Susceptibility And Its Mechanisms Of Different Populations Of Ditylenchus Destructor To Acetylcholinesterase Inhibitors

Acetylcholinesterase (AChE, EC 3.1.1.7) catalyzes the hydrolysis of the neurotransmitter acetylcholine at cholinergic synapses to keep the normal transmission of nerve impulses. It is the target of the organophosphate and carbamate compounds. When AChE is inhibited, acetylcholine builds up in the synapse, causing hyperexcitation and then desensitization of the muscle receptors, resulting in paralysis. Intensive use of these pesticides over the past years has led to the development of resistance in many target species. The efficacy of nematicides on the control of Ditylenchus destructor showed the tendency of decrease in the northern areas of China in the recent years. This study difinitizes the level of resistance of D. destructor to aldicarb, ethoprophos and abamectin, provides the molecular evidence that over-expression of AChE and AChE genes was associated with the resistance in D. destructor, with the evidences from toxicity assay, determination of enzyme activity, induction of nematicides to D. destructor, and mRNA expression of AChE genes.1. Four populations of Ditylenchus destructor were collected from different areas of Hebei Province and their susceptibilities to 3 different types of nematicides were tested with contact assays and sand column methods in laboratory. The contact assays results showed that the Changli populations were more sensitive to aldicarb and ethoprophos, the values of LC₅₀ were 891.73 mg/L and 743.82 mg/L, respectively. The other populations had lower susceptibilities to aldicarb and ethoprophos. The sus- ceptibilities of the three populations had 3.5～4.0 fold difference to aldicarb, and 1.6～3.8 fold to ethoprophos. The susceptibilities of the four populations to abamectin were no significant difference. The results of sand column assays showed that the movement of nematode could be inhibited by 5mg/L aldicarb and 50mg/L ethoprophos. The inhibition of 50 mg/L abamectin on nematodes movement was lower than that of aldicarb and ethoprophos.2. Different populations had different tolerance to salt concentration and temperature. ChangLi population had rather high tolerance at 46℃than that of other population. Funing population survived better than those from other area in 3%～5% salt solution.3. The AChE properties of four populations of D. destructor and susceptibilities to inhibitors were investigated. AChE was extracted in a direct method and ultracentrifugetion in sucrose gradients. AChE activities were determined by the amended spectrophotometric method of Ellman. Effect of inhibitors on AChE hydrolysis was analyzed. The results showed that there are two major class AChEs in acetylcholine hydrolysis at least. One class of AChE is highly sensitive to eserine (IC₅₀<10^-6 mol/L) and other class is less sensitive to eserine (IC₅₀>10^-4 mol/L). Inhibition of AChE activity of Changli population was higher than that of Zhuozhou, Lulong and Funing populations at the concentration 10^-6-10^-10 mol/L of eserine and at 10^-6-10^-10 mol/L of aldicarb. This susceptibilities difference of AChE of different populations to inhibitors may be due to the polymorphism of AChE and the multiple changes of different molecular polymorphism.4. Three new cDNA, named Dd-ace-1 (EF583059), Dd-ace-2 (EF583057) and Dd-ace-3 (EF583058) encoding acetylcholinesterases were isolated from D. destructor. The three full length cDNA, carrying the transspliced SL1 leader sequence, are 2196bp, 2425bp and 2517bp long with an open reading frame of 1908bp, 2205bp and 1836bp encoding 635, 734 and 611 amino acid residues, respectively. All of them shared of the characteristics of the AChE, the catalytic triads, the choline binding sit, 3 intra-chain disulfide bridges and aromatic residues lining the catalytic gorge. Phylogenetic analysis based on other nematodes and species AChEs showed that the deduced Dd-ACE-1, Dd-ACE-2 and Dd-ACE-3 formed a cluster with ACE-1s, ACE-2s and ACE-3s, respectively.5. Using the real-time quantitative PCR, the mRNA expression of three AChE genes in Funing, Lulong and Zhuozhou population was compared with those in Changli population. Expression level of AChE genes in Funing, Lulong and Zhuozhou population was higher than Changli population at different level. It provided the molecular evidence that over-expression of AChE genes was associated with decrease of susceptibilities of D. destructor to inhibitors.6. The induction of inhibitor to susceptive population provided stronger evidence that the level of AChE mRNA expression could be significantly enhanced after treated with sand column soaked with aldicarb and ethoprophos. But the susceptibilities of D. destructor to inhibitors could not be affected by the changes of expression of mRNA. The changing of movement percentage of different populations in sand column was observed.