Biochemical and molecular analyses of acetylcholinesterase from organophosphate-susceptible and resistant clones of the greenbug, Schizaphis graminum (Homoptera: Aphididae)

The structure-toxicity relationship of selected organophosphate (OP) insecticides was investigated in an OP-susceptible (OSS) and three resistant (OR-0, OR-1 and OR-2) clones of the greenbug, Schizaphis graminum (Rondani). The three OR clones were significantly resistant to all seven OPs examined (3- to 329-fold increase in resistance). Inhibition of acetylcholinesterase (ACNE) using both crude enzyme preparations and purified ACNE revealed that AChEs from the OR clones were significantly less sensitive to inhibition by six OP-oxon analogues (1.1- to 4.6-fold for crude ACNE from all three OR clones and 1.1- to 12.8-fold for ACNE purified from the OR-0 clone). ACNE from the OR clones also showed 1.8- to 2.3-fold higher specific activity than ACNE from the OSS clone in crude enzyme preparations. Results suggest that ACNE, with both reduced sensitivity and increased activity, is responsible for (e.g., OR-0) or contributes to (e.g., OR-1 and OR-2) OP resistance in the greenbug.; A full-length ACNE cDNA was isolated from a library constructed from the OSS greenbug clone. The cDNA (3,283 bp) contained a 2,028-by open reading frame encoding 676 amino acid residues. The deduced amino acid sequence was most similar (43% identity) to AChE1 of the nematode, Caenorhabditis elegans, and showed all the major features of ACNE. Sequence analysis of the ACNE cDNA fragments amplified from all three OR clones by RT-PCR did not show any difference from that of the OSS clone within the ACNE coding regions. Northern blot analysis of mRNA revealed a 3.7-kb transcript in all four clones and approximately 1.5-fold higher amount of mRNA in three OR clones than that in the OSS clone. However, Southern blot analysis suggested a single copy of this ACNE gene in the OSS and all three OR clones. Thus, the increased ACNE activity in the OR clones appeared to be due to increased expression of the ACNE gene, but the increased gene expression did not seem to be due to gene amplification. It is most likely that increased ACNE gene expression in all three OR clones is caused by increased rate of gene transcription and/or increased stability of ACNE mRNA.