Study on Salivary Glands α-amylase In Wheat Bug Eurygaster maura (Hemiptera: Scutelleridae)

α-amylase activity in the salivary glands of Eurygaster maura was determined by biochemical experiments. Some of adult insect was c olle ted and their salivary glands isolated and characterized. Enzyme samples from salivary glands of adults were prepared by the method of Cohen with slight modifications. α-Amylase activity was assayed based on Bernfeld met hod by the dinitrosalicylic acid (DNS) procedure. The activity of α-amylase in salivary glands was 0.050 U/insect. The optimum pH and temperature for the enzyme activ ity was determined to be 6.5-7 and 30-35oC, respectively. The enzyme activity was inhibited by addition of EDTA (Ethylenediamine tetraacetic acid) urea, CaCl2, MgCl2 and SDS but Mg2+, NaCl and KCl enhanced enzyme activity.


INTRODUCTION
Among the wheat pest in Iran, genera of Eurygaster sp. (Hemiptera: Scutelleridae) is the most economic pest. It's mainly injury is feeding of wheat seeds. This insect introduces its salivary enzymes into seed and after partially digestion, sucking digested material. Entrance of mentioned bugs salivary enzymes into the feeding seeds In addition of its direct injury to wheat seeds, causes decreasing of feeding seeds quality, has harmful medicine effects on consumers involved humans. E. maura is dominant wheat bugs in north of Iran particularly in Gorgan area, Golestan province. The insect is mainly found in wheat farm which causes severe damage to the vegetative growth stage of wheat in the early season. It also feeds on wheat grains in the late growth stage, thus damaged grains lose their bakery properties. In addition to direct damage to wheat grain it also inject salivary enzymes into the feeding seeds causing damage to seed quality, too. Injection of salivary enzymes into the wheat also produces hygienic problem for consumers. The most important times in the life cycle of E. maura are the period of late nymphal development and the intense feeding of the newly emerged adults. Nymphs in the early instars do not feed intensively. After the third instar, feeding is intensified and the damage to crops becomes obvious. The emerged adults start intense feeding on wheat grains [28] . During feeding, this pest with its piercing-sucking mouthparts injects saliva from salivary gland complexes into the grains to liquefy food. Then liquefied food is ingested and further digestion is made inside the gut [20] . Because of injecting enzymes into the grain during feeding, the enzymes degrade gluten proteins and cause rapid relaxation of dough which results in the production of bread with poor volume and texture [28] .
An understanding of how digestive enzymes function is essential when developing methods of insect control, such as the use of enzyme inhibitors and transgenic plants to control phytophagous insects [5,17,22] . For nearly all these strategies, having a strong understanding of the target pest's feeding is important.
Also, an understanding of the biochemistry and physiology of feeding adaptation is important.
Nothing is currently known about the properties of α-amylase of E. maura. The purpose of the present study is to identify and characterize the α-amylase activity of E. maura in order to gain a better understanding of the digestive physiology of wheat bug. This understanding will hopefully lead to new management strategies for this pest. Sample preparation: Enzyme samples from salivary glands of adults were prepared by the method of Cohen (1993) with slight modifications. Briefly, adults were randomly selected salivary gland complexes (SGC) from these individuals were removed by dissection under a light microscope in ice-cold saline buffer (0.006 M NaCl). The SGC was separated from insect's body, rinsed in ice-cold buffer, placed in a pre-cooled homogenizer and ground in one ml of universal buffer containing succinate, glycine, 2morpholinoethanesulfonic acid at pH 6.5 [18] .

Insects
The salivary glands was separated from the insect body, rinsed in ice-cold saline buffer, placed in a precooled homogenizer and ground in one ml of universal buffer. The homogenates from SGC were separately transferred to 1.5 mL centrifuge tubes and centrifuged at 15000×g for 20 min at 4°C. The supernatants were pooled and stored at -20°C for subsequent analyses.
Amylase activity assay: The α-Amylase activity was assayed by the dinitrosalicylic acid (DNS) procedure [6] , using 1% soluble starch (Merck, product number 1257, Darmstadt, Germany) as substrate. Ten microliters of the enzyme was incubated for 30 min at 35°C with 500 µL universal buffer and 40 µL soluble starch. The reaction was stopped by addition of 100 µL DNS and heated in boiling water for 10 min. 3, 5-Dinitrosalicylic acid is a color reagent that the reducing groups released from starch by αamylase action are measured by the reduction of 3, 5-dinitrosalicylic acid. The boiling water is for stopping the α-amylase activity and catalyzing the reaction between DNS and reducing groups of starch.
Then absorbance was read at 540 nm after cooling in ice for 5 min. One unit of α-amylase activity was  (Fig. 1). A blank without substrate but with α-amylase extract and a control containing no α-amylase extract but with substrate were run simultaneously with the reaction mixture. All assays were performed in duplicate and each assay repeated at least three times.

