Duplication of the Rdl GABA receptor subunit gene in an insecticide-resistant aphid, Myzus persicae

Resistance to cyclodiene insecticides is associated with replacements of a single amino acid (alanine 302) in a gamma-aminobutyric acid (GABA) recepto r subunit encoded by the single-copy gene Resistance to dieldrin (Rdl). Ala nine 302 is predicted to reside within the second membrane-spanning region of the Rdl receptor, a region that is thought to line the integral chloride ion channel pore. In all cyclodiene-resistant insects studied to date, thi s same alanine residue is replaced either by a serine, or, in some resistan t strains of Drosophila simulans, a glycine residue. Therefore, individuals can carry only two different Rdl alleles. In contrast, here we report the presence of up to four different Rdl-like alleles in individual clones of t he green peach aphid, Myzus persicae. In addition to the wild-type copy of Rdl gene (encoding A302 or allele A), M. persicae carries three other allel es with the following amino acid replacements: A302 --> Glycine (allele G), A302 --> Serine(TCG) (allele S) and A302 --> Serine(AGT)(allelle S'). Evid ence from direct nucleotide sequencing and Single Stranded Conformational P olymorphism (SSCP) analysis shows that at least three of these different Rd l alleles (i.e. A, G and S) are commonly present in individual aphids or ap hid clones. Southern analysis using allele-specific probes and analysis of sequences downstream of the exon containing the resistance-associated mutat ion confirm the presence of two independent Rdl-like loci in M. persicae. O ne locus carries the susceptible alanine (A) and/or resistant glycine (G) a llele while the other carries the two serine alleles (S or S'). Whereas res istance levels are correlated with the glycine replacement, the S allele wa s present in all aphid clones, regardless of their resistance status. These results suggest that target site insensitivity is associated with replacem ents at the first (A/G) but not the second (S/S') locus. Phylogenetic analy sis of nucleotide sequences indicates that both putative aphid Rdl loci are monophyletic with respect to other insect Rdl genes and may have arisen th rough a recent gene duplication event. The implications of this duplication with respect to insecticide resistance and insect GABA receptor subunit di versity are discussed.