Trakia Journal of Sciences, No 1, pp 27-37, 2014 Copyright © 2014 Trakia University Available online at: http://www.uni-sz.bg ISSN 1313-7050 (print) ISSN 1313-3551 (online) Original Contribution PARTIAL CHARACTERIZATION OF DIGESTIVE CARBOHYDRASES IN THE MIDGUT OF FIG TREE SKELETONIZER MOTH, CHOREUTIS NEMORANA HÜBNER (LEPIDOPTERA: CHOREUTIDAE) M. Gholamzadeh Chitgar1, M. Ghadamyari1*, M. Sharifi1, R. Hassan Sajedi2 1Department of Plant Protection, Faculty of Agricultural Science, University of Guilan, Rasht, Iran 2Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran ABSTRACT Choreutis nemorana (Lepidoptera: Choreutidae) is a generally common pest of fig tree in Iran causing noticeable distortion of the leaves. Application of transgenic plants expressing digestive enzyme inhibitors is one of the safe methods in integrated pest management (IPM) and blocking of carbohydrases by inhibitors represents a safe approach to pest management of C. nemorana. So, identification and characterization of the carbohydrases in gut extract of C. nemorana is necessary for achieving this goal. In this research, some enzymatic properties of α-amylase, α-/β-glucosidases and α-/β-galactosidases from gut of C. nemorana larvae were determined. The optimal pH for α-amylase, α-/β-glucosidases and α-/β- galactosidases was found 11, 6, 4, 5 and 6, respectively. Also, these enzymes reached the highest activity at 55°C for α-amylase, 35 and 45°C for α- and β-glucosidases and 45°C for α- and β-galactosidases, respectively. The zymogram pattern confirmed the presence 3, 2, 4 and 2 bands for α-/β-glucosidases and α-/β-galactosidases, respectively. Among α-/β-glucosidases and α-/β-galactosidases tested, α- galactosidase showed highest thermal stability at 50°C. Key words: Choreutis nemorana; α-amylase; α-/β-glucosidases; α-/β-galactosidases INTRODUCTION hydrolysis and converts them to maltose, Carbohydrates are essential for the majority of maltotriose and maltodextrins (3). Alpha- insects to produce nutrient materials needed for amylase is found in animals, microorganisms, growth, development and for the maintenance of bacteria, fungi and plants (4). These enzymes adult survival and reproduction (1). The nutritive play important roles in insect growth and value of carbohydrates depends on the development. Many authors have characterized availability of digestive enzymes to hydrolyze α-amylases from many different orders of insects complex carbohydrates to their constituent including Coleoptera, Lepidoptera and monomers which are then absorbed by midgut Hemiptera (5-8). In insects, α-amylases are epithelial cells. Many carbohydrases have been synthesized and secreted by midgut epithelial reported from salivary glands and midgut of cells and salivary glands, but these enzymes insects that among them only α-amylase act have been reported also from insect haemolymph preferentially on long α-1, 4-glucan chains (2). (8-11). This enzyme mediated starch and glycogen In addition to α-amylases, galactosidases and ________________________________ glucosidases are the major enzymes for *Correspondence to: Mohammad Ghadamyari, carbohydrate digestion in herbivorous insects. In Department of Plant Protection, Faculty of insects, digestive glucosidases and Agricultural Science, University of Guilan, Rasht, galactosidases are important for the hydrolysis of Iran. Tel.: 0098-131-6690009 (office); Fax: 0098- di and oligosaccharides (12). Alpha-glucosidase 131-6690281;E-mail: [email protected] or [email protected] (EC 3.2.1.20) is an exo-acting hydrolase which Trakia Journal of Sciences, Vol. 12, № 1, 201427 72 CHITGAR M. G., et al. removes glucose from 1, 4-alpha-glucosidic growth. For this reason, many studies have been linkages. This enzyme hydrolyzes sucrose, attempted to characterize the biochemical maltose and maltodextrins. Beta-glucosidase properties of digestive enzymes and the study of hydrolyzes aryl and alkyl β-glucosides and β1–4 the physiology and biochemistry of digestive linkages between two glucoses or cellobioses carbohydrases in the insect midgut is necessary (2). So far, biochemical properties of for achieving this goal. In our previous study, the glucosidases were studied from hypopharyngeal biochemical characterizations of different type of glands of honey bee, the midgut and salivary C. nemorana proteases were performed (19). glands of Glyphodes pyloalis Walker The present study reports some biochemical (Lepidoptera: Pyralidae) and midgut of properties of carbohydrases in the digestive Xanthogaleruca luteola Müll. (Coleoptera: system of C. nemorana. Chrysomelidae), Osphrantria coerulescens (Col.: Cerambycidae), Rhynchophorus MATERIAL AND METHODS ferrugineus Olivieri (Col.: Curculionide) (7, 13- Chemicals 16). Triton