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Distribution and dissemination of the Val1016Ile and Phe1534Cys Kdr mutations in Aedes aegypti Brazilian natural populations.

ABSTRACT: BACKGROUND: The chemical control of the mosquito Aedes aegypti, the major vector of dengue, is being seriously threatened due to the development of pyrethroid resistance. Substitutions in the 1016 and 1534 sites of the voltage gated sodium channel (AaNaV), commonly known as kdr mutations, confer the mosquito with knockdown resistance. Our aim was to evaluate the allelic composition of natural populations of Brazilian Ae. aegypti at both kdr sites. METHODS: The AaNaV IIIS6 region was cloned and sequenced from three Brazilian populations. Additionally, individual mosquitoes from 30 populations throughout the country were genotyped for 1016 and 1534 sites, based in allele-specific PCR. For individual genotypes both sites were considered as a single locus. RESULTS: The 350 bp sequence spanning the IIIS6 region of the AaNaV gene revealed the occurrence of the kdr mutation Phe1534Cys in Brazil. Concerning the individual genotyping, beyond the susceptible wild-type (NaVS), two kdr alleles were identified: substitutions restricted to the 1534 position (NaVR1) or simultaneous substitutions in both 1016 and 1534 sites (NaVR2). A clear regional distribution pattern of these alleles was observed. The NaVR1kdr allele occurred in all localities, while NaVR2 was more frequent in the Central and Southeastern localities. Locations that were sampled multiple times in the course of a decade revealed an increase in frequency of the kdr mutations, mainly the double mutant allele NaVR2. Recent samples also indicate that NaVR2 is spreading towards the Northern region. CONCLUSIONS: We have found that in addition to the previously reported Val1016Ile kdr mutation, the Phe1534Cys mutation also occurs in Brazil. Allelic composition at both sites was important to elucidate the actual distribution of kdr mutations throughout the country. Studies to determine gene flow and the fitness costs of these kdr alleles are underway and will be important to better understand the dynamics of Ae. aegypti pyrethroid resistance.

SUBMITTER: Linss JG

PROVIDER: S-EPMC3912884 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Distribution and dissemination of the Val1016Ile and Phe1534Cys Kdr mutations in Aedes aegypti Brazilian natural populations.

Linss Jutta Gerlinde Birggitt JG Brito Luiz Paulo LP Garcia Gabriela Azambuja GA Araki Alejandra Saori AS Bruno Rafaela Vieira RV Lima José Bento Pereira JB Valle Denise D Martins Ademir Jesus AJ

Parasites & vectors 20140115

<h4>Background</h4>The chemical control of the mosquito Aedes aegypti, the major vector of dengue, is being seriously threatened due to the development of pyrethroid resistance. Substitutions in the 1016 and 1534 sites of the voltage gated sodium channel (AaNaV), commonly known as kdr mutations, confer the mosquito with knockdown resistance. Our aim was to evaluate the allelic composition of natural populations of Brazilian Ae. aegypti at both kdr sites.<h4>Methods</h4>The AaNaV IIIS6 region was ...[more]

