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Chemosphere 90 (2013) 917–928
Contents lists available at SciVerse ScienceDirect
Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere
Modulation of immune-associated parameters and antioxidant responses
in the crab (Scylla serrata) exposed to mercury
Gopalakrishnan Singaram a,b,g,⇑, Thilagam Harikrishnan a,b, Fang-Yi Chen b, Jun Bo b, John P. Giesy b,c,d,e,f,g
a
Department of Zoology, The Presidency College, University of Madras, Chennai 600 005, India
State Key Laboratory of Marine Environmental Science, College of Oceanography and Environmental Science, Xiamen University, Xiamen 361005, China
c
Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5B3
d
Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5B3
e
Department of Zoology and Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA
f
Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong, China
g
School of Biological Sciences, Hong Kong University, Pokfulam Road, Hong Kong, China
b
h i g h l i g h t s
" A 14 d exposure of Hg induces physiological changes in the immune system of crab.
" The approach is based on utilizing the commercial crab Scylla serrata.
" Hg provokes immunomodulation, oxidative stress and antioxidant defences in crab.
" Correlation analysis between the responses was highly significant.
" First study to show Hg modulates immune functions and antioxidant system in crabs.
a r t i c l e
i n f o
Article history:
Received 24 November 2011
Received in revised form 24 May 2012
Accepted 22 June 2012
Available online 26 July 2012
Keywords:
Physiology
Enzyme
Biomarkers
Immunomodulation
Metal
Crustacean
a b s t r a c t
Organic and inorganic contaminants can suppress immune function in molluscs and crustaceans. It was
postulated that metals could modulate immune function in marine crabs. To test this hypothesis, sublethal effects of mercury (Hg) on cellular immune and biochemical responses of crabs were determined.
When crabs were exposed for 14 d to environmentally-relevant concentrations of Hg, changes in
immune-associated parameters including, total haemocyte count, lysosomal membrane stability, phenoloxidase, super oxide generation and phagocytosis were observed. Oxidative stress, as measured by lipid
peroxidation, antioxidant responses, including superoxide dismutase and catalase activities and glutathione-mediated antioxidant enzymes in serum, haemocyte lysate, gills, hepatopancreas and muscle were
assessed in crabs exposed to Hg. Exposure to Hg resulted in significantly lesser immune-associated
parameters in haemolymph and antioxidants in all tissues studied. Conversely, GST and phenoloxidase
activity, were greater in crabs exposed to Hg. Responses of antioxidant parameters (SOD, CAT and GPx)
were positively correlated with immune responses, including THC, superoxide and phagocytosis. These
results were postulated to be due to an immediate response of antioxidant defense to oxygen radicals
generated. Overall, the results suggest that 14 d exposure to environmentally realistic concentrations
of Hg causes immunomodulation and potentially harmful lessened antioxidant defenses of crabs.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Some aquatic organisms have the ability to live in contaminated
regions, due to inducible defense mechanisms that allow detoxification and excretion of contaminants and protection by antioxidants from oxidative stress (Bard, 2000; Geret and Bebianno,
2004). Biomarkers are biological parameters which represent
⇑ Corresponding author. Address: School of Biological Sciences, Hong Kong
University, Pokfulam Road, Hong Kong. Fax: +852 2559 8114.
E-mail address: sing_gopal@yahoo.com (G. Singaram).
0045-6535/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.chemosphere.2012.06.031
initial responses to environmental perturbations or contamination
(Bengtson and Henshel, 1996; Roy et al., 1996). Previous studies
have found a possible interaction between various contaminants
as well as diseases, and the stress that it causes in animals (Sinderman, 1993). Exposure of aquatic organisms to pollutants can promote increased production of reactive oxygen species (ROS)/
reactive nitrogen species (RNS). Thus, assessment of parameters
related to oxidative stress in specific sentinel organisms could be
included in studies of environmental pollution to predict the impact of pollutants present in the environment (Walker et al.,
1976; Pellerin-Massicote, 1994; Livingstone, 2001).
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Crustaceans are useful as integrating sentinels of exposure to
certain contaminants. The mud crab (Scylla serrata) is a useful sentinel, due to its biological and ecological characteristics (Elumalai
et al., 2002, 2007). The mud crab is important in the estuarine food
web because they are not only predators, but also scavengers and
prey themselves. Also, they are economically important as food for
humans.
Immune systems of crustaceans include both cellular and noncellular defense responses and circulating haemocytes are part of
the defense against potential pathogens. Since this is an early
internal defense by circulating haemocytes against pathogens,
any decrease in total haemocyte count (THC) or phagocytic activity
(PA) due to exposure to contaminants can impair the defensive response of the host against pathogens. Activated haemocytes can
undergo an ‘‘oxidative burst’’ by releasing ROS. Recent investigations of the crustacean immune system have found that ROSdependent immunity is important for survival (Ha et al., 2005).
