Molecular diagnostic for detecting the acetylcholinesterase mutations in insecticide-resistant populations of Colorado potato beetle, Leptinotarsa decemlineata (Say)

Abstract

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. The serine to glycine change at position 291 (S291G) in the AChE gene was found previously in an azinphosmethyl-resistant strain of CPB (AZ-R). PCR–RFLP assays were used to monitor the frequency of the S291G resistance mutation in resistant field populations of CPB, Aliabad, Bahar, Dehpiaz, and Yengijeh. The S291G mutation was detected in 66.6, 73.3, 53.3 and 26.6% of Bahar, Dehpiaz, Aliabad and Yengijeh populations, respectively. Moreover, only 25% of samples from the resistant field populations were homozygous for S291G mutation. 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. PCR–SSCP method was used to find sequence variation in the AChE gene. Based on the published nucleotide sequence information on AChE gene (GenBank L41180.1), five primer pairs were designed to amplify specific PCR products of 306bp (first fragment: codons 24–142), 370bp (second fragment: codons 141–261), 403bp (third fragment: codons 248–381), 335bp (fourth fragment: codons 376–486), and 335bp (fifth fragment: codons 488–598). No specific PCR product of desirable size for either the fourth and fifth fragments could be obtained. The DNA amplification products were subjected to SSCP analysis to identify the DNA sequence variations between the susceptible strain and resistant populations. Ninety-five beetles of susceptible strain (15 beetles) and four field populations (20 of each) were screened. Allelic differences were detected by distinctive electrophoretic patterns of each single strand. Nucleotide sequence variations in the different SSCP patterns were verified by direct DNA sequencing. Alignment of the AChE gene sequences with the sequencing results revealed 16 point mutations (V44G, E128D, R140G, I143F, T248S, F250S, G251C, I261M, S265I, S291G, G353R, L356R, E366K, S378E, S378R, E382D) from the field populations of CPB, which may contribute to the AChE insensitivity. Further analysis on the RNA level is necessary for clarification and validation of this contention.