Regulatory mechanisms involved in the control of CYP6M2 gene in insecticide resistant Anopheles gambiae (Diptera: culicidae)

Abstract

Cytochrome P450s, including CYP6M2 gene, are involved in the detoxification of permethrin. Some of these genes are regulated by CnCC / dKeap 1 and Spineless / Tango in Drosophila melanogaster. This mechanism is yet to be identified in Anopheles gambiae. Thus, we examine whether there is differential regulation of CYP6M2 gene between permethrin resistant Tiassalé and susceptible Kisumu strains of An. gambiae. Bioinformatics analysis was used to search for cis-acting elements within CYP6M2 (896 bp) region hypothesised to contain the promoter. Isolated and cloned CYP6M2 promoter reporter constructs were transfected into Anopheles gambiae Sua 5.1* cells to measure luciferase activity as a surrogate promoter activity. The WHO adult bioassay was used to expose adult females of the permethrin resistant Tiassalé and susceptible Kisumu strains of An. gambiae to discriminating doses of 0.75% permethrin. Uncharacterised strains from Auyo (Auyo-Nigeria) selected to 4% DDT and 0.1% Bendiocarb as recommended by WHO were also studied. Total RNA was isolated from the respective selected and unselected strains of An. gambiae and cDNA synthesised. Semi and Real time quantitative PCR (qPCR) using SYBR® Green were used to determine the gene expression and regulation levels. Results established the presence of putative AGAP010259 (AhR) and AGAP005300 (Nf2e1) cis-acting elements within Anopheles gambiae CYP6M2 promoters in silico. Luciferase reporter gene assays revealed no promoter activity as confirmed by using CYP9M10 promoter from Culex quinquefasciatus with a known promoter activity as control. There is higher expression of Nf2e1 than AGAP010259 and a variable expression of CYP6M2 in all the insecticide selected individuals, which may potentially be associated with insecticide resistance. This study provides useful information on our understanding of the regulatory mechanisms involved in insecticide resistance. These results have implications for the control of mosquito populations and the global spread of human, livestock and poultry diseases.

Date of Award	Dec 2014
Language	English
Awarding Institution	Abertay University
Sponsors	Tertiary Education Trust Fund (TETFUND) & University of Abuja
Supervisor	Yusuf Y. Deeni (Supervisor)