Multiple origins of knockdown resistance mutations in the Afrotropical mosquito vector Anopheles gambiae.
ABSTRACT: How often insecticide resistance mutations arise in natural insect populations is a fundamental question for understanding the evolution of resistance and also for modeling its spread. Moreover, the development of resistance is regarded as a favored model to study the molecular evolution of adaptive traits. In the malaria vector Anopheles gambiae two point mutations (L1014F and L1014S) in the voltage-gated sodium channel gene, that confer knockdown resistance (kdr) to DDT and pyrethroid insecticides, have been described. In order to determine whether resistance alleles result from single or multiple mutation events, genotyping of the kdr locus and partial sequencing of the upstream intron-1 was performed on a total of 288 A. gambiae S-form collected from 28 localities in 15 countries. Knockdown resistance alleles were found to be widespread in West Africa with co-occurrence of both 1014S and 1014F in West-Central localities. Differences in intron-1 haplotype composition suggest that kdr alleles may have arisen from at least four independent mutation events. Neutrality tests provided evidence for a selective sweep acting on this genomic region, particularly in West Africa. The frequency and distribution of these kdr haplotypes varied geographically, being influenced by an interplay between different mutational occurrences, gene flow and local selection. This has important practical implications for the management and sustainability of malaria vector control programs.
Project description:The effectiveness of insecticide-based malaria vector control interventions in Africa is threatened by the spread and intensification of pyrethroid resistance in targeted mosquito populations. The present study aimed at investigating the temporal and spatial dynamics of deltamethrin resistance in An. gambiae s.l. populations from North Cameroon. Mosquito larvae were collected from 24 settings of the Garoua, Pitoa and Mayo Oulo Health Districts (HDs) from 2011 to 2015. Two to five days old female An. gambiae s.l. emerging from larval collections were tested for deltamethrin resistance using the World Health Organization's (WHO) standard protocol. Sub samples of test mosquitoes were identified to species using PCR-RFLP and genotyped for knockdown resistance alleles (Kdr 1014F and 1014S) using Hot Ligation Oligonucleotide Assay (HOLA). All the tested mosquitoes were identified as belonging to the An. gambiae complex, including 3 sibling species mostly represented by Anopheles arabiensis (67.6%), followed by Anopheles coluzzii (25.4%) and Anopheles gambiae (7%). Deltamethrin resistance frequencies increased significantly between 2011 and 2015, with mosquito mortality rates declining from 70-85% to 49-73% in the three HDs (Jonckheere-Terstra test statistic (JT) = 5638, P< 0.001), although a temporary increase of mortality rates (91-97%) was seen in the Pitoa and Mayo Oulo HDs in 2012. Overall, confirmed resistance emerged in 10 An. gambiae s.l. populations over the 24 field populations monitored during the study period, from 2011 to 2015. Phenotypic resistance was mostly found in urban settings compared with semi-urban and rural settings (JT = 5282, P< 0.0001), with a spatial autocorrelation between neighboring localities. The Kdr 1014F allelic frequencies in study HDs increased from 0-30% in 2011 to 18-61% in 2014-2015 (JT = 620, P <0.001), especially in An. coluzzii samples. The overall frequency of the Kdr 1014S allele was 0.1%. This study revealed a rapid increase and widespread deltamethrin resistance frequency as well as Kdr 1014F allelic frequencies in An. gambiae s.l. populations over time, emphasizing the urgent need for vector surveillance and insecticide resistance management strategies in Cameroon.
