First report on knockdown resistance mutations in wild populations of Aedes aegypti from Argentina determined by a novel multiplex high-resolution melting polymerase chain reaction method

Abstract

Background: The mosquito Aedes aegypti is an urban vector of dengue and other arboviruses. During epidemics of these viruses, pyrethroid insecticides are used for the control of adult mosquitoes. The worldwide resistance of Ae. aegypti to these insecticides is a cause of failure of vector control campaigns. The primary target of pyrethroids is the voltage-gated sodium channel. Point mutations in the gene coding for this channel, called knockdown resistance (kdr) mutations, are associated with pyrethroid resistance. Two kdr mutations, V1016I and F1534C, have increased in frequency in natural populations of Ae. aegypti in the Americas during the last decade. Their association with pyrethroid resistance has been largely demonstrated in field populations throughout the Americas, and in in vitro assays. Diagnostics for kdr polymorphism allow early detection of the spread of insecticide resistance, which is critical for timely decisions on vector management. Given the importance of resistance management, high-throughput methods for kdr genotyping are valuable tools as they can be used for resistance monitoring programs. These methods should be cost-effective, to allow regional-scale surveys. Despite the extensive presence of Ae. aegypti and incidence of dengue in Argentina, the presence, abundance, and distribution of kdr mutations in populations of this mosquito have yet to be reported for the country.

Methods: Aedes aegypti samples were collected as immature stages or adults from Buenos Aires Metropolitan Area and northern localities of Tartagal (Salta Province) and Calilegua (Jujuy Province). Immature stages were maintained in the laboratory until they developed into adults. A high-resolution melting assay, based on an analysis of melting temperatures, was developed for the simultaneous genotyping of V1016I and F1534C kdr mutations. We used this method to infer the presence and frequencies of kdr alleles in 11 wild populations from Argentina.

Results: We demonstrated the presence of kdr mutations in Ae. aegypti in Argentina in regions where this species is under different selection pressures due to the use of pyrethroids. The populations under analysis are located in geographically distant regions of the species' distribution in Argentina: the northern provinces of Salta and Jujuy and the Buenos Aires Metropolitan Area. Higher frequencies of resistant-associated alleles were detected in the northern region. We report a multiplex high-throughput assay based on a high-resolution melting polymerase chain reaction method for the simultaneous genotyping of V1016I and F1534C kdr mutations. This assay was shown to be cost-effective, and thus provides an interesting molecular tool for kdr genotyping in A. aegypti control campaigns.

Conclusions: We report, to the best of our knowledge for the first time, the presence of kdr mutations in populations of Ae. aegypti from geographically distant locations of Argentina that differ with respect to their epidemiological situation and history of mosquito control. We have developed a high-throughput method for the genotyping of kdr mutations in Ae. aegypti from the Americas. Given its affordability and short running time, this method can be used in control campaigns to monitor the presence and spread of kdr alleles. The information provided here is relevant for the rational design of control strategies in the context of integrated vector management.

Keywords: Arbovirus; Dengue; Insecticide; Mosquito; Pyrethroid resistance; Vector management.

Fig. 1

Distribution of the knockdown resistance (kdr) alleles in Aedes aegypti in Tartagal and Calilegua National Park. Tartagal (Salta Province) and Calilegua National Park (Jujuy Province) (inset, upper left); Buenos Aires Metropolitan Region (inset, right)

Fig. 2

a–e Results of genotyping positions 1016 and 1534 in the voltage-gated sodium channel (Na_v) gene of Aedes aegypti (aedaenav) with high-resolution melting (HRM). Detection of kdr single nucleotide polymorphism by melt curve analysis. Alleles are distinguished by changes in the melting temperature (MT). a Raw results of multiplex HRM (mHRM). Peaks on the left and on the right indicate, respectively, IIS6 and IIIS6 Na_v segment amplicons. b, c Derivative and difference melting plots for IIS6 with variation in the 1016 position. d, e Derivative and difference melting plots for IIIS6 with variation in the 1534 position. Red kdr homozygous standard (R2R2), yellow heterozygous standard (SR2), green wild type homozygous standard (SS)

Fig. 3

a, b Changes in MTs with variation in the position of Na_v in Aedes aegypti. a Position 1016. Homozygous Val (VV), present in genotypes SS, SR1 and R1R1; heterozygous Val Ile (VI), present in genotypes SR2 and R1R2; homozygous Ile (II), present in genotype R2R2. b Position 1534. Homozygous Phe (FF), present in genotypes SS; heterozygous Phe Cys (FC), present in genotypes SR1 and SR2; homozygous Cys (CC), present in genotypes R1R1, R1R2 and R2R2. Different letters indicate significant differences (P < 0.05; ANOVA, Tukey’s multiple comparison test)