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
[alpha]-Amylases ([alpha]-1, 4-glucan-4-glucanohydrolases, EC
18.104.22.168) are hydrolytic enzymes that are widespread in nature, being
found in microorganisms, plants and animals. These enzymes catalyze the
hydrolysis of [alpha]-D-(1, 4)-glucan linkage in starch components,
glycogen and various other related carbohydrates (16), (30).
E. maura like other insect pests of wheat lives on a
polysaccharide-rich diet and depends to a large extent on the
effectiveness of its [alpha]-amylases for survival (23). It converts
starch to maltose, which is then hydrolyzed to glucose by an
[alpha]-glucosidase. In insects only [alpha]-amylases has been found to
hydrolyze long [alpha]-1, 4-glucan chains such as starch or glycogen
(33). Amylase activity has been described from several insect orders
including Coleoptera, Hymenoptera, Diptra, Lepidoptera and Hemiptera
(4), (23), (25), (31), (34).
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
Nothing is currently known about the properties of [alpha]-amylase
of E. maura. The purpose of the present study is to identify and
characterize the [alpha]-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
MATERIALS AND METHODS
Insects: The insects were collected from the Gorgan wheat farm of
Golestan Province, Iran and maintained on wheat plants in the laboratory
at 27[+ or -]2[degrees]C with 14 h light: 10 h dark cycle. Voucher
specimens are kept in the Entomological Laboratory, Plant Protection
Department, Tehran University (Fig. 1).
[FIGURE 1 OMITTED]
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, 2-morpholinoethanesulfonic acid at pH 6.5
The salivary glands was separated from the insect body, rinsed in
ice-cold saline buffer, placed in a pre-cooled 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 15000xg for 20
min at 4[degrees]C. The supernatants were pooled and stored at
-20[degrees]C for subsequent analyses.
Amylase activity assay: The [alpha]-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[degrees]C with
500 [micro]L universal buffer and 40 [micro]L soluble starch. The
reaction was stopped by addition of 100 [micro]L 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 [alpha]-amylase action
are measured by the reduction of 3, 5-dinitrosalicylic acid. The boiling
water is for stopping the [alpha]-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 [alpha]-amylase activity was defined as the amount of enzyme
required to produce 1 mg maltose in 30 min at 35[degrees]C. A standard
curve of absorbance against amount of maltose released was constructed
to enable calculation of the amount of maltose released during
[alpha]-amylase assays. Serial dilutions of maltose (Merck, Product
Number 105911, Mr 360.32 mg [mol.sup.-1]) in the universal buffer at pH
6.5 were made to give following range of concentrations of 2, 1, 0.5,
0.25, 0.125 mg [mL.sup.-1] (Fig. 1).
A blank without substrate but with [alpha]-amylase extract and a
control containing no [alpha]-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 [alpha]-amylase activity was examined using
[alpha]-amylase extracted from adult salivary glands. The effect of
temperature on [alpha]-amylase activity was determined either by
incubating the reaction mixture at 10, 15, 20, 25, 30, 35, 40, 45, 50,
55, 60 and 70[degrees]C for 30 min. The effect of temperature on
stability of amylase activity was tested by pre-incubation of the enzyme
at 10, 20, 30, 40, 50, 60 and 70[degrees]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 [alpha]-amylase was determined by
pre-incubation of enzyme at mentioned pH for 60 min prior to the assay.
Effect of activators and inhibitors on enzyme activity: To test the
effect of different ions on the enzyme, salivary glands were dissected
in distilled water. Enzyme assays were performed in the presence of
different concentrations of chloride salts of Na+(5, 10, 20 and 40 mM),
K+ (5, 10, 20 and 40 mM), Ca+2 (5, 10, 20 and 40 mM), Mg+2 (5, 10, 20
and 40 mM) and EDTA (0.5, 1, 2 and 4 mM), SDS (1, 2 and 4 mM) and urea
(0.5, 1, 2, 4, 6 and 8 M). These compounds were added to the assay
mixture and activity was measured after 30 min incubation period.