Project description:<h4>Background and methods</h4>A longitudinal Anopheles gambiae s.l. insecticide-resistance monitoring programme was established in four sentinel sites in Burkina Faso. For three years, between 2008 and 2010, WHO diagnostic dose assays were used to measure the prevalence of resistance to all the major classes of insecticides at the beginning and end of the malaria transmission season. Species identification and genotyping for target site mutations was also performed and the sporozoite rate in adults determined.<h4>Results</h4>At the onset of the study, resistance to DDT and pyrethroids was already prevalent in An. gambiae s.l. from the south-west of the country but mosquitoes from the two sites in central Burkina Faso were largely susceptible. Within three years, DDT and permethrin resistance was established in all four sites. Carbamate and organophosphate resistance remains relatively rare and largely confined to the south-western areas although a small number of bendiocarb survivors were found in all sites by the final round of monitoring. The ace-1R target site resistance allele was present in all localities and its frequency exceeded 20% in 2010 in two of the sites. The frequency of the 1014F kdr mutation increased throughout the three years and by 2010, the frequency of 1014F in all sites combined was 0.02 in Anopheles arabiensis, 0.56 in An. gambiae M form and 0.96 in An. gambiae S form. This frequency did not differ significantly between the sites. The 1014S kdr allele was only found in An. arabiensis but its frequency increased significantly throughout the study (P?=?0.0003) and in 2010 the 1014S allele frequency was 0.08 in An. arabiensis. Maximum sporozoite rates (12%) were observed in Soumousso in 2009 and the difference between sites is significant for each year.<h4>Conclusion</h4>Pyrethroid and DDT resistance is now established in An. gambiae s.l. throughout Burkina Faso. Results from diagnostic dose assays are highly variable within and between rounds of testing, and hence it is important that resistance monitoring is carried out on more than one occasion before decisions on insecticide procurement for vector control are made. The presence of 1014S in An. gambiae s.l., in addition to 1014F, is not unexpected given the recent report of 1014S in Benin but highlights the importance of monitoring for both mutations throughout the continent. Future research must now focus on the impact that this resistance is having on malaria control in Burkina Faso.
Project description:BACKGROUND:Malaria, one of the world's greatest public health challenges, is an endemic disease with stable transmission in Togo. Combating malaria requires an effective vector control. This study provides temporal data on insecticide resistance status in the major malaria vector Anopheles gambiae sensu lato (s.l.) from Togo. METHODS:Two to 5 days old females of An. gambiae s.l., originating from three localities (Baguida, Kovié, Kolokopé) were subjected to insecticide-impregnated papers during 3 years (2012, 2013, 2016) as follows: organochlorides (4% DDT), pyrethroids (0.05% deltamethrin, 0.75% permethrin, 0.05% lambdacyhalothrin), carbamates (0.4% bendiocarb and 0.1% propoxur), and organophosphates (5% malathion, 0.4% chlorpyrifos methyl, 1% fenitrothion) following the WHO standard protocol. Dead and surviving mosquitoes were stored separately in Eppendorf tubes containing silica gel for DNA extraction, species identification, and kdr and ace-1 genotyping. RESULTS:Knockdown times (KDT50 and KDT95) were high in An. gambiae s.l. The lowest KDTs were recorded at Baguida in 2013 for deltamethrin (KDT50?=?24.7, CI [22.4-27.12] and KDT95?=?90.78, CI [76.35-113.49]). No KDTs were recorded for DDT and in some instances for permethrin. In general, An. gambiae s.l. was resistant to most of the four classes of insecticides during the survey periods regardless of locality and year, except to chlorpyrifos methyl. In some instances, mosquitoes were fully susceptible to fenitrothion (Kolokopé: 100% and Kovié: 98.05%, CI [95.82-100.26]) and malathion (100% at both Kolokopé and Kovié) in 2013, and malathion only (Kolokopé; 100%) in 2016. Anopheles coluzzii, An. gambiae and Anopheles arabiensis were the three sibling species identified at the three localities with some hybrids at Baguida (2013), and Kovié (2012 and 2016), respectively. Anopheles gambiae was relatively dominant (61.6%). The kdr 1014F allele frequency was?>?0.9 in most of the cases, except at Kolokopé (f (1014F)?=?0.63, CI [0.55-0.71]) in 2013. The kdr 1014S allele frequency was below 0.02. The highest ace-1 frequencies were identified in An. gambiae at Baguida (2012: 0.52, CI [0.34-0.69] and 2013: 0.66, CI [0.46-0.86]). CONCLUSION:The resistance status is worrying in Togo and should be considered in future malaria vector resistance management programmes by decision-makers.