While this response is effective against pathogens, it can also result
in damage to tissues when ROS production is enhanced to a greater
level due to an imbalance between oxidant and antioxidant status
in the cell called ‘‘oxidative stress’’. Previously we have shown that
lipopolysaccharides (LPSs) modulate immune responses of crabs,
which leads to increases of antioxidant defense mechanisms such
as superoxide dismutase (SOD) and catalase (CAT) (Gopalakrishnan
et al., 2011), both of which participate in the innate immune defense against immuno-stimulants (Mathew et al., 2007; Ji et al.,
2009; Chen et al., 2010).
Mercury (Hg) is a non-essential metal, which has been identified as one of the adverse environmental pollutants to aquatic species (Day et al., 2007). Environmental concentrations of Hg have
been reported to range from 0.1 to 9.8 lg L1 (Bargagli et al.,
1998; Thilagam et al., 2008). Early studies of inorganic Hg have
been reported to be toxic to crustaceans even at small concentrations (Elumalai et al., 2007). Mercury caused mortality (Guilhermino et al., 1997), suppression of enzymes, induction of oxidative
stress (Elumalai et al., 2007) abnormalities during embryo development (Sánchez et al., 2005) and inhibition of acetyl choline
esterase (Devi and Fingerman, 1995; Frasco et al., 2005) in crustaceans. Though several studies are available on effects of mercury
on the crab S. serrata, these studies were restricted to histological
changes and enzyme activities (Krishnaja et al., 1987; Chen et al.,
2000) and there is a paucity of information on the effects of metals
on immunomodulation and antioxidant activity, including glutathione-mediated enzyme responses in crab. Therefore, a study of
the effects of Hg on immune function and antioxidant activities
of the mud crab S. serrata was conducted.
The objective of the present study was to determine whether a
14 d exposure to sublethal concentrations of Hg would induce
physiological changes in the immune system and to determine
the response of the antioxidants system. Specifically, changes in
immune parameters such as total haemocyte count (THC), membrane stability, phagocytosis, superoxide generation and phenoloxidase (PO) were measured. In addition, changes in lipid
peroxidation (LPO) and activities of antioxidant enzymes were
quantified in the haemocyte component, gill, hepatopancreas and
muscle. The present study is a first attempt to understand the effects of Hg on the immune functions and antioxidant system of
crabs exposed to Hg.
individuals each. Group I were reared in normal seawater. Group
II and III were exposed to seawater that contained sublethal concentrations of mercuric chloride (1.0 or 10 lg Hg L1). Similarly,
parallel duplicate tanks with six individuals were maintained for
all three groups. Water in both the control and treated groups were
renewed daily. The exposure was continued for 14 d at a constant
temperature of 28 ± 1 °C; salinity of 34 ppt; pH of 8.2 ± 0.1 and
with a photoperiod of 12 L:12 D. Following the exposure period,
three crabs from each duplicate chamber were collected for immunological, oxidative stress and antioxidant parameters after a period of 7 or 14 d. Methods for collecting haemolymph and
separation of haemocytes and preparation of serum and haemocyte lysate suspension (HLS) have been described previously (Liu
et al., 2010; Chen et al., 2010). Haemolymph from individual crabs
was examined and plasma, serum and haemocytes thus isolated
were not pooled.
3. Quantification of mercury
Concentrations of Hg were determined by use of automated
cold vapor atomic absorption spectrophotometer (AAS: Spectra
AA-10 Varian), according to the methods of Weltz and Schubert-Jacobs (1991). There was no difference between the nominal and
measured concentrations (Table 1) and the measured values were
in good agreement with the certified values (<10% deviation). Prior
to use of the instrument for measuring the actual concentration of
the mercury, the quality control of metal analysis was performed
by use of digestion blanks and reference material (Mussel, IAEA142). All glassware and equipment used were acid washed to avoid
contamination and to check for contamination, procedural blanks
were analyzed once for every three samples.
4. Immunological parameters
4.1. Total haemocyte count
The total number of haemocytes (THC) in haemolymph was
determined by use of a haemocytometer. Haemocytes in a mixture
of 20 lL of diluted haemolymph and trypan blue (Sigma, T-6164;
0.05% trypan blue in TBS; 50 mM Tris; 370 mM NaCl; pH 8.4), were
enumerated by use of a haemocytometer under a light microscope
at a magnification of 40.
4.2. Phenoloxidase assay
Activity of phenoloxidase in haemolymph plasma was assessed
by use of the procedure of Asokan et al. (1997). Briefly, 100 lL of
plasma were mixed with an equal volume of Tris buffered saline
(TBS; 50 mM Tris; 370 mM NaCl; pH 8.4) and incubated for
15 min at 22 °C. After incubation, 2 mL of 1 mg mL1 L-DOPA
(3,4-Dihydroxy-L-phenylalanine, Sigma Chemicals) was added
and further incubated for 5 min at 22 °C. All incubations were performed in the dark. After incubation, the optical density (O.D) of
samples was determined at a wavelength of 460 nm in a Shimadzu
Table 1
Nominal and measured concentration of mercury in exposure solution.
