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

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.Author summary: The mosquito taxa that make up the Culex pipiens complex are important vectors of the agents of several human diseases such as West Nile and St. Louis encephalitides, and lymphatic filariasis. They are also important vectors of avian malaria, which impacts livestock and wildlife. The development of effective strategies for the control of these mosquitoes requires knowledge of their origins, distribution, dispersal patterns, and the extent to which discreet taxonomic entities within the complex interbreed. To achieve these objectives, it is necessary to compare patterns of genetic diversity across many mosquito samples, which can be cost-prohibitive. To address this limitation, we developed a targeted, gene-based assay that allowed us to cost-effectively genotype a large number of genetic variants from a representative global sampling of individual Cx. pipiens complex mosquitoes. We show that this assay is a powerful tool for examining genetic structure and hybridization among populations. We also explore its utility for surveying alleles previously shown to be associated with insecticide resistance. Future use of this enrichment assay and the bioinformatics methods described here will allow researchers to study evolutionary patterns across the Cx. pipiens complex as well as monitor the presence of genetic variation that could affect control efforts.