Project description:Insecticide resistance has emerged as one of the major challenges facing National Malaria Control Programmes in Africa. A well-coordinated national database on insecticide resistance (IRBase) can facilitate the development of effective strategies for managing insecticide resistance and sustaining the effectiveness of chemical-based vector control measures. The aim of this study was to assemble a database on the current status of insecticide resistance among malaria vectors in Kenya.Data was obtained from published literature through PubMed, HINARI and Google Scholar searches and unpublished literature from government reports, research institutions reports and malaria control programme reports. Each data source was assigned a unique identification code and entered into Microsoft Excel 2010 datasheets. Base maps on the distribution of insecticide resistance and resistance mechanisms among malaria vectors in Kenya were generated using ArcGIS Desktop 10.1 (ESRI, Redlands, CA, USA).Insecticide resistance status among the major malaria vectors in Kenya was reported in all the four classes of insecticides including pyrethroids, carbamates, organochlorines and organophosphates. Resistance to pyrethroids has been detected in Anopheles gambiae (s.s.), An. arabiensis and An. funestus (s.s.) while resistance to carbamates was limited to An. gambiae (s.s.) and An. arabiensis. Resistance to the organochlorine was reported in An. gambiae (s.s.) and An. funestus (s.s.) while resistance to organophosphates was reported in An. gambiae (s.l.) only. The mechanisms of insecticide resistance among malaria vectors reported include the kdr mutations (L 1014S and L 1014F) and elevated activity in carboxylesterase, glutathione S-transferases (GST) and monooxygenases. The kdr mutations L 1014S and L 1014F were detected in An. gambiae (s.s.) and An. arabiensis populations. Elevated activity of monooxygenases has been detected in both An. arabiensis and An. gambiae (s.s.) populations while the elevated activity of carboxylesterase and GST has been detected only in An. arabiensis populations.The geographical maps show the distribution of insecticide resistance and resistance mechanisms among malaria vectors in Kenya. The database generated will provide a guide to intervention policies and programmes in the fight against malaria.
Project description:BACKGROUND:A longitudinal Anopheles gambiae s.l. insecticide resistance monitoring programme was established in four sentinel sites in Chad 2008-2010. When this programme ended, only sporadic bioassays were performed in a small number of sites. METHODS:WHO diagnostic dose assays were used to measure the prevalence of insecticide resistance to 0.1% bendiocarb, 4% DDT, 0.05% deltamethrin, 1% fenitrothion, and 0.75% permethrin in the main malaria vectors at the beginning and end of the malaria transmission season for three years 2008-2010, with subsequent collections in 2011 and 2014. Species and molecular identification of An. gambiae M and S forms and kdr genotyping was performed using PCR-RLFP; circumsporozoite status was assessed using ELISA. RESULTS:Between 2008 and 2010, significant changes in insecticide resistance profiles to deltamethrin and permethrin were seen in 2 of the sites. No significant changes were seen in resistance to DDT in any site during the study period. Testing performed after the period of routine monitoring had ended showed dramatic increases to DDT and pyrethroid resistance in 3 sites. No resistance to organophosphate or carbamate insecticides was detected. An. arabiensis was the predominate member of the An. gambiae complex in all 4 sites; adult collections showed temporal variation in species composition in only 1 site. Kdr analysis identified both 1014F and 1014S alleles in An. gambiae S only. Circumsporozoite analysis showed the highest vector infection rates were present in Donia, a site with extensive use of agricultural insecticides. CONCLUSIONS:During the monitoring gap of four years, significant changes occurred in resistance prevalence in 3 of the 4 sites (p = <0.001), endangering the efficacy of currently implemented malaria control interventions. Significant changes in insecticide resistance profiles and a lack of kdr resistance alleles in adult populations highlight the urgent need for comprehensive entomological monitoring to be implemented and sustained in country.
