Identifying permethrin resistance loci in malaria vectors by genetic mapping

Identification of the major loci responsible for insecticide resistance in malaria vectors would aid the development andimplementation of effective resistance management strategies, which are urgently needed to tackle the growing threat posedby resistance to the limited insecticides available for malaria control. Genome-wide association studies in the major malariavector, Anopheles gambiae, have been hindered by the high degree of within-population structuring and very low levels oflinkage disequilibrium hence we revisited the use of quantitative trait loci (QTL) mapping to study resistance phenotypesin this vector species. Earlier work, identified two major QTL associated with pyrethroid resistance in A. gambiae s.s. fromEast Africa using genetic crossing of laboratory-colonized resistant and susceptible strains. In this study, we report theresults from genetic mapping of pyrethroid resistance in three isofemale pedigrees established from wild-caught femaleA. gambiae s.s. mosquitoes from Benin. We identified two QTL on chromosomes 2L and 3R in these field populations, insimilar genomic locations to theQTLidentified in laboratory strains. The relative merits of two alternative study designs arediscussed and suggestions made for future genetic mapping studies of insecticide resistance in mosquitoes

Zoom in at African country level: potential climate induced changes in areas of suitability for survival of malaria vectors

Background: Predicting anopheles vectors’ population densities and boundary shifts is crucial in preparing formalaria risks and unanticipated outbreaks. Although shifts in the distribution and boundaries of the major malariavectors (Anopheles gambiae s.s. and An. arabiensis) across Africa have been predicted, quantified areas of absolutechange in zone of suitability for their survival have not been defined. In this study, we have quantified areas ofabsolute change conducive for the establishment and survival of these vectors, per African country, under twoclimate change scenarios and based on our findings, highlight practical measures for effective malaria control inthe face of changing climatic patterns. Methods: We developed a model using CLIMEX simulation platform to estimate the potential geographicaldistribution and seasonal abundance of these malaria vectors in relation to climatic factors (temperature, rainfalland relative humidity). The model yielded an eco-climatic index (EI) describing the total favourable geographicallocations for the species. The EI values were classified and exported to a GIS package. Using ArcGIS, the EI shapepoints were clipped to the extent of Africa and then converted to a raster layer using Inverse Distance Weighted(IDW) interpolation method. Generated maps were then transformed into polygon-based geo-referenced data setand their areas computed and expressed in square kilometers (km2). Results: Five classes of EI were derived indicating the level of survivorship of these malaria vectors. The proportionof areas increasing or decreasing in level of survival of these malaria vectors will be more pronounced in easternand southern African countries than those in western Africa. Angola, Ethiopia, Kenya, Mozambique, Tanzania, SouthAfrica and Zambia appear most likely to be affected in terms of absolute change of malaria vectors suitability zonesunder the selected climate change scenarios Conclusion: The potential shifts of these malaria vectors have implications for human exposure to malaria, asrecrudescence of the disease is likely to be recorded in several new areas and regions. Therefore, the need todevelop, compile and share malaria preventive measures, which can be adapted to different climatic scenarios,remains crucial

Variation in Arsenic metabolism in schistosomiasis associated bladder pathology in a rural community, Eggua, Ogun State, Nigeria

