And 13-Galactosidases from Red Palm Weevil, Rhynchophorus Ferrugineus (Olivier) (Col.: Curculionide)
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Plant Protect. Sci. Vol. 48, 2012, No. 2: 85-93 Biochemical Characterisation of a- and 13-Glucosidases and a- and 13-Galactosidases from Red Palm Weevil, Rhynchophorus ferrugineus (Olivier) (Col.: Curculionide) NASIR SABERI RISER', MOHAMMAD GHADAMYARI' and BEHNAM MOTAMEDINIYA2 'Department of Plant Protection, Faculty of Agriculture, University of Guilan, Rasht, Iran; 2Agricultural and Natural Resource Research Centre of Baluchestan, Baluchestan, Iran Abstract RISEH N.S., GHADAMYARI M., MOTAMEDINIYA B. (2012): Biochemical characterisation of a- and ii-glucosidases and a- and B-galactosidases from red palm weevil, Rhynchophorus ferrugineus (Olivier) (Col.: Curculionide). Plant Protect. Sci., 48: 85-93. The red palm weevil, Rhynchophorus ferrugineus (Olivier), is one of the most destructive pests of palm trees in the southeast of Iran. Digestion in the alimentary canal of the red palm weevil is facilitated by some carbohydrases. Results of the in vitro studies indicated the presence of a- and (3-glucosidases and a- and (3-galactosidases in the digestive system and haemolymph of this pest. In the digestive system of females, the activities of a-glucosidase and (3-galactosidase were higher than those of (3-glucosidase and a-galactosidase. Also, the specific activity of a- and (3-glucosidases and a- and (3-galactosidases in the female digestive system was much higher than that in larvae. Results showed that the highest activities of a- and (3-glucosidases were at pH 5 and of a- and (3-galactosidases at pH 4. The R. ferrugineus a- and (3-glucosidases and a- and (3-galactosidases have an optimum temperature activity at 50, 50-60, 40-60, and 40°C, respectively. A zymogram pattern in the native gel revealed that R. ferrugineus a- and (3-glucosidases and a- and (3-galactosidases in the digestive system showed 2, 3, 1 and 1 major bands, respectively. The activity of a-glucosidase in the digestive system of larvae and female adults was higher than that of the other carbohydrases. Therefore, it is the most important subject for further study and design of a new approach to the control of this pest using carbohydrase inhibitors. Keywords: carbohydrates; digestive system; palm trees Most living organisms are able to exploit en- dase), Pyrhocoridae zeamais (Coleoptera: Curcu- vironmental polysaccharides. a-Glucosidase lionidae), Apis mellifera (Hymenoptera: Apidae), (EC 3.2.1.20) is an enzyme that catalyses the hy- Drosophila melanogaster (Diptera: Drosophilidae), drolysis of 1,4-a-glucosidic linkages, releasing and Glyphodes pyloalis (Lep.: Pyralidae) (HUBER a-glucose. This enzyme strongly hydrolyses su- & MATHISON 1976; TANIMURA et al. 1979; BAKER crose, maltose, maltodextrin, and pNP-a-D-glu- 1991; SILVA & TERRA 1997; GHADAMYARI et al. copyranoside. It can be found in the alimentary2010). 13-Glucosidase hydrolyses 13 1-4 linkages canal, salivary secretions of insects (LAGADic &between two glucoses or glucose-substituted mol- CHARARAS 1988; RAMZI & HOSSININAVEH 2010) ecules (such as cellobiose) (TERRA et al. 1996). and hypopharyngeal glands of some insects, such In addition to the important digestive role of as Apis mellifera (BAKER & LEHNER 1972; TERRA et enzymes, they can also act as elicitors (trigger- al. 1996). So far, a-glucosidases have been isolateding agents of plant defence mechanisms) in plants and characterised from many insects including when they are encountered with the feeding dam- Dysdercus peruvianus (Hemiptera: Pyrrhocori-age of the insect pests (MATTIACCI et al. 1995). 85 Vol. 48, 2012, No. 2: 85-93 Plant Protect. Sci. a-D-Galactosidases (EC 3.2.1.22) are exo-actingdigestive system of insects but our knowledge of glycoside hydrolases that cleave a-linked galactosea- and P-galactosidases in the digestive system of residues from carbohydrates such as melibiose,insects is still rudimentary. The aim of the present raffinose, stachyose, and gluco- or galactomannansstudy was to determine biochemical characterisa- (METER & REID 1982). a-D-Galactosidases have tion of carbohydrate-hydrolysing enzymes in the been isolated from a variety of animal, plant anddigestive system of the red palm weevil in order microbial sources, but there is little information to gain a better understanding of the digestive about this enzyme in insects. 13-D-Galactosidase (EC physiology of this insect. 