Biologia 67/6: 1186—1194, 2012
Section Cellular and Molecular Biology
DOI: 10.2478/s11756-012-0121-y
Biochemical characterization of α- and β-glucosidases in alimentary
canal, salivary glands and haemolymph of the rice green caterpillar,
Naranga aenescens M. (Lepidoptera: Noctuidae)
Ameneh Asadi1, Mohammad Ghadamyari1*, Reza H. Sajedi2, Jalal J. Sendi1
& Mehrdad Tabari3
1
Department of Plant Protection, Faculty of Agricultural Science, University of Guilan, Rasht, Iran; e-mail:
[email protected]; [email protected]
2
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
3
Institute of Rice, Amol, Iran
Abstract: The biochemical properties of α- and β-glucosidase in salivary glands, alimentary canal and haemolymph of
Naranga aenescens larvae, one of the most damaging pests of the rice crop in Iran, were investigated. The specific activity of
α-glucosidases were 3.88, 2.74 and 1.58 µmol/min per mg protein in the alimentary canal, salivary glands and haemolymph
of last instar larvae, respectively. The specific activity of β-glucosidases were 1.27, 0.077 and 0.414 µmol/min per mg
protein in the alimentary canal, salivary glands and haemolymph of last instar larvae, respectively. The optimal pH for
α-glucosidases were 6.0, 6.0–8.0 and 6.0 and the maximum activity for β-glucosidases were obtained at pH 6.0, 5.0–7.0 and
5.0 in alimentary canal, salivary glands and haemolymph, respectively. The optimum temperatures for β-glucosidases were
determined at 55 ◦C in alimentary canal, 35–45 ◦C in salivary glands and 55 ◦C in haemolymph, whereas the α-glucosidases
reached their optimum at 45 ◦C in all three tissues. Effect of metal ions on the activity of α- and β-glucosidases showed
that K+ (20 mM) and Mg2+ (10 and 20 mM) increased N. aenescens α- and β-glucosidases activities from salivary glands,
while Ca2+ increased α- and β-glucosidases activities in haemolymph. In the presence of Fe2+ , Mn2+ , Hg+ and Zn2+ (10,
20 mM) and Hg2+ (20 mM), these enzymes from all tissues were completely inactivated. Km values were estimated for the
α-glucosidases as 3.96, 0.547 and 3.084 mM and for β-glucosidases as 1.93, 1.014 and 1.93 mM in the alimentary canal,
salivary gland and haemolymph, respectively. The zymogram analyses of N. aenescens crude extracts indicated the presence
of at least two isoforms for α-glucosidase and one isoform for β-glucosidase.
Key words: rice green caterpillar; α-glucosidase; β-glucosidase; alimentary canal; salivary glands; haemolymph.
Abbreviations: EDTA, ethylenediaminetetraacetic acid; 4-MUαG, 4-methylumbelliferyl-α-D-glucopyranoside; 4-MUβG,
4-methylumbelliferyl-β-D-glucopyranoside; PAGE, polyacrylamide gel electrophoresis; pNαG, p-nitrophenyl-α-D-glucopyranoside;
pNβG, p-nitrophenyl-β-D-glucopyranoside.
Introduction
α- and β-glucosidase secreted by insect midgut’s epithelial cells break down the polysaccharides into absorbable elements. Insect α-glucosidase (EC 3.2.1.20)
is an exo-acting hydrolase that exploits α-glucose
from the non-reducing end of oligosaccharides and
polysaccharides. This enzyme has been characterized
from bacteria, fungi, yeast, plants and animals (Vihinen & Mantsala 1989). So far, α-glucosidase was
studied from honey, hypopharyngeal glands of honey
bee, the midgut and salivary glands of Glyphodes pyloalis Walker (Lep.: Pyralidae) and midgut of Xanthogaleruca luteola Müll. (Col.: Chrysomelidae), Osphrantria coerulescens (Col.: Cerambycidae), Rhynchophorus ferrugineus Olivieri (Col.: Curculionidae)
(Huber & Mathison 1976; Ghadamyari et al. 2010;
Sharifi et al. 2011; Riseh et al. 2012). Although αglucosidases are found in four families of glycoside hydrolases (GHs): GH4, GH13, GH31, and GH97, based
on CAZy (Carbohydrate-Active enZymes) (Cantarel et
al. 2009) classification system, insect α-glucosidases are
found exclusively in family GH13 (Gabrisko & Janecek
2011).
