The nicotinic acetylcholine receptor subunit Mdα6 from Musca domestica is diversified via post-transcriptional modification

Recent studies showed that deletion of a nicotinic acetylcholine receptor (nAChR) subunit gene, D alpha 6 in Drosophila melanogaster results in a strain that is resistant to spinosad, indicating that D alpha 6 is important for the toxic action of this insecticide. To determine if spinosad resistance in house flies was due to a mutation(s) of Md alpha 6 (the orthologue of D alpha 6 from house flies), cDNAs were cloned and characterized from an insecticide-susceptible and a spinosad-resistant strain of the house fly, Musca domestica. The cDNAs contain a 1470-bp open reading frame encoding 490 amino acid residues, 415-bp 5' untranslated region (UTR) and a polymorphic 3'-UTR of -371 bp. The predicted mature protein possesses 468 amino acid residues, has the typical features of a nAChR a subunit and is 97% identical to Da6. Quantitative real-time PCR analysis revealed that Md alpha 6 was expressed in the head and the thorax at 1300- and 26-fold higher levels, respectively, than in the abdomen. There was no difference in the expression level of Md alpha 6 between spinosad-resistant and susceptible strains. Ten isoforms arising from alternative splicing were characterized, with isoform II being most common. A-to-I RNA editing was examined and found at 12 sites: editing at 11 of these sites resulted in an amino acid substitution. Md alpha 6 is linked to autosome 1 (spinosad resistance was previously shown to be linked to autosome 1). Single nucleotide polymorphisms, alternative splicing, mRNA levels and A-to-I RNA editing were compared between head and thorax and between insecticide- susceptible and spinosad-resistant strains. These comparisons indicate that Md alpha 6 is not responsible for spinosad resistance in house flies.