Musca domestica voltage gated sodium channel amplicon sequencing

Pyrethroid resistance, particularly knockdown resistance (kdr), is widespread in insect pest populations, but rarely has kdr been associated with field-level pest control failure. The prevailing understanding is that kdr can contribute to a resistant phenotype, but this knowledge has remained largely an academic pursuit and has not translated to tools and strategies needed to make rapid decisions for effective resistance management. As a first step to providing agricultural producers, pest control and public health agencies the operational tools needed to improve resistance assessment and use this information for improved management, we developed robust assays using the high specificity of rhPCR to reduce the time by about 80% and costs of resistance assessment by about 75% from the traditional Sanger based method used to assess kdr in Musca domestica. The ability to get an accurate answer at the first test is an important consideration for the use of an operational, rather than academic, research tool, so we used nanopore sequencing to confirm genotypes in a subset of samples and found the first pass genotyping accuracy of rhPCR method to be 76.9% versus 41.9% with the traditional Sanger method. To demonstrate the broad applicability and comparability of screening for kdr SNPs using rhPCR, we conducted the largest assessment of kdr genotypes of M. domestica in United States dairy operations to date and found similar kdr patterns to other recent published studies using traditional methods.