Project description:Population genetic structures of the two major malaria vectors Anopheles gambiae s.s. and An. arabiensis, differ markedly across Sub-Saharan Africa, which could reflect differences in historical demographies or in contemporary gene flow. Elucidation of the degree and cause of population structure is important for predicting the spread of genetic traits such as insecticide resistance genes or artificially engineered genes. Here the population genetics of An. gambiae s.s. and An. arabiensis in the central, eastern and island regions of Tanzania were compared. Microsatellite markers were screened in 33 collections of female An. gambiae s.l., originating from 22 geographical locations, four of which were sampled in two or three years between 2008 and 2010. An. gambiae were sampled from six sites, An. arabiensis from 14 sites, and both species from two sites, with an additional colonised insectary sample of each species. Frequencies of the knock-down resistance (kdr) alleles 1014S and 1014F were also determined. An. gambiae exhibited relatively high genetic differentiation (average pairwise FST?=?0.131), significant even between nearby samples, but without clear geographical patterning. In contrast, An. arabiensis exhibited limited differentiation (average FST?=?0.015), but strong isolation-by-distance (Mantel test r?=?0.46, p?=?0.0008). Most time-series samples of An. arabiensis were homogeneous, suggesting general temporal stability of the genetic structure. An. gambiae populations from Dar es Salaam and Bagamoyo were found to have high frequencies of kdr 1014S (around 70%), with almost 50% homozygote but was at much lower frequency on Unguja Island, with no. An. gambiae population genetic differentiation was consistent with an island model of genetic structuring with highly restricted gene flow, contrary to An. arabiensis which was consistent with a stepping-stone model of extensive, but geographically-restricted gene flow.
Project description:<b>Background</b>. The intensive use of insecticides in public health and agriculture has led to the development of insecticide resistances in malaria vectors across sub-Saharan Africa countries in the last two decades. The <i>kdr</i> target site point mutation which is among the best characterised resistance mechanisms seems to be changing its distribution patterns on the African continent. The 1014F <i>kdr</i> mutation originally described only in West Africa is spreading to East Africa while the 1014S <i>kdr</i> mutation originally described in East Africa, is spreading to West and Central Africa. However, the East-<i>kdr</i> mutation has not been reported in Côte d'Ivoire so far. <b>Methods</b>. Immature stages of <i>Anopheles gambiae s.l.</i> were collected from breeding sites at the outskirts of Yamoussoukro, Côte d'Ivoire. Emerging 3-5 day old adult female mosquitoes were tested for susceptibility to deltamethrin 0.05%, malathion 5%, bendiocarb 1% and dichlorodiphenyltrichloroethane (DDT) 4% according to WHO standard procedures. A total of 50 <i>An. gambiae s.l.</i> specimens were drawn at random for DNA extraction and identification down to the species level. A subsample of 30 mosquitoes was tested for the East-African <i>kdr</i> mutation using a Taqman assay. <b>Results</b>. The tested mosquito population appeared to be strongly resistant to deltamethrin (1.03% mortality), bendiocarb (38.46% mortality) and DDT (0% mortality) with probable resistance observed for malathion (92.47%). Among the 41 mosquitoes that were successfully characterized, <i>An. coluzzii</i> was predominant (68.3%) followed by <i>An. gambiae</i> <i>s.s.</i> (19.5%) and a few hybrids (7.3%). Out of 30 specimens genotyped for East-<i>kdr</i>, a single hybrid mosquito appeared to be heterozygous for the mutation. <b>Conclusion</b>. The present study revealed the presence of the East-<i>kdr</i> mutation in Côte d'Ivoire for the first time in <i>An. gambiae</i> and highlights the urgent need to start monitoring the allele and genotype frequencies.
Project description:The voltage gated sodium channel mutation Vgsc-1014S (kdr-east) was first reported in Kenya in 2000 and has since been observed to occur at high frequencies in the local Anopheles gambiae s.s.The mutation Vgsc-1014F has never been reported from An. gambiae Complex complex mosquitoes in Kenya.Molecularly confirmed An. gambiae s.s. (hereafter An. gambiae) and An. arabiensis collected from 4 different parts of western Kenya were genotyped for kdr from 2011 to 2013. Vgsc-1014F was observed to have emerged, apparently, simultaneously in both An. gambiae and An. arabiensis in 2012. A portion of the samples were submitted for sequencing in order to confirm the Vgsc-1014F genotyping results. The resulting sequence data were deposited in GenBank (Accession numbers: KR867642-KR867651, KT758295-KT758303). A single Vgsc-1014F haplotype was observed suggesting, a common origin in both species.This is the first report of Vgsc-1014F in Kenya. Based on our samples, the mutation is present in low frequencies in both An. gambiae and An. arabiensis. It is important that we start monitoring relative frequencies of the two kdr genes so that we can determine their relative importance in an area of high insecticide treated net ownership.
