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, ; 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 (: ), (EC 3.2.1.20) is an enzyme that catalyses the hy- (Diptera: ), drolysis of 1,4-a-glucosidic linkages, releasing and 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 (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 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 , 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. Optical density was measured at 405 nm using Statistical analysis. The data were compared a microplate reader (Stat Fax 3200, Awarenessby one-way analysis of variance (ANOVA) fol- Technology, Los Angeles, USA) after 10 minutes. lowed by Tukey's test when significant differences Controls without enzyme or without substratewere found at P = 0.05 using the SAS program, were included. A standard curve of absorbanceVersion 8.0. against the amount of p-nitrophenol released was constructed to enable the calculation of the amount ofp-nitrophenol released during the a- and RESULTS P-glucosidase and a- and P-galactosidase assays. Determination of pH optimum and effect of a- and 13-glucosidase activity temperature on a- and fl-glucosidase and a- and /3- galactosidase activity. The activity of a- and The specific activities of a- and P-glucosidases P-glucosidases and a- and P-galactosidases wasin the digestive system of females were 6.09- and

Table 1. The specific activities (mean ± SE) of some digestive in haemolymph, adult and larvae of Rhynchophorus ferrugineus

Activity (umol/min/mg protein) Enzymes larvae female male haemolymph a-Glucosidase 10.31± 0.3b* 62.85± 0.3a 3.03± 0.66c 0.12± 0.001d 13-Glucosidase 0.29± 0.005c 0.74± 0.011b 1.25± 0.015a 0.067± 0.01d a-Galactosidase 0.24± 0.04b 1.3± 0.02a 1.47± 0.04a 0.045± 0.01` 13-Galactosidase 0.43± 0.02c 11.09± 0.08a 6.07± 0.04b 0.14± 0.005d

"different letters indicate that the specific activity of enzymes is significantly different from each other by Tukey's test (P < 0.05)

87 Vol. 48, 2012, No. 2: 85-93 Plant Protect. Sci.

2.55-fold higher than those in the last larval instar,tive system of the last larval instar (Table 1). The respectively. Whereas the a- and P-glucosidasea-galactosidase activity in V2 section was higher activity in the digestive system of males was 0.29 - than in V1 and V3 sections of the last larval in- and 4.31-fold higher than in the digestive system ofstar. Also, the P-galactosidase activity in V1 was the last larval instar, respectively (Table 1). Also, 2.19-fold and 8.6-fold higher than in V2 and V3 the a-glucosidase activity in V1, V2 and V3 of theof the last larval instar, respectively (Figure 2). last larval instar was 8.14 ± 0.92, 9.6 ± 0.38, and 2.52 ± 0.36 1.tmol/min/mg proteins, respectively. The P-glucosidase activity in V2 was 1.39- and Effect of pH and temperature on a- and 8.34-fold higher than that in V1 and V3 of the last 13-glucosidase and a- and 13-galactosidase activity larval instar, respectively (Figure 2). The effects of pH on the hydrolytic activity toward pNaG, pNI3G, pNaGa, and pNI3Ga were a- and 13-galactosidase activity tested using 40mM phosphate-acetic-citric buffer (pH 2-12). Maximum activity in the digestive sys- The specific activity of a-galactosidase in the tem was observed at p1-15 for a- and P-glucosidases digestive system of adults and the last larval in- and at p1-14 for a- and P-galactosidase activity (Fig- star was determined and results showed that the ure 3). The R. ferrugineus a- and P-glucosidases specific activity of a-galactosidase in the diges-have an optimum temperature activity at 50°C tive system of females was 5.41-fold higher thanand 50-60°C, respectively. Also, the optimal that in the last larval instar. Whereas the activ-temperature for a- and P-galactosidases in the ity of P-galactosidase in the digestive system ofdigestive system was 40-60°C and 40°C, respec- females was 25.7-fold higher than in the diges- tively (Figure 4).