PMID: 24428880

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Project description:BackgroundAedes albopictus and Aedes aegypti are the major vectors of arboviral diseases. As effective vaccines are not available for most of the arboviral diseases, vector control by using insecticides play the key role to reduce the disease transmission. The emergence and spread of resistance to different classes of insecticides by the vectors is a major obstacle to control the disease transmission. Information about vector susceptibility to different insecticides and their mechanisms are very important for formulating proper vector control measures. The present study was designed to assess the susceptibility of Ae. aegypti against three different classes of adulticides, one larvicidal agent available and polymorphisms in the voltage-gated sodium channel (VGSC) gene related to insecticide resistance.MethodsImmature stages of Ae. aegypti were collected from three dengue endemic municipal areas of West Bengal and reared in the laboratory. Larvae and adults (F1 progeny) were used for insecticide bioassay as per WHO protocols. Knock down resistance gene (kdr) mutations were assessed by direct sequencing of PCR products.ResultsThe Ae. aegypti population was found to be susceptible to type II pyrethroids and malathion but highly resistant to DDT. A high rate of polymorphisms in the VGSC gene was observed among the collected mosquitoes. A double mutant V1016G + F1534C was found to be associated with DDT resistance but neither V1016G nor F1534C alone showed the same association. Association between the kdr mutations and the susceptibility status of pyrethroids could not be established due to very small sample size. A low to moderate level of resistance was noticed against temephos among the larval population based on WHO criteria.ConclusionThe replacement of DDT by type II pyrethroids for the management of dengue vectors is an appropriate decision taken by the national program which is supported by the findings of a higher level of resistance to DDT. Persistence of polymorphisms in the VGSC gene might be an indication of emergence of resistance against pyrethroid insecticides that should be monitored at a regular interval. Attempts should be made to determine the effectiveness of other larvicides for replacement of temephos if needed in future. Along with the chemical insecticides different biological vector control methods as well as biopesticides should also be used in vector control programmes.

Project description:Aedes aegypti is the primary vector of dengue, chikungunya, Zika, and urban yellow fever. Insecticides are often the most effective tools to rapidly decrease the density of vector populations, especially during arbovirus disease outbreaks. However, the intense use of insecticides, particularly pyrethroids, has selected for resistant mosquito populations worldwide. Mutations in the voltage gated sodium channel (NaV) are among the principal mechanisms of resistance to pyrethroids and DDT, also known as "knockdown resistance," kdr. Here we report studies on the origin and dispersion of kdr haplotypes in samples of Ae. aegypti from its worldwide distribution. We amplified the IIS6 and IIIS6 NaV segments from pools of Ae. aegypti populations from 15 countries, in South and North America, Africa, Asia, Pacific, and Australia. The amplicons were barcoded and sequenced using NGS Ion Torrent. Output data were filtered and analyzed using the bioinformatic pipeline Seekdeep to determine frequencies of the IIS6 and IIIS6 haplotypes per population. Phylogenetic relationships among the haplotypes were used to infer whether the kdr mutations have a single or multiple origin. We found 26 and 18 haplotypes, respectively for the IIS6 and IIIS6 segments, among which were the known kdr mutations 989P, 1011M, 1016I and 1016G (IIS6), 1520I, and 1534C (IIIS6). The highest diversity of haplotypes was found in African samples. Kdr mutations 1011M and 1016I were found only in American and African populations, 989P + 1016G and 1520I + 1534C in Asia, while 1534C was present in samples from all continents, except Australia. Based primarily on the intron sequence, IIS6 haplotypes were subdivided into two well-defined clades (A and B). Subsequent phasing of the IIS6 + IIIS6 haplotypes indicates two distinct origins for the 1534C kdr mutation. These results provide evidence of kdr mutations arising de novo at specific locations within the Ae. aegypti geographic distribution. In addition, our results suggest that the 1534C kdr mutation had at least two independent origins. We can thus conclude that insecticide selection pressure with DDT and more recently with pyrethroids is selecting for independent convergent mutations in NaV.

