ISJ 5: 179-yyy, 2008 ISSN 1824-307X

ISJ 5: 180-189, 2008 ISSN 1824-307X

RESEARCH REPORT

Lipase and invertase activities in midgut and salivary glands of Chilo suppressalis
(Walker) (Lepidoptera, Pyralidae), rice striped stem borer

A Zibaee, AR Bandani, S Ramzi

Plant Protection Department, Faculty of Agriculture, University of Tehran, Karaj 31584, Iran

Accepted November 4, 2008

Abstract

The rice striped stem borer, Chilo supprressalis, was introduced to Iran in 1973 where it is now
widely distributed and causes severe damages. Lipases, which catalyses the hydrolysis of fatty acid
ester bonds, are widely distributed among animals, plants and microorganisms. Invertases (β-
fructofuranosidase) are glycosidehydrolases that catalyze the cleavage of sucrose (β-D-
glucopyranosyl-S-D-fructofuranoside) into the monosaccharides glucose and fructose. Laboratory-
reared 4th instar larvae were randomly selected, their midgut and salivary glands were removed by
dissection under a light microscope and lipase and invertase activities were assayed. The activity of
lipase/invertase in the midgut and salivary gland were 0.49/0.27 and 0.35/0.23 µmol/min/mg protein,
respectively. The optimum pH and temperature for both the two enzymes were determined to be 10-
11 and 37-40 °C, which is consistent with pH and temperature values already observed in
Lepidoptera. The enzyme activity was reduced by addition of NaCl, KCl, MgCl2, SDS, urea and plant
extracts from Artemisia annua, but not by CaCl2 which enhanced enzyme activity. Pest control with
usage of resistant varieties of plants is one of the most important practices that are dependant on
inhibitors already present in nature. Hence, characterization of insect digestive enzymes, especially
examination of inhibition effects on enzyme activity, could be useful in developing new strategies for
pest control.

Key words: α-amylase; rice striped stem borer; midgut; salivary glands

Introduction

The rice striped stem borer (Chilo

supprressalis, Walker) is a cosmopolitan and
destructive pest in rice fields of the world (Zibaee et
al., 2008). This pest was introduced in Iran in 1973
and since then has been widely distributed in the
country rice fields. It causes severe damages in all
rice fields and its present density is superior than
the economic injury level (EIL) (Dezfoulian and
Moustofipoor, 1972). The chemical control by using
organophosphorus compounds, has been a
common control procedure, although other methods
based on agricultural practices such as ploughing,
usage of resistant varieties of plants, weed control
as overwintering sites and biological control with
Trichogramma spp. have been incorporated. In recent
___________________________________________________________________________

Corresponding author:
Ali Reza Bandani
Plant protection Department
College of agriculture and natural resources
University of Tehran, Karaj, 31584 Iran
E-mail: abandani@ut.ac.ir

years, resistant varieties and pheromones also have
been added to control the diffusion of C.
suppressalis like in other places of the world
(Muralidharan and Pasaalu, 2006). A study on 78
different varieties of rice showed that Binam with 15
% white head is the most resistant variety.
Germplasts studies showed that Khazar variety is
resistant to the first generation of rice striped stem
borer. However, it is susceptible to the second
generation.

Lipases (triacylglycerol–acyl-hydrolase EC
3.1.1.3), which catalyzes the hydrolysis of fatty acid
ester bonds, are widely distributed among animals,
plants and microorganisms (Naumff, 2001). It has
been showed that lipases can also hydrolyze a
variety of esters in organic solvent systems and thus
they can be widely used in many industrial areas,
e.g., dairy, food, detergent and biofuel industries
(Ishaaya and Swirski, 1970; Henrissat and Bairoch,
1993; Grillo et al., 2007). The most characteristic
property of lipases is that they act on substrate at
the interface between the aqueous and the lipid
phase (Grillo et al., 2007).

