Aquatic Toxicology 148 (2014) 104–112

Contents lists available at ScienceDirect

Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox

Transcriptomic responses to heat stress and nickel in the mussel Mytilus galloprovincialis Banni Mohamed a,b,∗ , Attig Hajer b , Sforzini Susanna a , Oliveri Caterina a , Mignone Flavio a , Boussetta Hamadi b , Viarengo Aldo a a Department of Environmental and Life Sciences, Università del Piemonte Orientale Vercelli Novara Alessandria, Via Michel 11, 15121 Alessandria, Italy b Laboratory of Biochemistry and Environmental Toxicology, ISA, Chott-Mariem, 4042 Sousse, Tunisia

a r t i c l e

i n f o

Article history: Received 21 June 2013 Received in revised form 31 December 2013 Accepted 5 January 2014 Keywords: Heat stress Nickel Transcriptomics Mytilus galloprovincialis

a b s t r a c t The exposure of marine organisms to stressing agents may affect the level and pattern of gene expression. Although many studies have examined the ecological effects of heat stress on mussels, little is known about the physiological mechanisms that maybe affected by co-exposure to heat stress and environmental contaminants such as nickel (Ni). In the present work, we investigated the effects of simultaneous changes in temperature and Ni supply on lysosomal membrane stability (LMS) and malondialdehyde accumulation (MDA) in the digestive gland (DG) of the blue mussel Mytilus galloprovincialis (Lam.). To elucidate how the molecular response to environmental stressors is modulated, we employed a cDNA microarray with 1673 sequences to measure relative transcript abundances in the DG of mussels exposed to Ni along with a temperature increase. A two-way ANOVA revealed that temperature and Ni rendered additive effects on LMS and MDA accumulation, increasing the toxic effects of metal cations. Ni loads in the DG were also affected by co-exposure to 26 ◦ C. In animals exposed only to heat stress, functional genomics analysis of the microarray data (171differentially expressed genes (DEGs)) highlighted seven biological processes, largely dominated by the up-regulation of folding protein-related genes and the down-regulation of genes involved in cell migration and cellular component assembly. Exposure to Ni at 18 ◦ C and 26 ◦ C yielded 188 and 262 DEGs, respectively, exhibiting distinct patterns in terms of biological processes. In particular, the response of mussels exposed to Ni at 26 ◦ C was characterized by the up-regulation of proteolysis, ribosome biogenesis, response to unfolded proteins, and catabolic-related genes, as well as the down-regulation of genes encoding cellular metabolic processes. Our data provide new insights into the transcriptomic response in mussels experiencing temperature increases and Ni exposure; these data should be carefully considered in view of the biological effects of heat stress, particularly in polluted areas. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Average surface marine water temperatures are expected to increase notably in the next decades (Meehl et al., 2007). Our knowledge of the ability of marine biota to cope and probably acclimate to increasing temperatures is very limited, but crucial to understanding how marine organisms in general and those inhabiting coastal zones in particular will respond to global warming. In addition to temperature increases, coastal areas are known to be

∗ Corresponding author at: Higher Institute of Agronomy, Life sciences, BP 47, Zone Touristique, Chott-Mariem 4042, Tunisia. Tel.: +390131360415; fax: +390131360390. E-mail address: m [email protected] (B. Mohamed). 0166-445X/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquatox.2014.01.004

subject to environmental contaminants, rendering the ecotoxicological issue more important. At the cellular and molecular levels, many studies have attempted to clarify the exact roles of genes encoding proteins associated with the stress response in heat-stressed organisms, such as heat-shock proteins and proteins involved in oxidative stress, proteolysis, and translocation (Farcy et al., 2009; Park et al., 2007). However, few investigations have attempted to address the response to increasing temperature by aquatic organisms exposed to toxic chemicals (Kamel et al., 2012; Negri et al., 2013; Silvestre et al., 2010; Verlecar et al., 2007), a scenario that may reflect risks to marine coastal areas. Nickel (Ni) is a metal of high environmental importance that has been shown to exert long-term harmful effects on aquatic biota (Kienle et al., 2009). Ni is often detected in the coastal environment as a result of industrial discharges from electroplating, smelting,

