important knowledge gaps that could help predicting malaria epidemiology in a context where the selection and spread of insecticide resistant vectors is ongoing.

Entomological longitudinal surveys in two contrasted eco-climatic settings in Cameroon reveal a high malaria transmission from Anopheles funestus associated with GSTe2 metabolic resistance.

BACKGROUND: The impact of metabolic resistance to insecticides on malaria transmission remains poorly characterised notably through application of entomological parameters. The lack of resistance markers has been one of the limiting factors preventing a robust assessment of such impact. To this end, the present study sought to investigate how the L119F-Gste2 metabolic gene influences entomological parameters underpinning mosquitos' propensity to transmit Plasmodium spp. METHODS: Longitudinal studies were carried out in Mibellon and Elende, two different eco-climatic settings in Cameroon and mosquitoes were collected using Human Landing Catch (HLC), Centre for Disease Control Light Trap (CDC-LT) and Pyrethrum Spray Catch (PSC) technics. Plasmodium sporozoite parasites were detected by TaqMan and Nested PCR, and blood meal origin by ELISA. The allele-specific PCR (AS-PCR) method was used to genotype the L119F-GSTe2 marker and association with malaria transmission was established by comparing key transmission parameters such as the Entomological Inoculation Rate (EIR) between individuals with different L119F-GSTe2 genotypes. RESULTS: An. funestus s.l was the predominant malaria vector collected during the entomological survey in both sites (86.6% and 96.4% in Elende and Mibellon, respectively) followed by An. gambiae s.l (7.5% and 2.4%, respectively). Sporozoite infection rates were very high in both collection sites (8.7% and 11% in Elende and Mibellon, respectively). An. funestus s.s exhibited a very high entomological inoculation rate (EIR) (66 ib/h/month and 792 ib/h/year) and was responsible for 98.6% of all malaria transmission events occurring in both sites. The Human Blood Index was also high in both locations (HBI = 94%). An. funestus s.s. mosquitoes with both 119 F/F (RR) and L119F (RS) genotypes had a significantly higher transmission intensity than their susceptible L/L119 (SS) counterparts (IRR = 2.2, 95%CI (1.1-5.2), p = 0.03; IRR = 2.5, 95% CI (1.2-5.8), p = 0.01 respectively). CONCLUSION: This study highlights the major role that An. funestus s.s plays in malaria transmission in Cameroon with an aggravation from GSTe2-based metabolic resistance.

An Africa-wide genomic evolution of insecticide resistance in the malaria vector Anopheles funestus involves selective sweeps, copy number variations, gene conversion and transposons.

Insecticide resistance in malaria vectors threatens to reverse recent gains in malaria control. Deciphering patterns of gene flow and resistance evolution in malaria vectors is crucial to improving control strategies and preventing malaria resurgence. A genome-wide survey of Anopheles funestus genetic diversity Africa-wide revealed evidences of a major division between southern Africa and elsewhere, associated with different population histories. Three genomic regions exhibited strong signatures of selective sweeps, each spanning major resistance loci (CYP6P9a/b, GSTe2 and CYP9K1). However, a sharp regional contrast was observed between populations correlating with gene flow barriers. Signatures of complex molecular evolution of resistance were detected with evidence of copy number variation, transposon insertion and a gene conversion between CYP6P9a/b paralog genes. Temporal analyses of samples before and after bed net scale up suggest that these genomic changes are driven by this control intervention. Multiple independent selective sweeps at the same locus in different parts of Africa suggests that local evolution of resistance in malaria vectors may be a greater threat than trans-regional spread of resistance haplotypes.

Can the use of larviciding with biological compounds contribute in increasing Anopheles gambiae s.l. susceptibility to pyrethroid in a population expressing high resistance intensity?

BACKGROUND: Larviciding using non-insecticide compounds is considered appropriate for controlling outdoor biting mosquitoes and for managing insecticide resistance. However, there is still not enough information on the influence of larviciding in managing pyrethroid resistance. In the present study, we checked whether the introduction of larviciding using the biolarvicide VectoMax G in the city of Yaoundé is contributing in restoring the susceptibility of An. coluzzii populations to pyrethroids. METHODOLOGY: The susceptibility status of field An. coluzzii population was evaluated at different time points before and during larviciding treatments. In addition, An. coluzzii larvae collected in the city of Yaoundé, were split into four groups and exposed to different selection regimes for many generations as follow; (i): deltamethrin 0.05%_only, (ii): Vectomax_only, (iii): Vectomax+deltamethrin 0.05%, (iv): VectoMax+deltamethrin 0.05% + susceptible. Life traits parameters were measured in the progeny and compared between colonies. The control was the susceptible laboratory strain "Ngousso". Kdr allele frequency and the profile of expression of different detoxification genes and oxidative stress genes was checked using qPCR analysis. Gene's expression was compared between the first and the last generation of each colony and in field populations collected before and during larviciding. RESULTS: An increase in mosquito susceptibility to deltamethrin and permethrin was recorded for the field populations after larviciding implementation. Resistance intensity to deltamethrin was found to decrease from high to low in field populations. Only the colony vectomax+deltamethrin+susceptible presented a high susceptibility to deltamethrin after 21 generations. The kdr gene frequency was found to be unchanged in the field population and laboratory colonies. A significant decrease in the overexpression profile of Gste2 was detected in field population after larviciding implementation. Other genes showing a similar pattern though not significant were Cyp6z1, Cyp6p1 and Cyp6g16. Concerning fitness only the colony vectomax+deltamethrin+susceptible was found to display a fitness profile similar to the susceptible colony with high fecundity, high hatching rate, short development time and long adult survival rate. CONCLUSION: The profile of the field population supported reversal of phenotypic resistance to pyrethroids however no reduction in the frequency of the kdr allele was recorded. Some detoxification genes were detected less overexpressed. The study suggest that reversal may take longer to achieve in a population expressing a very high resistance profile and under continuous insecticide selection pressure.