2. Materials and methods
Male mud crabs (S. serrata) weighing 250 ± 30 g, were collected
from a local crab farm and acclimatized to laboratory conditions
for 1 week. During acclimatization crabs were fed tilapia fish.
After acclimatization crabs were divided into three groups of six
Control
Low concentration
High concentration
Nominal concentration
(lg Hg L1)
Measured concentration
(lg Hg L1)
0
1.0
10
BDL
1.02 (0.06)
10.46 (0.67)
Values are given as mean ± S.D (in parenthesis) of six determination using samples
from different preparation. BDL- below detectable limit.
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G. Singaram et al. / Chemosphere 90 (2013) 917–928
UV-1700 spectrophotometer. Values were compared to a reagent
blank that contained 200 lL TBS and 2 mL L-DOPA. Protein content
of plasma was by the method of Lowry et al. (1951). The PO activity
was expressed as unit mg protein1 min1.
4.3. Superoxide anion generation assay
In vitro generation of superoxide anion (O
2 ) by haemocytes was
assessed by use of the nitro-blue tetrazolium reduction assay (NBT,
Sigma Chemicals) by use of the methods of Arumugam et al.
(2000). Suspensions of haemocytes were incubated with 125 lL
of 0.1% NBT for 15 min at 22 °C. At the end of the incubation, the
reaction was stopped by adding 460 lL of 70% methanol and centrifuged (1500 rpm, 10 min, 4 °C). The supernatant was discarded
and 4 mL of extraction fluid (6 mL KOH + 7 mL DMSO) were added
to the pellet to dissolve the insoluble formazan formed from reduction of NBT. Samples were further centrifuged (5000g for 15 min,
4 °C). The O.D. of the clear blue supernatant was measured at
625 nm using Shimadzu UV-1700 spectrophotometer, against a reagent blank consisting of 300 lL buffer, 125 lL NBT, and 4 mL
extraction fluid. Rate of generation of ROS was determined from
the O.D. at 625 nm/15 min.
4.4. Phagocytosis
Phagocytosis assays were performed on monolayers of yeast
cells as targets (Thiagarajan et al., 2006). A suspension of haemolymph (50 lL) was spread on a glass slide and the haemocytes
were allowed to adhere to the plate for 20 min at 25 °C. After
20 min, the monolayers were gently washed with TBS, to remove
unattached haemocytes and overlaid with 50 lL of 0.5% yeast cells
and the glass slides were further incubated for 15 min at 25 °C.
After rinsing with filtrated seawater, the slides were fixed with
2.5% glutaraldehyde for 5 min. Monolayers were washed with
TBS, overlaid with a cover slip, and observed using phase optics
of a Carl Zeiss Axioskop 2 plus microscope. Replicates were made
for each crab, and three counts of approximately 200 haemocytes
were made for each replicate. Results were expressed as percentage of phagocytic haemocytes (Equation 1).
% Phagocytosis ¼ ½ðPhagocytic haemocytesÞ=total haemocytes 100
ð1Þ
4.5. Lysosomal membrane stability
Haemolymph (100 lL) was pipetted into 0.5 mL a centrifuge
tube and aliquots (10 lL) of 0.33% neutral red (Sigma) solution in
TBS was added to each tube and the tube was incubated for 1 h
at 10 °C. Tubes were then centrifuged at 200g for 5 min and
washed twice in TBS. Aliquots (100 lL) of 1% acetic acid in 50% ethanol were added to all tubes. Tubes were covered with foil, incubated for 15 min at 20 °C and then the amount of neutral red in
the medium determined at 550 nm. The results were expressed
as O.D per mg1 mL1 haemocyte protein.
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peroxidation (LPO) was determined by measuring the of malondialdehyde (MDA) equivalents formed by reaction with thiobarbituric acid (Ohkawa et al., 1979) and absorbance was measured at
532 nm. SOD activity was measured as the degree of inhibition of
auto-oxidation of pyrogallol at an alkaline pH by the method of
Marklund and Marklund (1974). CAT activity was determined
according to the method of Sinha (1972). Dichromate in acetic acid
was reduced to chromic acetate when heated in the presence of
H2O2 with the formation of perchromic acid as an unstable intermediate. Chromic acetate was measured spectrophotometrically
at 570 nm. The reaction was allowed to continue for different periods of time and stopped by the addition of dichromate: acetic acid
mixture. The remaining H2O2 was determined by measuring the
chromic acetate spectrophotometrically. Activity was expressed
as l mol of H2O2 consumed min1 mg1 protein. Glutathione peroxidase (GPx) was assayed by measuring the amount of reduced
glutathione (GSH) consumed in the reaction mixture according to
the method of Rotruck et al. (1973). Concentrations of GSH were
estimated by the method of Moron et al. (1979) by reading the
O.D of the yellow substance formed when 5,50 -dithio-2-nitrobenzoic acid is reduced by glutathione at 412 nm. Glutathione -Stransferase (GST) activity of the fraction obtained with the substrate 1-chloro-2,4-dinitrobenzene was measured spectrophotometrically at 37 °C by following conjugation of the acceptor
substrate with glutathione as described in Habig et al. (1974)
and Jakoby (1985). Results were expressed as the formed conjugate
mg protein1 min1.
