Multiple origins of cyclodiene insecticide resistance in Tribolium castaneum (Coleoptera : Tenebrionidae)

The number of origins of pesticide resistance-associated mutations is impor tant not only to our understanding of the evolution of resistance but also in modeling its spread. Previous studies of amplified esterase genes in a h ighly dispersive Culex mosquito have suggested that insecticide resistance- associated mutations (specifically a single-gene duplication event) can occ ur a single time and then spread throughout global populations. In order to provide data for resistance-associated point mutations, which are more typ ical of pesticide mechanisms as a whole, we studied the number of independe nt origins of cyclodiene insecticide resistance in the red flour beetle Tri bolium castaneum. Target-site insensitivity to cyclodienes is conferred by single point mutations in the gene Resistance to dieldrin (Rdl), which code s for a subunit of a gamma-aminobutyric acid (GABA) receptor. These point m utations are associated with replacements of alanine 302 which render the r eceptor insensitive to block by the insecticide. We collected 141 strains o f Tribolium worldwide and screened them for resistance. Twenty-four strains contained resistant individuals. After homozygosing 23 of these resistance alleles we derived a nucleotide sequence phylogeny of the resistant strain s from a 694-bp section of Rdl, encompassing exon 7 (which contains the res istance-associated mutation) and part of a flanking intron, The phylogeny a lso included six susceptible alleles chosen at random from a range of geogr aphical locations. Resistance alleles fell into six clades and three clades contained both resistant and susceptible alleles. Although statistical ana lysis provided support at only the 5-6% level, the pattern of variation in resistance alleles is more readily explained by multiple independent origin s of resistance than by spread of a single resistance-associated mutation. For example, two resistance alleles differed from two susceptible alleles o nly by the resistance-associated mutation itself, suggesting that they form the susceptible ancestors and that resistance arose independently in sever al susceptible backgrounds. This suggests that in Tribolium Rdl, de novo mu tations for resistance have arisen independently in several populations. Id entical alleles were found in geographically distant regions as well, also implying that some Rdl alleles have been exported in stored grain. These di fferences from the Culex study may stem both from differences in the popula tion genetics of Tribolium versus that of mosquitoes and differences in mut ation rates associated with point mutations versus gene duplication events, The Tribolium data therefore suggest that multiple origins of insecticide resistance (associated with specific point mutations) may be more common th an the spread of single events. These findings have implications for the wa y in which we model the evolution and spread of insecticide resistance gene s and also suggest that parallel adaptive substitutions may not be uncommon in phyletic evolution.