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.

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.

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.

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.

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.

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.

Myxobolus makombensis n. sp. infection in African carp Labeobarbus batesii from the Makombè River, Cameroon: morphological and molecular characterization.

In this study, we examined myxozoan infections of Labeobarbus batesii sampled from the Makombè River in Cameroon. Fish were infected with Myxobolus makombensis n. sp. in the gill filament and M. dibombensis in the fins. Mature myxospores of M. makombensis n. sp. are pyriform in frontal view and biconvex in lateral view, with a truncated and slightly narrow anterior end. Spore dimensions (mean ± SD, with range in parentheses) are 17.5 ± 0.22 (16.2-18.9) µm length, 13.4 ± 0.25 (12-14.9) µm width, and 7 ± 0.21 (6.7-7.5) µm thickness, and spores exhibit a conspicuous anterior intercapsular appendix of 4.4 ± 0.18 (3.9-5.5) µm length. Myxospores have 2 pyriform polar capsules of unequal size; the larger one is 9.8 ± 0. 22 (8.2-10.9) µm long × 4.7 ± 0.15 (3.5-5.2) µm wide, and the smaller one is 8.8 ± 0.22 (7-10) µm long × 4.3 ± 0.12 (3.5-5.2) µm wide. Polar filaments possess 10 to 11 coils in the large polar capsule and 8 to 10 coils in the small polar capsule. Phylogenetic analysis of SSU rDNA sequences showed clustering of M. makombensis n. sp. close to M. dibombensis recently reported from the fins of the same host within a clade composed exclusively of parasites infecting cyprinid fishes.

Surveillance of Plasmodium malariae infection among inhabitants of rural areas in Ouidah-Kpomasse-Tori Bossito health district, Benin.

Among the Plasmodium species that infect humans, P. falciparum has been largely studied in malaria endemic areas. However, P. malariae infection is less documented among the human population. This study aimed to monitor the prevalence and distribution of P. malariae in Southern Benin. A cross-sectional survey was conducted in rural localities in the Ouidah-Kpomasse-Tori Bossito (OKT) health district in Southern Benin from June to October 2019. Socio-demographic data were collected using a questionnaire, while malaria infection data were obtained on the one hand by microscopy diagnosis and, on the other, by nested polymerase chain reaction (PCR). Based on microscopy, the prevalence of P. malariae mono-infection and coinfection of P. falciparum, P. malariae was respectively 2.3% and 1.2% in the OKT health district. This prevalence was higher (P < 0.01) than that reported by Damien et al. (2010) 10 years ago in the same study area with 0.7% and 0.3% of P. malariae and P. falciparum/P. malariae, respectively. Based on PCR analysis, P. malariae prevalence was 14.1%, including 5.2% of mono-infection and 8.9% of mixed infection with P. falciparum. Sub-microscopic Plasmodium infections were high (30.6%) and more pronounced in older participants (>20 years). The present study revealed that P. malariae increased in the OKT health district with a high prevalence of submicroscopic infection. Since our results provide valuable evidence of increasing P. malariae infection, the National Malaria Control Programs (NMCPs) must consider P. malariae when designing future measures for effective control and malaria treatment.

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.

Experimental Transmission of Plasmodium malariae to Anopheles gambiae.

Our current knowledge of the clinical burden, biology, and transmission of Plasmodium malariae is extremely scarce. To start addressing some of those questions, we experimentally infected Anopheles gambiae mosquitoes with fresh P. malariae isolates obtained from asymptomatic individuals in Lambaréné, Gabon. The proportion of mosquitoes infected via direct membrane feeding assay with either P. malariae monoinfections (16% [19 of 121]) or coinfections (28% [31 of 112]) was higher after serum replacement than in parallel groups without serum replacement (4% [4 of 102] and 4% [2 of 45], respectively; P < .01). Our results show that isolates from asymptomatic carriers can be used for experimental studies of P. malariae transmission.

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.

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.