Levels of Resistance to Pyrethroid among DistinctkdrAlleles inAedes aegyptiLaboratory Lines and Frequency ofkdrAlleles in 27 Natural Populations from Rio de Janeiro, Brazil

Abstract

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 Ilekdr) and NaVR2, with substitutions in both 1016 and sites (1016Ilekdr + 1534Cyskdr). 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.

On the use of inhibitors of 4-hydroxyphenylpyruvate dioxygenase as a vector-selective insecticide in the control of mosquitoes

“…A combination of different mechanisms, such as metabolic resistance, mutation of the target proteins and penetration factors (cuticular resistance) contribute to the resistance of insects to contact insecticides. Here we searched for cross-resistance using populations known to be resistant to organophosphates and pyrethroids, where both metabolic resistance (increased expression of detoxifying enzymes) and target-site mutations ( kdr ) are at play [24,25,27–30]. No evidence for cross-resistance to NTBC appeared using oral administration (Figure 4, Table 3), but when using the topical application assays a moderate (3.9 to 5.5 fold) but significant (p<0.001) increase in the LD50 was observed in Santarem, Nova Iguaçu and Oiapoque populations (Figure 5, Table 3).…”

“…aegypti populations, originally collected with ovitraps from different Brazilian cities: Santarém (Pará state), Nova Iguaçu (Rio de Janeiro state), and Oiapoque (Amapá state), were reared in the insectary at FIOCRUZ-RJ. A Rock-kdr strain (or R2R2 strain) that presents point mutations in the voltage-dependent sodium channel associated with pyrethroid resistance was also maintained in the FIOCRUZ-RJ insectary [24,25]. The Ae.…”

“…RR50 (Resistance rate 50: LD50 of strain or population studied/LD50 of Rockefeller strain) for deltamethrin was: Santarém population = 30.4, Nova Iguaçu population = 25.4, Oiapoque population = 143.9 [27–30]. In the case of Rock-kdr, a knockdown time assay with deltamethrin revealed that the time necessary to knockdown 95% of the Rock-kdr lineage was 6.7x longer than the time found for the Rockefeller susceptible strain [25].…”

Co-occurrence of kdr mutations V1016I and F1534C in pyrethroid-resistant Aedes aegypti (Diptera: Culicidae) populations from Costa Rica

“…aegypti populations, including a population from Guanacaste province, Costa Rica (Saavedra-Rodriguez et al 2007). Although, functional conformation experiments do not confirm the role of the V1016I mutation in pyrethroid resistance, many studies have correlated the presence of this mutation to phenotypic resistance (Deming et al 2016, Brito et al 2018, Maestre-Serrano et al 2019, Ryan et al 2019). In contrast, kdr mutation F1534C has been strongly associated with DDT and pyrethroid resistance and confirmed by functional experiments in Xenopus oocytes (Du et al 2013).…”

The need to harmonize insecticide resistance testing: methodology, intensity concentrations and molecular mechanisms evaluated inAedes aegyptipopulations in Central America and Hispaniola

“…Resistance to pyrethroids in mosquitoes have been widely associated with the F1534C (21, 23, 25, 27, 29, 30, 32, 39–41, 83, 88–101) and V1016I mutations (24, 25, 27, 29, 30, 32, 36, 38, 40, 41, 88, 93, 97, 100, 102, 103). The simultaneous presence of both mutations has been associated with enhanced tolerance to deltamethrin in the past (31). However, the relationship between both mutations in relation to resistance is not clear.…”