Biochemical Characterization of the α- amylase and α- β galactosidases in the Small Black and Yellow Wasp, Allantus viennensis Schr. (Hym.:Tenthredinidae)

Small black and yellow wasp, AllantusviennensisSchr. (Hym.:Tenthredinidae) is a serious pest of rose plants. Larvae feed on the leaves of rose bushes initially, the parenchyma of leaves and eventually the entire leaf except main rib.Carbohydrases such as amylases and galactosidases have main role in digestion and metabolism of carbohydrates in insects. The nutrients were used for growth, development, survival and reproduction of insects. Therefore, any interruption in enzymatic carbohydrate digestion and blocking of carbohydrases by inhibitors can deprive herbivorefrom utilizing the sources of carbohydrate energy efficiently. Transgenic plants expressing carbohydrase inhibitors have been considered as safe alternatives to chemical pesticides against herbivorous pests. Knowledge on digestive enzymes of insects is the first step to use plant origin inhibitors in pest control programs. In the present study,identification and characterization of α- amylase and α- β galactosidases in gut extract of A. viennensiswerestudied. The α-amylase activity was measured in the different larval stages (2-5 L) and three parts of gut including foregut, midgutand hindgut of fifth larval instar ofA. viennensis.10 μl of the enzyme was added to a tube containing 40 μl of universal buffer (50 mMsodium acetate-phosphate-glycine) and 50 μl of 1% (w/v) starch as substrate. The reaction was incubated at 30°C for 30 min and then was stopped by adding100 μl of dinitrosalycylic acid. Absorbance of product was measured at 540 nm with a Microplate Reader Model Stat Fax® 3200. One unit α-amylase was defined as the amount of the enzyme that liberated one micro mole of maltose from starch (as substrate) per minute. The activities of α and β-galactosidase were measured with pNαGa (p-nitrophenyl-α-D-galactopyranoside) and pNβGa (p-nitrophenyl-β-D-galactopyranoside) as substrates, respectively. 10 μl of the enzyme, 45 μL of substrate and 115 μL of universal buffer were incubated for 20 min at 35 °C. After incubation time, 600 μL of NaOH (0.25 M) was added to stop the reaction. P-nitrophenol absorbance was measured at 405 nm using a microplate reader. One unit enzyme is defined as the amount of the enzyme that liberates one micro mole of p-nitrophenol per minute. To obtain the optimal pH and temperature for the enzyme activity, various pHfrom 3.0 to 12.0 and different temperatures ranging from 10 to 70ºC were examined. To determine the kinetic valuesfor α-amylase, different concentrations of starch (0.05, 0.1, 0.25, 0.5 and 0.75) and for galactosidases different concentrations (2.5, 5, 10, 20, 40 and 80mM) of pNαGa and pNβGa were prepared. The Michaelis-Menten constant (Km) and maximal velocity (Vmax) of the enzymes were estimated from the Lineweaver-Burk plots. Electrophoresis was performed and appeared bands in the native gel were observed. The highest enzymes activity was obtainedin 5thinstarandas food absorption increased the enzyme activityenhanced. Also, the specific activity of enzyme in midgut was higher than that of foregut and hindgut.Midgut is the most important source of digestive enzymes as one of the main sites for the absorption of digested material. The greatestactivity of α-amylase in gut of A. viennensis was at alkaline pH (8).The high gut pH in A. viennensisis largely due to adaptation for feeding on rosecontaining tannins. Because tannin can bind with proteins in insect’s midgut at acidic pH values and then decrease the efficiency of food digestion. The highestactivities of α / β galactosidases in gut of A. viennensis were at pH 6. Optimal temperature for α-amylase, α and β galactosidases activity was obtained 50, 60 and 30°C, respectively. As calculated from Lineweaver-Burk plots, the Kmand Vmaxvalues for α-amylase were 1.478 mg/ml and 0.14 μmol min-1 mg-1 protein. The Km and Vmaxvalues for α and β galactosidase were 0.69 Mm, 0.41 Mm, 0.84 and 1.76 Mm min-1 ml-1, respectively. Zymogram pattern in the native gel revealed three, one and one bands for α-amylase, α and β galactosidases, respectively. According to the results, α-amylase, α and β galactosidasesexistedin gut of larvae of A. viennensis. These findings showed potential of digestive enzymes inhibitors for management of A. viennensispopulation and more research is needed.