Toxicological Properties Of Acetylcholinesterase In Model Insect Tribolium Castaneum (Herbst)

Tribolium castaneum(Herbst)(Coleoptera:Tenebrionidae) is a most important stored grain insect pest,which directly or indirectly causes a great deal of economic loss.Meanwhile,as a general model organism,T.castaneum has important research status in genetics,immunology,and other research fields.In 2008,the complete nucleotide sequence of T.castaneum was obtained by the international research group of 64 research teams from 14 countries.The results showed that the genome of T.castaneum includes approximate 2 billion nucleotide sequences and 16000 proteins, providing great help to the study of insect molecular ecology,toxicology,functional genomics,etc.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.AChE is the target of two major insecticide families,organophosphates (OPs) and carbamates.This project was undertaken to elucidate the toxicological role of AChE in T. castaneum.The study was supported by the program for New Century Excellent Talents in University(NCET-04-0854) and the National Natural Science Foundation of China(30570231).The main results are as follows:1 The toxicities of seven insecticides against T.castaneumThe efficacies of seven insecticides(including dichlorvos,aldicarb,carbosulfan, demeton-S-methyl,paraoxon-ethyl,abamectin and omethoate) against adults of T.castaneum were determined by residual insecticide-film method.The results showed that LC₅₀ of these insecticides against T.castaneum varied obiviously.Based on the linear regression between log dosage-probit, the toxicicities of organophosphorus insecticides(OPs) expressed better than carbamate insecticides. Among organophosphorus insecticides,paraoxon-ethyl was most efficient with least LC₅₀ of 295.31 mg/L.The contact toxicity of abamectin also exhibited good efficacy.For carbamate insecticides, the effects of aldicarb and carbosulfan were not ideal.The above results suggested that when miture application of different insecticides is applied,organophosphorus insecticides should be the main part in practice,and if so T.castaneum could be effectively controlled.2 Biochemical toxicology of AChE in T.castaneum2.1 Comparison of acetylcholinesterase activitiesAChE activity assay on T.castaneum revealed that head expressed highest and lowest value was recorded in abdomen.The protein content analysis showed a similar result.Compared by the results of K_m and V_m,the head of T.castaneum possess best affinity toward ATChI,moreover,the catalytic ability of it is better than that of the thorax and the abdomen.AChE from the head of this insect can be representative when carried out related research.It is better to choose the head and the thorax to do research about AChE of T.castaneum;according to the need, the abdomen can be made choice for selection.2.2 In vitro inhibition of insecticides to AChEThe inhibition of AChE in T.castaneum by dimedthyl paraoxon,propoxur,physostigmine, carbaryl and other inhibitors was studied.The results revealed that these insecticides showed strong inhibitory effects to ACHE,especially for organophosphorus insecticides(OPs),among which paraoxon-ethyl,demeton-S-methyl and dimedthyl paraoxon shows better effects.Carbamate insecticides play a considerable inhibitory effect on ACHE,and propoxur is better than carbosulfan. Omethoate and chlorpyrifos exhibited poor inhibitory effects on ACHE.3 Electrophoretic analysis of esterases and protein composition in T.castaneumProtein compositions of the head,thorax and abdomen of adults of T.castaneum were separated by SDS-PAGE and stained with coomassie brilliantblue R-250.The protein content in thorax was higher than those of head and abdomen,and the bands had some difference among them.By polyacrylamide gel electrophoresis(PAGE),esterase zymogram of three parts of T. castaneum was analyzed.The esterase isozyme of three parts were different from each other.In T. castaneum,the esterase isozyme was made up of mainly 2 bands- EST1 and EST2,and the content of EST1 is highest.Furthermore,the molecular weight(MW) of isozymes was greatly different.For the head,the proportion of higher MW is great;while for the thorax,the composing of lower MW were major.The inhibition pattern was assayed by PAGE.The order of their efficacy was:paraoxon＞chlorpyrifos＞carbosulfan＞malathion.The esterase isozyme of the head was relatively more sensitive than others.4 Molecular cloning and sequence analysis of cDNAs of AChE in T.castaneumAChE insensitivity is a frequent resistance mechanism in insects.To elucidate the molecular mechanism of AChE-mediated resistance,1682bp cDNA fragment of AChE gene in T.castaneum was generated by gene splicing method.This fragment consisted of 151 nucleotide acid of 5' untranslated regions(UTR) and an open reading frame of 1531bp nucleotides.Sequence Analysis of this fragment by the web of NCBI it was similar to predicted acetvlcholinesterase from T.castaneum. The corresponding codes to 3'-end also could be found.Then the full-length gene of AChE was generated by gene splicing method.The predicted amino acids contained all the typical residues of ACHE:the three residues(S256, E385,H498) that putatively form the catalytic triad,the oxy-anino hole(G169,G170,A257),and the sequence FGESAG,the conserved aromatic residues(W123,W165,Y181,W289,W348,F389, F389,Y392,W490) compared to Torpedo californica ACHE.Molecular phylogenetic tree of 51 AChE amino acid sequences from 37 species were constructed.Results showed that all AChE genes were divided into four distinct main groups:Insect Typeâ… AChE Gene,Vertebrate AChE Gene,Insect Typeâ…¡AChE Gene and Nematode AChE Gene. The AChE Gene from T.castaneum is an orthologous gene to D.melanogaster,that is,the Typeâ…¡AChE Gene.