Project description:BACKGROUND:Roraima, the northernmost State in Brazil, borders Venezuela and Guyana. Although mostly covered by the tropical forests, the urban centers of this state are highly infested with Ae. aegypti and are endemic for dengue, Zika and chikungunya. We accessed the insecticide resistance status of Ae. aegypti populations from the capital Boa Vista, two cities on international borders (Pacaraima and Bonfim) and Rorainópolis bordering Amazonas State, in order to evaluate the chemical control efficacy in these localities. METHODS:Tests with World Health Organization (WHO)-like tubes impregnated with the pyrethroid deltamethrin (0.05% and 0.12%) and the organophosphate malathion (0.7%) were conducted with Ae. aegypti from Boa Vista, Pacaraima, Bonfim and Rorainópolis, collected in 2016 and 2018. Genotyping of kdr mutations, related to resistance to pyrethroids, was performed for the SNP variations at sites 1016 and 1534 of the voltage gated sodium channel gene (NaV) with a TaqMan qPCR approach. RESULTS:Aedes albopictus was absent in our collections, and therefore only Ae. aegypti was tested. All Ae. aegypti populations were susceptible to 0.7% malathion in 2016; however, mortality dropped to under 90% in Bonfim and Pacaraima populations in 2018. All populations were resistant to 0.05% deltamethrin in both years. The time that 50% of females suffered knockdown (KdT50) under exposure to 0.05% deltamethrin was 3.3-5.9-fold longer in mosquitoes from the natural populations compared to the susceptible Rockefeller strain. Only the Pacaraima population (2018) remained resistant to 0.12% deltamethrin. Kdr genotyping revealed the absence of the wild-type NaVS haplotype (1016Val?+?1534Phe) in the populations from Roraima, indicating that all tested insects had a genetic background for pyrethroid resistance. The double kdr NaVR2 haplotype (1016Ile?+?15434Cys) was present in higher frequencies in all populations except for Rorainópolis, where this haplotype seems to have arrived recently. CONCLUSIONS:These results are important for the knowledge about insecticide resistance status of Ae. aegypti populations from Roraima and will help improve vector control strategies that may be applied to diverse localities under similar geographical and urban conditions.

Project description:Background:Several mutations in voltage gated sodium channel (NaV) have been identified in Aedes aegypti populations worldwide. However, only few are related to knockdown resistance to pyrethroids, most of which with variations in the 1016 and 1534 NaV sites. In Brazil, at least two NaV alleles are known: NaVR1, with a substitution in the 1534 (1016 Val+ + 1534 Ile kdr ) and NaVR2, with substitutions in both 1016 and sites (1016Ilekdr + 1534Cys kdr ). There is also the duplication in the NaV gene, with one copy carrying the substitution Ile1011Met, although its effects on pyrethroid resistance remain to be clarified. Our goals in this study were (1) to determine the role of each kdr NaV allele and the duplication on pyrethroid resistance and (2) to screen the frequency of the kdr alleles in 27 several natural Ae. aegypti populations from the metropolitan region of Rio de Janeiro. Methods:Pyrethroid resistance was evaluated by a knockdown time (KdT) assay, an adaptation of the WHO test tubes with paper impregnated with deltamethrin. We used laboratory-selected Ae. aegypti lineages: R1R1 and R2R2 (homozygous for the kdr NaVR1 and NaVR2 alleles, respectively), Dup (with duplication in the NaV gene), Rockefeller (the susceptibility reference control), and F1 hybrids among them. Genotyping of both 1016 and 1534 NaV sites was performed in 811 Ae. aegypti sampled from 27 localities from Rio de Janeiro (17), Niterói (6) and Nova Iguaçu (4) cities, Rio de Janeiro State, Brazil, with a TaqMan real time PCR approach. Results:The laboratory lineages R1R1, R2R2, and R1R2 were the only ones that needed more than 60 minutes to knock down all the insects exposed to the pyrethroid, being the KdT R2R2 > R1R2 > R1R1, corroborating the recessive nature of the kdr mutations. Frequency of kdr alleles NaVR1 and NaVR2 in field-caught mosquitoes varied from 0 to 52% and 43 to 86%, respectively, evidencing high levels of "resistant genotypes" (R1R1, R1R2, and R2R2), which together summed 60 to 100% in Ae. aegypti populations from Rio de Janeiro. Conclusions:The NaVR1 and NaVR2 kdr alleles confer resistance to the pyrethroid deltamethrin in homozygotes and R1R2 heterozygotes, being the R2R2 most resistant genotype. The allele containing duplication in the NaV gene, with a mutation in the 1011 site, did not confer resistance under the tested conditions. The frequencies of the "resistant genotypes" are elevated in Ae. aegypti natural populations from Rio de Janeiro.