Effect of pH and temperature on enzyme activity:
The effect of temperature and pH on α-amylase activity was examined using α-amylase extracted from adult salivary glands. The effect of temperature on α-amylase activity was determined either by incubating the reaction mixture at 10,15,20,25,30,35,40,45, 50, 55, 60 and 70°C for 30 min. The effect of temperature on stability of amylase activity was tested by preincubation of the enzyme at 10, 20, 30, 40, 50, 60 and 70°C for 30 min, followed by measurement of activity as mentioned before.
Optimal pH for amylase activity was determined using universal buffer with pH set at 2, 3, 4, 5, 5.5, 6, 6.5, 7, 7.5, 8, 9 and 10. Also, the effect of pH on stability of α-amylase was determined by preincubation of enzyme at mentioned pH for 60 min prior to the assay.
Protein determination: Protein concentration was measured according to the method of Bradford [9] , using bovine serum albumin (Bio-Rad, Munchen, Germany) as a standard.

Statistical analysis:
Data were compared by one-way analysis of variance (ANOVA) followed by Duncan multiple range test when significant differences were found at p = 0.05.

RESULTS
Standard curve: Protein concentration was measured according to the method of Bradford [9] , using bovine serum albumin (Bio-Rad, Munchen, Germany) as a standard (Fig. 2).
α-amylase activity: Studies showed that α-amylase activity is present in salivary glands of adult E. intergriceps. The activity of salivary glands enzyme was 0.050 U/insect ( Table 1).

Effect of pH and temperature on enzyme activity:
Similar to most insect α-amylases, which have optimal activities at neutral or slightly acid pH values, αamylase of E. maura showed an optimal pH of 6.5-7 (Fig. 3). The enzyme activity increased steadily from pH 2-7 and then decreased with increasing pH.
Pre-incubation of enzyme in different pHs for 1 h affected enzyme only in small scales (Fig. 3), showing both acidic and alkaline pHs have more or less the same effect on enzyme stability. Amylase was considerably active over a broad range of temperatures, with the optimum between 25-40°C (Fig. 4). Sensitivity of amylase to pre-incubation did not change significantly at pre-incubation temperature of 10-50°C, but the greatest sensitivity was found at higher temperatures (Fig. 4).   (Table 2), with the highest activity obtained with 20 mM Na ion concentration and with 40 mM K ion ( Table 2). Other two ions (Ca and Mg) had inhibitory effects that increased with increasing ion concentration ( Table 2). The inhibitory effect of Mg ion was stronger than Ca ion.

Effect of activators and inhibitors on enzyme activity: Na and K ions increased amylase activity only
Three other compounds, urea, SDS and EDTA, had an inhibitory effect on enzyme activity (Table 2). Inhibitory effects of SDS and EDTA at concentration of 1 mM were 3 and 2%, respectively.

DISCUSSION
The present study showed that the adult E. maura has αamylase activity in the salivary glands. The presence of the amylase activity in midgut of other phytophagous heteropterans has been reported [5,17,22] . The insects can digest polysaccharides partially by salivary secretions, which would be ingested along with partially digested starches to be used in the midgut [7] . Complete breakdown of starch should take place in the midgut where large amounts of amylase exist.
Amylases in insect are generally most active in the neutral to slightly acid pH condition [2] . Optimal pH values for amylases in larvae of several coleopterans were 4-5.8 and in Lygus spp. (Heteroptera) was 6.5 [34] . Optimum pH generally corresponds to the pH prevailing in the midguts from which the amylases are isolated.
The E. maura α-amylase has an optimum temperature activity of 30-35°C, which is consistent with the other reports [19,23] .
Mg and Ca ions have inhibitory effects on the αamylase activity of this insect. Also, there are reports that bacterial α-amylase (Thermus sp.) is not affected by Ca2+ [29] . However, it has been reported that αamylases are metalloproteins that require calcium for maximum activity. Calcium also affords stability for the amylases from a variety of sources, including insects, to both pH and temperature extremes [4] .
The other features of this enzyme, such as sensitivities to chelating agent (EDTA), urea and SDS, are that typical to many animal amylases [24,33] .