6. Statistical analyses
Statistical comparisons were performed by use of analysis of
variance (ANOVA) and SPSS software (Ver 10.0; SPSS). The experimental unit was individual crabs. The fact that multiple crabs were
exposed in each tank resulted in pseudo-replication, but for logistical reasons, it was not possible to house each crab in individual
chambers. While crabs were exposed in two different tanks for
each treatment, the six crabs were considered to be independent
replicates. Duplicate tanks were maintained for all concentrations
tested, the results reported as mean ± S.D. of six individuals per
group per time point (three crabs/tank) and the significance tested.
The data were first tested for normality and homogeneity using
Bartlett’s test. Since all data were normal, parametric statistics
were applied, by use of ANOVA, whether the groups differed and
if the ANOVA-calculated p value was significant (p 6 0.05). Tukey’s
multiple-comparison post hoc test was performed to identify statistical differences between exposed groups and control groups
(Zar, 1999). Principal Component Analysis (PCA) and a correlation
matrix were used to assess the interrelationships among the
parameters used. ‘‘Varimax Rotation’’ was used for extraction and
deriving factors in the PCA and the Pearson correlation coefficient
was used in the correlation matrix. Differences were statistically
significant when p < 0.01 and p < 0.05.
7. Results
5. Tissue preparation and enzyme determination
7.1. Immunomodulation
Haemolymph, gill, hepatopancreas and muscle were used for
determination of responses to oxidative stress by measuring measures of lipid peroxidation and activities of antioxidant enzymes.
Serum and HLS was prepared as mentioned above. Tissues were
homogenized (1:10 w/v) using a Potter–Elvjeham glass homogenizer in 50 mM sodium phosphate buffer, pH 7.0. Homogenates
were centrifuged at 4 °C for 15 min at 15 000g. Supernatants were
removed and used for determination of enzyme activity. Lipid
While crabs exposed to inorganic Hg for 14 d exhibited normal
behavior responding to stimuli and no mortality was observed
throughout the exposure period (14 d), the THC in the crabs exposed to both Hg concentrations was less than control group during the exposure periods. After 7 d, the THC of crabs exposed to the
lesser concentration of 1 lg Hg L1 was not significantly different
from the THC of controls. However, during both the exposure periods, crabs exposed to 10 lg Hg L1 resulted in 25% and 32%
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G. Singaram et al. / Chemosphere 90 (2013) 917–928
decrease in THC compared to that of the control group. These differences were statistically significant with respective control group
(Fig. 1A). Sub-lethal exposure to both concentrations of Hg for 7 or
14 d resulted in less phagocytosis relative to that of haemocytes of
the control crabs. After 14 d, the phagocytic response of haemocytes of crabs exposed to 10 lg Hg L1 was approximately 45% less
than that of the controls (Fig. 1B). After 7 or 14 d, generation of
super oxide anion by haemocytes was significantly (p < 0.05) less
in crabs exposed to both concentrations of Hg (Fig. 1C). The
amount of NBT reduction was significantly (p < 0.05) less in crabs
exposed to 1 lg Hg L1 for 14 d than in the controls (Fig. 1C). The
magnitude of NBT reduction in crabs exposed to 10 lg Hg L1
was significantly less than that of the controls after both 7 and
14 d of exposure.
Exposure to Hg resulted in greater PO activity in plasma
(Fig. 1D). Both concentrations of Hg caused similar effects on the
PO system and were both significantly (p < 0.05) greater than the
controls after 7 or 14 d of exposure.
Exposure to Hg resulted in significantly less membrane stability
of haemocytes after 7 and 14 d of exposure in both concentrations
of Hg (Fig. 1E). After 14 d of exposure, the membrane stability was
16% and 26% less in crabs exposed to 1 or 10 lg Hg L1, respectively than in the controls.
7.2. Oxidative stress and antioxidant parameters
Activities of enzymes involved in responding to oxidative stress
were affected by exposure to Hg. The activity of SOD was significantly less in HLS and serum of crabs exposed to both concentrations of Hg for 7 or 14 d (Fig. 2A and B), whereas the activity of
CAT in serum of crabs varied between the exposure periods. The
activity of CAT in HLS of crab exposed to the lesser concentration
Fig. 1. Effect of sublethal concentrations of mercury (Hg) on parameters associated with immune functions: (A) total haemocyte count [THC], (B) phagocytosis, (C) superoxide
generation, (D) phenoloxidase, and (E) membrane stability in S. serrata. Each bar represents mean ± standard error of six determinations using samples from different
preparations. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used. Significant differences between exposure groups and controls were
indicated with asterisks (⁄P < 0.05).
