Distribution of Insecticide Resistance Genetic Markers in the West Nile Virus Vector Culex pipiens from South-Eastern Romania

SIMPLE SUMMARY: Culex pipiens mosquitoes are the vectors of West Nile virus in south-eastern Romania, a region where significant outbreaks of infection with this virus have occurred since 1996. The mosquito control strategy in Romania consists of using chemical insecticides, usually after the onset of the first human infection cases, but with limited impact. The level of insecticide resistance in the mosquito populations in the investigated area has not been assessed previously. We screened mosquitoes for mutations associated with resistance to the most used categories of insecticides: organophosphates, carbamates, and pyrethroids. Prior to this, the biotype of each mosquito specimen was determined. Low-frequency resistance mutations to organophosphates and carbamates were detected in Culex pipiens molestus mosquitoes collected in urban areas. High frequencies of pyrethroid resistance mutations were found in the pipiens and molestus biotypes populations and also in the hybrids collected in urban and in intensive agriculture areas. We recommend limiting the use of pyrethroids, when possible, and continuous monitoring of insecticide resistance mutations in mosquito populations in south-eastern Romania. ABSTRACT: Culex pipiens pipiens and Culex pipiens molestus mosquitoes are the vectors of West Nile virus in south-eastern Romania, an area of intense circulation and human transmission of this virus. The level of insecticide resistance for the mosquito populations in the region has not been previously assessed. Culex pipiens mosquitoes collected between 2018 and 2019 in south-eastern Romania from different habitats were subjected to biotype identification by real-time PCR. Substitutions causing resistance to organophosphates and carbamates (F290V and G119S in acetylcholinesterase 1) and to pyrethroids (L1014F in voltage gated Na(+) channel) were screened by PCR or sequencing. Substitutions F290V and G119S were detected at very low frequencies and only in heterozygous state in Culex pipiens molestus biotype specimens collected in urban areas. The molestus biotype population analysed was entirely homozygous for L1014F, and high frequencies of this substitution were also found for pipiens biotype and hybrid mosquitoes collected in urban and in intensive agriculture areas. Reducing the selective pressure by limiting the use of pyrethroid insecticides only for regions where it is absolutely necessary and monitoring L1014F mutation should be taken into consideration when implementing vector control strategies.