Levels of cross-resistance to pyrethroids conferred by the Vssc knockdown resistance allele 410L+1016I+1534C in Aedes aegypti

Abstract

Aedes aegypti is a primary vector of viral pathogens and is responsible for millions of human infections annually that represent critical public health and economic costs. Pyrethroids are one of the most commonly used classes of insecticides to control adult A. aegypti. The insecticidal activity of pyrethroids depends on their ability to bind and disrupt the voltage-sensitive sodium channel (VSSC). In mosquitoes, a common mechanism of resistance to pyrethroids is due to mutations in Vssc (hereafter referred as knockdown resistance, kdr). In this study, we found that a kdr (410L+V1016I+1534C) allele was the main mechanism of resistance in a pyrethroid-resistant strain of A. aegypti collected in Colombia. To characterize the level of resistance these mutations confer, we isolated a pyrethroid resistant strain (LMRKDR:RK, LKR) that was congenic to the susceptible Rockefeller (ROCK) strain. The full-length cDNA of Vssc was cloned from LKR and no additional resistance mutations were present. The levels of resistance to different pyrethroids varied from 3.9- to 56-fold. We compared the levels of resistance to pyrethroids, DCJW and DDT between LKR and what was previously reported in two other congenic strains that share the same pyrethroid-susceptible background (the ROCK strain), but carry different kdr alleles (F1534C or S989P + V1016G). The resistance conferred by kdr alleles can vary depending on the stereochemistry of the pyrethroid. The 410L+1016I+1534C kdr allele does not confer higher levels of resistance to six of ten pyrethroids, relative to the 1534C allele. The importance of these results to understand the evolution of insecticide resistance and mosquito control are discussed.

Fig 1. Protocol for the isolation of the congenic strain containing the 410L+1016I+1534C kdr allele introgressed into the genetic background of the ROCK strain.

Number of heterozygous (+/kdr) males included in each backcross varied depending on the number of females added to the cage (approximately half the number of females).

Fig 2. The Vssc cDNA from the ROCK and LKR strains was obtained by PCR and cloning of four overlapping fragments.

The overlapping pieces and relative position of primers (blue arrows) are shown in the diagram. List of the primers, sequences and purposes are also included.

Fig 3. Structures of the insecticides tested.

Stereochemistry is not indicated for all structures. All of the insecticides shown are pyrethroids, except for DDT and indoxacarb.

Fig 4. Levels of resistance levels conferred to insecticides targeting the VSSC in the LKR, 1534C:ROCK and KDR:ROCK strains having the 410L+1016I+1534C, 1534C and 989P+1016G alleles, respectively.

RRs for KDR:ROCK are calculated from the values in Table 3 or were previously published [25] and are given here for comparison purposes. RRs for 1534C:ROCK for bioallethrin, cyhalothrin, etofenprox, flumethrin, DDT and DCJW were previously reported [24] and are given here for comparison purposes (those that were not tested are indicated by a #). Bars with different letters are significantly different based on non-overlap of the 95% confidence intervals.

Fig 5. Levels of resistance levels conferred to 1R-cis αS cypermethrin, alpha-cypermethrin (mix of the 1R-cis αS and 1S-cis αR isomers), and cypermethrin (mix of eight isomers) in the LKR, 1534C:ROCK and KDR:ROCK strains having the 410L+1016I+1534C, 1534C and 989P+1016G alleles, respectively.

The RR for KDR:ROCK to cypermethrin was previously published [25] and is given here for comparison purposes. Bars with different letters are significantly different based on non-overlap of the 95% confidence intervals.

Fig 6. Levels of resistance levels conferred to permethrin, cis-permethrin, and trans-permethrin in the LKR, 1534C:ROCK and KDR:ROCK strains having the 410L+1016I+1534C, 1534C and 989P+1016G alleles, respectively.

The RRs for KDR:ROCK to permethrin, cis-permethrin, and trans-permethrin were previously published [25] and given here for comparison purposes. Bars with different letters are significantly different based on non-overlap of the 95% confidence intervals.

Fig 7. Levels of resistance levels conferred to tefluthrin and 1R-trans tefluthrin in the LKR and KDR:ROCK strains having the 410L+1016I+1534C and 989P+1016G alleles, respectively.

The RR for KDR:ROCK to tefluthrin was previously published [25] and is given here for comparison purposes. Bars with different letters are significantly different based on non-overlap of the 95% confidence intervals.