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G. Singaram et al. / Chemosphere 90 (2013) 917–928
of Hg for 7 d was not significantly different from that of the control
group. Activity of CAT in serum was less in crabs exposed to either
concentration for either duration (Fig. 2C and D). Activities of SOD
in gill, hepatopancreas and muscle were 34–44, 21–57 and 38–45%
less, respectively, in crabs exposed to Hg, relative to that in the
controls (Fig. 3A). Activity of CAT was 26%, 31% and 21% in gill,
hepatopancreas and muscle, respectively, after 14 d of exposure
to 10 lg Hg L1 (Fig. 3B).
Exposure to Hg resulted in statistically significant oxidative
stress, as determined by concentrations of LPO in HLS, serum, muscle, hepatopancreas and gills of crabs (Figs. 2 and 3). LPO of HLS was
greater in crabs exposed to Hg and was greater after 14 d than it
was after 7 d. LPO in serum was not affected by exposure to Hg
for 7 or 14 d (Fig. 2E and F). The magnitude of LPO in gills were
21% and 34% greater in crabs exposed to either concentration after
7 d and approximately 49% and 51% greater in crabs exposed for
14 d. A similar pattern was observed for the hepatopancreas. In
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muscle, the magnitude of LPO was 25% and 39% greater, after 14 d
exposure to the lesser and greater concentrations of Hg, respectively (Fig. 3C).
Concentration of GSH in HLS and serum of crabs were significantly less than that of controls after 14 d exposure to either concentration of Hg (Fig. 4A and B). Activity of GPx activity in HLS and
serum was not significantly different from that of the controls after
7 d of exposure to Hg. However after 14 d exposure the activity of
GPx was less in HLS of crabs exposed to either concentration of Hg.
Activity of GPx in serum was significantly less only when crabs
were exposed to the greater concentration of Hg for 14 d (Fig. 4C
and D). The response of GST activity in serum and HLS of crab exposed to both concentrations of Hg were similar after 7 d exposure
(Fig. 4E and F). After 14 d GST in HLS in crabs exposed to either
concentration of Hg was significantly greater than in the controls.
Glutathione-mediated antioxidant enzyme activities were significantly affected by exposure to Hg (Fig. 5A–C). Concentrations
Fig. 2. Effect of sublethal concentrations of mercury (Hg) on antioxidant and oxidative stress parameters in serum and HLS of S. serrata: (A) HLS SOD, (B) serum SOD, (C) HLS
CAT, (D) serum CAT, (E) HLS LPO, and (F) serum LPO. Each bar represents mean ± standard error of six determinations using samples from different preparations. One-way
analysis of variance (ANOVA) followed by Tukey’s post hoc test was used. The significant difference between control and exposure groups were indicated with asterisks
(⁄P < 0.05).
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G. Singaram et al. / Chemosphere 90 (2013) 917–928
of GSH in gill, hepatopancreas and muscle of crabs were 29%, 46%
and 36% less, respectively, relative to that of controls, when crabs
were exposed to 1 lg Hg L1 for 14 d (Fig. 5A). The activity of GPx
was significantly less in HP and muscle after 7 or 14 d exposure
to the lesser concentration of Hg. However, there was significantly
less GPx activity in gill only after 14 d of exposure (Fig. 5B). After
7 d, there was significantly more GST activity in all tissues when
crabs were exposed to the greater concentration. Activity of GST
was 25%, 23% and 29% greater in gill, hepatopancreas and muscle,
respectively, when the crabs were exposed for 14 d to 10 lg Hg L1
than in the same tissues of controls.
7.3. Correlations among parameters
Statistically significant (p < 0.01 and p < 0.05) associations were
observed between parameters of the immune system and antioxidant enzymes in haemolymph of crab (Table 2). There were statistically significant correlations between THC and other parameters
Fig. 3. Effect of sublethal concentrations of mercury (Hg) on parameters related to antioxidant and oxidative stress in different tissues of crab: (A) SOD, (B) CAT, and (C) LPO.
Each bar represents mean ± standard error of six determinations using samples from different preparations. One-way analysis of variance (ANOVA) followed by Tukey’s post
hoc test was used. The significant difference between control and exposure groups were indicated with asterisks (⁄P < 0.05).
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G. Singaram et al. / Chemosphere 90 (2013) 917–928
studied with correlation coefficients greater than 0.9. A statistically
significant, positive correlation was found between parameters
studied except phenoloxidase. Correlations between parameters
associated with the response of the immune system and measured
antioxidant parameters in serum and HLS were also statistically
significantly. Correlations between HLS, LPO and the immuneassociated parameters and in most cases were statistically significant. Except for activity of phenoloxidase, all the parameters associated with the immune system were negatively correlated with
HLS and concentration of GST in serum (Table 2). Magnitudes of
LPO were negatively correlated with activities of both SOD and
CAT in components of haemolymph and super oxide anion generation in haemocytes (Table 2). The lesser activity of SOD was probably responsible for the subsequent increase in concentrations of
MDA. However, the correlation between the MDA and SOD in the
haemolymph component was not statistically significant.
