Purification and characterization of acetylcholinesterase in Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae).

Red palm weevil (RPW), Rhynchophorus ferrugineus, is one of the most destructive pests of cultivated palm trees. The application of synthetic insecticides is currently a main strategy for RPW control. In this study we estimated the distribution of acetylcholinesterase (AChE), as a detoxifying enzyme and the target site of inhibition by insecticides, using ASChI as substrate in different organs of the pest including whole gut, cuticle, fat body, head and haemolymph. The activity ranged from 314.9 to 3868 U in individual organs while the specific activity ranged from 99 to 340.8 U/mg proteins; the cuticle had the highest enzyme level. During larval development, the 11th instar larvae had the highest enzyme content with 5630 U in the cuticle, with a specific activity of 140 U/mg protein. The two major AChE isoenzymes were purified by chromatography on gel filtration and ion exchange columns. They had specific activities of 3504.3 and 2979 U/mg protein, molecular weights of 33 and 54 kDa and activation energies of 8.3 and 4.4 kcal/mol, respectively. Both isoenzymes had monomeric forms, optimum activity at pH 8.0 and 40 °C, were completely inhibited by Hg2+ and Cu2 and showed similar trends towards the inhibitors eserine, BW284C51 and iso-OMPA. The catalytic properties were compared with those previously recorded for different insect species. This work will pave the way for more studies for improving the understanding of insecticide resistance and developing the field application of synthetic insecticides for controlling R. ferrugineus to ensure successful application.

Acetylcholinesterases from entomopathogenic nematode Heterorhabditid bacteriophora: Susceptibility to insecticides and immunological characteristics.

Acetylcholinesterases (AChEs) from the infective juveniles (IJs) of entomopathogenic nematode (EPN) have been investigated with respect to their susceptibility to insecticides and immunological characteristics, aiming at nominating the most compatible insecticide(s) to be used in conjunction with the most insecticide-tolerant EPN strain before incorporation in integrated pest management (IPM) programs. The inhibition kinetics of two purified AChE isoenzymes, AChEAII and AChEBI isolated from Heterorhabditid bacteriophora EM2 strain, by different insecticides revealed that the insensitivity to inhibition by such insecticides could be arranged in a descending order as; methomyl>carbofuran>acetamiprid>oxamyl>malathion. Except for malathion, the insecticides competitively inhibited AChEs with Ki values ranging from 0.1 to 15mM and IC50 values from 1.25 to 23mM. The two AChE isoforms are several folds less sensitive to inhibition by methomyl and carbofuran compared to those previously reported for other insect species. AChEBI was used as an immunogen to raise anti-AChEBI antisera in rabbits. The prepared antisera cross-reacted with AChEs of five different heterorhabditid nematode strains implying the presence of common epitopes shared along all the examined strains. Such studies could aid in the rational selection of the compatible insecticide(s) and the prepared polyclonal anti-AChE antisera would be a valuable immunodiagnostic tool for evaluating the most insecticide-tolerant EPN strain(s) in IPM programs.

The activity of detoxifying enzymes in the infective juveniles of Heterorhabditis bacteriophora strains: Purification and characterization of two acetylcholinesterases.