180

To date, many research groups have carried
out the isolation and purification of lipases from
various sources, mainly microorganisms, fish, fungi,
milk and plants (Cherry and Crandall, 1932;
Henrissat and Bairoch, 1993; Degerli and Akpinar
2002; Grillo et al., 2007). However, lipid
biochemistry studies in insects is time-consuming
and moved on very slowly due to high diversity of
insects and changes in the lipid composition and
lipophorin present in hemolymph during
metamorphosis from larva to pupa (Degerli and
Akpinar, 2002). Recently, lipid mobilization and
transport in insects is under investigation, especially
lipases and lipophorin (a reusable lipoprotein
particle in insect systems) because of their roles in
energy production and transport of lipids at flying
activity (Ayre, 1967). Although stored lipids in
vertebrate adipose tissue are released as free fatty
acids, in insects most fatty acids are released as
1,2-diacylglycerols and mobilization of lipid reserves
from insect fat body is under the control of
adipokinetic hormone (Grillo et al., 2007).

Invertases (β-fructofuranosidase, EC3.2.1.26)
also termed fructosidase, saccharase, or sucrase,
are glycosidehydrolases (EC 3.2.1) that catalyze the
cleavage of sucrose (β-D-glucopyranosyl-S-D-
fructofuranoside) into the monosaccharides, glucose
and fructose (Henrissat and Bairoch, 1993; Sturm
and Tang, 1999; Naumoff, 2001). Invertase, thus,
appears to be a particularly important enzyme for
plants and animals. Given this general importance,
a surprisingly limited number of studies have tried to
quantify invertase activity in ants (Ayre, 1963, 1967;
Ricks and Vinson, 1972) or other animals (Martinez
del Rio, 1990; Zhang et al., 1993). This might be
due to the particular methodological problems
arising from the quantification of invertase in
animals whose carbohydrate metabolism is highly
active.

Digestion is a phase of insect physiology on
which little research has been performed, despite
the economic importance of the food of insects and
the fact that the most important control measures
involve the action of digestive juices on poisons
taken into the digestive tract. A better understanding
of enzyme catalysis is essential in order to develop
methods of insect control (Bandani et al., 2001;
Ghoshal et al., 2001; Maqbool et al., 2001). The
purpose of the present study is to identify and
characterize the lipase and invertase activities from
midgut and salivary glands (SG) of rice striped stem
borer larvae to gain a better understanding of the
digestive physiology. This understanding will
hopefully lead to new management strategies for
control of this pest.

Materials and Methods

Insects

To decrease the side effects of laboratory mass
culture, 400 pupae were collected from fields and
reared on the same variety seedling (Taroum) as
sampling sites. Insects were reared based on the
method mentioned by Kammano and Sato (1985) in
28 ± 1 °C, light cycle 16L:8D and RH > 80 %. When
the larvae grow up to 4th instar larvae, 30 larvae
were randomly selected for biochemical analysis.

For 4th instar determination, Dayer's formula was
used which had been described by Majidi et al.
(2002).

Sample preparation and enzyme assays

Briefly, larvae were randomly selected and total
midgut and SG were removed by dissection under a
stereo microscope in ice-cold saline buffer (6 μM
NaCl). The midgut and SG were separated from the
insect’s body, rinsed in ice-cold buffer, placed in a
pre-cooled homogenizer and ground in 1 ml of
universal buffer containing succinate, glycine and 2-
morpholinoethanesulfonic acid (pH 7.2)
(Hosseinkhani and Nemat-Gorgani, 2003). The
homogenates from both preparations (midgut and
SG) were separately transferred to 1.5 ml centrifuge
tubes and centrifuged at 20,000 x g for 20 min at 4
°C. The supernatants were pooled and stored at -20
°C for subsequent analyses.