In order to characterize relationships between parameters associated with the immune system, antioxidant enzymes that respond
to oxidative stress and the measured in the HLS, the entire set of
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parameters was also subjected to PCA. The rotated component matrix, developed by PCA is given (Fig. 6). The dimensions of the parameters were reduced from 17 original variables to two principal
components. Approximately 88% of the variation was accounted
for by the first two components. Of this variation, component 1 accounted for 78.34% while component 2 accounted for approximately 9.81% of the variation. Significant correlations between
variables and axes are indicative of a good representation of these
variables with PCA. As a consequence, the relationship between
parameters associated with oxidative stress, as measured by LPO
and parameters associated with the immune system in the HLS
were found to be more pronounced than the LPO observed in serum. However, a strong correlation was observed between the serum based antioxidant and parameters associated with an immune
response.
In order to compare the present results with crabs in which an
immune response was stimulated by LPS. To characterize the relationships between parameters associated with the immune response and parameters associated with antioxidant responses,
Fig. 4. Effect of sublethal concentrations of mercury (Hg) on glutathione mediated antioxidant parameters in serum and HLS in S. serrata: (A) HLS GSH, (B) serum GSH, (C) HLS
GPx, (D) serum GPx, (E) HLS GSH, and (F) serum GSH Each bar represents mean ± standard error of six determinations using samples from different preparations. One-way
analysis of variance (ANOVA) followed by Tukey’s post hoc test was used. The significant difference between control and exposure groups were indicated with asterisks
(⁄P < 0.05).
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G. Singaram et al. / Chemosphere 90 (2013) 917–928
such as activities of SOD and CAT in immunocytes of the crab
challenged with LPS, immune parameters and the antioxidant
parameters in crabs stimulated with LPS were also subjected to
PCA analysis (Data not shown). The dimension of parameters
associated with both immune and antioxidant responses of
immunocytes of the crab was reduced from 13 to 2 components.
Approximately 80% of the variation was accounted for by the first
two components. Of this variation, component 1 accounted for
63.55% of the variation while component 2 accounted for approximately 16.75% of the variation.
8. Discussion
While this was the first study to examine effects of chronic
exposure to Hg on immune and antioxidant competence of the
crab S. serrata, a similar study by Krishnaja et al. (1987) examined
effects of Hg on other aspects of the physiology of S. serrata. However, the results reported from that study were restricted to histology. The study upon which we report here differs from the earlier
study in that the effects of sublethal concentrations of Hg on
immunological and antioxidant parameters were investigated.
Fig. 5. Effect of sublethal concentrations of mercury (Hg) on glutathione-mediated antioxidant enzymes in tissues of crab: (A) GSH, (B) GPx, and (C) GSH. Each bar represents
mean ± standard error of six determinations using samples from different preparations. One-way analysis of variance followed by Tukey’s post hoc test was used. The
significant difference between control and exposure groups were indicated with asterisks (⁄P < 0.05).
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.841*
.896**
.931**
.809*
.773*
1.000
.909
.969**
.947**
.901**
.834*
1.000
.945
.942**
.907**
.953**
1.000
SERUM-LPO
.171
.206
.028
.171
.225
.228
.043
.039
.103
.536
.116
.400
.302
.525
.309
1.000
.801
.792*
.790*
.807*
.710
.770*
.707
.574
.578
.853*
.867*
.796*
.714
.920**
1.000
.799
.756*
.721
.797*
.669
.754*
.733*
.638
.622
.909**
.762*
.813*
.672
1.000
*
HLS-GST
SERUM-GST
*
**
HLS-GSH
SERUM-GSH
*
*
HLS-GPx
SERUM-GPx
*
**
SERUM-SOD
HLS-SOD
**
**
SERUM-CAT
HLS-CAT
*
**
.971
1.000
1.000
PA
THC
SO
PO
LMS
HLS-CAT
SERUM-CAT
HLS-SOD
SERUM-SOD
SERUM-GPx
HLS-GPx
SERUM-GSH
HLS-GSH
SERUM-GST
HLS-GST
SERUM-LPO
HLS-LPO
Correlation
Correlation is significant at the 0.01 level.
Correlation is significant at the 0.05 level.
.970
.980**
1.000
.999
.970**
.968**
1.000
**
LMS
**
PO
**
SO
**
THC
PA
Table 2
Correlations between measured parameters in S. serrata exposed to sublethal concentrations of mercury.
**
HLS-LPO
G. Singaram et al. / Chemosphere 90 (2013) 917–928
925
Exposure of S. serrata to sublethal concentration of Hg not only
influenced the immunological parameters but also the antioxidant
enzymes and other parameters.
In crustaceans the role of PO in immune reactions has been
extensively reviewed by Söderhäll and Cerenius (1998). PO is present in the plasma of crustaceans as an inactive proPO. Upon activation, the active form of the enzyme, which is responsible for
melanin deposition, is released into the haemolymph (Cerenius
and Soderhall, 2004). Any modulation of this defense enzyme
could have an effect on survivability of animals upon challenge
with infectious pathogens (Gopalakrishnan et al., 2011). An increase in PO, due to toxicant exposure, has been previously reported (Coles et al., 1994). Similarly in the present investigation,
exposure to Hg caused an increase of PO in haemolymph plasma.