Project description:BACKGROUND:Insecticide resistance threatens malaria control in sub-Saharan Africa. Knockdown resistance to pyrethroids and organochlorines in Anopheles gambiae sensu lato (s.l.) is commonly caused by mutations in the gene encoding a voltage-gated sodium channel which is the target site for the insecticide. The study aimed to examine risk factors for knockdown resistance in An. gambiae s.l. and its relationship with malaria infection in children in rural Gambia. Point mutations at the Vgsc-1014 locus, were measured in An. gambiae s.l. during a 2-year trial. Cross-sectional surveys were conducted at the end of the transmission season to measure malaria infection in children aged 6 months-14 years. RESULTS:Whilst few Anopheles arabiensis and Anopheles coluzzii had Vgsc-1014 mutations, the proportion of An. gambiae sensu stricto (s.s.) mosquitoes homozygous for the Vgsc-1014F mutation increased from 64.8 to 90.9% during the study. The Vgsc-1014S or 1014F mutation was 80% higher in 2011 compared to 2010, and 27% higher in the villages with indoor residual spraying compared to those without. An increase in the proportion of An. gambiae s.l. mosquitoes with homozygous Vgsc-1014F mutations and an increase in the proportion of An. gambiae s.s. in a cluster were each associated with increased childhood malaria infection. Homozygous Vgsc-1014F mutations were, however, most common in An. gambiae s.s. and almost reached saturation during the study meaning that the two variables were colinear. CONCLUSIONS:As a result of colinearity between homozygous Vgsc-1014F mutations and An. gambiae s.s., it was not possible to determine whether insecticide resistance or species composition increased the risk of childhood malaria infection.
Project description:BACKGROUND:Mutations in the voltage-gated sodium channel at codon 1014 confer knock-down resistance (kdr) to pyrethroids in a wide range of insects. Anopheles gambiae exhibits two mutant alleles at codon 1014, serine and phenylalanine; and both are now widespread across Africa. Existing screening methods only allow for one resistant allele to be detected per assay. A new locked nucleic acid (LNA) qPCR assay was developed for the simultaneous detection of both mutant alleles and the wild type allele in a single assay. This tri-allelic detection assay was assessed as part of a study of the insecticide resistance in An. gambiae sensu stricto (s.s.) in the previously un-sampled area of Nord Ubangi, Democratic Republic of the Congo. METHODS:Samples from three sites were tested for insecticide susceptibility using WHO bioassays, with and without the synergist PBO preceding pyrethroid exposures, and were subsequently analysed for frequency and resistance-association of the Vgsc-1014 and Vgsc-N1575Y mutations. Results from the LNA-kdr 1014 assay were compared to results from standard TaqMan-kdr assays. RESULTS:Anopheles gambiae sensu lato (s.l.) was by far the predominant vector captured (84%), with only low frequencies of Anopheles funestus s.l. (9%) detected in Nord Ubangi. Molecular identification found An. gambiae s.s. to be the principal vector (99%) although Anopheles coluzzii was detected at very low frequency. Anopheles gambiae were susceptible to the carbamate insecticide bendiocarb, but resistant to DDT and to the pyrethroids permethrin and deltamethrin. Susceptibility to both pyrethroids was partially restored with prior exposure to PBO suggesting likely involvement of metabolic resistance. Anopheles gambiae s.s. was homozygous for kdr resistant alleles with both the L1014F and L1014S mutations present, and the N1575Y polymorphism was present at low frequency. The LNA-kdr assay simultaneously detected both resistant alleles and gave results entirely consistent with those from the two TaqMan-kdr assays. CONCLUSION:This study provides rare data on insecticide resistance and mechanisms in Anopheles from the centre of Africa, with the first detection of N1575Y. Nord Ubangi populations of An. gambiae s.s. show insecticide resistance mediated by both metabolic mechanisms and Vgsc mutations. The LNA-kdr assay is particularly suitable for use in populations in which both 1014S and 1014F kdr alleles co-occur and provides robust results, with higher throughput and at a quarter of the cost of TaqMan assays.