Project description:Pyrethroids are the most used insecticide class worldwide. They target the voltage gated sodium channel (NaV), inducing the knockdown effect. In Aedes aegypti, the main dengue vector, the AaNaV substitutions Val1016Ile and Phe1534Cys are the most important knockdown resistance (kdr) mutations. We evaluated the fitness cost of these kdr mutations related to distinct aspects of development and reproduction, in the absence of any other major resistance mechanism. To accomplish this, we initially set up 68 crosses with mosquitoes from a natural population. Allele-specific PCR revealed that one couple, the one originating the CIT-32 strain, had both parents homozygous for both kdr mutations. However, this pyrethroid resistant strain also presented high levels of detoxifying enzymes, which synergistically account for resistance, as revealed by biological and biochemical assays. Therefore, we carried out backcrosses between CIT-32 and Rockefeller (an insecticide susceptible strain) for eight generations in order to bring the kdr mutation into a susceptible genetic background. This new strain, named Rock-kdr, was highly resistant to pyrethroid and presented reduced alteration of detoxifying activity. Fitness of the Rock-kdr was then evaluated in comparison with Rockefeller. In this strain, larval development took longer, adults had an increased locomotor activity, fewer females laid eggs, and produced a lower number of eggs. Under an inter-strain competition scenario, the Rock-kdr larvae developed even slower. Moreover, when Rockefeller and Rock-kdr were reared together in population cage experiments during 15 generations in absence of insecticide, the mutant allele decreased in frequency. These results strongly suggest that the Ae. aegypti kdr mutations have a high fitness cost. Therefore, enhanced surveillance for resistance should be priority in localities where the kdr mutation is found before new adaptive alleles can be selected for diminishing the kdr deleterious effects.

Project description:BACKGROUND:Aedes aegypti is a globally distributed vector of human diseases including dengue, yellow fever, chikungunya, and Zika. Pyrethroid insecticides are the primary means of controlling adult A. aegypti populations to suppress arbovirus outbreaks, but resistance to pyrethroid insecticides has become a global problem. Mutations in the voltage-sensitive sodium channel (Vssc) gene are a major mechanism of pyrethroid resistance in A. aegypti. Vssc resistance alleles in A. aegypti commonly have more than one mutation. However, our understanding of the evolutionary dynamics of how alleles with multiple mutations arose is poorly understood. METHODOLOGY/PRINCIPAL FINDINGS:We examined the geographic distribution and association between the common Vssc mutations (V410L, S989P, V1016G/I and F1534C) in A. aegypti by analyzing the relevant Vssc fragments in 25 collections, mainly from Asia and the Americas. Our results showed all 11 Asian populations had two types of resistance alleles: 1534C and 989P+1016G. The 1534C allele was more common with frequencies ranging from 0.31 to 0.88, while the 989P+1016G frequency ranged from 0.13 to 0.50. Four distinct alleles (410L, 1534C, 410L+1534C and 410L+1016I+1534C) were detected in populations from the Americas. The most common was 410L+1016I+1534C with frequencies ranging from 0.50 to 1.00, followed by 1534C with frequencies ranging from 0.13 to 0.50. Our phylogenetic analysis of Vssc supported multiple independent origins of the F1534C mutation. Our results indicated the 410L+1534C allele may have arisen by addition of the V410L mutation to the 1534C allele, or by a crossover event. The 410L+1016I+1534C allele was the result of one or two mutational steps from a 1534C background. CONCLUSIONS/SIGNIFICANCE:Our data corroborated previous geographic distributions of resistance mutations and provided evidence for both recombination and sequential accumulation of mutations contributing to the molecular evolution of resistance alleles in A. aegypti.

Dataset Information

Distribution and dissemination of the Val1016Ile and Phe1534Cys Kdr mutations in Aedes aegypti Brazilian natural populations.

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Distribution and dissemination of the Val1016Ile and Phe1534Cys Kdr mutations in Aedes aegypti Brazilian natural populations.

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