3.2.1.23) is a hydrolase enzyme that catalyses the hydrolysis of P-galactosides into monosaccharides (SEZGINTURK & DIKKAYA 2008). Also, our knowl- MATERIAL AND METHODS edge of this enzyme in insects is still rudimentary. The damage caused by R. ferrugineus was re- Insect. The insect was collected from palm trees ported for the first time in 1990 on traditionalin Sistan and Baluchestan province of Iran. The date palm groves in Saravan region (Sistan and last larval instar and adults were randomly selected Baluchestan province, Iran). This pest is a seriousfor measuring the enzyme activity. pest of various palm species including dates in Iran. Chemicals. p-Nitrophenol and bovine serum al- The immature stages develop within the tree trunk, bumin were purchased from Merck (Darmstadt, destroy its vascular system and eventually cause the Germany). p-Nitrophenyl-a-D-glucopyranoside collapse and death of the tree (Ju et al. 2011). The (pNaG), p-nitrophenyl-P-D-glucopyranoside red palm weevil has been managed in Iran through(pN(3G), p-nitrophenyl-a-D-galactopyranoside employing an integrated pest management (IPM) ( pNaGa), p-nitrophenyl-P-D-galactopyranoside strategy comprising several tactics such as mass (pN (3G a), 4- methylumb elliferyl- 13-D -glucopyrano si- trapping with pheromone-baited traps. Althoughde (4-MU(3G), 4-methylumbelliferyl-a-D-gluco- these tactics have been widely used, controlling this pyranoside (4-MUaG), 4-methylumbelliferyl-P-D-ga- pest has not been very effective yet when there is lact op yr a no side (4-MU(3Ga) and 4-methyl-um- an outbreak of the red palm weevil. Currently, some belliferyl-P-D-galactopyranoside (4-MUaG a) were of the major aspects in pest control are to achieve obtained from Sigma (St. Louis, USA). the selective inhibition of the digestive enzymes Sample preparation. Larvae and adults were of many insect pests. Carbohydrases are activeimmobilised on ice and dissected under a stereo upon a large range of substrates and in some casesmicroscope in ice-cold saline buffer. Digestive they are implicated in plant resistance to insectsystems were removed and their contents were predation. Besides hydrolysing several carbohy-eliminated. Digestive systems of the last larval drates, thus liberating monosaccharides that caninstar were divided into three distinct divisions be absorbed, they can cleave plant toxic glycosides. (V1, V2 and V3) (Figure 1). The samples were These, after being hydrolysed by P-glucosidase, cantransferred to a freezer (-20°C). The sample was form cyanide or other toxic compounds such as homogenised in cold double-distilled water using inhibitors of glucose 6-phosphate dehydrogenasea hand-held glass homogeniser and centrifuged at (DESROCHES et al. 1997) or trehalase (NARANo 10 000 rpm for 10 min at 4°C. Also, the haemo- & SACKTOR 1984). As the first step to achievinglymph was collected from larvae. Small incisions this it is necessary to characterise carbohydrate were made in the soft cuticle anterior to the second hydrolases of the digestive system. The study ofthoracic segment of larvae and the haemolymph insect digestive enzymes is important becausewas collected with a 75 1.t1 glass capillary tube. the gut is the major interface between the insect Then the haemolymph was quickly added to a and its environment. To understand how diges- tube containing an anticoagulant solution (glucose tive enzymes act on their substrate in insects, it 1.0 g; citrate 0.4 g; NaC1 0.21 g; double-distilled is essential to develop methods of insect control. water was added to 50 ml). After collecting, the So far, research has been done on proteases in thesamples were transferred to a freezer (-20°C). digestive system of the red palm weevil (ALAR- Determination of a- and /3- glucosidase and CON et al. 2002) but works on carbohydrases ofa- and fl-galactosidase activity, Activities of a- and R. ferrugineus have been missing. Also, there areP-glucosidases and a- and P-galactosidases activi- some researches on a- and P-glucosidases in the ties were measured with pNaG, pNI3G, pNaGa, and 86 Plant Protect. Sci. Vol. 48, 2012, No. 2: 85-93 determined at several pH values using 40mM glycine-phosphate-acetic-citric buffer. The ef- fect of temperature on a- and P-glucosidase and a- and P-galactosidase activities were measured using the homogenate of adults by incubating the reaction mixture at 20, 30, 40, 50, 60, and 70°C for 30 min, followed by measurement of the activity. Protein concentration. Protein concentration was estimated by the Bradford method (BRADFORD 1976), using bovine serum albumin as standard. Polyacrylamide gel electrophoresis and zymo- gram analysis. Non-denaturing polyacrylamide gel electrophoresis (PAGE) was carried out as de- scribed by DAVIS (1964). For a zymogram analysis of a- and P-glucosidases and a- and P-galactosidases, the samples were mixed with sample buffer and applied onto polyacrylamide gel (4% and 10% po- lyacrylamide for the stacking and resolving gels, Figure 1. Different parts (V1, V2 and V3) of the alimentary respectively). Electrophoresis was performed with canal in the larvae of Rhynchophorus ferrugineus 100 V at 4°C. Afterwards, the gel was immersed in 3mM 4-MUI3G, 4-MUaG, 4-MUI3Ga and 4-MUaGa p1\113Ga as substrate, respectively. Homogenates in 0.1M sodium acetate (pH 5.0) for 10 min at room were incubated for 20 min at 37°C with 45 IA oftemperature to develop bands showing a- and substrate (25mM) and 115 IA of 40mM glycine- P-glucosidase and a- and P-galactosidase activities, phosphate-acetic-citric buffer. The reaction was respectively. The blue-fluorescent bands appeared stopped by addition of 600 IA of NaOH (0.25M). in a few minutes under UV.