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.

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.

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.

Temporal variation of high-level pyrethroid resistance in the major malaria vector Anopheles gambiae s.l. in Yaoundé, Cameroon, is mediated by target-site and metabolic resistance.

Constant assessment of insecticide resistance levels is mandatory to implement adequate malaria control tools, but little information is available on the annual dynamics of resistance. We, therefore, monitored variations in resistance in Anopheles gambiae s.l. over four seasons during 2 years in two localities of Yaoundé: urban Etoa-Meki and peri-urban Nkolondom. Mosquitoes were collected seasonally at larval stage and reared to adults for insecticide susceptibility tests and molecular analysis of resistance mechanisms. Anopheles coluzzii was found in Etoa-Meki and An. gambiae in Nkolondom. Low mortalities to pyrethroids were observed (permethrin <10%, deltamethrin <21%), and resistance extended to 5× and 10× diagnostic doses, revealing a marked increase compared to previous studies. A seasonal variation in resistance was observed with the highest levels within dry seasons in Etoa-Meki and rainy seasons in Nkolondom. The 1014F kdr allele shows a high frequency (0.9), associated with overexpression of metabolic genes (Cyp6M2, Cyp6P4, Cyp9K1, Cyp6Z1, and Cyp6Z2) varying significantly seasonally. This study reveals an escalation in resistance to pyrethroids in Yaoundé's malaria vectors with seasonal variations. An adequate choice of the implementation period of punctual vector control actions according to the resistance profile will help to potentiate the desired effect and thus improve its efficiency.

Seasonal variation of microbiota composition in Anopheles gambiae and Anopheles coluzzii in two different eco-geographical localities in Cameroon.

Understanding the environmental factors affecting the microbiota in malaria vectors may help in the development of novel vector control interventions, similar to paratransgenesis. This study evaluated seasonal and geographical variations in the microbial community of the two major malaria vectors. Adult Anopheles mosquitoes were collected across two different eco-geographical settings in Cameroon, during the dry and wet seasons. DNA was extracted from the whole individual mosquitoes from each group and processed for microbial analysis using Illumina Miseq sequencing of the V3-V4 region of the 16S rRNA gene. Data analysis was performed using QIIME2 and R software programs. A total of 1985 mosquitoes were collected and among them, 120 were selected randomly corresponding to 30 mosquitoes per season and locality. Overall, 97 bacterial taxa were detected across all mosquito samples, with 86 of these shared between dry and wet seasons in both localities and species. There were significant differences in bacterial composition between both seasons, with a clear separation observed between the dry and wet seasons (PERMANOVA comparisons of beta diversity, Pseudo-F = 10.45; q-value = 0.01). This study highlights the influence of seasonal variation on microbial communities and this variation's impact on mosquito biology and vectorial capacity should be further investigated.

Infestation rates, seasonal distribution, and genetic diversity of ixodid ticks from livestock of various origins in two markets of Yaoundé, Cameroon.

Little is known about the impact of ticks on livestock and humans in Cameroon. This study aimed to determine the prevalence, seasonal variation, and genetic diversity of hard ticks in the country. Ticks were collected during a cross-sectional survey on domestic livestock in two markets of Yaoundé in 2019 and 2020 and identified using morphological keys, 16S ribosomal DNA, (16S rDNA), and the cytochrome c oxidase subunit 1 (Cox1) genes. The infestation rates were 39.18%, 11.53%, and 2.74% in cattle, sheep, and goats respectively. Three genera of ticks were identified, Rhipicephalus, Amblyomma, and Hyalomma comprising eleven tick species. The main species were Rhipicephalus decoloratus (30.25%), R. microplus (24.43%), and Amblyomma variegatum (12.96%). Rhipicephalus spp. (81.31%) and Amblyomma variegatum (51.54%) were abundant during the rainy season, while Hyalomma spp. (83.86%) during the dry season (p-value <0.00001). Cox1 and 16S rDNA analysis showed a high level of genetic diversity among tick species with sequences close to those observed across Africa. Phylogenetic analysis revealed that our R. microplus belong to clade A and we identified R. sanguineus s.l. as R. linnea. This study shows a high tick infestation rate in cattle, while low in small ruminants with an extensive diversity of tick species, including several known vectors of important tick-borne diseases.