Exposure to toxic inorganic Arsenic (iAs) in areas endemic for urogenital schistosomiasis may confer increased risk for bladder cancer. The severity of the adverse effects of iAs however depends on its metabolism, which is highly variable among individuals. Genetic polymorphism in Arsenic (+3) Methyl Transferase enzyme, accounts significantly for these variations. To investigate the relationship of AS3MT gene polymorphisms and Arsenic metabolism to schistosomiasis and/or associated bladder pathology, 119 individualsfrom Eggua in southwest Nigeria were recruited for this study. Screening for schistosomiasis and bladder pathology was done by microscopy and ultrasonography respectively. Wagtech Digital Arsenator was used to assess total urinary arsenic concentrations and thus determine the level of arsenic exposure. The single nucleotide polymorphism AS3MT/Met287Thr T>C (rs11191439) was genotyped using Alelle-Specific PCR. Of the participants who tested positive for schistosomiasis, 33.3% exhibited bladder pathology. Total urinary arsenic concentration in 80% of the participants was above the WHO limit of 0.05mg/L. The Met287Thr allelic distribution conformed to the Hardy-Weinberg equilibrium (X2= 0.161, P> 0.05). Observed allelic frequencies were 0.96 and 0.04 for wild-type T and mutant C alleles respectively. There was no significant relationship between AS3MT SNP, arsenic concentrations and schistosomiasis associated bladder pathology. In conclusion, the community is highly exposed to arsenic, although with a possible genetic advantage of increased AS3MT catalytic activity. However, we see the need for urgent intervention as inter-individual differences in arsenic metabolism may influence the bladder pathology status of individuals in the community. And although urogenital schistosomiasis is waning in Eggua, it is not known what synergy the infection and high arsenic exposure may wield on bladder pathology

Zoom in at African Country level: Potential Climate Induced Changes in Areas of Suitability for Survival of Malaria Vectors

Predicting anopheles vectors’ population densities and boundary shifts is crucial in preparing for malaria risks and unanticipated outbreaks. Although shifts in the distribution and boundaries of the major malaria vectors (Anopheles gambiae s.s. and An. arabiensis) across Africa have been predicted, quantified areas of absolute change in zone of suitability for their survival have not been defined. In this study, we have quantified areas of absolute change conducive for the establishment and survival of these vectors, per African country, under two climate change scenarios and based on our findings, highlight practical measures for effective malaria control in the face of changing climatic patterns. We develop a model using CLIMEX simulation to estimate the potential geographical distribution and seasonal abundance of these malaria vectors in relation to climatic factors 9temperature, rainfall and relative humidity). The model yielded an eco climatic index (EI) describing the total favorable geographical locations for the species. The EI value were classified and exposed to a GIS package. Using ArcGIS, the EI shape points clipped to the extent of Africa and then converted to a raster layer using inverse Distance Weighted (IDW) interpolation method. Generated maps wre then transformed into polygon-based geo-referenced data set and areas computed and expressed in square kilometers (km2). Five classes of EI were derived indicating the level of survivorship of these malaria vectors. The proportion of areas increasing or decreasing in level of survival of these malaria vectors will be more pronounced in eastern and southern African countries than those in western Africa. Angola, Ethiopia, Kenya, Mozambique, Tanzania, South Africa and Zambia appear most likely to be affected in terms of absolute change of malaria vectors suitability zones under the selected climate change scenarios. The potential shifts of these malaria vectors have implications for human exposure to malaria, as recrudescence of the disease is likely to be recorded in several new areas and regions. Therefore, the need to develop, compile and share malaria preventive measures, which can be adapted to different climatic scenarios, remains crucial. \u

Expression of the cytochrome P450s, CYP6P3 and CYP6M2 are significantly elevated in multiple pyrethroid resistant populations of Anopheles gambiae s.s. from Southern Benin and Nigeria

