α-amylase ACTIVITY IN THE SALIVARY GLANDS AND THE MIDGUT OF APODIPHUS AMYGDALI GERMAR (HEMIPTERA: PENTATOMIDAE)

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1 Trki Journl of Sciences, No 2, pp , 216 Copyright 216 Trki University Aville online t: ISSN (print) ISSN (online) doi: /tjs Originl Contriution α-amylase ACTIVITY IN THE SALIVARY GLANDS AND THE MIDGUT OF APODIPHUS AMYGDALI GERMAR (HEMIPTERA: PENTATOMIDAE) S. Rmzi 1, A. Ziee 2 * 1 Plnt Protection Reserch Deprtment, Horticulturl Science Reserch Institute, Te Reserch Center, Agriculturl Reserch Eduction nd Extension Orgniztion (AREEO), Lhijn, Irn. 2 Deprtment of Plnt Protection, Fculty of Agriculturl Sciences, University of Guiln, Rsht, Irn ABSTRACT In the current study, ctivities of α-mylse were termined nd chrcterized in the slivry glnds nd the midgut of n orchrd pest, Apodiphus mygdli. It ws found tht ctivity of the α-mylse in midgut ws sttisticlly higher thn tht of slivry glnds. Optiml ph of mylolytic ctivities were otined to e 8 for slivry glnds nd 5 for midgut. The enzymes extrcted from slivry glnds nd midgut hd the highest ctivities t tempertures of 45 nd 4 C, respectively. Mono- nd di-vlent ctions significntly chnged mylolytic ctivities in the midgut nd slivry glnds of A. mygdli. In cse of slivry glnds, N +, K + nd Mg 2+ showed no effects ut C 2+ nd Cu 2+ incresed the enzymtic ctivity. N + nd Mg 2+ cresed midgut α-mylse of A. mygdli ut C 2+ showed dverse results. Liewever-Burk nlysis of the enzyme in slivry glnds nd midgut reveled V mx of nd 5.88 (U/mg protein) s well s K m of 6.85 nd 2.58 (%), respectively. Since α-mylse hs criticl role in crohydrte digestion of insects, those hve een trgeted y severl reserches to velop inhiitors in resistnt vrieties. But chrcteriztion of the enzymes is the initil nd mjor step to rech such n ojective. Key words: Apodiphus mygdli, α-mylse, chrcteriztion INTRODUCTION Digestion in insects is multiple steps tht ingested mcromolecules re roken down s monomers to e sored vi epithelil cells of midgut. Severl mcromolecules such s crohydrtes, proteses nd lipids re ingested tht require relevnt enzymes, crohydrses, proteses nd lipses, to e digested. Crohydrses re divid into severl enzymes like mylses, β-glucnses, Xylnses nd Pectinses, Chitinses nd Lysozymes, α-glucosidses, β-glucosidses nd β-glctosidses, Trehlses, Acetylhexosminidses, β-fructosidses nd α-glctosidses (1). In cse, α-mylse seems to e more crucil since they re the first enzymes tht encounter crohydrtes in the midgut ecuse the enzymes rek mcromolecule from internl onds. α- Amylses (EC ) re type of hydrolyzes tht ctlyze reking-down of strch nd glycogen from inner long α-1,4-glucn chins *Corresponnce to: Arsh Ziee, Deprtment of Plnt Protection, Fculty of Agriculturl Sciences, University of Guiln, Rsht, Irn , rsh.ziee@gmx.com, rsh.ziee@guiln.c.ir (2). Generl properties of the enzymes re moleculr weight of 48 6 kd, pi vlues of , nd K m vlues with solule strch round.1% (1). Menwhile, the enzyme is clcium-pennt nd it is ctivted y chlori with displcement of the ph optimum (1). Apodiphus mygdli Germr (Hemipter: Penttomide) is n hemiptern pest of fruit trees y wi distriution in Europe nd Middle Est (3). Host trees contins plum, pricot, pple, olive, per nd pistchio in ddition to non-fruit trees like poplr, pine, plne-tree, elm nd willow rk (3). A. mygdli utilize slivry secretions to liquefy plnt tissues, pump the liquid food to its midgut where the min digestive process re m y recruiting relevnt enzymes. Feeding ctivities of the pest cuses host wekness nd ttrcts other insects to feed on host plnts. Feeding on fruits cuses complete grdtion nd yield loss (4). Sprying of chemicl insecticis leds to severe concerns like resistnt of pest to used chemicls, environmentl pollution, effects on Trki Journl of Sciences, Vol. 14, 2,

