| Dendrolimus superans (Butler)(Lepidoptera:Lasiocampidae) named Siberian caterpillar in English, is one of important defoliator to boreal conifer, which periodically outbroke and led to serious losses to the forestry production. Presently, researches on D. superans (Butler) focused on its living habits, prediction the forecast, chemical control, biological control, physical and mechanical prevention and treatment, and induced resistance utilization, etc. The studies on the enzymology and biochemical toxicology have not been reported to date. In this paper, activity distribution characteristics of acetylcholinesterase, carboxylesterase, glutathione S-transferase, superoxide dismutase and catalase in D. superans6th instar larvae were systematically analyzed on the biochemical perspective. The optimal reaction condition of acetylcholinesterase was established by orthogonal experiment. Sensitivities of five enzymes to several insecticide preparations and raw insecticides were compared in vitro and the activity variations of five were analyzed in vivo. Acetylcholinesterase and carboxylesterase were purified by a series steps of centrifugation, ammonium sulfate precipitation, ion exchange chromatography, affinity chromatography, etc. Enzymatic properties of purified and unpurified carboxylesterases had been investigated. All the results will provide a theoretical basis for the selection of efficient, low-residue, environment-friendly insecticide, and the studies on biochemical toxicology of D. superans.1. The impact of enzyme and substrate concentration, reaction time, reaction temperature and the pH on acetylcholinsterase activity was analyzed by orthogonal designed experiment, and the optimum conditions for acetycholinsterase had been ultimately determined as0.2g/mL enzyme concentration,0.8mmol/L substrate concentration, pH7.5reaction system,35℃bath temperature and5min reaction time.2. Activity distribution of AChE, CarE, GST, SOD and CAT in four tissues (head, midgut, fat body and epidermis) of the D. superans6th instar larvae were characterised. AChE distributed relatively uniformly, activity in the fat body was slightly higher than in other tissues, accounting for29.01%of the body, while activity in the epidermal content slightly less than other tissues, accounting for21.16%. Activity of CarE mainly concentrated in the midgut, accounting for61.31%of the body, while the head had the least, only accounting for6.37%of the body. The fat body and the head had the largest and the least activity of GST, accounting for36.15%and6.58%of the body, respectively. The most activity of SOD was detected in the head, accounting to39.79%of the body, while the least was detected in the epidermis, accounting to15.30%. For the activity of CAT, the head accounted for3.66%and the fat body accounted for38.17%of the body, which were the tissues with the highest and the lowest activity of CAT, respectively.3. The influence of several insecticide preparations and raw insecticides on five enzymes from D. superans (Butler)6th instar larvae had been measured in vitro. Results showed that among insecticide preparations, two organophosphates (dichlorvos and phoxim) had significantly stronger inhibition to AChE activity than any other tested insecticide preparations, whose IC50values were1.050×10-9and7.144×10-8mg/mL respectively; phoxim and dichlorvos also showed strongest inhibition to CarE activity; dichlorvos had the strongest inhibition to GST activity (IC50=8.737×10-6mg/mL), followed by lambda-cyhalothrin with an IC50of7.174×10-3mg/mL; cypermethrin and lambda-cyhalothrin showed the strongest inhibition to SOD activity, whose IC50werel.879×10-1and6.115×10-1mg/mL respectively. Whereas dichlorvos and phoxim showed the weakest inhibition to SOD activity. Moreover, these two insecticide formulations showed inducing activation as the concentration dropped below0.05mg/mL; diflubenzuron and avermectin showed strongest inhibition CAT activity, with IC50values of2.233and7.672mg/mL, respectively. Among raw insecticides, two organophosphorus (dichlorvos, omethoate) showed stronger inhibition than any other raw insecticides to AChE, whose IC50values were4.762×10-3and2.369×10-3mg/mL respectively. Raw dichlorvos and omethoate had the strongest inhibition to CarE activity. Two raw pyrethroid insecticides (beta-cyfluthrin and lambda-cyhalothrin) showed the strongest inhibition to GST activity. The raw lambda-cyhalothrin had the strongest inhibition to in vitro SOD activity, while raw imidacloprid had the weakest inhibition. Raw abamectin and lambda-cyhalothrin showed the strongest inhibition to CAT activity, and inhibition by the raw omethoate was the weakest.4. After being treated with low-dose insecticde preparations and raw insecticdes through topical application, AChE、CarE、GST、SOD and CAT activity of D. superans (Butler) larvae susceptible strain had been investigated in vivo. Results showed that the activities of the five enzymes changed correspondingly after being treated with either insecticde preparations or raw insecticdes, and there was not a linear relationship between treatment time and the specific activity of the enzyme. At the same time point, the specific activities of five enzymes were closely related to the kind, mechanism and treated time of insecticide.5. AChE and CarE of D. superans (Butler)6th instar larvae were separated and purified by a series of setps of ammonium sulfate precipitation, gel chromatography and affinity chromatography, and the biochemical characteristics of the crude and purified CarE were analyzed. Results revealed that AChE had been purified successfully from the crude protein. However, the fold purification of60%ammonium sulfate precipitation and Sephadex G-25gel filtration chromatography were1.645and11.297, respectively, indicating a low purification efficiency. The purification of AChE by CEA affinity chromatography was much better than by Procainamide affinity chromatography. The specific activity of the purified AChE reached162.866μmol/min-mg protein, purified-fold attained to115.344, and the yield was16.510%.CarE from D. superans6th instar larvae was purified by gradient ammonium sulfate precipitation, gel chromatography, DEAE cellulose ion exchange chromatography and hydroxyapatite affinity chromatography, and the final purified CarE was obtained with138.348-fold and2.782%-yield to the crude homogenate. The molecular weight of CarE was estimated to be84.78kD approximately by SDS-PAGE. Km values of crude CarE were8.601×10-5and3.775×10-4mol/L respectively by using alpha-NA and beta-NA as the substrate of kinetic analysis of CarE from D. superans larvae, which were1.989-fold and2.337-fold of the purified. CarE, while the Kcat values of purified CarE were5.824×101and2.306×102min-1respectively, which were106.084-fold and135.012-fold of crude one. Both the crude and the purified CarE of D. superans6th instar larvae were sensitive to dichlorvos, avermectin and lambda-cyhalothrin in vitro. Dichlorvos showed the strongest inhibition to the activity of CarE, and the IC50value to the purified CarE was1.105×10-4mg/mL, which was33.394-fold of the crude one. However, the IC50values of avermectin and lambda-cyhalothrin to the crude CarE were1.259×10-2and4.529×10-1mg/mL, which were1.228-fold and23.214-fold of the purified CarE respectively. |
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