A gene-based capture assay for surveying patterns of genetic diversity and insecticide resistance in a worldwide group of invasive mosquitoes

Abstract

Understanding patterns of diversification, genetic exchange, and pesticide resistance in arthropod disease vectors is necessary for effective population management. With the availability of next-generation sequencing technologies, one of the best approaches for surveying such patterns involves the simultaneous genotyping of many samples for a large number of genetic markers. To this end, the targeting of gene sequences of known function can be a cost-effective strategy. One insect group of substantial health concern are the mosquito taxa that make up the Culex pipiens complex. Members of this complex transmit damaging arboviruses and filariae worms to humans, as well as other pathogens such as avian malaria parasites that are detrimental to birds. Here we describe the development of a targeted, gene-based assay for surveying genetic diversity and population structure in this mosquito complex. To test the utility of this assay, we sequenced samples from several members of the complex, as well as from distinct populations of the relatively under-studied Culex quinquefasciatus. The data generated was then used to examine taxonomic divergence and population clustering between and within these mosquitoes. We also used this data to investigate genetic variants present in our samples that had previously been shown to correlate with insecticide-resistance. Broadly, our gene capture approach successfully enriched the genomic regions of interest, and proved effective for facilitating examinations of taxonomic divergence and geographic clustering within the Cx. pipiens complex. It also allowed us to successfully survey genetic variation associated with insecticide resistance in Culex mosquitoes. This enrichment protocol will be useful for future studies that aim to understand the genetic mechanisms underlying the evolution of these ubiquitous and increasingly damaging disease vectors.

Fig 1. Results of Principal Component Analyses.

Shown are the first and second principal components (PC1 & PC2) for all the Cx. pipiens complex samples (left panel) and just the Cx. quinquefasciatus samples (right panel). These analyses were performed with neutral, segregating variants. Taxonomic and population memberships were based on prior designations and collection location respectively.

Fig 2. All complex ADMIXTURE results.

Shown are the percent ancestry assignments (Q) for K values 2 through 7 based on our analysis of admixture. Sample designations are given on the left along with taxonomic designations.

Fig 3. Cx. quinquefasciatus ADMIXTURE results.

Shown are the percent ancestry assignments (Q) for K values 2 through 7 based on our analysis of admixture. Sample designations are given on the left along with population designations.

Fig 4. Maximum-likelihood phylogeny of samples.

A maximum-likelihood analysis of all samples using a transversional model of mutation with a gamma distribution of rate heterogeneity TVM + Γ; Tavaré, 1986 [71]. 100 bootstrap replicates of the analysis were performed and the bootstrap support for major nodes are shown in red. The colors correspond to the different taxonomic designations.