Aryl and alkyl β-glucosides can be hydrolyzed by
β-glucosidase (EC 3.2.1.21) (β-d-glucoside glucohydrolase) and led to glycon and aglycone release (Reese
1977). These enzymes have been studied in plants,
fungi, bacteria and animals (Woodward & Wiseman
1982). In insects, hemicelluloses and cellulose can be hydrolyzed enzymatically by some specific enzymes to diand oligo-β-saccharides which digestive β-glucosidases
* Corresponding author
c 2012 Institute of Molecular Biology, Slovak Academy of Sciences
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Biochemical characterization of α- and β -glucosidases in Naranga aenescens
are important for their hydrolysis (Terra & Ferreira
1994). Due to involvement of β-glucosidases in interactions between insect and host plant, β-digestive glucosidases have been studied in many insect species
from different orders (Ferreira & Terra 1983; Santos
& Terra 1985; Sharifi et al. 2011; Riseh et al. 2012). βGlucosidases are reported in midgut and salivary glands
of lepidopteran insect, such as G. pyloalis (Ghadamyari
et al. 2010), Thaumetopoea pityocampa Schiffermuller
(Lep.: Notodontidae) (Pratviel-Sosa et al. 1986), Parnassius apollo L. (Lep.: Papilionidae) (Nakonieczny et
al. 2006), Bombyx mori (Lep.: Bombycidae) (Byeon et
al. 2005), Spodoptera frugiperda Smith (Lep.: Noctuidae), Erinnyis ello Linnaeus (Lep.: Sphingidae), and
Diatraea saccharalis Fabricius (Lep.: Pyralidae) (Santos & Terra 1985; Ferreira et al. 1998).
Naranga aenescens Moore (Lep.: Noctuidae), commonly known as the rice green caterpillar, can cause significant economic damage to rice when too abundant.
This pest feeds on rice’s leaves and the times of high
densities also feeds on panicle rachis near the developing kernels causing these kernels to dry before filling.
This insect was reported in Guilan and Mazanderan
provinces of Iran in 1986 and was widely distributed
in all paddy fields (Abivardi 2001). Chemical control is
still the predominant method for control of rice green
caterpillar in Iran and fenitrothion and chlopyrifos was
recommended by Iranian Plant Protection Organization for its control. Application of these insecticides
against N. aenescens is extremely hazardous to farmers, consumers and environment. The gut physiology of
this pest can be important subject for study and design of new approach for its control, such as transgenic
plants and synthetic inhibitors.
In our previous study, the biochemical characterizations of different isoforms of N. aenescens α-amylases
were performed (Asadi et al. 2010). The present paper
delivers the report on the biochemical properties of αand β-glucosidases in salivary glands, alimentary canal
and haemolymph of N. aenescens larvae.
Material and methods
Chemicals
p-Nitrophenol and bovine serum albumin were purchased
from Merck (Merck, Darmstadt, Germany). p-Nitrophenylα-D-glucopyranoside (pNαG), p-nitrophenyl-β-D-glucopyranoside (pNβG), 4-methylumbelliferyl-α-D-glucopyranoside
(4-MUαG) and 4-methylumbelliferyl-β-D-glucopyranoside
(4-MUβG) were obtained from Sigma (Sigma, St Louis, MO,
USA).
Insect
Insect was collected from rice seedling of Oryza sativa L.
variety ‘Hashemi’ in the northern provinces of Iran. Last
instar larvae were randomly selected for measuring the enzyme activity.
Sample preparation
Last instar larvae were immobilized on ice and dissected
under a stereo microscope in ice-cold saline buffer and
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whole of the alimentary canal and salivary glands were removed. The gastrointestinal tract examined without the
contents and malpigian tube and unwanted tissues. Also,
the haemolymph was collected from last instar larvae. The
larvae were bent backwards, and were holding the head and
tail of the larvae with one hand. The hemolymph was collected by cutting the larval proleg with a 75 µL glass capillary tube. After collecting, the samples were transferred to
the freezer (−20 ◦C). Also, digestive systems were divided
into three distinct divisions (Fig. 1).
The whole of the alimentary canal, three sections of alimentary canal and salivary glands were homogenized in cold
double-distilled water (140 µL and 40 µL double-distilled
water for a gut in α- and β- glucosidase, respectively, 3 µL
double-distilled water for a pair of salivary glands) using
a hand-held glass homogenizer. The homogenate was centrifuged at 13000×g for 15 min at 4 ◦C and supernatants
were collected and stored at −20 ◦C for subsequent analyses.
