New SNP Markers to Identify Culex Complex Species (Diptera: Culicidae) by High-Resolution DNA Melting Analysis

The members from Culex pipiens complex are the primary vector of various arboviruses including West Nile virus and Eastern equine encephalitis in the United States. Within Cx. pipiens complex, there are three biotypes that differ largely in habitat, bloodmeal preference, mating behavior, and overwintering strategy. The three biotypes are Cx. pipiens form molestus (F.) (Diptera: Culicidae), Cx. pipiens f. pipiens (L.) (Diptera: Culicidae), and Cx. pipiens quinquefasciatus (Diptera: Culicidae) (also known as Cx. quinquefasciatus). It is clear that additional genetic markers, molecular markers based on single nucleotide polymorphisms (SNPs), were identified from the Cx. pipiens complex species. Genomic DNA was extracted from adult females of f. pipiens and f. molestus and was amplified by polymerase chain reactions. Thirty out of the 100 primer pairs showed amplification and were used for SNP identification. All thirty genes contained biotype-specific SNPs: 10 were located on the Cx. pipiens complex genetic map from previous genetic studies. We also tested a high-resolution DNA melting analysis as a biotype identification method by examining the SNPs in the two genes (CPIJ005487 and CPIJ002074). Our method provides a high confidence for biotype determination among the three Cx. pipiens complex mosquitoes.Data deposition: NCBI SNP Database (ssSNP accession nos.: ss947848635-ss947848878).