Date Published: January 29, 2019
Publisher: Public Library of Science
Author(s): Corey L. Campbell, Karla Saavedra-Rodriguez, Tristan D. Kubik, Audrey Lenhart, Saul Lozano-Fuentes, William C. Black, Luciano Andrade Moreira.
Association mapping of factors that condition pyrethroid resistance in Aedes aegypti has consistently identified genes in multiple functional groups. Toward better understanding of the mechanisms involved, we examined high throughput sequencing data (HTS) from two Aedes aegypti aegypti collections from Merida, Yucatan, Mexico treated with either permethrin or deltamethrin. Exome capture enrichment for coding regions and the AaegL5 annotation were used to identify genes statistically associated with resistance. The frequencies of single nucleotide polymorphisms (SNPs) were compared between resistant and susceptible mosquito pools using a contingency χ2 analysis. The -log10(χ2p value) was calculated at each SNP site, with a weighted average determined from all sites in each gene. Genes with -log10(χ2p value) ≥ 4.0 and present among all 3 treatment groups were subjected to gene set enrichment analysis (GSEA). We found that several functional groups were enriched compared to all coding genes. These categories were transport, signal transduction and metabolism, in order from highest to lowest statistical significance. Strikingly, 21 genes with demonstrated association to synaptic function were identified. In the high association group (n = 1,053 genes), several genes were identified that also genetically or physically interact with the voltage-gated sodium channel (VGSC). These genes were eg., CHARLATAN (CHL), a transcriptional regulator, several ankyrin-domain proteins, PUMILIO (PUM), a translational repressor, and NEDD4 (E3 ubiquitin-protein ligase). There were 13 genes that ranked among the top 10%: these included VGSC; CINGULIN, a predicted neuronal gap junction protein, and the aedine ortholog of NERVY (NVY), a transcriptional regulator. Silencing of CHL and NVY followed by standard permethrin bottle bioassays validated their association with permethrin resistance. Importantly, VGSC levels were also reduced about 50% in chl- or nvy-dsRNA treated mosquitoes. These results are consistent with the contribution of a variety of neuronal pathways to pyrethroid resistance in Ae. aegypti.
The major arbovirus vector Aedes aegypti continues to pose significant threats to human health in tropical and subtropical urban areas [1,2]. In southern Mexico, where dengue is hyperendemic , insecticides used for Ae. aegypti control include organophosphates for larval control and pyrethroids for adults . The voltage-gated sodium channel (Vgsc, aaeNAV, LOC5567355) is one target of pyrethroid toxicity . Increased resistance in Ae. aegypti to pyrethroid insecticides has been documented in many locations [6–9], and is associated with the presence of specific VGSC alleles. In addition, multiple lines of evidence indicate that metabolic resistance mechanisms may be equally important as target-site resistance [10–13]. Metabolic resistance to pyrethroids is generally described as a number of Vgsc-independent mechanisms derived from gene duplication, transcript overexpression, sequestration or increased reduction/oxidation (redox) activity. Cytochromes P450 (CYP), episilon class glutathione S-transferases (eGST) and esterases (EST) are major contributors to metabolic resistance, which are stimulated in response to oxidative stress [14–16].
We identified gene subsets with significant enrichment in populations resistant to pyrethroids. Importantly, genes were also identified that genetically, functionally or physically interact with Vgsc in other model organisms or in cell culture. Genes which were present in the high association set and for which multiple lines of evidence support genetic or physical interactions with VGSC are highlighted in Fig 5 [25,26,28,32,33,37]. We also found that aedine transcriptional regulators CHL and NVY are associated with permethrin resistant phenotypes. This is consistent with the hypothesis that transcriptional processes are under selective pressure. Both CHL and NVY are located on the p arm of chromosome 3 (red arrows, Fig 3) near VGSC within the area of a proposed selective sweep. The functional associations described in this report are consistent with the idea that specific genes within the VGSC-proximal cluster are associated with resistant phenotypes, are under selective pressure, and are not selected merely due to chromosomal proximity.