2 non-trget orgnisms, chemicl residues in griculturl products nd etc. This point goes to e more criticl when direct sprying re m on trees where their fruits re consumes y humn. Hence, doption of other control procedures seems to e importnt. One of the promising pest control could e velopment of resistnt vrieties y using inhiitors of digestive enzymes. α-amylse is one of the trgeted digestive enzymes to velop inhiitors. There re Six different α-mylse inhiitors in insect control including lectinlike, knottin-like, cerel-type, Kunitz-like, c- purothionin-like, nd thumtin-like tht my e useful in pest control (5). These inhiitors show structurl diversity, different mos of inhiition, nd different specificity profiles ginst diverse rnge of α-mylses (5). To rech such n ojective, chrcteriztion of trget enzymes is mndtory to sign or screen of n inhiitor. So, the ojective of the current study were to termine mylolytic ctivity in the slivry glnds nd the midgut of A. mygdli, optiml ph nd temperture for enzymtic ctivity, effects of ctions nd kinetic study. MATERIALS AND METHODS Fifth nymphl instrs of A. mygdli ws collected from elm trees in Shirz (Frs province) nd trnsferred to lortory. Then, the nymphs were rered on elm leves t 28±1 C, 7% of humidity nd 16 L:8D photoperiods. Adults were rndomely selected nd used for iochemicl experiments. Smple preprtion Dissection of dults were crried out y the method of Cohen (6). Slivry glnds nd midgut of A. mygdli were removed y dissection using stereomicroscope in ice-cold sline uffer (NCl, 1 mm). Removed tissues were rinsed in 1mL of ice-cold distilled wter in the portions of five midgut nd ten slivry glnds. Tissues were ground y n homogeniszer nd centrifuged in 13, rpm for 2 min t 4 C. Superntnt ws crefully removed nd trnsferred to new tues nd stored t -2 C for susequent experiments. α-amylse ssy The method scried y Bernfeld (7) ws used to termine α-mylse ctivity in the slivry glnds nd the midgut of A. mygdli. Briefly, 1 µl of the enzyme were incuted for 3 min t 35 C with 5 μl of universl uffer (2 mm, Glycin, Succinte nd 2- morpholinoethn sulfuric cid, ph 7) nd 4 μl of solule strch (1%). The rection ws stopped y dding of 1 μl dinitroslicylc cid nd heting in oiling wter for 1 min prior to red sornce t 545 nm. One unit of α-mylse ctivity ws fined s the mount of enzyme required to produce 1 mg mltose in 3 min t 35 C. A lnk without sustrte ut with α-mylse extrct nd control contining no α-mylse extrct ut with sustrte were mesured t the sme time s the rection mixtures. Determintion of optiml temperture nd ph of the enzyme The effects of temperture nd ph on α- mylse ctivities in A. mygdli were termined y incution of the rection mixture in vrious temperture nd ph sets. The effect of temperture ws termined y incuting the rection mixture t 15, 2, 25, 3, 35, 4, 45, 5, 55 nd 6 7 C for 3 min. Optiml ph ws termined using universl uffer with ph set t 3, 4, 5, 6, 7, 8, 9, 1, 11, nd 12. Other steps were crried out s scried erlier. Effect of different ctions on α-mylse ctivity Different concentrtions of mono- nd divlent ctions (, 1, 3 nd 5 mm) were ssyed to find their possile effects on the enzymtic ctivity. Used ctions were N+, K+, C2+, Cu2+ nd Mg2+. Rection mixture contined 5 μl of universl uffer, 2 μl of sustrte, 2 μl of ion concentrtion nd 1 μl of the enzyme. The experiment continued s scried erlier. Determintion of kinetic vlues Different concentrtions of strch (.2,.4,.6,.8 nd 1%) were prepred nd enzymtic ctivity ws termined from smples of slivry glnds nd midgut. Otined dt were inserted in Sigm-Plot softwre to clculte V mx nd K m vlues. Protein ssy Protein concentrtions were ssyed ccording to the method scried y Lowry et l. (8). Sttisticl nlysis All dt were compred y one-wy nlysis of vrince followed y Tukey s test when significnt differences were found t p.5 nd mrked in figures nd tles with letters. RESULTS AND DISCUSSION Adults of A. mygdli ws dissected unr stereomicroscope reveling n limentry cnl consists four-sectioned midgut (V 1 -V 4 ) (Figure 1). V1, V3 nd V4 re the slenr sections ut V2 is ulk-like section (Figure 1). Results of the iochemicl experiments reveled significnt presence of α-mylse in the slivry glnds nd the midgut of A. 184 Trki Journl of Sciences, Vol. 14, 2, 216