Determination of α- and β-glucosidase activities and protein
concentration
Specific activities of α- and β-glucosidase in alimentary
canal, salivary glands and haemolymph were determined
in the supernatants. All assays were performed at 35 ◦C
in 40 mM glycine-phosphate-acetate mixed buffer (pH 6.0
for α-glucosidases, pH 5.0 for β-glucosidases) in five replicates. Activities of α- and β-glucosidases were measured
with pNαG and pNβG as substrates, respectively. Ten µL
supernatants were incubated for 20 min at 35 ◦C with 45 µL
of pNαG (25 mM) or pNβG (25 mM) and 115 µL of 40 mM
mixed buffer (final volume of the incubation mixture was
170 µL). The pNαG and pNβG hydrolyses were stopped
by adding 600 µL of NaOH (0.25 M) and hydrolysis was
measured by colorimetric detection of p-nitrophenol release
at 405 nm using a microplate reader (Awareness, Stat Fax
3200, USA) after 10 min. Standard curves using different concentrations of p-nitrophenol were included to enable
quantification. Protein concentration was determined by the
Bradford method (Bradford 1976) using bovine serum albumin as standard.
Polyacrylamide gel electrophoresis and zymogram analysis
Zymogram analyses were carried out using non-denaturing
polyacrylamide gel electrophoresis (PAGE) based on the
method of Davis (1964) modified by Riseh et al. (2012).
The enzyme samples were mixed with non-denaturing sample buffer (without mercaptoethanol) and applied onto 10%
(w/v) polyacrylamide gel. Electrophoresis was performed
with constant voltage (100 V) in a refrigerator (at 4 ◦C). After the electrophoresis is complete, the gel was submerged in
3 mM 4-MUαG and 4-MUβG in 0.1 M sodium acetate (pH
5.0) for 15 min at 26±2 ◦C to develop fluorescents bands corresponding to α- and β-glucosidases activities, respectively.
The blue-fluorescent bands were appeared under UV and
photographed with gel documentation apparatus (Uvitec
Cambridge).
Effects of pH and temperature of enzyme activities
The activities of α- and β-glucosidases in the alimentary
canal, salivary gland and haemolymph were determined at
room temperature at different pH values of 40 mM glycinephosphate-acetate mixed buffer (pH 3.0–12.0). Also, the activities of the enzymes were measured at several temperatures (15–85 ◦C) in 40 mM glycine-phosphate-acetate mixed
buffer, pH optimum.
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A. Asadi et al.
Fig. 1. Alimentary canal and salivary glands of N. aenescens.
Table 1. Comparison of α- and β-glucosidase activity in different
tissues of last instar larvae of N. aenescens.a
Activity (µmol/min/mg protein)
Tissue
α-Glucosidase
Alimentary canal
Salivary gland
Haemolymph
Foregut
Midgut
Hindgut
3.88
2.74
1.58
0.51
3.08
0.31
±
±
±
±
±
±
0.03
0.02
0.02
0.22
0.23
0.01
a
c
d
e
b
f
β-Glucosidase
1.272
0.077
0.414
0.268
0.716
0.307
±
±
±
±
±
±
0.015 a
0.01 f
0.007 c
0.139 e
0.088 b
0.06 d
a
Different letters indicate that the specific activity of enzymes is
significantly different from each other by Tukey’s test (p < 0.05).
Effect of metal ions on enzyme activities
Enzyme assays were performed in the presence of different concentrations of chloride salts of Zn2+ , Mg2+ , Ca2+ ,
K+ , Co+2 , Ba+2 , Fe+2 , Mn+2 , Hg+2 , Hg+ and EDTA (10
and 20 mM) for α- and β-glucosidases. The activities of
enzymes were measured by adding ions in 40 mM glycinephosphate-acetate mixed buffer (optimum pH), and activity
was measured after 20 min pre-incubation. A control (no
added ions) and blank (without enzyme) included ion were
also measured.
Kinetic parameters of α- and β- glucosidase
The Michaelis-Menten constant (Km ) and the maximum
velocities (Vmax ) of α- and β-glucosidase were determined
by Lineweaver-Burk plots. The homogenate was incubated
in an appropriate buffer (optimum pH) at 35 ◦C with
pNαG and pNβG in final concentrations ranging from
5 to 80 mM. The experiments were performed in triplicate.
Statistical analysis
Data were compared by one-way analysis of variance
(ANOVA) followed by Tukey’s test using SAS program version 8 (SAS Institute 1997).
Results
α- and β-glucosidase activity
Studies showed that both α- and β-glucosidase are
present in the alimentary canal, salivary glands and
haemolymph of last instar larvaeN. aenescens. The activity of α-glucosidase in the alimentary canal was 1.41and 2.45-fold higher than its activity in salivary glands
and haemolymph of last instar larvae, respectively (Table 1). The specific activity of β-glucosidases were 1.27,
0.077 and 0.414 µmol/min per mg protein in the alimentary canal, salivary glands and haemolymph of
last instar larvae, respectively. The alimentary canal
of N. aenescens was divided into three main sections:
foregut, midgut and hindgut (Fig. 1) and the α- and
β-glucosidase specific activities of each of this section were measured for the last larval instar as indicated in Material and methods section. Under these
conditions, the corresponding α-glucosidases specific
activities were 0.268 µmol/min per mg protein for
forgut, 0.716 µmol/min per mg protein for midgut, and
0.307 µmol/min per mg protein for hindgut (Table 1).
