Deciphering pyrethroid resistance in Cx. pipiens (L): Implications of cytochrome P450; expression profiling and regulatory microRNA

Abstract

Over the past decades, the extensive use of pyrethroids insecticides for vector control has resulted in the development of insecticide resistance. Cytochrome P450 has been recognized to play a critical role in the metabolic detoxification of insecticides. In the current study, Culex pipiens mosquitoes were collected from Giza Governorate in Egypt and tested for insecticide susceptibility against deltamethrin. First detection of Knockdown resistance gene (Kdr) mutations in field collected mosquitoes was performed. Activities of cytochrome oxidase P450 detoxification enzyme that synchronized with the resistance development, was assessed. Expression profiles of cytochrome P450s and their putative corresponding regulating miRNAs, which was previously reported in Cx. pipiens pallens were evaluated in pyrethroid resistant field-collected Cx. pipiens using RT-qPCR and stem-loop RT-qPCR, respectively. Specific stem-loop reverse transcription primers and forward primers were designed for miRNAs profiling. Our results elucidated the pyrethroid resistance development and revealed its relation to the metabolic and target site modification mechanisms with a first report of L1014F-kdr mutation detection. RT-qPCR results have showed an up-regulation in the expression of the studied P450 transcripts. Negative correlations were found between the expression of P450s and their regulatory miRNAs except for CYP9J35, where positive correlation was found with its corresponding miR-13. Interestingly, our data was the first to detect negative correlation between miR-285 and its putative CYP6Cp1 target gene. These findings highlighted the significance of identifying P450 gene along with regulatory miRNAs as a key mechanism implicated in pyrethroid resistance in field Culex vector population. The elucidation of this mechanism would shed light on the development of insecticide resistance and would help in shaping strategies to combat such vectors.