B. Mohamed et al. / Aquatic Toxicology 148 (2014) 104–112

mining and refining operations, and other industrial emissions (Vijayavel et al., 2009). Relative to other divalent metals, knowledge about Ni toxicity on various species and its mode of action is very limited (Attig et al., 2010; Dondero et al., 2011). Mussels, including Mytilus galloprovincialis, are well studied at the molecular, cellular, tissue, and organismal levels. These filterfeeding organisms are usually used in large monitoring programs to evaluate the accumulation of contaminants in their tissues and the consequent effects on biological processes (Banni et al., 2007, 2011; Canesi et al., 2011; Viarengo et al., 2007). The digestive gland of mussels has been used as a model system for studying the transcriptomic response to several environmental stressors and for the analysis of biomarkers and chemical loads in this organism (Banni et al., 2007, 2011; Viarengo et al., 2007; Dondero et al., 2011). Changes in biomarkers spanning the molecular to organismal levels are often assessed to evaluate the physiological status of organisms exposed to environmental stressors (Viarengo et al., 2007). The present investigation had two main objectives. First, we sought to identify the effects of an 8 ◦ C temperature increase and a sublethal Ni concentration on two sensitive biomarkers of the M. galloprovincialis digestive gland: lysosomal membrane stability (LMS) and oxidative stress, as judged by malondialdehyde (MDA) accumulation (Moore, 1976; Viarengo etal., 2007). Second, we used a DNA microarray with ∼1673 genes to analyze the digestive gland transcriptomic response of the mussels to temperature increases and sublethal concentrations of Ni. The microarray data were confirmed by quantitative reverse-transcription PCR (qRT-PCR).The effects of Ni were related to the levels of Ni loads in the digestive gland tissues. 2. Materials and methods 2.1. Animals and treatments Specimens of M. galloprovincialis (Lam.), 5–6 cm shell length, were purchased from an aquaculture mussel farm in Bizerte Lagoon (Bizerte, Tunisia) in October 2011. These specimens were transferred to aquaria at a density of 1 animal/L in clean, aerated seawater collected offshore. Experiments were carried out at two temperatures (18 ◦ C and 26 ◦ C). The mussels were acclimated for 6 days, sufficient for stabilizing mussel physiology at the control temperature of 18 ◦ C (Dondero et al., 2011; Negri et al., 2013). After acclimation, groups of mussels were kept in 20-L polypropylene plastic vessels (four replicates per treatment) and exposed semi-statically to 135 ␮g/L Ni for 4 days. This exposure level is the effective concentration (EC25) for observing a change in digestive gland LMS (Dondero et al., 2011). One set of animals was maintained in seawater with no addition of Ni at the two temperatures. Seawater at the desired temperature was renewed every day, and Ni was added together with a commercial algal preparation (30 mg animal-1 day-1) (Liquifry; Interpret Ltd.). Only female individuals (scored by microscopic inspection of gonad biopsies) were selected for subsequent analysis to avoid gender-based biases in gene expression. After exposure to heat and Ni, digestive glands were rapidly removed, frozen in liquid N2 , and stored at −80 ◦ C for MDA and chemical analysis. A second set of tissues was kept at −20 ◦ C in an RNA-preserving solution (RNA Later; Sigma-Aldrich) for transcriptome analysis, and a third set was mounted on aluminum chucks and frozen in super-cooled N-hexane as previously described (Moore, 1976) for histochemical determination of LMS. 2.2. Chemical analysis Ni levels were determined in the mussel digestive gland (∼0.5 g of a 1:1 homogenate in double-distilled water) by inductively