X-100, bovine serum albumin, 3, 5- dinitrosalicylic acid (DNS) and starch were Alpha-D-galactosidases (EC 3.2.1.22) are exo- purchased from Merck (Merck, Darmstadt, acting hydrolases which catalyze the removal of Germany). P-nitrophenyl-α-D-glucopyranoside galactose from 1, 4-α-galactosidic linkages in (pNαG), p-nitrophenyl-β-D-glucopyranoside melibiose, raffinose, stachyose, and gluco- or (pNβG), p-nitrophenyl-α-D-galactopyranoside galactomannans (17). Beta-galactosides (pNαGa), p-nitrophenyl-β-D-galactopyranoside available in insect food can be converted to (pNβGa), 4-methylumbelliferyl-β-D- monosaccharides by beta-D-galactosidase (EC glucopyranoside (4-MUβG), 4- 3.2.1.23). Compared to glucosidases, little methylumbelliferyl-α-D-glucopyranoside (4- attention has been paid to the biochemistry of MUαG), 4-methylumbelliferyl-α-D- galactosidases in the digestive system of insect galactopyranoside (4-MUαGa) and 4- (15). Up to now, biochemical properties of α- methylumbelliferyl-β-D-galactopyranoside (4- and ß-galactosidases have been characterized in MUβGa) were obtained from Sigma (Sigma, St some coleopteran insects (7, 15, 16). Louis, MO, USA). The fig tree skeletonizer moth, Choreutis Insects nemorana Hübner (Lepidoptera: Choreutidae) C. nemorana larvae were collected from the fig distributed in the Canary Islands and Madeira, trees in Guilan province, Iran. Insect rearing was through the Mediterranean region and north performed on fig leaves at optimum rearing Africa to Asia (18). This insect is the most conditions of 25 ± 2 °C, 60 ± 10% RH with a destructive pest of fig tree in Guilan province, photoperiod of 16 h light and 8 h dark. Last Iran. The larvae of this insect feed on leaves instar larvae were randomly selected 24h after causing extensive defoliation and at times of molting for the measuring enzyme activity. severe infestation, damage to fig leaves significantly reduced photosynthesis and led to Sample preparation and enzyme assays huge loss of the crop. They are protected by a 300 last instar synchronized larvae (after last web of silken threads and feeds on upper molting) were randomly selected for gut epidermal and parenchymal cells (18) and this extraction. The individuals were immobilized on damage causes the removal of chlorophyll from ice and digestive systems of them (without the leaves reducing photosynthesis. Also, contents) were removed by dissection under a penetration of pesticides in this protected area is dissecting microscope in ice-cold saline buffer. considered to be hindered. Then, tissues were transferred to a freezer (-20 °C). 10 larval guts were put in each tube and Insect control strategies interfering with considered as one replicate. For measuring carbohydrate digestion are known proposed as a enzyme activities, the sample was homogenized practical and safe method for control of in cold double-distilled water using a hand-held herbivorous pest. The plant expressing glass homogenizer and centrifuged at 10,000 carbohydrase inhibitors can reduce insects’ rpm for 10 min at 4°C. survival and reproduction and retard their Tr28 Trakia Journal of Sciences, Vol. 12, № 1, 2014 Trakia Journal of Sciences, Vol. 12, № 1, 2014 CHITGAR M. G., et al. Determination of enzyme activities and the enzymes were determined by incubating the protein concentration reaction mixture at different temperatures Alpha-amylase activity was determined at room ranging from 15 to 65 ºC for 30 min. Enzyme temperature (26±2 °C) in 40 mM glycine- activity was measured by the standard assay phosphate-acetate mixed buffer based on the method as previously described. method of Asadi et al. (5). The soluble fraction (10 μl) was added to a tube containing 40 μl of Thermostability the buffer and 50 μl of 1 % (w/v) starch and Thermal stabilities of alpha/beta glucosidases incubated for 30 min. The DNS method and alpha-beta galactosidases were determined at according to Bernfeld (20) was used for their optimal pH by incubating the enzymes at estimation of the reducing sugar obtained from 45, 55, 50 and 50 ºC, respectively for series of the reaction of amylase. Absorbance was time intervals, followed by cooling on ice, and measured at 545 nm with a Microplate Reader determining residual activity under above assay Model Stat Fax® 3200 (Awareness Technology conditions. Inc.). Control without enzyme including buffer and starch was used in the experiment. Also, Polyacrylamide gel electrophoresis and separate incubation considered as negative zymogram analysis control by adding enzymes to the reaction Non-denaturing polyacrylamide gel mixture (buffer + starch) immediately after electrophoresis (PAGE) for galactosidases and addition of the DNS. glucosidases was carried out at constant voltage (100V)