Biochemical And Toxicological Properties And CDNA Cloning Of Acetylcholinesterase In Tribolium Castaneum (Herbst)

Acetylcholinesterase (AChE; EC 3.1.1.7) is a key enzyme catalyzing the hydrolysis of the neurotransmitter acetylcholine in the nervous system, thereby ending transmission of nerve impulses at cholinergic synapses, and AChE is the target of two major insecticide families, organophosphates (OPs) and carbamates. To elucidate the role of AChE and the mechanism of AChE-mediated resistance, the biochemical and toxicological characterizations and gene cloning of AChE were investigated in the resistant (HSTC-R and HYTC-R) and susceptible strains (ABTC-S) of Tribolium castaneum (Herbst). This study was supported by theiprogram for New Century Excellent Talents in University(NCET-04-0854), and main results were summarized as follows:The biochemical and toxicological characterizations of AChE in 10-d larvae, 20-d larvae, pupae and adults of T. castaneum were investigated by the microplate reader method. The results showed that compared to their susceptible counterparts, the contents of protein in 10-d and 20-d larvae of the two resistance strains were significantly higher than that of the susceptible strain (P＜0.05). The activities per insect and specific activities of AChE in immature period (10-d larvae, 20-d larvae and pupae) of HSTC-R strain and HYTC-R strain were significantly reduced (P＜0.05). The comparison of the apparent Michaelis constant (K_m) showed that larvae period of the resistance strains increased significantly, indicating that AChE might have changed in quality. However, AChE K_m of 10-d larvae of HYTC-R strain has no obvious change. The maximal velocities (V_max) in immature period of HSTC-R strain increased significantly (P＜0.05), showing that the catalytic activity of AChE in HSTC-R strain was significantly higher than that in the susceptible strain. The V_max values in HYTC-R strain were increased little, but not significant. The inhibition kinetics of 5 inhibitors (Eserine, malaoxon, carbaryl, paraoxon-ethyl and demeton-S-methyl) to AChE in vitro revealed that the sensitivities of the resistance strains AChE to those inhibitors were reduced, furthermore, HSTC-R strain was more insensitive. It could be concluded that the target resistance existed in the two resistant strains of T. castaneum, and the resistance was correlated with the quality change of AChE. The structure modification leads to reduced affinity to substrate and insensitivity to insecticides.To elucidate the molecular mechanism of AChE-mediated resistance, two cDNA fragments of AChE gene in T. castaneum were amplified with the degenerate primers from the conserved peptide sequences of AChEs in insect and acari species by reverse transcription-polymerase chain reaction (RT-PCR) method. Two fragments of 150 bp (GenBank accession number: EF532798) and 207 bp were obtained. The 150 bp deduced amino acid sequence of AChE consisted of 50 amino acid residues, which shared numerous similarities with AChE of Leptinotarsa decemlineata AChE precursor (90%, Q27677), Cydia pomonella AChE2 (88%, ABB76665), Bombyx mori AChE typeⅠ(88%, BAF33337), Plutella xylostella AChE (88%, AAL33820), Helicoverpa assulta AChE (88%, AAV65638), H. armigera AChE (88%, AAM9Q333) and Torpedo californica AChE (56%, P04058). The sequence analysis indicated there is high degree of amino acid sequence homology between T. castaneum and other species. In addition, it included several characteristic sequences of AChE. Hence, it could be concluded that the fragment encoded the AChE in T. castaneum. However, The 207 bp deduced amino acid sequence of AChE consisted of 69 amino acid residues, which shared no similarities with AChE of other species, hence, it could be concluded that the fragment encoded the AChE in T. castaneum. The phenomenon may result from degeneracy of primer.The biochemical results indicated that the resistances of T. castaneum were linked with AChE. The findings had great theoretical and practical significance in the physiological and biochemical mechanisms to insecticides, the cross-resistance among OPs and non-OPs, the strategies for the delaying and management of resistance. In addition, the cDNA cloning of AChE fragment in T. castaneum has-provided some basic information on the complete sequence and the molecular mechanism of altered AChE. This will contribute to development of molecular diagnostic technique for T. castaneum resistance in field, further understanding of the molecular toxicological characteristics, and paving the way for designing new insecticides and developing new strategies for pest management.