Zymogram analysis
Zymogram patterns using native PAGE for N. aenescens alimentary canal showed at least one isozyme
for β-glucosidase and two isozymes for α-glucosidase
(Fig. 2).
Effect of pH and temperature on α- and β-glucosidase
activities
The optimum pH for α-glucosidases were 6.0–7.0 in
alimentary canal, 6.0–8.0 in salivary glands and 6.0
in haemolymph (Fig. 3). The optimum pH for βglucosidases were 6.0 in alimentary canal, 5.0–7.0 in
salivary glands and 5.0–6.0 in haemolymph (Fig. 4).
All these α-glucosidases exhibited their maximum activities at 45 ◦C from all three tissues (Fig. 3). Maximum
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Biochemical characterization of α- and β-glucosidases in Naranga aenescens
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Fig. 2. Zymogram of α- and β-glucosidases from N. aenescens.
Extract was applied onto gel. α, equivalent 1; β1, equivalent 0.66;
β2, equivalent 0.75; β3, equivalent 1; β4, equivalent 2 midgut
extract.
Fig. 4. Effect of pH (a) and temperature (b) on activity of βglucosidase extracted from alimentary canal (•), salivary glands
() and haemolymph (+) of N. aenescens larvae.
Effects of metal ions and EDTA on the α- and βglucosidase activities
Effect of metal ions on the activities of α- and βglucosidases showed that K+ (20 mM) and Mg2+ (10,
20 mM) increased N. aenescens α- and β-glucosidase
activities from salivary glands and Ca2+ increased both
α- and β-glucosidase activities in haemolymph. In the
presence of Fe2+ , Mn2+ , Hg+ and Zn2+ (10, 20 mM)
and Hg2+ (20 mM), a complete inactivation has been
observed for these enzymes from all tissues (Tables 2
and 3).
Fig. 3. Effect of pH (a) and temperature (b) on activity of αglucosidase extracted from alimentary canal (•), salivary glands
() and haemolymph (+) of N. aenescens larvae.
β-glucosidase activities were found at temperature 55 ◦C
in alimentary canal and haemolymph and 35–45 ◦C in
salivary glands (Fig. 4).
Kinetic parameters
Kinetic parameters of α- and β-glucosidases in the alimentary canal, salivary glands and haemolymph were
measured by pNαG and pNβG substrates at optimal
pH and 35 ◦C. The Km values of α-glucosidases in the
alimentary canal, salivary gland and haemolymph were
3.96, 0.55 and 3.08 mM, respectively. Also, the Km values of β-glucosidases in the alimentary canal, salivary
gland and haemolymph were obtained as 1.93, 1.01 and
1.93 mM, respectively (Table 4).
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A. Asadi et al.
Table 2. Effect of various metal ions and EDTA (10 mM) on relative N. aenescens α- and β-glucosidase activities.a
Relative activity (%) (mean ± SE)
Reagents
Control
K+
Ca2+
Co2+
Ba2+
Fe2+
Mg2+
Mn2+
Zn2+
Hg2+
Hg+
EDTA
Alimentary canal
Salivary gland
Haemolymph
α-Glucosidase
β-Glucosidase
α-Glucosidase
β-Glucosidase
α-Glucosidase
β-Glucosidase
100d
110.2 ± 0.5b
93.5 ± 0.5e
30 ± 0.6i
59.5 ± 0.5g
6 ± 0.5k
129.8 ± 0.2a
52.5 ± 2.8h
53.9 ± 0.8h
106 ± 0.5c
15.2 ± 3.1j
84.4 ± 0.2f
100d
110.75 ± 2b
95.97 ± 1.7e
73.4 ± 2.7g
81.19 ± 2.1f
49.1 ± 0.2i
117 ± 2a
72.47 ± 2.1j
79.81 ± 2.3f
65.1 ± 2.7h
3.15 ± 2.3j
106.7 ± 2.3c
100c
104.6 ± 1.4b
141.5 ± 2.7a
97.4 ± 1.8d
51.3 ± 2.4h
80.2 ± 2.7f
103.2 ± 1.4b
78.2 ± 0.7g
48.15 ± 2.8i
91.99 ± 1.9e
30 ± 1.6j
101 ± 1.7c
100d
103.6 ± 2c
166 ± 2a
91.66 ± 0.3e
58 ± 2.3i
63 ± 2.6h
115 ± 2.9b
58.2 ± 2i
83 ± 1.2f
69 ± 1.7g
14.3 ± 2.7j
62.3 ± 2h
100ab
108 ± 0.7a
122 ± 0.8abc
32 ± 2.5cd
47 ± 0.7bcd
4.7 ± 0.7d
129 ± 1.4a
4.6 ± 0.4d
34.7 ± 1.3cd
99 ± 2.9ab
10 ± 0.7d
73.7 ± 2.4abc
100a
103 ± 1.2a
111 ± 0.5a
87.7 ± 0.9a
91 ± 2.4a
9.3 ± 2.7b
109.6 ± 2a
22.7 ± 2.3b
78.2 ± 2.7a
98.5 ± 0.3a
1.1 ± 0.5b
97.7 ± 0.8a
a
Different letters indicate that the specific activity of enzymes is significantly different from each other by Tukey’s test (p < 0.05).
