Insecticide-resistant populations of Colorado potato beetle (CPB), with insensitive acetylcholinesterase (AChE) have recently been reported from commercial potato fields of Hamedan province in west of Iran. The objective of this study was to clarify the molecular mechanism of this insensitivity. T-ARMS PCR assays were used to monitor the frequency of the R30K and I392T resistance mutations in resistant field populations of CPB, Aliabad, Bahar, Dehpiaz, and Yengijeh. Only 21/6% and 1/66% of samples from the resistant field populations were homozygous for R30K and I392T mutations, respectively. There was no significant correlation between the mutation frequencies and resistance levels in the resistant populations, indicating that other mutations may contribute to this variation. Based on the published nucleotide sequence information on AChE gene (GenBank L41180.1), two primer pairs were designed to amplify specific PCR products of 1067 bp (first fragment: codons 1 through 348), and 537 bp (second fragment: codons 420 through 590). Alignment of the AChE gene sequences with the sequencing results revealed 64 point mutations from the field populations of CPB, that likely contribute to the AChE insensitivity.
