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

Document Type : Research Article

Authors

1 guilan

2 Plant Protection Research Department, Mazandaran Agricultural and Natural Resources Research and Education Center

3 University of Guilan

Abstract

 
Introduction: 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.
Materials and Methods: 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.
Results and Discussion: 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.
Conclusions: 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.

Keywords


1- Aghaali N., Ghadamyari M., Ajamhasani M., and Mohammadi Khoramabadi A. 2011. Biochemical characterization of digestive α-amylase from rosaceae branch borer, Osphranteria coerulescens Redt. (Col.: Cerambycidae). p. 338. Global Conference on Entomology, 5 – 9 March. 2011. Chiang Mai, Thailand.
2- Aghaali N., Ghadamyari M., and Ajamhasani M. 2012. Biochemical characterization of glucosidases and galactosidases from rosaceae branch borer, Osphranteria coerulescens Redt. (Col.: Cerambycidae). Romanian Journal of Biochemistery, 49 (2): 125–137.
3- Alfonso J.F., Ortego F., Sanchez-Monge R., Garcia-Casado G., Pujol M., Castanera P., and Salcedo G. 1997. Wheat and barley inhibitors active towards α-amylase and trypsin- like activities from Spodoptera frugiperda. Journal of Chemical Ecology, 23: 1729-1741.
4- Asadi A., Ghadamyari M., Sajedi R.H., Jalali J., and Tabari M. 2010. Biochemical characterization of midgut, salivary glands and haemolymph α-amylases of the rice green caterpillar, Naranga aenescens L. (Lep.: Noctuidae). Bulletin of Insectology, 63(2): 175-181.
5- Baker J.E. 1989. Interaction of partially- purified amylase from larval Anagastia kuehniella (Lepidoptera: Pyralidae) with amylase inhibitors from wheat. Comparative Biochemistry and Physiology, 93 B: 239 - 246.
6- Baker J.E. 1991. Purification and partial characterization of alfa amylase allozymes from the lesser grain borer, Rhizoperta dominica. Insect Biochemistry, 21: 303-311.
7- Berenbum M. 1980. Adaptive significant of midgut pH in larval Lepidoptera. American Nautralist, 115: 295-302
8- Bernfeld P. 1955. Amylase, α and β. Methods in Enzymology, 1: 149- 151.
9- Boyd D.W. 2002. Digestive enzymes and stylet morphology of Deraeocoris nigritulus (Uhler). (Hemiptera: Miridae) reflect adaptations for predatory habits. Annual of the Entomological Society of America, 96: 667- 671.
10- Bradford M. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72: 248- 254.
11- Chapman R.F. 1998. The Insects Structure and Function. 4th ed. Cambridge University Press, p. 782.
12- Christopher M.S.M., and Mathavan S. 1985. Regulation of digestive enzyme activity in the larvae of Catopsilia crocale. Journal of Insect Physiology, 31: 217-221.
13- Davis B.J. 1964. Disc electrophoresis II. Method and application to human serum proteins. Annals of the New York Academy of Sciences, 12: 404- 427.
14- Dow J.A. 1984. Extremely high pH in biological systems: a model for carbonate transport. American Journal of Physiology, 246: 633-635.
15- Franco O.L., Rigden D.J., Melo F.R., and Grossi- de-sa M.F. 2002. Plant α-amylase inhibitors and their interaction with Insect α- amylases, structure, function and potential for crop protection. European Journal of Biochemistry, 269: 397-412.
16- Garcia-Olemd F., Sanchez-mong G.R., Gomez L., Royo J., and Carbonero P. 1987. Plant proteinaceous inhibitor of proteinases and α-amylas. Plant Molecular Cell Biology, 4: 275-335.
17- Ghanbarinezhad, R., Ghadamyari, M., and Sajedi, R. 2014. Biochemical characterization of α-amylase in Epilachna chrysomelina (Col.: Coccinellidae). Iranian Journal of Plant Protection Science, 45 (2): 251-263. (In Persian).
18- Ghanbarinezhad, R., Ghadamyari, M., Sajedi, R., and GholamzadehChitgar, M. 2015. Biochemical characterization of galactosidases in Epilachna chrysomelina (Col.: Coccinellidae). Journal of Plant Protection, 38 (3): 13-24. (In Persian).
19- Gholamzadeh Chitgar M., Ahsaei S.M., Ghadamyari M., Sharifi, M., Hosseini Naveh V., and Sheikhnejad H. 2013. Biochemical characterization of digestive carbohydrases in the rose sawfly, Arge rosae Linnaeus (Hymenoptera: Argidae). Journal of Crop Protection, 2 (3): 305-318.
