5042
Manal Magdy Eldin Mohamed Ghanem
Biochemical and toxicological studies on esterases-as insecticide detoxifying enzymes-in entomopathogenic nematodes
Acetylcholinesterase (AChE), Detoxifying enzymes, Entomopathogenic nematodes, Heterorhabditis bacteriophora, Inhibition, Insecticides, Purification
Detoxifying enzymes including glutathione-S-transferases (GSTs), acetylcholinesterases (AChEs), carboxylesterases (CoEs), using two substrates α-naphthyl and P-nitrophenyl acetates, were investigated in the IJs of six different strains of heterorhabditid nematodes. They are ranged from 22.2-64.6, 158-550, 46.2-135.6 and 60-275 units/105 IJs, respectively. Heterorhabditis bacteriophora EM2 has the highest enzyme levels. AChE is the predominant detoxifying enzyme and might play the most important role in the detoxification of insecticide(s). Two AChEs, AChEAII and AChEBI, are purified to homogeneity with specific activities 1207 and 1560 units/mg protein,respectively. The native molecular weights were found to be 180 and 68 kDa, and confirmed by SDS-PAGE where they are found in dimeric and monomeric forms, respectively. Both isoenzymes showed optimum activity at pH 8.5 and 35oC. Their enzymatic activities were completely lost by 1mM Hg+2 and Ni+2 and greatly enhanced up to 6.1-fold by Mn+2. The substrate specificity, the relative efficiency ofsubstrates hydrolysis, substrate inhibition and inhibition specificity clearly indicated that they are true AChEs. Kinetics and toxicological characteristics in vitro for AChEs by different insecticides revealed that the median inhibition concentrations (IC50) ranged from 1.25 to 23 mM and the potency of resistance against different insecticides could be arranged as methomyl> acetamiprid> carbofuran> eserine> deltamethrin> oxamyl> malathion. Except for malathion, they are competitively inhibited AChEs with Ki values ranged from 0.10 to 15 mM. Antiserum against the promising AChEBI has been prepared. It cross-reacted with AChEs ofH. bacteriophora EM2 and EM1 with 88 and 43%, respectively. The results strengthen the possibility that H. bacteriophora EM2 could be defined as insecticide(s)-resistant strain. The levels of detoxifying enzymes and insensitive AChEs represented as mechanisms of environment adaptation and contributed in insecticide resistance. Anti-AChEBI could be used as a marker for identification insecticide(s)-resistant EPN strain. The kinetics and inhibition kinetic parameters were compared with those earlier reported for different insect species as target hosts for H. bacteriophora EM2.
2015
Ph.d
Helwan
Science