(3-galactosidase a-galactosidase 0.30 - a

0.25 -

0.20 -

0.15 - b 0.10 - 0.5 - c 0.05 - 0 II 0 1i 1.8 - (3-glucosidase 12 - a-glucosidase 1.6 - a 1.4 - 10 - 1.2 = 8- 1.0 - Figure 2. Comparison of the activity 0.8 - 6- of a- and (3-glucosidases and a- and 0.6 - 4- (3-galactosidases extracted from different parts of the digestive sys- 0.4 - c 2- tem of Rhynchophorus ferrugineus 0.2 - larvae. Different letters indicate that 0- 0-I1i the activity of enzymes in different V1 V2 V3 V1 V2 V3 tissues is significantly different from Tissue Tissue each other by Tukey's test (P < 0.05)

88 Plant Protect. Sci. Vol. 48, 2012, No. 2: 85-93

-A- a-glucosidase 100 -A- a-galactosidase -El- a-glucosidase -El- a-galactosidase 80-

:E 60

crs

40 - ad

20 -

0 0 20 40 60 80 100 - -A- a-galactosidase 100 -A- a-glucosidase -El- a-galactosidase -El- a-glucosidase 80- 80

60 - 60

40 - 40

20 - 20

0- 0 2 4 6 8 10 12 20 40 60 80 pH Temperature (°C)

Figure 3. Effect of pH on the activity of a- and (3-glucosi- Figure 4. Effect of temperature on the activity of a- and (3-glu- dases, a- and (3-galactosidases extracted from the digestive co sidases and a- and (3-galactosidases extracted from the system of Rhynchophorus ferrugineus digestive system of Rhynchophorus ferrugineus. Highest activity at optimal temperature was taken as 100%; Rela- Zymogram analysis tive activity (%) = (absorbance at respective temperature/ absorbance at optimal temperature) x 100 The crude extracts of R. ferrugineus were ana- lysed by native PAGE. After a- and P-glucosidase and a- and (3- galactosidase activity staining, 4, 4, lymph of R. ferrugineus. The specific activities of 2, and 1 isoforms of a- and P-glucosidases and a- a- and P-glucosidases and a- and P-galactosidases and P-galactosidases in the digestive system werefrom the digestive system of females were higher clearly detected, respectively (Figure 5). However, than those of larvae, males and haemolymph of as depicted in Figure 5, the intensity of two, onethe last larval instar (Table 1). In the digestive and one bands was high for a- and P-glucosidasessystem of females the activities of a-glucosidase and (3- galactosidase, respectively. and (3- galactosidase were much higher than those of P-glucosidase and a- galactosidase (Table 1). Specific activities of a- and P-glucosidases and DISCUSSION a- and P-galactosidases in the female digestive system were much higher than in larvae and males. The data revealed that a- and P-glucosidases andThe female needs a lot of energy for reproduction, a- and P-galactosidases are present in the digestive which may be one of the reasons why this stage system of adults, the last larval instar and haemo- shows a high level of a- and P-glucosidase and

89 Vol. 48, 2012, No. 2: 85-93 Plant Protect. Sci.

a-glu 13-glu 13-gala a-gala Figure 5. Zymogram of a- and (3-glucosidases and a- and (3-galactosidases in the digestive system of CM. the last larval instar of Rhynchophorus ferrugineus

4 a-glu - a-glucosidase; 13-glu - 13-glucosidase; 13-gala - 13-galactosidase; a-gala - a-galactosidase Two and three major bands for a- and 13-glucosidases and one major band for a- and 13-galactosidases were detected at different protein concentrations (1.83, 1.22, and 0.61 mg/ml)