20- Gholamzadeh Chitgar, M., Ghadamyari, M., Sharifi, M., and Hassan Sajedi, R. 2014. Partial characterization of digestive carbohydrases in the midgut of fig tree skeletonizer moth, Choreutis nemorana Hubner (Lepidoptera: Choreutidae). Trakia Journal of Sciences, 1: 27-37.
21- Hori K. 1968. Some properties and developmental changes in occurrence of the salivary amylase of the cabbage bug, Eurydema rugosa Motschulsky (Hemiptera: Pentatomidae). Applied Entomology and Zoology, 3: 198-202.
22- Hori K. 1973. Studies on enzymes, especially amylases, in the digestive system of the bug, Lygus disponsi and starch digestion in the system. Research Bulletin Onihiro University, 8: 173-260.
23- Hosseini R., and Sahragard A. 2003. Study on morphological Characters and some features of biology and spatial distribution pattern of rose minor leaf eating sawfly, Allantus viennensis (Schr.) (Hym.; Tenthredinidae) in Guilan University. Journal of Agricultural Sciences and Natural Resources, 10(2): 103-115.
24- Jahanjo F., Ghadamyari M., Hosseini R., and Sajedi R.H. 2013. Biochemical characterization of digestive α-β glucosidases in Allantus viennensis (Hym.: Tenthredinidae). Iranian Plant Protection Journal, 44 (1): 141-151. (In Persian).
25- Jaimand K., Rezaee M.B., Tabaei Aghdaei S.R., Nadery Hajibagher Kandy M., and Meshkizadeh S. 2012. Dtermination of tannins in rose water, wastewater and petal residue of Rosa damascena Mill. Iranian Journal of Medicinal and Aromatic Plants, 27( 2): 348-357.
26- Keith J.L., and Peterman B.F. 1979. Temperature effect in enzyme kinetics. Methods in Enzymology, 63: 234-257.
27- MacGregor‌ E., and Sevensson B. 2001. Biochimica Relationship of sequence and structure to specificity in the α- amylase family of enzymes. Biochemical et Biophysical Acta, 1546: 1-20.
28- Meier H., and Reid J.S.G. 1982. Reserve polysaccharides other than starch in higher plants. P. 418-471. In: Loewus F.A., Tanner W. (ed.) Encyclopedia of Plant Physiology. Springer Verlag, New York.
29- Ramzi S., and Hosseininaveh V. 2010. Biochemical characterization of digestive α-amylases, α-glucosidase and β-glucosidases in pistachio green stink bug, Brachynema germari Kolenati (Hemiptera: Pentatomidae). Journal of Asia- Pacific Entomology, 13(3): 215-219.
30- Saberi Riseh N., Ghadamyari M., and Motamediniya B. 2012. Biochemical characterization of α and β-glucosidases and α- and β-galactosidases from red palm weevil, Rhynchophorus ferrugineus Olivieri (Col.: Curculionide). Plant Protection Science, 48: 85.93.
31- Saberi Riseh, N., and Ghadamyari, M. 2012. Biochemical characterization of α-amylases from gut and hemolymph of Rhynchophorus ferrugineus Olivieri (Col.: Curculionidae) and their inhibition by extracts from the legumes Vigna radiata L. and Phaseolus vulgaris L. Invertebrate Survival Journal, 9: 72-81.
32- Sexena K.N. 1954. Physiology of the alimentary canal of Leptocorisa varicornis fabr. (Hemiptera: Coreidae). Journal of the Zoological Society of India, 6: 111-112.
33- Sharifi M., Gadamyari M., Mahadavi M., and Fetemeh S. 2011. Biochemical characterization of digestive carbohydrases from Xanthogaleruca luteola and inhibition of its α-amylase by inhibitors extracted from the common bean. Archive Biological Sciences Belgrade, 63 (3): 705-716.
34- Sharifi M., Gholamzadeh Chitgar M., Gadamyari M., Sajedi R.H., and Amini S. 2012. Characterisation of digestive protease in the rose sawfly, Arge rosae Linnaeus (Hymenoptera: Argidae). Archives of Phytopathology and Plant Protection, 45(10): 1170-1182.
35- Sharma H.C., and Ortiz R. 2000. Transgenics, pest management, and the environment. Current Science, 79: 421–437.
36- Silva C.P., Terra W.R., Xavier-Filho J., Grossidesa M.F., Lopes A.R., and Pontes E.G. 1999. Digestion in larvae of Calosobruchus maculates and Zabrotes subfasciatus (Coleoptera: Bruchidae) with emphasis on alpha amylase and oligosaccharidase. Insect Biochemistry and Molecular Biology, 29: 355-366.
37- Terra W.R., and Ferreira C. 1994. Insect digestive enzymes: properties, compartmentalization and function. Comparative Biochemistry and Physiology, 109: 1- 62.