Control was measured without adding any compounds.
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.
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).
[FIGURE 2 OMITTED]
[alpha]-amylase activity: Studies showed that [alpha]-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 [alpha]-amylases, which have optimal activities at neutral or
slightly acid pH values, [alpha]-amylase 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.
[FIGURE 3 OMITTED]
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[degrees]C (Fig. 4). Sensitivity of
amylase to pre-incubation did not change significantly at pre-incubation
temperature of 10-50[degrees]C, but the greatest sensitivity was found
at higher temperatures (Fig. 4).
[FIGURE 4 OMITTED]
Effect of activators and inhibitors on enzyme activity: Na and K
ions increased amylase activity only a little (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.
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.
The present study showed that the adult E. maura has
[alpha]-amylase 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 [alpha]-amylase has an optimum temperature activity of
30-35[degrees]C, which is consistent with the other reports (19), (23).
Inhibitors and activators used were chosen for comparison with
reported values (2), (24), (33), (35). Data showed that NaCl activated
the enzyme. Similarly, in Lygus hesperus Knight and L. lineolaris
(Palisot de Beauvois), [alpha]-amylases were activated by NaCl (1),
(34). Cohen and Hendrix found that some homopterans'
[alpha]-amylase is also Cl-activated (13). Amylase activation by Cl- is
characteristic has been reported in many mammals and bacteria (29),
(33), nematodes (23), as well as other insects. However, the amylases in
some insect species, e.g., Callosobruchus chinensis (Linaeus)
(Coleoptera: Bruchidae), Bombyx mori (Linnaeus) (Lepidoptera:
Bombycidae), are inhibited by Cl- (33). Potassium ions have been shown
to have more or less the same effect on [alpha]-amylase as Cl-ions.
Mg and Ca ions have inhibitory effects on the [alpha]-amylase
activity of this insect. Also, there are reports that bacterial
[alpha]-amylase (Thermus sp.) is not affected by Ca2+ (29). However, it
has been reported that [alpha]-amylases 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).
We thanks to M.R. Ahmadi for collecting and rearing insects. This
research was supported by University of Tehran Grant (no. 31303).
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Mohammad Mehrabadi and Ali R. Bandani
Department of Plant Protection, School of Plant Protection and
Horticultural Sciences, Agriculture Campus, University of Tehran, Karaj,
Corresponding Author: Mohammad Mehrabadi, Department of Plant
Protection, School of Plant Protection and Horticultural Sciences,
Agriculture Campus, University of Tehran, Karaj, Iran
Table 1: The activity of [alpha]-amylase in adults of E. maura
Stage Activity per ml enzyme ([mu]mol Unit Activity ([mu]mol
[min.sup.-1] [mL.sup.-1], [min.sup.-1] [u.sup.-1],
Mean[+ or -]SE) Mean[+ or -]SE
Adult 0.00050 [+ or -] 0.020 0.0050 [+ or -] 0.023
Sample size, n = 10
Table 2: Relative activity of E. maura [alpha]-amylase toward different
compounds (a). Values are means[+ or -]S.E. (Standard error), n = 3
Compound Concentration Relative activity(%)
Control -- 100
NaCl 5 mM 100
10 mM 100
20 mM 105
40 mM 99
CaCl2 5 mM 99
10 mM 102
20 mM 95
40 mM 96
KCl 5 mM 102
10 mM 100
20 mM 104
40 mM 108
MgCl2 5 mM 90
10 mM 83
20 mM 77
40 mM 70
EDTA 0.5 mM 98
1 mM 6
2 mM 94
4 mM 93
SDS 1 mM 97
2 mM 94
4 mM 22
Urea 0.5 M 98
1 M 95
2 M 91
4 M 85
6 M 58
8 M 20
(a) The enzyme was pre-incubated for 10 min at 35[degrees]C with listed
compounds at the final concentration indicated prior to substrate
addition. Activity in absence of compounds was taken as 100%. Each value
represents the average of three independent experiments.