Molecular Approaches to Insect Resistance Management

Abstract

Three DNA-based genotyping techniques, bi-directional PCR amplification of specific allele (bi-PASA), single stranded conformational polymorphism (SSCP) and minisequencing, have been developed and compared for the detection of the S291G (insensitive acetylcholinesterase) and L1014F (insensitive sodium channel) mutations associated with azinphosmethyl and permethrin resistance, respectively, in the Colorado potato beetle (CPB). Extraction of genomic DNA from individual neonates that were hatched from previously collected egg masses is the most efficient and reliable means to obtain suitable templates in terms of convenience, economy, speed, and DNA quality. bi-PASA, employing two allele-specific primers, appears to be the most efficient and rapid genotyping method for the simultaneous detection of both resistant/susceptible homozygous (SS, RR) and heterozygous (SR) alleles. Its resolution, however, is strongly dependent on the quality of template genomic DNA. SSCP also allows unambiguous genotyping, including the detection of heterozygous alleles, is less dependent on template DNA quality, however, it requires a longer processing time. Minisequencing is amenable to a 96-well microtiter plate format for the processing of a large number of samples and allows direct detection of resistant/susceptible homozygous alleles but is not as efficient as the PASA and SSCP in detecting heterozygous alleles. In considering the advantages and disadvantages of each technique, DNA-based genotyping is best employed in combinations, with the bi-PASA as the primary method and the SSCP and minisequencing as the secondary validating methods. These methods are rugged, rapid, cost-effective and capable of resolving SS, RR and SR individuals. The availability of such DNA-based genotyping techniques, using neonate genomic DNA as templates, will enable the precise monitoring of the resistant and susceptible allele frequencies, including those of heterozygote individuals, in field populations of CPB. Additionally, an immunoassay based on antibodies raised against esterases associated with resistance has been developed and is considered to be more efficient than a bioassay or enzyme assay because of its higher sensitivity and specificity.