Lipase activity

The enzyme assays were carried out as
described by Tsujita et al. (1989). Thirty µl of gut
and salivary glands tissue extracts and 100 µl of p-
nitrophenyl butyrate (50 mM), as substrate, were
incorporated, mixed thoroughly and incubated at 37
°C. For negative control tubes, samples (midgut and
salivary glands) were placed in a boiling water bath
for 15 min to destroy the enzyme activity and then
cooled. After 1 min, 100 µl distilled water were
added to each tube (control and treatment) and
absorbance was read at 405 nm. One unit of enzyme

0.1

0.2

0.3

0.4

0.5

0.6

Midgut Salivary glands

A
ct

iv
ity

o
f l

ip
as

e
(µ

m
ol

/m
in

/m
g

pr
ot

ei
n)

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

Midgut Salivary glandsA
ct

iv
ity

o
f I

nv
er

ta
se

(
µm

ol
/m

in
/m

g
pr

ot
ei

Fig. 1 Activity level of lipase (up) and invertase
(down) in 4th instar larvae midgut and salivary
glands of rice striped stem borer.

181

http://www.citeulike.org/user/biblio24/author/Tsujita

y = 0.0091x + 0.0556
R2 = 0.9693

0.1

0.2

0.3

0.4

0.5

0.6

0 10 20 30 40 50

Concentration of p-nitrophenol

A
b

so
rb

an
ce

a
t

5
n

y = 0.2011x + 0.008
R2 = 0.9922

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 0.5 1 1.5 2 2.5 3 3.

Concentration of Glucose

A
b

so
rb

an
ce

a
t

34
0

Fig. 2 Standard calibration curve for the
determination of p-nitrophenol and glucose released
in the lipase (up) and invertase (down) assay.

will release 1.0 nmol of p-nitrophenol per min at pH
7.2 at 37 °C using p-nitrophenyl butyrate as
substrate. Standard curve was used to calculate the
specific activity of enzyme.

Invertase activity

Samples were transferred to 100 ml flasks and
1 ml toluene was added to arrest the enzyme
activity. After 15 min, 6 ml of 0.2 M glycine buffer
(pH 7.2) containing 18 mM sucrose was added to
the samples and the flasks were closed with cotton
plugs then held for 24 h at 30 °C. Samples were
passed through Whatman filter paper and glucose in
the filtrate was assayed at 340 nm (Nelson, 1994).

Kinetic parameters measurements

Twenty µl of appropriately diluted enzyme
preparation was used in each assay. Final
concentrations for substrate were 20, 30, 40, 50 and
60 mM for lipase and 0.09, 0.18, 0.36, 0.72 and
1.54 mM for invertase, respectively. The Michaelis
constant (Km) and the maximum velocity (Vmax) were
estimated by Sigmaplot software version 11 (Systat
Software Inc., Chicago, IL, USA) and the results of
Km and Vmax were the means ± SE of three
replicates for every population.

Effect of pH and temperature on enzyme activity

The effect of temperature and pH on lipase and
invertase activity were examined using enzyme
extractions from the larval midgut and SG. The

effect of temperature on enzymes activity was
determined by incubating the reaction mixture at 20,
25, 30, 35, 37, 40, 45, 50, 55, 60 and 70 °C for 24 h,
followed by measurement of activity. Optimal pH for
their activities was determined using universal buffer
with pH set at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13.

Effect of activators and inhibitors on enzyme activity

To test the effect of different ions on the
enzymes, midguts and SG 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), Ca2+ (5, 10, 20 and 40 mM), Mg2+ (5,
10, 20 and 40 mM), and ethylenediaminetetraacetic
acid (EDTA; 0.5, 1, 2 and 4 mM), sodium
dodecylsulfate (SDS; 1, 2 and 4 mM), urea (0.5, 1,
2, 4, 6 and 8 mM) and Artemisia annua extract (10,
15 and 25 % concentrations). These compounds
were added to the assay mixture, and activity was
measured after 30 min incubation. A control was
also measured (no compounds added).