3 Specific ctivity (U/mg protein) mygdli dults so tht the enzyme hd higher ctivity in the midgut thn the slivry glnds (Figure 2). The otined results were expectle since A. mygdli fed on plnt tissues in which strch is the min storge components. Menwhile, mylse of hemiptern slivry glnds hs mjor role in liquefying of plnt tissues nd the finl nd complete digestion of strch is m in midgut (9, 1). Severl studies hve reported presence of mylses in slivry glnds nd midgut of hemipterns. Mehrdi et l. (11) termined mylolytic ctivity in the slivry glnds nd the midgut of Eurygster integriceps Puton (Hemipter: Scutelleride). Ziee et l. (9) nd Sorkhi-Adolmleki et l. (12) reported mylolytic ctivity in sliv nd midgut of Andrllus spinins Fricius (Hemipter: Penttomide). Figure 1. Morphology of the limentry cnl in the dults of Apodiphus mygdli.,7,6 *,5,4,3,2,1 Slivry Glnd Midgut Figure 2. Comprison of mylolytic ctivity in the slivry glnds nd the midgut of A. mygdli. Sttisticl differences hve een shown y sterisk (t-test, p.5). Trki Journl of Sciences, Vol. 14, 2,

4 Specific ctivity (U/mg protein) Specific ctivity (U/mg protein) Enzymes showed vrious rection towrd sustrtes nd inhiitors when they re exposed in vrious ph nd temperture. Our results indicted tht α-mylse in the slivry nd the midgut of A. mygdli showed the highest ctivities in phs 8 nd 5, respectively (Figure 3; F: F: 74.32, Pr>F:.1; F: 13.68, Pr>F:.2). Menwhile, ctivity of the enzyme from oth sources incresed from temperture 15 C to optiml vlue then shrply cresed so tht Optiml temperture of the slivry nd midgut α-mylses were found 45 nd 4 C, respectively (Figure 4; F: 97.32, Pr>F:.1; F: 64.56, F:.1). Bndni et l. (13) found optiml ph of 6.5 nd optiml temperture of 25-4 C for purified α-mylse in the midgut of E.integriceps. Bezdi et l. (14) monstrted optiml ph of 4.5 so slivry α-mylse ctivity in E. integriceps. Zeng nd Cohen (15) reported optiml ph of 6 for α-mylse of Lygus herperus nd L. lineolris. Ziee et l. (9) nd Sorkhi-Adolmleki et l. (12) found optiml phs of 8 nd 7 for slivry nd midgut α-mylse of A. spinins. In cse of temperture, otined vlues re similr to finding on other hemipterns like E. integriceps, Lygus spp nd A. spinins (9-15).,8,7 Slivry glnds,6,5,4,3 c,2,1 e ph rnge,6,5 Midgut,4,3,2, ph rnge Figure 3. Optiml ph termintion of α-mylse in the slivry glnds nd the midgut of A. mygdli. Sttisticl difefrences hve een shown y vrious letter (Tukey test, p.5). 186 Trki Journl of Sciences, Vol. 14, 2, 216

5 Specific ctivity (U/mg protein) Specific ctivity (U/mg protein) Slivry glnds 2,5 2 1,5 1 c c,5 d e Temperture ( C) Midgut 2 1,8 1,6 1,4 1,2 1,8,6,4,2 c ef f f Temperture ( C) Figure 4. Optiml temperture ( C) termintion of α-mylse in the slivry glnds nd the midgut of A. mygdli. Sttisticl difefrences hve een shown y vrious letter (Tukey test, p.5). In gro-ecosystems, mny fertilizers re used to improve qulity nd quntity of griculturl products. These compounds could ffect vrious physiologicl processes of insects i.e. enzymtic ctivities. Moreover, mny enzymes recruit ions in their ctive site or ions could serve s cofctors nd increse or sometimes crese the enzymtic ctivity. In the current study, Mon- nd di-vlent ctions significntly chnges mylolytic ctivities in the midgut nd slivry glnds of A. mygdli. In cse of slivry glnds, N +, K + nd Mg 2+ showed no effects ut C 2+ nd Cu 2+ incresed the enzymtic ctivity (Tle 1). N + nd Mg 2+ cresed midgut α-mylse of A. mygdli ut C 2+ showed dverse results (Tle 1). Tken collectively, it cn e conclud tht C 2 hd criticl role in mylolytic ctivity of A. spinins slivry glnds nd midgut. Similr results hve een oserved in cse of E. integriceps nd A. spinins (9, 12, 13). Linwever-Burk nlysis to show kinetic prmeters of slivry nd midgut α-mylse in A. spinins reveled V mx of nd 5.88 (U/mg protein) s well s K m of 6.85 nd 2.58 (%), respectively (Figure 5). K m hs n inverse reltionship with the sustrte concentrtion to sturte ctive sites of the enzyme. On the other hnd, lower K m revel stronger inding of enzyme to sustrte for grdtion. Moreover, higher vlue of V mx monstrtes ility of the enzyme to rech the highest velocity for sustrte gredtion. In our study, α-mylse of slivry glnds hd the highet V mx ut α-mylse of the midgut hd the lower K m. It could e conclud tht higher velocity of slivry α-mylse enle insect to fster liquefction of plnt tissues ut lower K m of midgut α-mylse enle the insect to efficient digestion of ingested crohydrtes. Trki Journl of Sciences, Vol. 14, 2,