Background: Insecticide resistance in Anopheles mosquitoes is threatening the success of malaria control programmes. This is particularly true in Benin where pyrethroid resistance has been linked to the failure of insecticide treated bed nets. The role of mutations in the insecticide target sites in conferring resistance has been clearly established. In this study, the contribution of other potential resistance mechanisms was investigated in Anopheles gambiae s.s. from a number of localities in Southern Benin and Nigeria. The mosquitoes were sampled from a variety of breeding sites in a preliminary attempt to investigate the role of contamination of mosquito breeding sites in selecting for resistance in adult mosquitoes. Results: All mosquitoes sampled belonged to the M form of An. gambiae s.s. There were high levels of permethrin resistance in an agricultural area (Akron) and an urban area (Gbedjromede), low levels of resistance in mosquito samples from an oil contaminated site (Ojoo) and complete susceptibility in the rural Orogun location. The target site mutation kdrW was detected at high levels in two of the populations (Akron f = 0.86 and Gbedjromede f = 0.84) but was not detected in Ojoo or Orogun. Microarray analysis using the Anopheles gambiae detox chip identified two P450s, CYP6P3 and CYP6M2 up regulated in all three populations, the former was expressed at particularly high levels in the Akron (12.4-fold) and Ojoo (7.4-fold) populations compared to the susceptible population. Additional detoxification and redox genes were also over expressed in one or more populations including two cuticular pre-cursor genes which were elevated in two of the three resistant populations. Conclusion: Multiple resistance mechanisms incurred in the different breeding sites contribute to resistance to permethrin in Benin. The cytochrome P450 genes, CYP6P3 and CYP6M2 are upregulated in all three resistant populations analysed. Several additional potential resistance mechanisms were also identified that warrant further investigation. Metabolic genes were over expressed irrespective of the presence of kdr, the latter resistance mechanism being absent in one resistant population. The discovery that mosquitoes collected from different types of breeding sites display differing profiles of metabolic genes at the adult stage may reflect the influence of a range of xenobiotics on selecting for resistance in mosquitoes

Evidence of a multiple insecticide resistance in the malaria vector Anopheles funestus in South West Nigeria.

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

Acetylcholinesterase (ace‑1R) target site mutation G119S and resistance to carbamates in Anopheles gambiae (sensu lato) populations from Mali

Open Access Journal; Published online: 05 Jun 2020Background The long-lasting insecticidal nets (LLINs) and indoor residual spraying of insecticide (IRS) are major malaria vector control strategies in Mali. The success of control strategies depends on a better understanding of the status of malaria vectors with respect to the insecticides used. In this study we evaluate the level of resistance of Anopheles gambiae (sensu lato) to bendiocarb and the molecular mechanism that underlies it. Methods Larvae of An. gambiae (s.l.) were collected from breeding habitats encountered in the three study sites and bioassayed with bendiocarb. The ace-1 target site substitution G119S was genotyped using a TaqMan assay. Results The three species of the An. gambiae complex in Mali, i.e. An. arabiensis, An. coluzzii and An. gambiae (s.s.) were found in sympatry in the three surveyed localities with different frequencies. We observed a resistance and suspicious resistance of the three species to bendiocarb with a mortality rate ranging from 37% to 86%. The allelic frequency of the G119S mutation was higher in An. gambiae (s.s.) compared to the other two species; 42.86%, 25.61% and 16.67% respectively in Dangassa, Koula, and Karadié. The allelic frequency of G119S in An. coluzzii ranged from 4.5% to 8.33% and from 1.43% to 21.15% for An. arabiensis. After exposure to bendiocarb, the G119S mutation was found only in survivors. The survival of Anopheles gambiae (s.l) populations from the three surveyed localities was associated with the presence of the mutation. Conclusions The study highlights the implication of G119S mutation in bendiocarb resistance in An. gambiae (s.s.), An. arabiensis and An. coluzzii populations from the three surveyed localities

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

Typologie Des Gîtes Larvaires Et Résistance Des Vecteurs Du Paludisme A La Deltaméthrine Dans les Milieux Urbain Et Rural Du Département De l’Atlantique Au Sud Du Bénin: Données Préliminaires

