PROTEIN MAP OF GUT IN ADULT SUNN PEST, EURYGASTER INTEGRICEPS PUT. (HEM.: SCUTELLERIDAE): TWO-DIMENSIONAL ELECTROPHORESIS TECHNIQUE

Gut is main part of digestive system in sunn pest. Digestion and absorption of food materials is the main role of gut and proteins are key molecules for such a function. In this study protein pattern of gut in new adult insects was investigated by Two-dimensional gel electrophoresis in which 100 protein spots could identified by CBB staining. Seventy one protein spots were tagged by molecular weight and isoelectric point. These proteins were divided into different functional category based to their physiological role. These proteins were belong to musculature proteins; carbohydrate, lipid and protein metabolism; energy metabolism; immune related proteins and nutrition proteins. This is the first report of 2-DE pattern of gut in sunn pest and with these ongoing studies, our aim will be to develop organ-related proteome map in sunn pest. KEY WORD: Gut, protein, proteomics, sunn pest, digestion. Sunn pest, Eurygaster integriceps is a key pest against wheat and barley in Middle east countries such a Iran, Iraq, Turkey and Syria (Javaheri et al., 2009). This insect is monovoltine and has two different phases in life cycle. Sunn spend two to three months as active form and feed from wheat. Then, immigration phase occurs in which they immigrate to wintering sites and diapauses take place. This insect has five insatr nymphs that 1st instars cannot feed and feeding startfrom end of 2nd instars (Critchley, 1998). The most of nutrition performed in adult insects and 5th insatrs. In iran, chemical control against this insect performed in adult stage and sometimes in 5th instars. Digestive system of hemiptera consisted of two organs, salivary gland and gut (Saxena, 1963). These insects have extra oral digestion in which salivary gland injects some of saliva to tissue plant and after liquefy substrates, pumped them to gut. Main digestion of food materials performed in gut to prepare for absorption (Habibi et al., 2008). Gut of sunn pest like the other hemipterous bugs consisted of three parts, foregut, midgut and hindgut in which midgut divided to four parts. The largest part of gut in hemiptera is midgut. Gut is a barrier to enthomopathogenic agents that entered from oral (Pauchet et al., 2008). For example Bt toxins act in gut of insects and disturbed ion balances in epitheliums cells (Hakim et al., 2010). Also some of digestive insecticides and plant toxins like plant protease inhibitors acts in gut and interrupt feeding process (Jouanian et al., 1998; Alborn et al., 1997). Using of oral toxins that posses selectivity effects only against of target insects are new strategies for pest management. Efficiency of gut in digestion and immunity system is related to expressed proteins in it (Pauchet et al., 2008). Proteins are product of genes activity which _____________Mun. Ent. Zool. Vol. 7, No. 1, January 2012__________ 230 causes an action cell. The study of protein is a critical step in understanding physiological role of a selected organ (Liu et al., 2010; Alborn et al., 2007). Proteomics is a new technique for protein researchers. In this technology at first, total proteins separated by two dimensional electrophoresis and then some (Proteomics analysis) or all of them (Protein mapping) were identified by mass spectrometry. Protein mapping of gut were studied in several insects such a Helicoverpa armigera (Pauchet et al., 2008), Bombyx mori (Zhang et al., 2011), Aedes aegypti (Popva-butler and Dean, 2008), Tribolium castaneum (Moris et al., 2009) and Spodoptera littoralis (Liu et al., 2009). In this study, we separated gut proteins from new adult of sunn pest and identified some of them according to molecular weight and isoelectric point. To our knowledge this is the first report of sunn pest proteome analysis. MATERIAL AND METHODS Insects Adult insect collected from wheat farm around Tabriz area in summer 2010 and transferred to insectary room for rearing. Insects was reared on wheat var. Alvand in 27°C±1 and humidty 40% with 16:8 (L:D) photoperiod regime. Gut of adults dissected under stereomicroscope and washed with PBS (pH= 6.9). After dissection, guts were transferred to micro tube contains ice PBS and cocktail of protease inhibitors and kept in -80°C until use. Protein extraction A portion including three guts in one ml PBS was homogenized and centrifuged at 30000 g, 30 min, 4°C to remove insoluble materials. Gut proteins were precipitated by 10% trichloroacetic acid and then washed by 100% acton three times and pellets were solubilized in lysis buffer (7 M Urea, 2 M thiourea, 2% CHAPS, 60 mM DDT and 1% ampholyte (pH:3-10)). Insoluble material was removed after two times centrifugation (20000g, 20min, 25°C). Total protein was determined according to Bradford method using protein dye reagent and bovine serum albumin as standard. Twodimensional polyacrylamaide gel electrophoresis (2-DE) A total of 600 μg of extracted proteins were separated in the first dimension by isoelectric focusing (IEF) tube gels and in the second dimension by SDS-PAGE. An IEF tube gel of 11 cm length and 3 mm diameter was prepared. IEF gel solution consisted of 8 M urea, 3.5% polyacrylamide, 2% NP-40, 2% ampholines (pH 3.5–10.0 and pH 5.0–8.0), ammonium persulfate and TEMED. Electrophoresis was carried out at 200 V for 30 min, followed by 400 V for 17 h and 600 V for 1 h. After IEF, SDS–PAGE in the second dimension was performed using 15% polyacrylamide gels with 5% stacking gels. The gels were stained with Coomassie brilliant blue (CBB), and image analysis was performed. The position of individual proteins on gel was evaluated automatically with Melanie 7 software. The pI and Mr of each protein were determined using 2D-PAGE markers (BioRad, Hercules, CA, USA). The information of gut proteins from other insects available in data banks were used for identifiing of gut proteins in sunn pest.