Effect of A. annua extract on enzymatic parameters
of Invertase and lipase

For this experiment, 20 µl of appropriately
diluted enzyme preparation was used in each
assay. Final concentrations for substrate were 20,
30, 40, 50 and 60 mM for lipase and 0.09, 0.18,
0.36, 0.72 and 1.54 mM for invertase, respectively.
Finally, 20 % of plant extract added to each well. Km
and Vmax were estimated by Sigmaplot software
version 11 (Systat Software Inc.) and the results of

0
0.2
0.4
0.6
0.8

3 4 5 6 7 8 9 10 11 12 13

A
ct

iv
ity

o
f L

ip
as

e
(µ

m
ol

/m
in

/m
g

pr
ot

ei
n )

Midgut Salivary glands

0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9

3 4 5 6 7 8 9 10 11 12

In
ve

rt
as

e
ac

tiv
ity

(µ

m
ol

/m
in

/m
g

pr
ot

ei
n )

Midgut Salivary glands

Fig. 3 Effect of pH on activity of lipase (up) and
invertase (down) extracted from midgut and salivary
gland of rice striped stem borer.

182

0
0.1
0.2
0.3
0.4
0.5
0.6

20 30 35 37 40 45 50 55 60 70

Tem perature (°C)

A
ct

iv
it

y
o

f
L

ip
as

e
(µ

o
l/m

in
/m

g
p

ro
te

in
)

Midgut Salivary glands

Results

Lipase and Invertase activities

Studies showed that lipase and invertase are
present in the midgut and salivary glands of adult C.
suppressalis (Fig. 1). The activity of lipase was
0.486 µmol/min/mg protein and 0.27 µmol/min/mg
protein in midgut and SG, respectively. The invertase
activity in midgut and SG was 0.35 and 0.23
µmol/min/mg protein, respectively. There was a
significant difference in the degree of enzyme
activity between midgut and salivary glands (Figs 1,
2).

Effect of pH and temperature on enzyme activity

The in vitro evaluation of C. suppressalis lipase and invertase from midgut and SG indicated that
enzyme activity increased steadily from pH 3 to 11
and from 3 to 10, respectively. After reaching the
threshold pH level, enzyme activity decreased with
the increasing of pH and there were significant
differences among measured values for each pH
(Fig. 3). Both enzymes were considerably active
over a broad range of temperatures. As the results
show, the optimum temperature for lipase and
invertase activities were 37 and 40 °C for midgut
and SG, respectively (Fig. 4).

-0.1
0

0.1
0.2
0.3
0.4
0.5
0.6

20 30 35 37 40 45 50 55 60 70

Tem perature (° C)

A
ct

iv
it

y
o

f
In

ve
rt

as
e

(µ
m

o
l/m

in
/m

g
p

ro
te

in
)

Midgut Salivary glands

Effect of activators and inhibitors on enzyme activity

Several molecules and chemical compunds
affects the activity of lipase and invertase in midgut
and SG of rice striped stem borer, although they
had a similar effect on both enzymes (Tables 1 and
2). Activity level of enzymes in midgut and SG
elevated due to increasing of CaCl2 and EDTA
concentrations for lipase and just CaCl2 for
invertase (Table 1 and 2). Activity level of enzyme
decreased in presence of NaCl, KCl, EDTA, MgCl2,
SDS, urea and A. annua extract in both midgut and
SG (Tables 1 and 2).

Fig. 4 Effect of temperature on activity of lipase (up)
and invertase (down) extracted from midgut and
salivary gland of rice striped stem borer.

Km and Vmax were the means ± SE of three
replicates for every population. For determination of
A. annua extract effect on enzymatic parameters of
lipase and invertase, 20 % of plant extract was
added to each well.

Kinetic Parameters

As can be seen in Table 3, lipase Vmax of
midgut and SG were 0.5 and 0.35 µmol/min/mg
protein, respectively. Lipase Km of midgut and SG
were 15 and 19 mM, respectively. Invertase Vmax
was 0.9 and 0.5 µmol/min/mg protein in midgut and
SG, respectively, while invertase Km was 0.31 and
0.39 mM in midgut and SG (Table 3, Fig. 5).

Polyacrylamide Gel Electrophoresis (PAGE)

In order to determine the molecular mass of
native lipase, native polyacrylamide disc-gel
electrophoresis was carried out using the method of
Parish and Marchalonis (1970) using 2.7 % and 7.7
% polyacrylamide for the stacking and resolving
gels, respectively. The gel was stained with 1.5 %
(w/v) Coomassie Brilliant Blue G-250 and distained
in glacial acetic acid-methanol-water 7.5: 5.0: 87.5.

Effect of A. annua extract on enzymatic parameters
of invertase and lipase

Enzymes parameters changed due to using of
A. annua extract. As it is shown in Table 4, lipase
kinetic parameter, Vmax-Km were 0.35 µmol/min/mg
protein-37.5 mM in midgut and 2.12 µmol/min/mg
protein-21 mM in SG. As well as invertase is
concerned, Vmax-Km were 0.49 µmol/min/mg protein-
0.30 mM in midgut and 0.81 µmol/min/mg protein-
0.92 mM in SG (Table 4, Fig. 6).

Protein determination

Protein concentration was measured according
to the method of Bradford (1976), using bovine
serum albumin (Bio-Rad, München, Germany) as a
standard.

Statistical analysis
Native PAGE Data were compared by one-way analysis of

variance (ANOVA) followed by Tukey's studentized
test when significant differences were found at
P=0.05. Enzyme kinetic parameters were analyzed
by using the Sigmaplot software version 11 (Systat
Software Inc.).

Analysis of midgut and SG lipase and invertase
enzyme from homogenates of C. suppressalis by
vertical slab electrophoresis on 8 % polyacrylamide
gels indicated one band in all samples except for
midgut's lipase (Fig. 7).

183

Table 1 Relative activity of C. suppressalis lipase toward different compounds

Compounds Concentration (mmol/l)
Relative activity

(Midgut)
Relative activity
(salivary gland)

Control - 100 100

NaCl 5 102.77* 189.47*
10 77.77* 157.9*
20 33.33* 89.47*
40 6.38* 52.63*

CaCl2 5 50* 57.89*
10 77.77* 121.05*
20 88.88* 173.68*
40 119.44* 210.52*

KCl 5 97.3* 100*
10 76.54* 87.36*
20 55.89* 68.66*
40 32.77* 45.25*

MgCl2 5 247.22* 421.05*
10 208.33* 363.15*
20 91.66* 236.84*
40 44.44* 131.57*

EDTA 0.5 38.88* 48.84*
1 72.22* 73.68*
2 105.55* 157.89*
4 231.55* 207.89*

SDS 2 113.88* 194.73*
4 80.55* 121.05*
6 44.44* 52.63*
8 26.66* 45.26*

Urea 1000 116.66* 205.23*
2000 102.77* 142.1*
4000 69.44* 115.78*
5000 27.77* 89.47*
6000 0.036* 31.57*

Plant extract 10 % 78* 86*
15 % 59* 63*
25% 34* 39*

*P < 0.05 vs control

184

Table 2 Relative activity of C. suppressalis invertase in presence different compounds

Compounds

Concentration
(mmol/l)

Relative activity
(Midgut)

Relative activity
(salivary gland)

Control - 100 100

NaCl 5 102.77* 189.47*
10 77.77* 157.9*
20 33.33* 89.47*
40 6.38* 52.63*

CaCl2 5 50* 57.89*
10 77.77* 121.05*
20 88.88* 173.68*
40 119.44* 210.52*

KCl 5 97.3* 100*
10 76.54* 87.36*
20 55.89* 68.66*
40 32.77* 45.25*

MgCl2 5 247.22* 421.05*
10 208.33* 363.15*
20 91.66* 236.84*
40 44.44* 131.57*

EDTA 0.5 38.88* 48.84*
1 72.22* 73.68*
2 105.55* 157.89*
4 231.55* 207.89*

SDS 2 113.88* 194.73*
4 80.55* 121.05*
6 44.44* 52.63*
8 26.66* 45.26*

Urea 1000 116.66* 205.23*
2000 102.77* 142.1*
4000 69.44* 115.78*
5000 27.77* 89.47*
6000 0.036* 31.57*

Plant extract 10 % 78* 86*
15 % 59* 63*
25% 34* 39*

*P < 0.05 vs control

185

Lipase-Midgut

1/S

-0.05 0.00 0.05 0.10 0.15

1/
V

Lipase-Salivary glands

1/S

-0.05 0.00 0.05 0.10 0.15

1/
V

Invertase-Salivary glands

1/S

-4 -2 0 2 4 6 8 10 12

1/
V

Invertase-Midgut

1/S

-4 -2 0 2 4 6 8 10 12

1/
V

Fig. 5 Lineweaver-Burk plot (Vmax and Km) of lipase and invertase extracted from 4th instar larvae of rice striped
stem borer.

Discussion

The present study shows that the larvae of C.

suppressalis present lipase and invertase activities
both in the midgut and in the SG. Reports
concerning lipase characterization have been
obtained from several species of insects. Metcalf
(1945) found amylase, protease, and lipase to be
absent from the SG of the mosquito Anopheles
quadrimaculatus while he observed that invertase is
present in both midgut and crop homogenates. Fisk
and Shambaugh (1954) found no activity of lipase in
the SG of A. quadrimaculatus. A total body lipase
was partially purified from abdomen homogenate of
Gryllus campestris L. (Orthoptera, Gryllidae)
(Orscelik et al., 2007).

Current study showed that both lipase and
invertase are present in C. suppressalis and that
the optimal pH for both enzymes are in alkaline
condition (around pH 10). Optimal temperatures
for both enzymes are 37-40 °C in midgut and SG,

respectively. Ishaaya and Swirski (1970)
demonstrated that the optimum pH and temperature
for invertase activity in the hemipteron
Chrysomphalus aonidum are 5.5 and 30 °C,
respectively. Degleri and Anuipor (2002) showed
that the optimal pH of lipases activity in the teleost
fish Cyprinion macrostomus is 7.5. Studies on lipase
properties of yeasts revealed that optimum pH and
temperature for lipases activity are 7.5-8.2 and 30-
40 °C, respectively (Vakhlu and Kour, 2006). The
utilization of dietary lipids was studied in adult
females of the blood-sucking bug Rhodnius prolixus
with the use of radiolabeled triacylglycerol (Grillo et
al., 2007). These researches indicates that lipase
activity is affected by pH and shows an optimal
activity at a pH of 7.0-7.5. The optimal pH generally
reflects the pH of the environment in which the
enzyme normally functions. One way in which pH
affects reactions rates is by altering the charge state
of the substrate or of the active site of the enzyme.
Extreme pHs can also disrupt the hydrogen bonds

186

Lipase-Midgut

1/S

.08 -0.06 -0.04 -0.02 0.00 0.02 0.04 0.06 0.08 0.10 0.12

1/
V

Invertase-Midgut

1/S

.0 -0.5 0.0 0.5 1.0 1.5 2.0

1/
V

0.0

0.2

0.4

0.6

0.8

1.0

Invertase-Salivary glands

1/S

-4 -2 0 2 4 6 8 10 12

1/
V

Fig. 6 Lineweaver-Burk Plot (Vmax and Km) of lipase and invertase extracted from 4th instar larvae of rice striped
stem borer due to using A. annua extract.

that hold the enzyme in its three-dimensional
structure, denaturing the protein (Zeng et al., 2000).
Biological reactions occur faster with increasing
temperature up to the point of enzyme denaturation,
above which temperature enzyme activity and the
rate of the reaction decreases sharply (Applebaum
1985; Agblor et al., 1994; Zeng et al., 2002).

Current study results showed that Ca2+ ions
have activatory effects on the lipase and invertase
activities of rice striped stem borer. Podolor and
Applebaum (1971) reported that Ca2+ ions have
activatory effects on the lipase and invertase
activities of the coleopteron Callosobruchus
chinensis. These results showed that these
enzymes are metalloproteins which require calcium
for maximum activity.

Researches have shown a significant
correlation between the activity level of digestive
enzymes in the hemolymph and the midgut. Saleem

and Shakoori (1987) showed that sublethal
concentrations of pyrethroids decrease amylase
activity in larval gut of the beetle Tribolium
castaneum. Lee et al. (1994) showed that some
insect growth regulators decreased the activity level
of alpha-amylase and esterase in treated larvae of
C. suppressalis. Ascher and Ishaaya (2004) showed
that the activity level of this enzyme increased 30 %
in the noctuid moth Spodoptera littoralis treated with
phentine acetate compared with control. Shekari et
al. (2008) suggest that its activity level decreases 24
h after treatment and sharply increases at 48 h.

Digestive enzyme inhibitors occur naturally in
many food plants and are particularly abundant in
cereals and legumes (Franco et al., 2002). Insects
gain access to food sources when they evolve
enzymes that are not affected by inhibitors present
in the food source, and plants become resistant
when they evolve inhibitors effective against these

187

Table 3 Kinetic parameters of lipase and invertase enzymes extracted from midgut and SG of 4th instar larvae of
C. suppressalis

Midgut* SG* Enzymes

Vmax
(µmol/min/mg protein)

Km
(mM)

Vmax
(µmol/min/mg

protein)

Km
(mM)

Lipase 0.5 ± 0.06 15 ± 3.74

0.35 ± 0.09

19 ± 6.45

Invertase 0.9 ± 0.036

0.31 ± 0.047

0.5 ± 0.078

0.39 ± 0.77

*Means ± SE, N = 3

Table 4 Kinetic parameters of lipase and invertase enzymes extracted from midgut and SG of 4th instar larvae of
C. suppressalis after exposure to A.annua extract

Midgut* SG* Enzymes
Vmax

(µmol/min/mg protein)
Km

(mM)
Vmax

(µmol/min/mg protein)
Km

(mM)
Lipase 0.35 ± 0.087 37.5 ± 15.24

2.12 ± 0.68 21 ± 5.8

Invertase 0.49 ± 0.087

0.30 ± 0.063

0.81 ± 0.021

0.92 ± 0.77

*Means ± SE, N = 3

insect enzymes. When the action of digestive
enzymes is inhibited, insect’s nutrition is impaired,
growth and development are retarded and
eventually death occurred due to starvation. Genes
encoding for digestive enzyme inhibitors have been
used to make transgenic crops by gene transfer
technology. In transgenic pea expressing the α-
amylase inhibitor, the expression of digestive
enzyme inhibitors makes plants harmful to target
insects and pests, interfering with their digestive and

Fig. 7 Native-PAGE gel electrophoresis of midgut
and SG from C. suppressalis.

absorption processes, whereas neither anti-
nutritional nor toxic effects were observed in rats
(Pusztai et al., 1999). The primary reason for
producing insect-resistant transgenic crops is to
reduce the use of chemical pesticides, which by one
side lowers production costs and in the meantime
reduces the insecticide loads in the environment.
Making insect-resistant plants requires the
characterization of α-amylase and other digestive
enzymes of the target insect and the identification of
suitable inhibitors from plants or other sources.

In our opinion, the purification and
characterization of more insect digestive enzymes
will greatly facilitate the understanding of the
mechanisms responsible for this selectivity and will
help to design new and more specific strategies for
insect control.

Acknowledgment

This research was supported by a University of
Tehran grant. We are really appreciated Mr Belbasi
and Ramadzan-Khani for their assistances. We
have special thanks to three anonymous Reviewers
for their helpful comments that resulted in a
significant improvement of the article.

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