Biochemical characterization of digestive α-amylase and α-/β-glucosidases in the fall web worm, Hyphantria cunea Drury (Lepidoptera:Arctidae)

Amylases and glucosidases are the major enzymes for carbohydrate digestion in herbivorous insects. Any interruption in enzymatic carbohydrate digestion and blocking of carbohydrases by inhibitors can deprive insect from utilizing the sources of carbohydrate energy efficiently. It can reduce insects’ survival and reproduction and retard their growth. The control strategies interfering with carbohydrate digestion are known to have been proposed as a practical and safe method for control of herbivorous pests. Therefore, in this research, enzymatic properties of α-amylase and α-/β-glucosidases from digestive system oflarvae of the fall web worm, Hyphantria cunea Drury were determined in order to better understand the nutritional physiology of the pest.
The α-amylase activity was determined with 1% (w/v) starch as substrate. Absorbance of product was measured at 540 nm with a Microplate Reader Model Stat Fax® 3200. The activities of α-/β-glucosidases were measured with pNαG (p-nitrophenyl-α-D-galactopyranoside) and pNβG (p-nitrophenyl-β-D-glucopyranoside) as substrates, respectively. P-nitrophenol absorbance was measured at 405 nm. To obtain the optimal pH and temperature for the enzyme activity, various pH 4.0 to 12.0 and different temperatures ranging from 15 to 75ºC were examined. The enzymes activity was assayed in the presence of chemicals including EDTA, Hg2Cl2, ZnCl2, CoCl2, FeCl2, MgCl2, KCl, BaCl2, CaCl2 and MnCl2 at two concentrations of 10 and 20 mM. Electrophoresis was performed and the realized bands in the native gel were observed.
The mean specific activities of α-amylase and α-/β-glucosidases in gut of fifth larval instar were obtained as 9.1, 3.6 and 6.8 μmol/min/mg protein, respectively. Maximum activity of α-amylase in salivary gland and gut of fifth instar larvae was at pH 10.0 and α/β-glucosidases in gut and hemolymph was at pH of 8, 7, 8 and 8, respectively. The optimal temperature of α-amylase in gut and salivary gland was at 55 and 45°C. The values for α and β-glucosidases in gut and hemolymph were 35, 45, 45 and 35°C, respectively. The highest inhibition effect of enzyme activity was caused by Na and Co2 ions (at concentrations of 10 and 20 mM, respectively) on α-amylase activity, Fe2 (concentration of 20 mM) on α-glucosidases activity and Mn2 (concentration of 10 mM) on β-glucosidases activity. Mn2 (at concentrations of 10 and 20 mM), Ba2 (at concentration of 20 mM) and Mg2 (at concentration of 10 mM)significantly increased the activity of α-amylase, α–glucosidase and β–glucosidase, respectively. The zymogram pattern showed the presence of 2 bands for α-amylase and α-/β-glucosidases in gut of fifth instar larvae of H. cunea.
According to the results, α-amylase, α and β glucosidases are present in gut of larvae of H. cunea. These findings can be used in future research about using digestive enzymes inhibitors for management of H. cunea.