6 Tle 1. Effects of mono- nd di-vlent ctions on α-mylse ctivities in the slivry glnds nd the midgut of A. mygdli. Ction Concentrtion Slivry glnds Midgut N + Control 1±1.59 1± ± ± ± ± ± ±2.43 K + Control 1± ± ± ± ± ± ± ±5.49 C 2+ Control 1±5.51c 1± ±1.78c 86± ± ± ± ±1.34 Mg 2+ Control 1±5.41 1± ±4.82 9± ± ± ± ±6.19 Cu 2+ Control 1±3.14 1± ± ± ± ± ± ±.89 *. Sttisticl differences hve een shown y vrious letters (Tukey test, p.5). Figure 5. Linwever-Burk plots sjowing kinetic prmeters of α-mylse in the slivry glnds nd in the midgut of A. mygdli. CONCLUSIONS Results of the current study clerly picted presence of α-mylse s one of the mjor enzymes in the midgut of A. mygdli. Determintion of mylolytic ctivities in insects is one of the min steps to velop sfe nd efficient pest control. Finding of enzyme properties will e helpful to otin n inhiitor to crese enzymtic ctivity leding to ml-nutrition of trget pest. In cse, screening of vrious medicinl plnts will e helpful to extrct inhiitors like lectins to inhiit mylolytic ctivity in A. mygdli nd suppress popultion outreks of the pest. REFERENCES 1. Terr, W. R. nd Ferrier, C., Biochemistry of digestion. In: Gilert LI, Editor. Insect moleculr iology nd iochemistry, volume. pp Elsevier, Ntion, J.L., Digestion. In: Insect Physiology nd Biochemistry CRC Press, Boc Rton 27 63, Muhmmed, S. H. nd Al-Irqi, R. A., The iology of the stink ug Apodiphus 188 Trki Journl of Sciences, Vol. 14, 2, 216

7 mygdli Germr (Hemipter: Penttomide). Mesopotomi. J Agri., 38:1 11, Schuh RT, Slter JA True ugs of the world. Ithc (NY): Cornell University Press. 5. Frnco, O.L., Ridgen, D.J., Melo, F.R., Bloch, C. Jr., Silv, C.P. nd Grossi--S, M.F., Activity of whet α-mylse inhiitors towrds ruchid α-mylses nd structurl explntion of oserved specificities. Eur. J. Biochem., 267 (8): , Cohen, A. C., Orgniztion of digestion nd preliminry chrcteriztion of slivry trypsin like enzymes in predceous Heteroptern, Zelus rendii. J. Insect Physiol., 39: , Bernfeld, P., Amylses, α nd β. Method. Enzym., 1: , Lowry, O. H., Roserough, N. J., Frr, A. L. nd Rndll, R. J., Protein mesurement with the folin-phenol regent. J. Biol. Chem., 193: , Ziee, A., Hod, H. nd Fzeli-Dinn, M., Purifiction nd iochemicl properties of slivry α-mylse in Andrllus spinins Fricius (Hemipter: Penttomide). Invert. Surv. J., 9: 48-57, Somdr, K. nd Shrivstv, M., Digestive enzymes in the gut nd slivry glnd of the lrve of Chilo uricilius Ddgn. Cell Mol. Life Sci., 36: , Mehrdi, M., Bndni, A. R., Sdti, F. nd Rvn, S., Sunn pest, Eurygster integriceps Putton (Hemipter: Scutelleride), digestive α-mylse, α- glucosidse nd β-glucosidse. J. Asi- Pcific. Entomol., 12: 79 83, Sorkhi-Adolmleki, A., Ziee, A., Hod, H. nd Fzeli-Dinn, M., Purifiction nd chrcteriztion of digestive α- mylse in predtory ug, Andrlus spinins Fricius (Hemipter: Penttomide). J. Insect Sci., 14(65). Aville online: Bndni, A.R., Kzzzi, M. nd Mehrdi, M., Purifiction nd chrcteriztion of midgut -mylses of Eurygster integriceps. Entomol. Sci., 12: 43 5, Bezdi, M.S., Pourd, R.F., Sghi, H. nd Golmohmmdi, G., Some properties of α-mylse in the slivry glnds of Eurygster integriceps Puton (Hemipter: Scutelleride). Munis. Entomol. Zool., 3: , Zeng, F. nd Cohen, A.C., Prtil chrcteriztion of -mylse in the slivry glnds of Lygus hesperus nd L. lineolris. Comp. Biochem. Physiol. B., 126: 9 16, 2. Trki Journl of Sciences, Vol. 14, 2,