La lutte antilarvaire récemment recommandée par l’OMS, requiert une connaissance approfondie de la distribution et de la typologie des gîtes larvaires des vecteurs du paludisme. L’objectif de cette étude est d’identifier les différents gîtes larvaires des anophèles et leur mécanisme de résistance à la deltaméthrine. Des prospections larvaires ont été effectuées en 2017 durant les saisons pluvieuses et sèches dans trois communes au sud du Bénin. Les moustiques issus de l’émergence des larves ont été soumis à la deltaméthrine et au bendiocarb selon le protocole de l’OMS. L’identification moléculaire des anophèles et le génotypage de la mutation Kdr ont été réalisés par PCR et l’expression des oxydases, des estérases α et β, et des GST ont été mesurées. Les prospections larvaires ont permis de répertorier 37 gîtes larvaires regroupés en 13 types. La majorité des gîtes étaient anthropiques. La densité larvaire variait d’un type de gîtes à l’autre. An. coluzzii et An. gambiae étaient les deux vecteurs du paludisme vivant en sympatrie dans lestroissites d’étude. Ils sont fortement résistants à la deltaméthrine avec la présence de la mutation kdr à des fréquences très élevées et une augmentation des activités des estérases dans les populations d’anophèles collectés à Zè et des GST à Abomey-Calavi et Allada. La prolifération des vecteurs du paludisme serait imputable à l’insalubrité de l’environnement immédiat et aux activités anthropiques qui créent et assurent le maintien des gîtes larvaires. Ces données pourraient servir au renforcement des stratégies de lutte contre le paludisme déjà en cours. Anopheles larval control, recently recommended by WHO, requires a deep knowledge of the distribution and typology of larval breeding sites. The objective of this study is to identify the different larval habitats colonized by Anopheles and their insecticide resistance mechanism. Larval surveys were carried out in three Districts in south of Benin in 2017, during the rainy and dry seasons. Mosquitoes breeding sites have been characterized and mapped. Mosquitoes from the emergence of larvae were tested to deltamethrin and bendiocarb according to the WHO protocol. The molecular identification of anopheles and the genotyping of the kdr mutation were performed by PCR and the expression of oxidases, esterases, and GSTs was measured. Larval surveys have identified 37 breeding sites categorized into 13 types. Most of the larval habitats were anthropogenics. An. coluzzii and An. gambiae were the two malaria vectors found in sympatric in the three study sites. These two vectors were highly resistant to deltamethrin with the presence of the kdr L1014F mutation at very high frequencies and an increase in esterase activities in anopheline populations collected in Zè and GST in Abomey-Calavi and Allada. The proliferation of malaria vectors is attributable to the unhealthy environment and human activities that create and maintain mosquito breeding. This study highlighted diversity in the type of breeding site of An. gambiae s.s in the Atlantic Department, suggesting the adaptation of this species in its environment. These results could be used to develop an antilarval control strategy in Abomey-Calavi, Zè and in Allada

Capture of high numbers of Simulium vectors can be achieved with Host Decoy Traps to support data acquisition in the onchocerciasis elimination endgame

Onchocerciasis elimination is within reach in many countries but requires enhanced surveillance of the Simulium vectors of Onchocerca volvulus. Collection of sufficient numbers of adult Simulium to detect infective O. volvulus larvae is hindered by limited sampling tools for these flies. Here, we tested for the first time the Host Decoy Trap (HDT), an exposure free method previously developed for Anopheles vectors of malaria parasites, as a potential sampling tool for adult Simulium. In three replicates of a randomized Latin square experimental design, the HDT was compared to Human Landing Catches (HLC) and the Esperanza Window Trap (EWT). A total of 8,531 adult S. damnosum sensu lato blackflies (S. squamosum group) were found in catches from the three different trapping methods. The HDT (mean catch 533 ± 111) caught significantly more S. squamosum than the EWT (mean catch 9.1 ± 2.2), a nearly 60-fold difference. There was no significant difference between the HLC (mean catch 385.6 ± 80.9) and the HDT. Larvae indistinguishable from those of O. volvulus were dissected from 2.86% of HDT samples (n = 70) and 0.35% of HLC samples (n = 285); a single infective third-stage larvae (L3) was found during dissection of a sample from the HDT. Owing to its very high capture rate, which was comparable to the HLC and significantly greater than EWT, alongside the presence of infected flies in its catch, the HDT represents a potentially valuable new tool for blackfly collection in elimination settings, where thousands of flies are needed for parasite screening