Investigation of cis-regulation of metabolic insecticide resistance related gene expression in Anopheles gambiae using allelic imbalance
Malaria control relies on insecticides targeting the mosquito vector, but is being increasingly compromised by insecticide resistance. Elevated expression of metabolic enzymes frequently drives resistance. In diploids, gene expression is regulated both in cis, by regulatory sequences on the same chromosome, and by trans acting factors, affecting both alleles equally. Differing levels of transcription can be caused by mutations in cis-regulatory modules, but few cis-regulatory modules controlling the expression of genes that determine insecticide resistance have been identified. Genes potentially under differential cis-regulation between bendiocarb resistant and susceptible Anopheles gambiae strains were identified by counting transcripts produced from maternal and paternal alleles in F1 hybrids of these strains (allelic specific expression). Cis regulatory module sequences controlling gene expression in insecticide resistance relevant tissues such as midgut, Malpighian tubules and legs were predicted using a previously established machine learning method. These predictions included CRM proximal to both genes under differential cis regulation and genes that show consistent differential expression patterns in multiple resistant Anopheles strains. Overall design: Crosses were performed between Anopheles gambiae  insecticide susceptible Kisumu strain (origin Kisumu, Kenya, 1970) and bendiocarb and pyrethroid resistant Anopheles gambiae Nagongera strain (origin Tororo, Uganda, 2013). In three crosses the mother was from Nagongera strain (3 biological replicates Wilding_1, Wilding_2 and Wilding_3) and in the other three then mother was from Kisumu strain (3 biological replicates Wilding_4, Wilding_5 and Wilding_6). RNA was extracted from a pool of 10 whole sugar fed adult F1 female progeny from each cross, 3-5 days after eclosion. All mosquitoes were raised in an insectary with temparature 26°C and 70% relative humidity. We first compared the gene expression between the reciprocal crosses (mother from Kisumu strain  vs mother from Nagongera strain). We then analyzed expression of genes from each parental allele within each sample (allele specific expression)