1.83 1.220.61 1.83 1.22 0.61 1.83 1.22 0.611.83 1.22 0.61 a- and P-galactosidase activity compared with lar-pl-I of the three major a-glucosidases found in vae and males. The activity of a- and (3- glucosidases the gut of Bruchus afinis (Coleoptera: Bruchidae) and a- and P-galactosidases was also character- adults coincided well with the pl-I of the midgut ized by a zymogram analysis after native PAGE where most of the digestive processes are known which allowed the visualisation of the enzyme to occur (LAGADIC & CHARARAS 1988). Similarly, activity in situ. The results indicated 4, 4, 2, and digestive a-glucosidase from D. maculatus exhib- 1 isoforms of a- and (3- glucosidases and a- andited its optimal activity at pH values close to the P-galactosidases in the crude digestive system of themidgut p1-I (CHINNERY 1971). In B. afinis adults, last larval instar, respectively (Figure 5). In other alimentary carbohydrates are hydrolysed by highly coleopteran insects, P-glucosidase in the digestive active a-glucosidase (LAGADIC & CHARARAS 1988) system of Xanthogaleruca luteola and Osphranteria where pH would optimise their activities toward coerulescens (unpublished data) has three and foura number of various sugars present in the food of isoforms, respectively. Also, P-galactosidase in thethis bruchid (LUCA 1966). digestive system of X. luteola and 0. coerulescens The pH optima of a-galactosidase in the midgut has one and six isoforms, respectively. of Odontotermes obesus (Isoptera: Termitidae) were Results showed that the highest activities of5.2. Also, in the hindgut the pH optimum of this a- and (3- glucosidases were at pl-I 5 and of a- and insect was 8.4 for a-galactosidase (SINGH 1976). In P-galactosidases at pl-I 4 (Figure 3). Alpha- and non-coleopteran insects, BURTON (1975) showed (3- glucosidases and a- and P-galactosidases in that the optimal pl-I was 5.5 for the a-glucosidase insects are generally the most active in neutral tofrom both the ventriculus and the salivary glands slightly acid pH conditions (LAGADIC 1994; GH- of Helicoverpa zea (Lep.: Noctuidae). Also, GHA- ADAMYARI et al. 2010; RAMZI & HOSSEININAVEH DAMYARI et al. (2010) showed that the optimal pH 2010). Optimal pH values for glucosidases in larvaevalues for a- and (3- glucosidases activity in the of several coleopterans were 3.0-6.5 (CHINNERYmidgut and salivary glands of G. pyloalis were 1971; GATEHOUSE & ANSTEE 1983; LAGADIC & 7.5, 5.5, 8-9 and 8-9, respectively. CHARARAS 1988; LAGADIC 1994). Maximum ac- Comparing activities against p-nitrophenyl gly- tivity in the digestive system of X. luteola wascosides of glucose and galactose, the ratios of observed at p1-I 5 and 4 for a-glucosidase and 13-glucosidase/13-galactosidase were 0.67 and 0.066 a-galactosidase, respectively, whereas the optimal for larvae and females of R. ferrugineus, respec- p1-I for P-glucosidase and P-galactosidase activitytively, when the activities were determined in was 6 and 3, respectively (unpublished data). Simi- the whole digestive system. Whereas the ratio larly, a-glucosidase of Callosobruchus maculatus of a-glucosidase/a-galactosidase was as 42.95 was the most active at pH 5.6 (GATEHOUSE &and 48.34 for larvae and females of R. ferrugi- ANSTEE 1983). In other coleopterans, like in manyneus, respectively. These results showed that the insects, the optimal pH for a-glucosidase activityP-galactosidase and a-glucosidase activities in is generally in the slightly acid region. Maximalthe digestive system of larvae and females were activities of sucrose hydrolysing a-glucosidasehigher than P-glucosidase and a-galactosidase from the midgut of Dermestes maculatus adults activities. Among the a- and P-glucosidase and a- occurred at pH 6.3 (CHINNERY 1971). The optimal and P-galactosidase activities tested, the activities

