Molecular analysis of kdr-like resistance in a permethrin-resistant strain of Colorado potato beetle

Abstract

Resistance to the rapid paralytic effects of pyrethroids in the permethrin-resistant (PE-R) strain of the Colorado potato beetle (CPB) is phenotypically similar to that of the kdr trait of the house fly, which is caused by a single amino acid substitution (L1014F) in the Vssc1 voltage-sensitive sodium channel gene of that species. To test the hypothesis that the kdr-like trait of the PE-R strain of the CPB is caused by a similar mechanism, we amplified a 370-bp segment of the CPB ortholog of the house fly Vssc1 gene and employed that segment as a hybridization probe in a restriction fragment length polymorphism (RFLP) analysis of the linkage between this CPB sodium channel gene and the kdr-like resistance trait. These experiments identified an EcoRI RFLP that was specifically associated with the resistance trait when present, but this analysis was confounded by the absence of the diagnostic EcoRI fragments in all analyzed individuals of either the PE-R or the susceptible (SS) strain and by the presence in some individuals of an additional EcoRI polymorphism that was not correlated with resistance. To identify sodium channel sequence polymorphisms associated with the kdr-like resistance trait, we determined the sequence of cDNA clones comprising a 3262-bp segment of the CPB sodium channel gene that included five sites at which resistance-associated polymorphisms have been identified in other insect species. Sequence analyses of individual CPB sodium channel cDNAs revealed a single amino acid substitution in the PE-R strain corresponding to the L1014F mutation in the house fly Vssc1 sodium channel. Direct comparison of cDNA and genomic DNA sequences from individual insects showed that whereas some SS individuals carried both resistant and susceptible alleles, only the wildtype allele was detectably transcribed. These results imply that a mutation in the CPB sodium channel gene corresponding to the L1014F mutation found in kdr house flies is the cause of the kdr-like resistance trait in the PE-R strain.