All metal ion were added as chloride salts.
Table 3. Effect of various metal ions and EDTA (20 mM) on relative N. aenescens α- and β-glucosidase activities.a
Relative activity (%) (mean ± SE)
Reagents
Control
K+
Ca2+
Co2+
Ba2+
Fe2+
Mg2+
Mn2+
Zn2+
Hg2+
Hg+
EDTA
Alimentary canal
Salivary gland
Haemolymph
α-Glucosidase
β-Glucosidase
α-Glucosidase
β-Glucosidase
α-Glucosidase
β-Glucosidase
100ab
100.5 ± 0.6a
95.6 ± 0.8b
11.6 ± 1.8ef
7.3 ± 0.4f
2.8 ± 0.4g
103.4 ± 0.5a
19.3 ± 0.5d
14.8 ± 0.4e
86.6 ± 0.8c
2.8 ± 0.9g
66.5 ± 1.5c
100c
105.83 ± 1.3b
110.53 ± 2.3a
65.64 ± 2.6h
82.8 ± 1.5e
9.8 ± 1.8j
111.8 ± 0.3a
54 ± 1i
67.7 ± 1.3g
79.3 ± 0.7f
7.79 ± 0.9k
96.2 ± 2.7d
100d
121.8 ± 1.7b
155.6 ± 0.8a
34.5 ± 0.9g
29.6 ± 1.4h
71.2 ± 0.6f
106.5 ± 1.5c
19.26 ± 1j
35.12 ± 1.3g
12 ± 1.2k
27.39 ± 0.4i
98.1 ± 0.8e
100d
179 ± 2.4c
243 ± 2.7a
78.18 ± 0.9f
53.6 ± 0.9h
46.3 ± 2.4i
229.9 ± 2.4b
3 ± 2k
86.3 ± 2.4e
10.9 ± 2.3j
10.6 ± 2.7j
63.6 ± 0.9g
100d
118 ± 2.4b
112 ± 2.1c
38 ± 0.4h
43 ± 2g
7.5 ± 2.5k
131 ± 1.3a
14 ± 1.7j
27.8 ± 1.3i
84.2 ± 1.6e
5.2 ± 1.3l
45.7 ± 1.5f
100e
110.5 ± 0.6b
119.8 ± 1.5a
107.7 ± 1.1c
107.7 ± 1.1c
6.8 ± 2.7g
104.3 ± 0.7d
13 ± 0.8i
90.8 ± 0.8g
83.5 ± 2.9h
1.1 ± 0.9k
96.2 ± 2.4f
a Different letters indicate that the specific activity of enzymes is significantly different from each other by Tukey’s test (p < 0.05). All
metal ion were added as chloride salts.
Table 4. Kinetic parameters of alimentary canal, salivary gland and haemolymph α- and β-glucosidases from N. aenescensa .
Parameter
Enzyme
Km
Vmax
Km
Vmax
α-Glucosidase
α-Glucosidase
β-Glucosidase
β-Glucosidase
Alimentary canal
3.96
0.786
1.927
0.143
±
±
±
±
0.005 a
0.02 a
0.04 a
0.02 c
Salivary gland
0.547
0.006
1.014
0.195
±
±
±
±
0.003 c
0.013 c
0.04 b
0.03 b
Haemolymph
3.084
0.117
1.927
0.291
±
±
±
±
0.02 b
0.015 b
0.003 a
0.06 a
a Different letters indicate that the kinetic parameters of enzymes are significantly different from each other by Tukey’s test (p < 0.05).
Km and Vmax are expressed as mM and mM/min per mg protein, respectively.
Discussion
α- and β-glucosidase activities were detected in the
alimentary canal, salivary glands and haemolymph of
last larval instar of N. aenescens using pNαG and
pNβG as substrates (Table 1). Our results showed that
there is a significant difference in the specific activity
of α- and β-glucosidase in alimentary canal, salivary
gland and haemolymph of last instar N. aenescens.
