A NEW MULTIPLEX SNP GENOTYPING ASSAY TO SIMUTANEOUSLY SCREEN FOR EIGHT VOLTAGE-GATED SODIUM CHANNEL MUTATIONS IN AEDES AEGYPTI

Abstract

Aedes aegypti has been implicated as the vector responsible for transmission of dengue, chikungunya, and Zika viruses during disease outbreaks in Florida within the past 15 years. Recently, locally acquired dengue cases have increased dramatically, with more than 450 cases documented in Florida since 2019. This mosquito is known to be resistant to pyrethroid-based insecticides in Florida. Resistance of insects to pyrethroids due to knockdown resistance (kdr) is the result of single nucleotide polymorphisms (SNPs) in the voltage-gated sodium channel gene (vgsc). Recently, two novel SNPs, F174I and E478K, and four known SNPs, V410L, S723T, D1763Y, and Q1853R were reported circulating in Floridian Ae. aegypti populations for the first time. The present study provides a more comprehensive estimate of these SNP frequencies through the screening of a larger number of Floridian Ae. aegypti samples using a new custom multiplex SNP assay we developed using the Agena Biosciences iPLEX Assay platform to facilitate the rapid screening of multiple SNPs at an affordable cost. Our assay was successful in screening 162 Ae. aegypti mosquitoes for 8 SNPs from 4 counties in Southern Florida (Broward, Collier, Palm Beach, and Monroe Counties). This new assay can be used for studies examining the association between genetic mutations and pyrethroid-resistant phenotypes in Ae. aegypti populations such as increased time of survival after insecticide exposure.