90 Plant Protect. Sci. Vol. 48, 2012, No. 2: 85-93 of (3- glucosidase and a-galactosidase were very ity at 30°C and 35°C for the midgut and salivary low in the digestive system of larvae, females and glands, respectively. males. The ratio of (3- glucosidase /(3- galactosidase Results showed that glucosidase and galactosidase was reported as 88.5 in Rhynchosciara americanaactivity is present in all regions of the alimentary Wiedemann (Diptera: ) (TERRA et al. canal of the last larval instar of R. ferrugineus 1979), 105 in Stomoxys calcitrans (DELOACH & (Figure 2). Our results illustrated that there is SPOTES 1984), 58 in the midgut tissue of Rhod- a significant difference in the activity of a- and nius prolixus (TERRA et al. 1988), and 2.5 in (3- glucosidases and (3- galactosidases in V1, V2, and C. maculates (GATEHOUSE et al. 1985). Also, in V3 of the last larval instar (Figure 2). The rank D. peruvianus, the 13-glucosidase/13-galactosidase order from the highest to the lowest (3- galactosidase ratio in the midgut tissue was 28.7, but in the whole activity was V1 > V2 > V3 whereas the a- and midgut (epithelium plus luminal contents) the(3- glucosidase activities in V2 were much higher ratio was 3.0, suggesting a major contribution ofthan in V1 and V3. Also, results showed that the (3- galactosidase activity by the seed meal present ratio of a-glucosidase/a-galactosidase in V1 was in the gut lumen (SILvA & TERRA 1997). The ratios higher than in V2 and V3. The alimentary canal of a-glucosidase/a-galactosidase found in adultsof the pistachio green stink bug, B. germari, was and the last larval instar of X. luteola were 7.5 and divided into four distinct divisions (v1, v2, v3, and 3.5, respectively (unpublished data). FERREIRA v4) by RAMZI and HOSSEININAVEH (2010) and et al. (1998) reported that high (3- glucosidaseactivities of a-amylase and a- and (3- glucosidase activity was found in the foliage feeder Abrac- were measured in these parts. Their results showed ris flavolineata (Orthoptera: Acrididae), in the that the highest a-amylase and a- and (3- glucosidase stored plant product feeder Tenebrio molitor (Co-activities were in section v3, whereas the lowest leoptera: Tenebrionidae) and in the pollen-feeder activities were in section v4. Also, results of the bipunctata (Hymenoptera: Apidae). in vitro studies on certain digestive enzymes from Whereas our results showed that the (3- glucosidase the alimentary canal of 0. obesus workers indi- activity in the digestive system of larvae, malescated the presence of a-galactosidase both in the and females and in the haemolymph of larvaemid- and hindgut of this termite (SINGH 1976). was very low. Our results coincided well with low The activity of a- and (3- glucosidases and a- and (3- glucosidase activity found among predaceous (3- galactosidases was also characterised by a zy- insects exemplified by Pheropsophus aequinoc-mogram analysis after native PAGE which allowed tialis (Coleoptera: Carabidae) and Pyrearinus visualisation of the enzyme activity in situ under termitilluminans (Coleoptera: Elateridae) (FER- UV. The results indicated the presence of at least REIRA et al. 1998). Also, (3- glucosidase activity 2, 3, 1 and 1 major bands for a- and (3- glucosidases is low in the decaying plant-feeder R. americana and a- and (3- galactosidases in the digestive system, and in feeders of plant aerial parts, exemplified by respectively. The activity of glucosidases from the Spodoptera frugiperda (Lep.: No ctuidae), Erinnyisalimentary canal of the last larval instar on the ello (Lep.: Sphingidae) and Diatraea saccharalisgel was much higher than that of galactosidases. (Fabricius) (FERREIRA et al. 1998). Our results Also, as depicted in Figure 5, the intensity of bands showed that a-galactosidase activity is relatively concerning a-galactosidase activity was lower than low in the digestive system of larvae, males andfor a- and (3- glucosidases. The zymogram pattern females. The a- and (3- glucosidase and a- andin the native gel revealed that Xanthogaleruca (3- galactosidase activities were determined at dif- luteola (Col.: Chrysomelidae) (3- glucosidase and ferent temperatures ranging from 20°C to 80°C. (3- galactosidase in the digestive system had three The maximum activity of a- and (3- glucosidases and one isoforms, respectively (SHARIFI et al. 2011) and a- and (3- galactosidases in R. ferrugineus in- and in this insect (3- glucosidase activity was much cubated at different temperatures was observed higher than that of (3- galactosidase in crude extract. at 50, 50-60, 40-60, and 40°C, respectively, which PAGE analysis of a- and (3- glucosidase activities is consistent with a- and (3- glucosidases activitiesin gel revealed the presence of one band in the in G. pyloalis (45°C) (GHADAMYARI et al. 2010). salivary gland and midgut of Lygaeus pandurus RAMZI and HOSSEININAVEH (2010) showed that (Hemiptera: Lygaeidae) (VATANPARAST et al. 2011). a- and (3- galactosidase of Brachynema germari Glucosidases and galactosidases play an impor- (Hemiptera: Pentatomidae) has an optimal activ- tant role in insect digestion. Our results showed