The specific activity of the α- and β-glucosidase in
the alimentary canal of N. aenescens was much higher
than that in the salivary glands and haemolymph,
which is consistent with α- and β-glucosidase activity in G. pyloalis (Ghadamyari et al. 2010) and Hyphantria cunea (Drury) (Lep.: Pyralidae) (our unpublished data). In contrast, β-glucosidase activity in the
salivary glands of Nasutitermes takasagoensis is more
than 66% of total activity in the digestive system
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Biochemical characterization of α- and β-glucosidases in Naranga aenescens
(Tokuda et al. 1997). Previous works showed the presence of α- and β-glucosidase activities in the salivary glands of other phytophagous lepidopterans insect
(Santo & Terra 1985; Franzl et al. 1989; Ferreira et
al. 1997; Marana et al. 2000; Ghadamyari et al. 2010).
The specific β-glucosidase activity in midgut of the
sugar cane borer D. saccharalis was determined to be
0.021 µmol/min per mg protein with p-nitrophenyl-βglucoside as the substrate (Azevedo et al. 2003). Also,
Ghadamyari et al. (2010) showed that the specific βglucosidase activity in midgut of G. pyloalis 5th instar
larvae was 0.998±0.01 µmol/min per mg protein when
pNβG was used as the substrate. Therefore, the specific β-glucosidase activity in N. aenescens was higher
than that in some lepidopterian insects, such as G. pyloalis, S. frugiperda, E. ello and D. saccharalis (Santos & Terra 1985; Ferreira et al. 1998; Ghadamyari
et al. 2010). In contrast to our results, very high βglucosidase activity was reported in Apollo butterfly,
Parnassius apollo ssp. Frankenbergeri (Nakonieczny et
al. 2006), that allows this insect to hydrolyze at least
some compounds containing β-glycosidic bonds in its
diet. Similar to our results, β-glucosidase activity in
midgut of Neotermes koshunensis (Isoptera: Kalotermitidae) was higher than its activity in salivary glands
(Tokuda et al. 2002). It seems the activities of these
enzymes vary depending on insect species, glycosidic
bonds available in diets and host plant foods, e.g., in
blood-sucking insect Rhodnius prolixus α-glucosidase is
involved in heme detoxification (Mury et al. 2009).
The specific activities of α- and β-glucosidase measured in different sections of last larval instar (Table 1)
indicate a higher activity in midgut than that determined in foregut and hindgut. In particular, the βglucosidase activity in midgut was 2.64-fold and 2.33fold higher than its activity in foregut and hindgut of
last larval instar, respectively (Table 1). Similar to our
results, Sharifi et al. (2011) showed that the α- and
β-glucosidase activity in the midgut of X. luteola was
higher than that in foregut and hindgut of last larval
instar.
The ratios of α-glucosidase/β-glucosidase were 3
in alimentary canal, 35.58 in salivary glands and 3.8 in
haemolymph, based on assay results using pNαG and
pNβG as substrates. These results demonstrated that
the α-glucosidase specific activities in digestive system,
salivary glands and haemolymph of 5th instar larvae
were higher than β-glucosidase.
α- and β-glucosidases in alimentary canal, salivary
glands and haemolymph of N. aenescens showed their
maximum activity at neutral to slightly acidic pH conditions. Optimal pH for disaccharidases and specific
glucosidases in lepidopteran insect was reported as 6.0–
8.0 for example, 5.3 for trehalase in Galleria mellonella
L. (Lep.: Pyralidae) (Janda 1985), 6.0 for β-glucosidase
in the larvae of silkworm (Byeon et al. 2005), 4.5–5
for β-glucosidase in Zygaena trifolii Esper (Lep.: Zygaenidae) (Franzl et al. 1989), 4.9–5.6 for α-glucosidase
in the larvae of Apollo butterfly (Nakonieczny et al.
2006) and 8.0 for α-glucosidase in 3rd instar larvae
1191
of Earias vitella (Lep.: Noctuidae) (Tripathi & Krishna 1988). Also, maximum salivary β-glucosidase activity in N. koshunensis was reported as 5.6 (Ni et al.
2007). Ghadamyari et al. (2010) reported that the optimal pH for α- and β-glucosidase activities extracted
from midgut and salivary glands of the lesser mulberry pyralid were 7.5, 5.5, 8–9 and 8–9, respectively.
Also, the X. luteola α- and β-glucosidases have highest activity at pH 5.0 and pH 6.0, respectively (Sharifi et al. 2011). The differences between the α- and βglucosidase optimal pH between insect species may refer to their phylogenetic relation or response to different
diets (Nakonieczny et al. 2006). Also the origin of the
α- and β-glucosidase, i.e. digestive system or salivary
glands may justify these differences, for example, the
deduced amino acid sequences of β-glucosidase in the
midgut of Nasutitermes takasagoensis are 87–91% identical to those of the salivary β-glucosidases (Tokuda
et al. 2009). Recent evolutionary studies on insect αglucosidases indicate that the similarity between orthologues from different species is higher than that of paralogues from one species (Gabrisko & Janecek 2011).