It has been previously shown that intermediates of PO, such as quinones, and semiquinones, generate superoxide anion during redoxcycling of these intermediates (Nappi et al., 1995). Thus, it is plausible that greater activities of PO due to exposure to Hg could, in
turn, result in greater concentrations of free radicals that could
produce oxidative stress and other free radical-mediated cellular
damage (Thiagarajan et al., 2006). Whether the change in activity
of PO due to exposure to Hg is an adaptive response of crabs to
an infection remains to be elucidated. A recent study of a crustacean demonstrates that haemocytes are also involved in production of PO (Matozzo and Marin, 2010) and several studies have
found a significant relationship between THC and activity of PO
(Cheng et al., 2005). Similarly, in the present study the correlation
between THC and activity of PO was inversely proportional to THC.
Hence, it could be hypothesized that the rise in PO activity was a
physiological response of crabs to an increase in immunosurveillance to compensate for the lesser THC (Hauton et al., 1997).
Changes in concentrations of superoxide generation produced
by haemocytes that was caused by exposure to contaminants have
been well documented in invertebrates (Pipe et al., 1999; Wootton
et al., 2003; Thiagarajan et al., 2006). Generation of O
2 following
exposure to a metal (Thiagarajan et al., 2006) could be attributed
to direct or indirect interactions of metals with the cytoskeleton
(Gomez-Mendikute et al., 2002; Gomez-Mendikute and Cajaraville,
2003). Perhaps, disruption of cytoskeleton proteins could in turn
affect the assembly of the NADPH-oxidase complex in the plasma
membrane which could result in non-activation of the complex.
Such an alteration in haemocyte activation and subsequent O
2
generation could cause crabs to be susceptible to infection over
prolonged periods of exposure and greater oxidative stress. In the
present study exposure of S. serrata to Hg for either 7 or 14 d leads
to a reduction in NBT and such a reduction may not be possible to
distinguish whether the decrease in superoxide anion resulted
from decreased activity of NADPH Oxidase, which is responsible
for superoxide anion generation, or due to increase in antioxidant
activity which are responsible for scavenging the superoxide anion
and moreover the greater decrease in superoxide production will
decrease the immunity of the crab.
Phagocytosis is an immune reaction of both invertebrates and
vertebrates and is a major effector mechanism of cellular immune
components of crustaceans. Phagocytosis is commonly monitored
to assess effects of chemicals on immune function of animals and
cellular integrity (Fournier et al., 2000). Chronic exposure to the
concentrations of Hg studied here caused significant effects on
phagocytosis of yeast cells by haemocytes of S. serrata. The observation that exposure to Hg resulted in less phagocytosis is consistent with the results of studies in which invertebrates were
exposed to toxicants including Hg (Brousseau et al., 2000; Mattozzo et al., 2001; Thiagarajan et al., 2006).
Stability of the membranes as measured by retention of neutral
red has been used as an integrative biomarker of cytotoxicity to
haemocytes. The lesser stability of membranes in haemocytes of
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G. Singaram et al. / Chemosphere 90 (2013) 917–928
crabs exposed to Hg is evidence of damage to membranes of haemocytes. Haemocytes with intact membranes will retain neutral
red after initial uptake and damage to membranes increases the
rate of leakage as measured by retention time. Stability of membranes is important in maintenance and functioning of the cellular
processes, thus any destruction in the membrane of haemocytes
will affect the phagocytic capability and reduce immunocompetence and overall fitness. The strong, positive correlations between
the membrane stability and phagocytic activity of haemocytes that
were observed in this study are consistent with Hg causing damage
to membranes.
The lesser protein contents of the hepatopancreas, muscle and
gill of crabs exposed to Hg observed in the present study could
be attributed to interference with and/or modulation of their participation in various biological processes that were altered by
exposure to Hg. Mercury is known to induce oxidative stress by
triggering ROS through the mitochondrial electron transport chain
(Lund et al., 1991; Pourahmad et al., 2003). In the present study
generation of free radicals in response to Hg should be scavenged
by the various antioxidant systems to serve as a protective response to detoxify the ROS generated. Organisms are equipped
with a cascade of enzymes to counteract free radicals produced
either during normal metabolism or due to exposure of chemicals
such as ions (Halliwell and Gutteridge, 1985).
SOD is the first antioxidant enzyme which scavenges superoxide radicals (O
2 ), and CAT is responsible for detoxification of
H2O2 formed as a result of the reaction catalyzed by SOD. The lesser activities of SOD and CAT observed in the present study are
consistent with ROS being generated during the response to Hg.