91 Vol. 48, 2012, No. 2: 85-93 Plant Protect. Sci. that these enzymes are important in the initial and GATEHOUSE A.M.R., ANSTEE J.H. (1983): The presence and final phases of food digestion of R. ferrugineus. The partial characterization of carbohydrase enzymes in the glucosidase and galactosidase activity in females gut of Callosobruchus maculatus. Cellular and Molecular was higher than in larvae and males. Among the Life Sciences, 39: 1013-1015. carbohydrases tested in this study, a-galactosidases GATEHOUSE A.M.R., FENTON K.A., ANSTEE J.H. (1985): and a-glucosidases showed the lowest and the Carbohydrase and esterase activity in the gut of larval highest specific activities in crude extracts and Callosobruchus maculatus. Experientia, 41: 1202-1205. gel, respectively. Also, the carbohydrase specific GHADAMYARI M., HOSSEININAVEH V., SHARIFI M. (2010): activities in the haemolymph of red palm weevil Partial biochemical characterization of a- and 13-gluco- were lower than in digestive systems. sidases of lesser mulberry pyralid, Glyphodes pyloalis Walker (Lep.: Pyralidae). Comptes Rendus Biologies, 333: 197-204. Acknowledgments. We express our gratitude to the research HUBER R.E., MATHISON R.D. (1976): Physical, chemical council of the University of Guilan and Ministry of Science, Re- and enzymatic studies on the major sucrose on honey searches and Technology for financial support of this project. bees (Apis melifera). Canadian Journal of Biochemistry, 54: 153-164. Ju R.T., WANG F., WAN F.-H., Bo L. (2011): Effect of host References plants on development and reproduction of Rhyncho- phorus ferrugineus (Olivier) (Coleoptera: Curculionidae). ALARCON F. J., MARTINEZ T.F., BARRANCO P., CABELLO T., Pest Science, 84: 33-39. DIA z M., MOYANO F. J. (2002): Digestive proteases during LAGADIC L. (1994): Some characteristics of digestive a-gly- development of larvae of red palm weevil, Rhynchophorus cosidases from adults of Bruchus afinis Friilich, in rela- ferrugineus (Olivier 1790) (Coleoptera: Curculionidae. tion with intestinal pH. Comparative Biochemistry and Insect Biochemistry and Molecular Biology, 32: 265-274. Physiology, 108: 249-253. BAKER J.E. (1991): Properties of glycosidases from the LAGADIC L., CHARARAS C. (1988): Etude des activites osi- maize weevil, Sitophilus zeamais. Insect Biochemistry, dasiques digestives d'adultes de Bruchus afinis en tours 21: 615-621. d'hivernation en Clevage artificial et en conditions semi- BAKER R.J., LEHNER Y. (1972): A look at honey bee gut naturelles. Entomologia Experimentalis et Applicata, 48: functions. Bee Journal, 112: 336-338. 247-255. BRADFORD M.M. (1976): A rapid and sensitive method LUCA Y. (1966): Alimentation imaginale des bruchidts for the quantitation of microgram quantities of protein (Col.). Parasitica, 22: 26-54. utilizing the principle of protein-dye binding. Analytical MATTIACCI L., DICKE M., POSTHUMUS M.A. (1995): 13-Gluco- Biochemistry, 72: 248-254. sidase: an elicitor of herbivore induced plant odour that BURTON R.L. (1975): Carbohydrate digestion in the adult attracts host-searching parasitic wasps. Proceedings of Heliothis zea. Journal of Insect Physiology, 21: the National Academy of Sciences USA, 92: 2036-2040. 1855-1857. MEIER H., REID J.S.G. (1982): Reserve polysaccharides other CHINNERY J.A.B. (1971): Carbohydrases of the midgut of than starch in higher plants. In: LOEWUS F.A, TANNER W. the leather beetle Dermestes maculatus. Journal of Insect (eds): Encyclopedia of Plant Physiology. Springer Verlag. Physiology, 17: 47-61. New York: 418-471. DAVIS B.J. (1964): Disc electrophoresis II. Method and ap- NAKANO M., SACKTOR B. (1984): Renal trehalase: two plication to human serum proteins. Annals of the New subsites at the substrate binding site. Biochimica et Bio- York Academy of Sciences, 121: 404-427. physica Acta, 791: 45-49. DELOACH J.R., SPATES G.E. (1984): Glycosidase activity RAMZI S., HOSSEININAVEH V. (2010): Biochemical char- from midgut region of Stomoxys calcitrans (Diptera: acterization of digestive a-amylase, a-glucosidase and Muscidae). Insect Biochemistry, 14: 169-173. 13-glucosidase in pistachio green stink bug, Brachynema DESROCHES P., MANDON N., BEAEHR J.C., HUIGNARD J. germari Kolenati (Hemiptera: Pentatomidae). Journal of (1997): Mediation of host plant use by a glucoside in Cal- Asia Pacific Entomology, 13: 215-219. losobruchus maculatus F. (Coleoptera: Bruchidae). Jour- SEZGINTURK M.K., DINcKAYA E. (2008): 13-Galactosidase nal of Insect Physiology, 43: 439-446. monitoring by a biosensor based on Clark electrode: Its FERREIRA C., TORRES B.B., TERRA W.R. (1998): Substrate optimization, characterization and application. Biosens specificities of midgut 13-glycosidases from insects of dif- Bioelectron, 23: 1799-1804. ferent orders. Comparative Biochemistry and Physiology, SILVA C.P., TERRA W.R. (1997): a-Galactosidase activity 119B: 219-225. in ingested seeds and in the midgut of Dysdercus peru-

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Corresponding author: Dr. MOHAMMAD GHADAMYARI, University of Guilan, Faculty of Agricultural Science, Department of Plant Protection, Rasht, Iran tel.+ 98 1316 690 009; e-mail: [email protected] or [email protected]

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