Frequency of sodium channel genotypes and association with pyrethrum knockdown time in populations of Californian Aedes aegypti

Abstract

Background: Since their detection in 2013, Aedes aegypti has become a widespread urban pest in California. The availability of cryptic larval breeding sites in residential areas and resistance to insecticides pose significant challenges to control efforts. Resistance to pyrethroids is largely attributed to mutations in the voltage gated sodium channels (VGSC), the pyrethroid site of action. However, past studies have indicated that VGSC mutations may not be entirely predictive of the observed resistance phenotype.

Methods: To investigate the frequencies of VGSC mutations and the relationship with pyrethroid insecticide resistance in California, we sampled Ae. aegypti from four locations in the Central Valley, and the Greater Los Angeles area. Mosquitoes from each location were subjected to an individual pyrethrum bottle bioassay to determine knockdown times. A subset of assayed mosquitoes from each location was then analyzed to determine the composition of 5 single nucleotide polymorphism (SNP) loci within the VGSC gene.

Results: The distribution of knockdown times for each of the five Californian populations sampled was non-parametric with potentially bimodal distributions. One group succumbs to insecticidal effects around 35-45 min and the second group lasts up to and beyond the termination of the assay (120+ min). We detected 5 polymorphic VGSC SNPs within the sampled California populations. One is potentially new and alternatively spliced (I915K), and four are documented and associated with resistance: F1534C, V1016I, V410L and S723T. The Central Valley populations (Clovis, Dinuba, Sanger and Kingsburg) are fairly homogenous with only 5% of the mosquitoes showing heterozygosity at any given position. In the Greater LA mosquitoes, 55% had at least one susceptible allele at any of the five SNP loci. The known resistance allele F1534C was detected in almost all sampled mosquitoes (99.4%). We also observe significant heterogeneity in the knockdown phenotypes of individuals with the identical VGSC haplotypes suggesting the presence of additional undefined resistance mechanisms.

Conclusions: Resistance associated VGSC SNPs are prevalent, particularly in the Central Valley. Interestingly, among mosquitoes carrying all 4 resistance associated SNPs, we observe significant heterogeneity in bottle bioassay profiles suggesting that other mechanisms are important to the individual resistance of Ae. aegypti in California.

Keywords: Aedes aegypti; IPLEX genotyping; Pyrethroid; Resistance; Voltage gated sodium channel California..

Fig. 1

Topology of the mosquito sodium channel. The Ae. aegypti reference sequence was translated in CLC Main Workbench, Version 7. The resulting amino acid sequence was aligned to the Musca domestica (ANW06229) and Drosophila melanogaster (AAB59195) reference protein sequences. SNP annotations were transferred to Musca to determine the Musca protein position, and structural annotations were transferred from Drosophila to Aedes. The topology of the sodium channel was illustrated using Protter version 1 [49]. The sodium channel protein contains four homologous repeat domains (I–IV). Each repeat domain has six α-helical transmembrane segments (1–6, 7–12, 13–18, 19–24). Filled circles represent the five SNPs assayed in this study. Ref. = Reference. Alt. = alternate. aa = amino acid

Fig. 2

Locations from which Ae. aegypti were analyzed. Cities where founders for each lab strain were collected. Individuals were collected from the 5 cities we labeled on the map. Each lab strain was reared from individuals collected at various sites in these cities and reared together to increase specimen numbers.

Fig. 3

Distribution of knockdown times for each population. Each sample is represented by a circle. Black filled circles indicate genotyped samples, while the white circles represented samples that were not genotyped. Differences between group medians were determined by using the Mood’s median test followed by fdr correction. Letters indicate statistically significant differences.

Fig. 4

Median knockdown time for each present genotype. a Frequency of the 8 observed genotypes by population and their respective median knockdown times. b Kaplan-Meier analysis of observed knockdown between the 8 genotypes within all populations. C. P-values for log-rank comparisons between each genotype.

Fig. 5

Kaplain-Meier survival curve analysis of the homozygous resistant genotype. A comparison of knockdown between the homozygous resistant phenotypes from all 5 populations tested. ***P < 0.001, **P < 0.01, *P < 0.05.