Dataset Information

Kdr genotyping in Aedes aegypti from Brazil on a nation-wide scale from 2017 to 2018.

ABSTRACT: Insecticide resistance is currently a threat to the control of Aedes agypti, the main vector of arboviruses in urban centers. Mutations in the voltage gated sodium channel (Na_V), known as kdr (knockdown resistance), constitute an important selection mechanism for resistance against pyrethroids. In the present study, we investigated the kdr distribution for the Val1016Ile and Phe1534Cys alterations in Ae. aegypti from 123 Brazilian municipalities, based on SNP genotyping assays in over 5,500 mosquitoes. The alleles Na_VS (1016Val⁺?+?1534Phe⁺), Na_VR1 (1016Val⁺?+?1534Cys^kdr) and Na_VR2 (1016Ile^kdr?+?1534Cys^kdr) were consistently observed, whereas kdr alleles have rapidly spread and increased in frequency. Na_VS was the less frequent allele, mostly found in Northeastern populations. The highest allelic frequencies were observed for Na_VR1, especially in the North, which was fixed in one Amazonian population. The double kdr Na_VR2 was more prevalent in the Central-west and South-eastern populations. We introduce the 'kdr index', which revealed significant spatial patterns highlighting two to three distinct Brazilian regions. The 410L kdr mutation was additionally evaluated in 25 localities, evidencing that it generally occurs in the Na_VR2 allele. This nationwide screening of a genetic mechanism for insecticide resistance is an important indication on how pyrethroid resistance in Ae. aegypti is evolving in Brazil.

SUBMITTER: Melo Costa M

PROVIDER: S-EPMC7414026 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Kdr genotyping in Aedes aegypti from Brazil on a nation-wide scale from 2017 to 2018.

Melo Costa Monique M Campos Kauara Brito KB Brito Luiz Paulo LP Roux Emmanuel E Melo Rodovalho Cynara C Bellinato Diogo Fernandes DF Lima José Bento Pereira JBP Martins Ademir Jesus AJ

Scientific reports 20200806 1

Insecticide resistance is currently a threat to the control of Aedes agypti, the main vector of arboviruses in urban centers. Mutations in the voltage gated sodium channel (Na<sub>V</sub>), known as kdr (knockdown resistance), constitute an important selection mechanism for resistance against pyrethroids. In the present study, we investigated the kdr distribution for the Val1016Ile and Phe1534Cys alterations in Ae. aegypti from 123 Brazilian municipalities, based on SNP genotyping assays in over ...[more]

PMID: 32764661

Similar Datasets

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: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: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:Recent outbreaks of locally transmitted dengue and Zika viruses in Florida have placed more emphasis on integrated vector management plans for Aedes aegypti (L.) and Aedes albopictus Skuse. Adulticiding, primarily with pyrethroids, is often employed for the immediate control of potentially arbovirus-infected mosquitoes during outbreak situations. While pyrethroid resistance is common in Ae. aegypti worldwide and testing is recommended by CDC and WHO, resistance to this class of products has not been widely examined or quantified in Florida. To address this information gap, we performed the first study to quantify both pyrethroid resistance and genetic markers of pyrethroid resistance in Ae. aegypti and Ae. albopictus strains in Florida. Using direct topical application to measure intrinsic toxicity, we examined 21 Ae. aegypti strains from 9 counties and found permethrin resistance (resistance ratio (RR) = 6-61-fold) in all strains when compared to the susceptible ORL1952 control strain. Permethrin resistance in five strains of Ae. albopictus was very low (RR<1.6) even when collected from the same containers producing resistant Ae. aegypti. Characterization of two sodium channel kdr alleles associated with pyrethroid-resistance showed widespread distribution in 62 strains of Ae. aegypti. The 1534 phenylalanine to cysteine (F1534C) single nucleotide polymorphism SNP was fixed or nearly fixed in all strains regardless of RR. We observed much more variation in the 1016 valine to isoleucine (V1016I) allele and observed that an increasing frequency of the homozygous V1016I allele correlates strongly with increased RR (Pearson corr = 0.905). In agreement with previous studies, we observed a very low frequency of three kdr genotypes, IIFF, VIFF, and IIFC. In this study, we provide a statewide examination of pyrethroid resistance, and demonstrate that permethrin resistance and the genetic markers for resistance are widely present in FL Ae. aegypti. Resistance testing should be included in an effective management program.

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: 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.

Dataset Information

Kdr genotyping in Aedes aegypti from Brazil on a nation-wide scale from 2017 to 2018.

Publications

Kdr genotyping in Aedes aegypti from Brazil on a nation-wide scale from 2017 to 2018.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets