Vol.3, No.3, 218-226 (2011) Natural Science
http://dx.doi.org/10.4236/ns.2011.33028
Heat shock proteins development in different stages of
Schistocerca gregaria as response to heavy metals
intoxication
Hesham A. Yousef1*, Amira Afify1, Afaf Abdel Meguid1, Hany M. Hassan2
1Entomology Department, Faculty of Science, Cairo University, Egypt; *Corresponding Author:
2Agriculture Research Center, Ministry of Agriculture, Egypt
Received 31 December 2010; revised 14 February 2011; accepted 16 February 2011.
ABSTRACT
The induction of heat shock proteins in different
stages of S. gergaria exposed to long and short
term contamination with heavy metals, Cd and
Pb in food was determined, revealing a promi-
nent variable effect in response to the term of
exposure and the type of contamination. HSP 70
was specially quantified and characterized to
reveal the probability of using HSP as a bio-
marker for pollution.
Keywords: Hsps; Hsp70; Heavy Metals; Cd; Pb;
S. Gergaria
1. INTRODUCTION
Heavy metals are among the most problematic causes
of water, soil and plant pollution. They enter the ground
water and surface water through seepage from household
waste, and a number of industries. In the case of soil
pollution, terrestrial insects, including orthopteran spe-
cies, may provide good samples to evaluate the mutagenic
effects of some environmental contaminants, and may be
considered suitable bioindicators [1-4].
Cadmium and lead are the most dangerous, carcino-
genic, widespread heavy metals, released and accumu-
lated in the environment. Their toxicity for epigeic insect
depends mainly on the way of their entrance to the body
cavity and the most important is their intake with food.
Insects are able to accumulate metals in intracellular
granules and/or to some extent control this route, by
selecting the quality of food, mainly plants containing
less metal, by avoiding plant parts which are more con-
taminated, or simply by rejecting metals with excreta
[5,6]. Insects, which develop partly in the soil, are also
exposed directly to metal ions present in the soil water.
Among them are many grasshopper species which lay
egg-pods directly in the soil.
Exposure of organisms to different environmental
pollutants has been found to induce a variety of stress
response including the heat shock proteins [7]. Under
normal, unstressed conditions, the constitutively present
stress proteins are essential for cell viability, they par-
ticipate in protein folding and assembling, metabolic
processes, and cell growth and development [8].
Cytoprotective functions and immune response have
been attributed to HSPs and, in particular to the HSP 70
family [9-11].
The aim of the present work was to determine the in-
duction of heat shock proteins in S. gergaria exposed to
heavy metals, Cd and Pb, with special quantifying and
characterizing the HSPs 70 to reveal the probability of
using HSP as a biomarker for pollution.
2. MATERIALS AND METHODS
2.1. Colonization of S. Gregaria
Locusts were obtained from the laboratory colonies
maintained at the Entomology Department, Faculty of
Science, Cairo University. The locusts were reared in
wooden cages at (32 ± 2)˚C, 50% - 60% RH and 16 hrs
day light. A daily supply of fresh grass, clover plant was
supplied to the locusts. Packed moist sterilized sand in
suitable glass containers about 7 cm in diameter and 10
cm deep were prepared for egg-laying.
2.2. Heavy Metals Treatment
Living individuals of S. gregaria of the 4th, 5th instars,
and newly emerged (NEA) (4 days old) and mature (15
days old) adult (MA), fed on treated clover (their stems
were previously immersed for 24 hrs in distilled water
containing 25mg and 50mg/L of CdCl2 and PbCl2, to
allow the clover to absorb contaminated water) or on
untreated clover, were collected from their respective
cage.

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2.3. Sample Preparation for SDS-PAGE and
Elisa Test
Living individuals of S. gregaria of the 4th, 5th instars,
and newly emerged (4 days old) and mature (15 days old)
adult, fed on treated clover (their stems were previously
immersed for 24 hrs in distilled water containing 25 mg
and 50 mg/L of CdCl2 and PbCl2, to allow the clover to
absorb contaminated water) and untreated clover were
collected from their respective colony.
One g of each instar was homogenized in 1 ml of sa-
line solution using Teflon in glass homogenizer. The
body extract was centrifuged at 12,000 rpm for 15 min at
4˚C, using Kubota cooling high speed centrifuge. Total
protein concentration was determined Spectrophoto-
metrically according to the method described by Brad-
ford [12], using bovine serum albumin as standard. The
optical density of the protein sample was measured at
595 nm versus the blank.
2.4. SDS-Polyacrylamide Gel
Electrophoresis
SDS-PAGE was performed in a slab gel apparatus
utilizing the discontinuous system described by Laemmli
[13].
250 µg total protein of each sample (diluted 1:1 (vol:
vol) with treatment buffer and boiled in water bath for
2.5 minutes) were loaded with a drop of tracking dye in
each lane. Similarly protein marker was loaded on the
gel beside the samples. Electrophoresis was running at
80 volt and 30 mA, the gels were stained for 0.5 h in
COBB stain, and excess dye was removed by placing the
gel in distaining solution. The gels were scanned, using
the densitometric scanner by which the relative concen-
tration and Mwt values of each characteristic band can
be deduced using protein molecular weight marker
(28.5-116 kDa) including B-galactosidase, Phosphory-
lase b, Bovine serum albumin, Alcohol dehydrogenase
and carbonic anhydrase with the molecular weights: 116,
97.4, 66.2, 37.6 and 28.5 kDa respectively.
2.5. Detection of the Hsp 70 Using the
ELISA Method
The components of Stress Xpress TM Hsp 70 ELISA
kit wer used at room temperature. Lyophilized recombi-
nant Hsp 70 standard was centrifuged before and after
rehydration. Dilute rehydrated recombinant Hsp 70
standard and samples in sample diluents. Add 100 µl
prepared standards and samples in duplicate to wells of
Hsp 70 immunoassay plate. Cover immunoassay plate;
incubate plate at room temperature for 2 hours. Wash
wells with 1X Wash Buffer. Add 100 µl diluted Biotin
Anti-Hsp 70 to each well. Cover immunoassay plate.
Incubate plate at room temperature for 1 hour. Wash
wells 6X with 1X Wash Buffer. Add 100 µl diluted
Avidin-HRP Conjugate to each well. Cover immunoas-
say plate. Incubate plate at room temperature for 1 hour.
Wash wells 6X with 1X Wash Buffer. Add 100 µl TMB
Substrate to each well, incubate at room temperature for
10 min, and add 100 µl Acid Stop Solution to each well.
Measure absorbance at 450 nm, and plot the Hsp 70
standard curve and calculate hsp 70 sample concentra-
tions.
3. RESULTS AND DISCUSSION
3.1. Effect of Cd & Pb on 4th Instar Nymph
Proteins
The SDS-PAGE analysis of the whole body proteins
of 4th instar S. gregaria exposed for long term (from
newly hatched 1st nymphal instar) to low and high con-
centrations of Cd and Pb, showed that the most promi-
nent variations appeared in the protein bands, Mw.61
and 49 KDa (group 60 and 50), revealing that the highest
amount of induced proteins were expressed in these
groups (Figure 1(a)).
The short term exposure of the 4th instar S. gregaria to
heavy metals (4 days before collecting) showed an in-
crease in concentration of the protein bands, Mw. 88, 77
and 57 KDa, in relation to control. Also, the protein
bands, Mw. 67, 53, 45 KDa, almost appeared in case of
Cd and in Pb treatment (Figure 1(b)). This result re-
vealed the induction of new sets of proteins belong to
groups 60, 50, 40 KDa, and increase of protein concen-
trations belonging to groups 80 and 70 KDa (Figure
1(b)).
3.2. Effect of Cd & Pb on 5th Instar Nymph
Proteins
The long term exposure of 5th instar nymph to heavy
metals showed the appearance of protein band, Mw. 26
KDa, only in the two treatments of high & low Pb con-
centrations (Figure 2(a)). A prominent variation was
observed including the increase of concentrations of
protein bands (67 and 34 KDa) after low Cd treatment,
protein bands (52 and 39 KDa) after treatment of high
Cd. Also, Low and high concentration of lead treatment
has inducing effect by expression of new protein band
(Mw. 26 KDa).
SDS-PAGE of the whole body of 5th instar, S. gre-
garia exposed to short term contamination with heavy
metals showed disappearance of proteins with Mw. 121,
97, 73, 65 and 58 KDa in low Pb treatment, and appear-
ance of protein band, Mw 44 KDa. Newly expressed
protein bands, Mw. 47 and 36 KDa were newly expressed

H. A. Yousef et al. / Natural Science 3 (2011) 218-226 220
(a)
(b)
Figure 1. SDS-PAGE of whole body tissue proteins of 4th in-
star S. gregaria under normal and heavy metal stress condi-
tions for (a) long term exposure and (b): short term exposure.
Lane 1: Mw. marker; Lane 2: normal; Lane 3: low Cd; Lane 4:
high Cd; Lane 5: low Pb and Lane 6: high Pb.
after all treatments, which were absent in control.
On the other hand, the short exposure to heavy metals
revealed that, induction of new set of proteins as in band,
125 KDa after high Cd, bands, Mw. 47 & 36 KDa after
Cd and Pb treatments. Increased concentration of other
protein bands, were observed as in bands, 34 and 24
KDa (Figure 2(b)).
It is clear that analysis of the SDS-PAGE of the whole
body proteins of 5th instar S. gregaria after long and
short exposure to heavy metals revealed the increase in
concentration of proteins with small Mw families which
(a)
(b)
Figure 2. SDS-PAGE of whole body tissue proteins 5th instar
of S. gregaria under normal and heavy metal stress conditions
for (a): long term exposure and (b): short term exposure. Lane
1: Mw. marker; Lane 2: normal; Lane 3: low Cd; Lane 4: high
Cd; Lane 5: low Pb and Lane 6: high Pb.
may be included in small Hsps 20, 30 & 50 KDa. There
was a low increase in concentration of protein bands,
Mw. 77, 61 & 57 kDa observed in treatments of short
exposure to heavy metals and in low Cd concentration in
long term exposure to heavy metals.
3.3. Effect of Cd & Pb on Newly Emerged
Adult (NEA) Proteins
The proteins of the whole body tissue of NEA after
long exposure of heavy metals revealed appearance of

H. A. Yousef et al. / Natural Science 3 (2011) 218-226
221221
protein band, Mw 7 KDa and disappearance of bands,
Mw 117 and 99 KDa in treatment of high Cd, and low
Pb concentration.
Figure 3(a) showed that, there was a high expression
of proteins, as in bands with Mw. 77 and 57 KDa, espe-
cially in high Cd and low Pb treatments. The protein
bands, Mw. 34, 15 & 12 KDa over expressed in the Cd
and Pb treatments in relation to the normal specimen.
Also, proteins, Mw. 117 and 99 KDa disappeared in
treatment of high Cd and low Pb concentrations. On the
other hand, the protein band, Mw.7 KDa appeared only
in the two previous treatments. Unlike that of long term
exposure, short term exposure caused over-expression in
some proteins as in the band with Mw. 55 KDa under the
effect of high Cd concentration, and the two treatments
of high and low Pb concentrations, also band, Mw. 12
KDa was over expressed in all treatments than control.
The analysis of the whole body proteins of NEA, S.
gregaria separated by electrophoresis, after short term
exposure of heavy metals revealed the higher induction
of protein bands with Mw. 57, 15 and 12 KDa. High Cd
and low Pb treatments caused more induction of the
proteins, Mw. 34 and 24 KDa (Figure 3(b)).
The long term exposure of NEA, S. gregaria to heavy
metals (Figure 4(a)) induced over expression of the
protein band with Mw. 72 KDa (except in the low Cd
treatment) and protein bands (Mw. 49& 12 KDa). On the
other hand, lower expression in the protein band (Mw.
32 KDa) was observed.
All the bands are similar in all treatments and control.
The main difference between them was in the concentra-
tions of bands, Mw. 49 and 32 KDa. There was over
expression in all treatments than control but in band, Mw.
39 KDa, there was down expression in the treatment of
low concentration Cd & Pb with the same ratio than con-
trol (Figure 4(b)).
The analysis of the whole body proteins of mature
adult S. gregaria separated by electrophoresis showed
that the most prominent variations due to the effect of
heavy metals after short exposure were observed in the
protein bands with Mw. 72 KDa. This reflects the high-
est induction of this protein band in all treatments in
relation to the control (about 4-6 folds).
Stressful conditions are frequently accomplished by
disturbances and alterations of cellular functions at
various levels with the resulting situation associated with
the increase in stress proteins [14]. These functions in-
clude the folding and maintenance of structural proteins
in various intracellular compartments, refolding the mis-
folded proteins, translocation of proteins across mem-
branes, prevention of protein aggregation, and degrada-
tion of unstable proteins [9,15], help in the adaptation of
insects frequently, or continuously exposed to severe
stress [16-18], involved in generating immune response
[11]. Hsp 70 is also known to facilitate antigen presenta-
tion in cells such as macrophages and dendrites [10].
In the present work, proteins from the whole body of
S. gregaria were examined in the term of heavy metal
contamination in both long term exposure (heavy metal
accumulation), and short term exposure through the de-
velopmental stages from 4th instar to fully mature adult.
SDS-PAGE and their analyses were obtained, revealing
prominent changes in certain major proteins reflecting
the elevation of the present polypeptides or expression of
(a)
(b)
Figure 3. SDS-PAGE of whole body tissue proteins of NEA S.
gregaria, under normal and heavy metal stress conditions for
(a): long term exposure and (b): short term exposure. Lane 1:
M.wt marker; Lane 2: normal; Lane 3: low Cd; Lane 4: high
Cd; Lane 5: low Pb and Lane 6: high Pb.

H. A. Yousef et al. / Natural Science 3 (2011) 218-226 222
(a)
(b)
Figure 4. SDS-PAGE of whole body tissue proteins of mature
adult S. gregaria, under normal and heavy metal stress condi-
tions for (a): long term exposure and (b): short term exposure.
Lane 1: Mw. marker; Lane 2: normal; Lane 3: low Cd; Lane 4:
high Cd; Lane 5: low Pb and Lane 6: high Pb.
new sets of proteins (Hsps), in different molecular weight
groups. These Hsps ranged from high (90 KDa) to low
molecular weight (12KDa). Based on the criterion that
the synthesis (appearance) or elevations of these poly-
peptides were stimulated after heat shock (or environ-
mental stress as heavy metal contamination), they are
referred to as heat shock proteins (Hsps) or stress pro-
teins [19]. They belong to the most well known families
of Hsp 100, Hsp 90, Hsp 70, Hsp 60, Hsp 40, and the
small Hsps (below 30KDa) [20].
The obvious increase of small Hsps levels may have
resulted from the characteristic and relatively stable
breakdown products of Hsp 70, i.e. , they are proteolyti-
cally reduced to small Hsps [21,22].
Hsps, which are a quite divergent domain consist of
80–100 amino acids and can form high molecular weight
polymeric structures that may be necessary for function
within the cell. During cellular stress, Hsps bind to un-
folded or damaged proteins, inhibit their aggregation and
maintain their client protein in a soluble and folding
competent state such that they can be refolded to their
native conformation by other ATP-dependent chaperones
[23].
Hsps 52-57 and 62-66 KDa were almost the major
proteins stimulated by the contamination with cadmium
and lead metals; their expression elevated or newly syn-
thesized. Another prominent induction in small Hsps;
12-15 and 24 KDa were observed by the heavy metal
stress (Figures 1-4). Cadmium was reported to be in-
duced the expression of heat shock proteins (Hsps) and
metallothioneins [24-26].
Also it was very clear that short exposure to the heavy
metals was more effective than long exposure in eleva-
tion or inducing protein synthesis and rather a decrease
in some protein levels (Figures 1-4). The lack of inten-
sive synthesis of stress proteins indicates that, in S. gre-
garia, the protective role of these Hsps against the action
of toxic substances does not function or alternatively
insects living in the contaminated area or exposed to
toxic materials for long times have adapted to the level
of existing contamination and not only do not synthesis
more Hsps but rather decrease synthesis [27,4].
Adaptive changes in Hsp expression during several
days also support the ecological significance of Hsps in
natural populations [28,29]. In different species of Dro-
sophila, it was shown that expression of Hsp 70 was
lower in lines frequently or continuously exposed to
sever stress (16-18]. The interpretation was that the costs
of Hsp expression in populations frequently exposed to
stress outweighed the benefits and that stress adaptation
was achieved through some other means. The same pat-
tern was subsequently found in natural population of
Drosophila [30] and in soil invertebrates exposed to
heavy metals [31]. According to these findings, the ad-
aptation role of Hsps in connection to environmental
stress resistance seems to occur during periods of rela-
tively rare, unexpected extreme stress exposures and not
during every day environmental fluctuations. Other
mechanisms of adaptation to stressful conditions are
selected for under chronic stressful environmental con-
ditions [17,32].
It was suggested that the low Hsp induction was
caused by a more pronounced need for chaperones in
order to maintain optimal cell function and homeostasis.

H. A. Yousef et al. / Natural Science 3 (2011) 218-226
223223
The up-regulation of Hsps in addition to being an im-
portant part of the response to sudden extreme stress
exposures is of ecological relevance on a much wider
scale with respect to less severe but regular incidences of
stress [33].
In conclusion, heat shock proteins are important in re-
lation to stress resistance and adaptation to the environ-
ment. The regulation of Hsp is influenced by both envi-
ronmental and genetic stress factors.
3.4. Estimation of Hsp 70
Data analysis of the Hsp 70 detected, using ELISA
technique with the monoclonal antibody (anti-Hsp70),
revealed that the onset development of Hsp expression at
the different instars of S. gregaria, in both the control
and the insects exposed to heavy metals exhibit Hsp 70.
The Hsp 70 expressed in the 4th instar are shown in
(Table 1), in the long term exposure to heavy metals Cd
and Pb in both low and high concentrations. The in-
creases of the expressed Hsp 70 were significant at the
high Cd, and low Pb concentrations (about 2 folds to
control). A non significant change in the low Cd, and
high Pb concentrations were observed. On the other
hand, the short term exposure to heavy metals showed
significant increase in the expressed Hsp 70 with respect
to the control.
The highest increase in Hsp expression was observed
in the treatment with low Cd concentration at short term
exposure (5 times more than control) and in contrast, the
lowest expression in Hsp 70 was observed in the treat-
ment with low Cd concentration at long term exposure
(19.8% to control). At the short term exposure, it is clear
that, the expression level of Hsp 70 at high concentration
of Cd and Pb was the same, but at long term exposure,
the high Cd concentration has an effect nearly equal to
the effect of low Pb concentration.
The concentration of Hsp 70 detected in all treatments
in 5th instar exposed to heavy metals for long term in-
creased significantly with respect to control, with an
exception (insignificant increase) in low Pb concentra-
tion (Table 1). The present data revealed that the Hsp 70
was highly expressed in insects exposed to heavy metals
at short term exposure, and the highest increase of Hsp
70 expression was in low Pb concentration at short term
exposure (12.27 folds more than control). At the 5th in-
star, the effect of low Pb concentration was higher than
low Cd concentration, but high Cd concentration was
higher than high Pb concentration especially after short
term exposure.
Newly emerged adult S. gregaria was the stage at
which all treatments gave significant results of Hsp 70
elevation (Table 1), the highest Hsp 70 concentration
expressed was observed at the treatment of high concen-
tration of Cd at short term exposure (307.5%) (3 folds).
The first decrease in Hsp70 concentration was observed
in the treatment of low Pb at long term exposure (64.4%)
(The decrease by 40%), the data shows fluctuations.
In the mature adult S. gregaria, Hsp 70 concentration
increased significantly in the long term exposure to high
Cd concentration and low Pb concentration (204.6% &
123.5%) (2 folds and 23.5%), but the increment of Hsp
70 was insignificant at low Cd concentration (Table 1).
The surprise was appeared in the high concentration of
Pb; at which a high decrease in Hsp 70 concentration
was observed (96.6% decrease of the control). The short
term exposure of this stage caused obvious increment of
Hsp 70, but it was lower than that of other stages.
In the present work, the non stressed (control) insects
expressed Hsp 70, which was considered previously as
normal component of larval imaginal discs and found in
normal Drosophila embryos [34], and in addition to the
growing evidence that Hsps are present under non-heat
shock conditions in specific developmental stages/cell
types in different organisms [20].
In the present study, all stages showed higher Hsp 70
expression after short term exposure than that expressed
after long term exposure. This was obvious in 5th instar
were the highest concentration of Hsp 70 was after short
term exposure and the lowest Hsp 70 expression after
long term exposure and this may be due to the fact that
Table 1. Concentrations of Hsp 70 in the body of different instars S. gregaria after long term and short term exposure to heavy metals.
Long term exposure Short term o exposure
Stage Control
Low Cd High Cd Low Pb High Pb Low Cd High Cd Low Pb High Pb
4th 1.06 + .075 1.27 ± 0.1452.27 ± 0.044* 2.3 ± 0.058*1.35 ± 0.11 5.7 ± 0.12*3.4 ± 0.12* 4.5 ± 0.18* 3.4 ± 0.13*
5th 0.59 + 0.06 1.35 ± 0.145*1.05 ± 0.05* 0.677 ± 0.0772.23 ± 0.09* 6.65 ± 0.11*5.24 ± 0.09* 7.24 ± 0.06* 4.3 ± 0.08*
NEA 1.74 + 0.08 2.25 ± 0.09*2.37 ± 0.1* 1.12 ± 0.122*2.46 ± 0.053*4.3 ± 0.13*5.35 ± 0.06* 3.35 ± 0.07* 3.75 ± 0.09*
MA 1.036 + 0.1 1.063 ± 0.072.12 ± 0.09* 1.28 ± 0.09*0.035 ± 0.008*4.25 ± 0.07*3.5 ± 0.14* 4.85 ± 0.11* 4.4 ± 0.11*
Significant at *P < 0.05; in all cases significance was tested with respect to 0 (control) using t-test, (N = 3).

H. A. Yousef et al. / Natural Science 3 (2011) 218-226 224
5th instar in locusts eat large quantities, so after short
exposure the shock of heavy metal in large quantity in
short time leads to rapid response from the cells to ex-
press more Hsp 70 concentration to resist the heavy
metal stress but at long period of exposure the cells may
be adapted to the heavy metal stress after this long time
and the expression of Hsp 70 becomes normal and near
to the control results [26,4].
Adaptive changes in Hsp expression over days also
support the ecological significance of Hsps in natural
populations [27,28]. In different species of Dro sophila, it
was shown that expression of Hsp 70 was lower in lines
frequently or continuously exposed to severe stress
[15-17]. The interpretation was that the costs of Hsp
expression in populations frequently exposed to stress
outweighed the benefits and that stress adaptation was
achieved through some other means. The same pattern
was subsequently found in natural population of Droso-
phila [29] and in soil invertebrates exposed to heavy
metals [30]. According to these findings, the adaptation
role of Hsps in connection to environmental stress resis-
tance seems to occur during periods of relatively rare,
unexpected extreme stress exposures and not during
every day environmental fluctuations. Other mechanisms
of adaptation to stressful conditions are selected for un-
der chronic stressful environmental conditions [17,32].
Induction of Hsp 70 in response to exposure to heavy
metals and physical stress has been reported in some
invertebrates [29]. In Tigriopus brevicornis exposed to
metals such as copper, nickel, silver and zinc leads to
induction of metallothionein-like proteins [35]. In mol-
lusks, the Hsp 70 biomarker has shown a good correla-
tion with the level of pollution [36]. Hsp 70 levels were
induced in samples collected from contaminated sites
[37]. In vitro, studies have demonstrated that Hsp 70
induction is a cytoprotective response of cells to toxic
chemical exposure [38,39].
Insects living in contaminated environment have
adapted to the level of existing contamination and not
only do not synthesize more Hsp 70 proteins in response
to heavy metal exposure, but rather decrease synthesis.
While in other studies, the concentration of Hsp 70 de-
tected in animals cells exposed to toxic substances was
higher than in the control [37,40]. However, an increase
in concentration of Hsp70 has not been observed in all
species examined [29] or only at low concentrations of
heavy metal exposure [37] and rather after short term
than after chronic exposure [41].
The present work, based on the available data reveals
that Hsps are ecologically relevant for all life stages.
Correlation of Hsp expression levels and stress resis-
tance might or might not exist between life stages.
Probably the up-regulation of Hsps in addition to be-
ing an important part of the response to sudden extreme
stress exposures is of ecological relevance on a much
wider scale with respect to less severe but regular inci-
dences of stress. Also the expression level of Hsps, in
each species and population is a balance between bene-
fits to resistance and costs, due to the impacts on growth,
developmental rate and fertility that up-regulation of
Hsp promotes.
The data presented in this work suggests the ability of
using the induction and development of Hsp 70 in S.
gregaria as a good biomarker for environment pollution
by the heavy metals (Cd & Pb) at low contamination
irrespective to the insect stage.
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