The infectivity and detoxifying enzyme activities including glutathione-S-transferase (GST), acetylcholinesterase (AChE) and carboxylesterase (CaE) are investigated in the infective juveniles (IJs) of six different strains of Heterorhabditis bacteriophora as a biocontrol agent against insect pests. The specific activities ranged from 10.8-29.8 and 50-220units/mg protein for GST and AChE, respectively; and from 24.7-129 and 22.6-77.3units/mg protein for CaE as estimated by P-nitrophenyl and α-naphthyl acetates, respectively. H. bacteriophora EM2 strain has the highest infectivity and the highest enzymatic activities as well. AChE is the predominant detoxifying enzyme that might imply its major role in the detoxification of insecticide(s). The isoenzyme pattern demonstrated two major slow-moving isoforms in all EPN strains examined. Purification of two AChE isoforms, AChEAII and AChEBI, from H. bacteriophora EM2 strain is performed by ammonium sulfate precipitation, gel filtration on Sephacryl S-200 and chromatography on DEAE-Sepharose. AChEAII and AChEBII have specific activities of 1207 and 1560unit/mg protein, native molecular weights of 180 and 68kDa, and are found in dimeric and monomeric forms, respectively. Both isoforms showed optimum activity at pH8.5 and 35°C. AChEBI exhibited higher thermal stability and higher activation energy than AChEAII. The enzymatic activities of purified AChEs are completely inhibited by Hg(+2) and Ni(+2) and greatly enhanced by Mn(+2). The substrate specificity, the relative efficiency of substrates hydrolysis, substrate inhibition and inhibition by BW284C51, but not by iso-OMPA, clearly indicated that they are true AChEs; their properties are compared with those recorded for insects as target hosts for H. bacteriophora EM2.

Purification and characterization of an acetylcholinesterase from the infective juveniles of Heterorhabditis bacteriophora.

Acetylcholinesterases (AChEs) have been estimated in the infective juveniles (IJs) of eight different strains of heterorhabditid nematodes. The enzyme content ranged from 45.6 to 421.3 units/10(5) IJs with specific activity 34.0 to 82.6 units/mg protein. The isoenzyme patterns revealed the existence of two-slow-moving isoforms. Heterorhabditis bacteriophora AChE1A has been purified from the IJs of the heterorhabditid nematode strain of the highest enzymatic activity to homogeneity by ammonium sulfate precipitation, gel filtration on Sephacryl S-200 and DEAE-Sepharose. The specific activity of the purified enzyme was 1378.1 units/mg protein with purification fold 17.5 over crude extract. The enzyme has a pH optimum at 7.5. The optimum temperature for enzyme activity and stability was 35 degrees C. The activation energy was calculated to be 9.0 kcal/mol. The enzyme hydrolyzes acetylthiocholine (AcSCh), propionylthiocholine (PrSCh), S-butyrylthiocholine (BuSCh) and benzoylthiocholine (BzSCh) iodides with relative rate 100, 74.6, 41.7 and 22.2%, respectively. It displayed an apparent Michaelis-Menten behavior in the concentration range from 0.1 to 2 mM for the three former substrates with Km values 0.27, 0.42 and 0.59 mM, respectively. H. bacteriophora ChE1A is an AChE since it hydrolyzed AcSChI at higher rate than the other substrates and displayed excess substrate inhibition with AcSChI at concentrations over 2 mM. It was inhibited by eserine and BW284C51, but not by iso-OMPA. Its biochemical properties were compared with those reported for different species of insects as target hosts for heterorhabditid nematodes and animal parasitic nematodes.

Purification and characterization of alpha-amylase from the infective juveniles of the nematode Heterorhabditis bacteriophora.

Glycogen content and alpha-amylase activity were estimated in the infective juveniles (IJs) of Heterorhabditis bacteriophora at different times of storage. The glycogen content declined from 5.8 to 2.5 ng/IJ during storage for 40 days at 27 degrees C. The change in glycogen content coincided with the change of alpha-amylase activity during storage. alpha-Amylase was purified from IJs at zero time of storage by ion exchange chromatography and gel filtration. Ion exchange chromatography resolved alpha-amylase into three isoenzymes. The major isoenzyme alpha-amylase I had the highest specific activity and was purified to homogeneity. A molecular mass of 46-47 kDa was estimated for both the native and denatured enzyme, suggesting that the enzyme is monomeric. The Km values were 6.5 and 9.6 mg/ml using starch and glycogen as substrates, respectively. alpha-Amylase I showed optimum activity at pH 7.0 and had an optimum temperature of 40 degrees C. The enzyme was unstable at temperatures above 40 degrees C. The enzyme activity was severely inhibited by EDTA, p-CMB and iodoacetic acid, but potentiated by CaCl2 and NaCl. These results are discussed and compared with previously reported alpha-amylases in the insect hosts of the parasite.