Secondary metabolites such as tannins in plants
mediate defences against insect herbivores that impair
digestive processes in the insect gut. These compounds
bind to proteins available in insect’s gut at acidic and
lower pH and these binding led to reduction in efficiency of digestion (Dow 1984). Therefore, the high
pH of the insect gut environment is an adaptation to
feed on plant containing high concentration of tannins
(Chapman 1998). It seems the pH value in digestive
system of this pest is alkali, such as known for other
lepidopteran insect; however, the gut pH in this pest
was not studied.
Like most enzymatic reactions, the rate of pnitrophenyl release from pNαG and pNβG by N. aenescens α- and β-glucosidases increases as the temperature is raised. In the case of these enzymes, such
as for many enzymes, activities are adversely affected
by high temperatures. As shown in Figs 3 and 4, the
reaction rate increases with temperature to a maximum level at 45–55 ◦C, then abruptly declines with further increase of temperature due to denaturation of αand β-glucosidases at temperatures above 55 ◦C. The
N. aenescens α- and β-glucosidases exhibit an optimum activity at temperature 45–55 ◦C, which is consistent with other reports. Ghadamyari et al. (2010)
reported that optimal α- and β-glucosidase activity in
midgut of G. pyloalis was observed at 45 ◦C. The enzyme activity at temperatures ranging from 15 to 45 ◦C
increased steadily until that achieving the maximum
activity at 45–55 ◦C (Figs 3 and 4). At 55 ◦C, the α- and
β-glucosidase activity dropped sharply to about 20%
and 40% of the maximal value. Most insect α- and βglucosidases exhibit temperature optima ranging from
20 to 50 ◦C (Huber & Mathison 1976; Ghadamyari et al.
2010; Riseh et al. 2012). Franzl et al. (1989) reported
a temperature optimum for Z. trifolii β-glucosidase at
40 ◦C. Also, in the larvae of B. mori, the highest βglucosidase activity was obtained at 35–50 ◦C (Byeon
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1192
et al. 2005). Sharifi et al. (2011) showed that the X.
luteola α- and β-glucosidases have an optimum temperature activity at 60 and 50 ◦C, respectively. Ni et
al. (2007) showed that the optimum pH and temperature for N. koshunensis β-glucosidase was 45 ◦C and
the enzyme was active from 20 ◦C up to 45 ◦C. Highest
β-glucosidase activity of the palm weevil R. palmarum
L. was attained at 55 ◦C (Yapi et al. 2009).
The activity of α- and β-glucosidase was also
characterized by activity staining after native PAGE
which allowed visualization of the blue fluorescent
bands under UV. The results indicated that α- and βglucosidases in the alimentary canal of N. aenescens
showed two isoforms and one isoform, respectively. In
Athalia rosae, there are three isoforms of β-glucosidase
in the alimentary canal and two isoforms in the
haemolymph (our unpublished data). Ferreira et al.
(2003) reported that the midgut of the yellow mealworm Tenebrio molitor L. (Coleoptera: Tenebrionidae) larvae possesses four β-glycosidases. Three βglycosidases, named βGly1, βGly2 and βGly3, were
isolated from midgut tissues of the sugar cane borer,
D. saccharalis (Azevedo et al. 2003). Also, two βglycosidases were purified from S. frugiperda midgut
(Marana et al. 2000). The results of Riseh et al.
(2012) indicated two and three isoforms of α- and βglucosidases in crude digestive system extract of last
larval instar of R. ferrugineus, respectively. In the digestive system of other coleopteran insect, X. luteola, the
β-glucosidase has three isoforms (Sharifi et al. 2011).
Zymogram analysis of α- and β-glucosidase activities
in the gut of Osphranteria coerulescens (Redt) showed
that these activities correspond to three and four major
bands from this insect’s digestive system, respectively
(our unpublished results).
The effect of several metal ions and EDTA on the
α- and β-glucosidase activities in the alimentary canal,
salivary glands and haemolymph of N. aenescens last
instar larvae was studied at their optimum pH. Effect
of ions on the activity of α- and β-glucosidases showed
that K+ (20 mM) and Mg2+ (10, 20 mM) increased N.