The lesser activity of CAT could also be attributed to greater production of superoxide anion radical, which has been reported to inhibit CAT activity (Kono and Fridovich, 1982). In contrast, the
activity of SOD in hepatopancreas in fresh water crabs exposed
to chromium and cadmium and marine blue crab Callinectes sapidus exposed to copper were greater following exposure (Brouwer
and Brouwer, 1998). Oxyradicals in combination with H2O2 can result in production of hydroxyl radicals through the Haber–Weiss
reaction, which results in more LPO. The significantly less activities
of both enzymatic and concentrations of non-enzymatic antioxidants that were observed in HLS, serum and the tissues of S. serrata
exposed to Hg may be an adaptive response of crabs to counteract
oxidative stress.
The greater concentration of MDA, lesser activities of GPx and
concentrations of GSH observed in crabs exposed to Hg in the current study is consistent with GPx and GSH being antioxidants that
can be depleted during scavenging of ROS/RNS. Such depletion of
antioxidant defenses due to exposure to Hg could result in greater
susceptibility to lipid peroxidation. The significantly lesser activity
of GPx and correspondingly greater concentration of MDA observed in this study are consistence with glutathione being involved in detoxification of electrophiles. A lesser concentration of
GSH is consistent with greater utilization of GSH by GPx to catalyze
the reduction of H2O2 to H2O, and O2. The lesser activity of GPx can
be attributed to lesser concentrations of glutathione (Kappus,
1985). The greater GST activity following exposure to Hg demonstrates activation of detoxification mechanisms. Since GST is involved in detoxification and lipid peroxidation processes, these
findings suggest interference of Hg with at least one of the processes. Hg has been shown to cause oxidative stress in several species (Zaman et al., 1994), hence it can be hypothesized that the
greater activity of GST observed in crabs exposed to Hg might be
an adaptive response to oxidative stress.
Loading values calculated during the PCA were used to examine
the contribution of each biomarker to the overall variance explained by each of the components. The magnitude of the loading
values for each marker is indicative of its significance to the final
component. The most significant loadings to components 1 and 2
were likely to be associated with the immediate response to the
contaminant, which includes superoxide (SO), serum CAT, HLS
SOD, PA, PO, serum SOD, THC followed by the other antioxidant responses. Serum LPO deviated significantly from components 1 and
2. The resulting PCA model also showed a trend with duration of
exposure. For instance the stability of THC membranes, superoxide
and phagocytosis were grouped together in crab exposed to Hg. PO,
HLS GST and LPO of HLS were grouped together. Based on this result, it was postulated that duration of exposure was important in
Fig. 6. Rotated component matrix developed by Principal Component Analysis (PCA) of the biomarker.
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G. Singaram et al. / Chemosphere 90 (2013) 917–928
determining THC and magnitude of reduction in membrane stability. Both of these parameters have a direct impact on phagocytosis,
which may also have an impact by decreasing the production of
superoxide. The immune system of crustaceans consists of a complex defense system and that the antioxidant system is involved in
preventing impairment of immune function. Also, the antioxidants
produced by haemocytes are likely involved with the immune response and might be compensated for by other defense mechanisms. In order to investigate trends of the immune system and
subsequent antioxidant responses of crabs that had been stimulated with LPS, PCA was used. The results of this analysis showed
that most of the significant value loading to component 1 and 2
are associated with the immediate response to nonself challenge
similar to that of Hg exposure. From these comparison it can be
concluded that antioxidants were involved in responding to oxidative stress caused by either LPS or Hg.
The data presented in this report demonstrate that exposure of
the crab S. serrata to Hg caused drastic changes in antioxidant-related parameters, especially the strong inhibition of SOD and CAT.
Furthermore exposure to environmentally relevant concentrations
of Hg modulated the immune response in crabs. Exposure to Hg
suppressed immune responses such as THC, superoxide generation, phagocytosis, PO activity and membrane stability. Considering all of the biomarkers investigated in this study, it was
concluded that the detoxification system of S. serrata is interactive
and complex. Free radicals of oxygen were produced as a mediator
of Hg toxicity in S. serrata and therefore dynamic processes in the
crabs were utilized to eliminate Hg mediated toxicity and production of oxygen free radicals. However, data presented here represent the immunomodulation of immune associated parameters
and the antioxidant response due to mercury exposure for 14 d
and our future study will focus on the information concerning
the modulation of molecular mechanisms of immune response
due to mercury under a longer-term chronic exposures followed
by the pathogenic challenge to establish the changes in gene
expression and protein synthesis in response to pollutants or
pathogens. Furthermore, the insight gained from the information
generated in this study will benefit future studies and evaluation
of potential functions of antioxidant and immunomodulation
against microbial infection commonly found in crab farming and/
or the polluted environments.
Acknowledgements
We thank Prof. Kejian Wang of the State Key Laboratory of Marine Environmental Science, College of Oceanography and Environmental Science, Xiamen University, Xiamen 361005, China for
critical review of this manuscript. Prof. Giesy was supported by
the Canada Research Chair program, an at large Chair Professorship
at the Department of Biology and Chemistry and State Key Laboratory in Marine Pollution, City University of Hong Kong, The Einstein
Professor Program of the Chinese Academy of Sciences and the Visiting Professor Program of King Saud University.
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