aenescens α- and β-glucosidase activities from salivary
glands, Ca2+ increased α- and β-glucosidases activities
in haemolymph and Ba2+ decreased even completely
N. aenescens α- and β-glucosidases activities from all
three tissues, except the β-glucosidase extracted from
haemolymph. Also, EDTA decreased α-glucosidase activity from haemolymph and salivary glands and βglucosidase activity extracted from alimentary canal of
N. aenescens. Our results showed that N. aenescens
α- and β-glucosidases extracted from salivary glands
and α-glucosidase from haemolymph require calcium
for maximum activity. Mahboobi et al. (2011) showed
that activity of β-glucosidase in midgut ofAelia acuminata L. (Hemiptera: Pentatomidae) increased with addition of Na+ , Mg2+ , K+ , and Ca2+ and decreased in
the presence of sodium dodecyl suplhate, urea, Cu2+ ,
and Tris. Yapi et al. (2009) showed that CuCl2 , ZnCl2
and FeCl3 had inhibitory effect on β-glucosidase activity in digestive fluid of the palm weevil R. palmarum
A. Asadi et al.
larvae, whereas BaCl2 , MgCl2 , MnCl2 , SrCl2 and CaCl2
had no effect on the enzyme activity. Ghadamyari et
al. (2010) showed that the CaCl2 (40 mM) decreased
midgut β-glucosidase activity, whereas α-glucosidase
activity was significantly increased at this concentration and the α-glucosidase activity, in contrast to βglucosidase, was enhanced with increasing EDTA concentration.
Our results demonstrated that the Km values
of the α- and β-glucosidase in alimentary canal and
haemolymph were higher than that of salivary gland.
The Km and Vmax values of the β-glucosidase extracted from midgut of G. pyloalis were reported as
0.99 mM and 0.30 µmol/min per mg protein, respectively (Ghadamyari et al. 2010). Most insect α- and
β-glucosidases show Km values in the range 0.24–3 mM
(Ferreira & Terra 1983; Cuevas et al. 1992). Histidine,
aspartic and glutamic acid are conserved amino acid
residues at active sites of insect α-glucosidase. However,
some of these key residues were not detected in yeast
α-glucosidase. This structural difference between αglucosidases from different sources may led to changes
in the active site geometry and substrate specificity
as well as the catalytic efficiency (Mury et al. 2009).
The amino acid sequences of α- and β-glucosidase were
identified in some insects, but up to now there is no
information about this topic in lepidopteran α- and βglucosidases where amino acid sequences need to be analyzed. Research on substrate specificities of midgut βglycosidases from insects of different orders showed the
evolutionary trend from multiple enzymes with different substrate specificities to a single enzyme that is able
to hydrolyse all the β-glycosides within the same site
(Ferreira et al. 1998).
Many flavonoids, alkaloids, terpenoids, anthocyanins, glycosides and phenolic compounds, isolated from
plants, showed inhibitory effect on α-glucosidase (Kumar et al. 2011). It was also shown that insect βglucosidases which hydrolyze the plant glucosinolates
and cyanogenic glycosides have optimum pH 4.0–6.2
(Yu 1989). Plant secondary metabolites including alkaloids, terpenes, steroids, iridoid glycosides, aliphatic
molecules and phenolics are mediated in defences
against herbivores pest either by repellence or by impairing digestive processes in the insect gut (Hsiao
1985). These compounds confer resistance to various
host-plant species against herbivores pest. Glycosides
are secondary metabolites that can confer resistance to
plants against pests. So far, DIMBOA (a glycoside) isolated from corn seedlings has been shown to possess insecticidal properties and retard the larval development
of European corn borer, Ostrinia nubilalis (Hubner)
(Klun et al. 1967). Also, there is a correlation between
concentration of this metabolite in plant tissues and
level of resistance to corn leaf aphid. DIMBOA showed
decrease in reproductive potential, increase in mortality and feeding deterrents on aphids (Long et al. 1977).
Some Phaseolus varieties contain high concentration of
glycosides were resistant to a Mexican bean beetle, Epilachna varivestris (Nayar & Frankel 1963).
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Biochemical characterization of α- and β -glucosidases in Naranga aenescens
In conclusion, several investigations indicate the
main role of β-glucosidases in insect-host plant interaction and plant resistance to pests. The results showed
that α- and β-glucosidases play a fundamental role in
digestion of rice leaves in N. aenescens and the specific β-glucosidase activity in N. aenescens was higher
than that in some lepidopterian insects, such as G. pyloalis, S. frugiperda, E. ello and D. saccharalis. Since
the application of pesticides against this pest led to
contamination of drinking water and environment, one
area that can be considered in the development of new
insecticidal agents is based on the physiology and biochemistry of carbohydrases from the alimentary canal
of this pest. Therefore, the discovery of novel inhibitors
for α- and β-glucosidases available in plants can contribute to managing this pest via pest-resistant transgenic plants.
Acknowledgements
The authors express their gratitude to the Research Council of the University of Guilan and Ministry of Science, Researches, and Technology, Islamic Republic of Iran, for financial support during the course of this project.
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Received December 5, 2011
Accepted September 7, 2012
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