105

coupled plasma mass spectrometry (VG Plasma Quad 3; VG Elemental). Samples (n = 10) were added to 5 mL of concentrated 65% nitric acid and placed in a microwave oven for mineralization. The samples were then filtered on a nitrocellulose membrane (0.45 ␮m). Procedure validation was performed using Std CRM 145 R reference material containing known amounts of metal. 2.3. MDA determination Digestive gland tissues (0.5–1 g; n = 10) were homogenized in two volumes of buffer containing 20 mM Tris HCl (pH 7.4) and 0.1% mercaptoethanol. The homogenate was centrifuged at 18,000 × g (4 ◦ C) for 20 min. MDA concentration was determined as described by Gérard-Monnier et al. (1998). 2.4. LMS assay LMS was determined based on the period of acid labilization required to produce the maximum staining intensity (Moore, 1976) the labilization period is the time required to fully labilize the responsive fraction of lysosomal hydrolase in the digestive gland cells. LMS (n = 10) was evaluated in cryostat sections (10 ␮m) from five digestive glands. These sections were obtained with a Leica cryostat at −27 ◦ C, as described by Moore (1976). Lysosome staining intensity was determined by studying the slide at 400× magnification with an inverted Axiovert microscope (Zeiss) connected to an Axiocam digital camera (Zeiss). Digital image analysis was carried out using the Scion Image software package (Scion Corp. Inc.) from 8-bit grayscale images. 2.5. Microarray hybridization and analysis Competitive dual-color microarray hybridization was performed with the Mytarray V1.1 platform (Venier et al., 2006); fluorescence-labeled cDNA probes were obtained by direct labeling in the presence of modified Cy3- and Cy5-dCTP (Perkin Elmer). The procedure was carried out as described previously (Dondero et al., 2011) using 0.5 ␮g of an anchored oligodT(19)VN. Total RNA was extracted from female individual digestive gland pieces using acid phenol-chloroform precipitation according to Chomczynski and Sacchi (1987), with TRI-Reagent (Sigma-Aldrich). RNA was further purified by precipitation in the presence of 1.5 M LiCl2, and the quality of each RNA preparation was confirmed by UV spectroscopy and TBE agarose gel electrophoresis in the presence of formamide. Laser scanning of microarrays was performed with an Agilent G2565CA scanner (Agilent Technologies, Inc.) at 5-␮m resolution. Sixteen-bit TIFF images were analyzed with Genepix 6.0 (Axon) to extract raw fluorescence data from each spot. The experimental design accounted for two complete “triangular loops” in which each RNA sample from the tissue of mussels exposed to temperature (18 ◦ C and 26 ◦ C)and/or Ni (135 ␮g/L) was hybridized with RNA from mussels exposed to the physiological temperature (18 ◦ C). Direct comparisons were performed between RNA samples obtained from Ni-exposed and unexposed animals at 18 ◦ C and 26 ◦ C to complete the triangular loop and to cover all conditions (Banni et al., 2011; Dondero et al., 2011). Each experimental condition included at least four biological replicates of RNA samples from individual female animals using the day-swap procedure, for a total of 24 experiments. Computational and statistical analyses of microarray data were performed using the Linear Mode for Microarray Analysis software (Smyth, 2004). Offset background subtraction, loess normalization, and least-squares regression were employed, along with moderated t-tests and empirical Bayes statistics. Gene expression (in the rest of the text, gene expression is used as a synonym of transcription, although it is acknowledged that translation and mRNA and

106

B. Mohamed et al. / Aquatic Toxicology 148 (2014) 104–112

protein stabilities also regulate gene expression) was considered to be significantly different in the test condition versus the reference condition when the log-odd value (B) was higher than 0. The analysis procedure was carried out essentially as described by Dondero et al. (2011). Microarray data were clustered with the Genesis software (D’haeseleer, 2005; Sturn et al., 2002). MIAME-compliant microarray data, including a detailed description of the experimental design and each hybridization experiment, were deposited in the Gene Expression Omnibus under identifier “GSE48062”. The following link provides access to the deposited data http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE48062

0.1 ␮M of each dual-labeled probe (hexachlorofluorescein/BH1 for actin and Texas Red/BH2 for 18S rRNA) and 0.1 ␮M and 0.4 ␮M of forward and reverse primer, respectively, for 18S rRNA and actin (Table S1). For all Taqman assays, the thermal protocol was as follows: 30 s at 95 ◦ C, followed by 40 cycles of 10 s at 95 ◦ C and 20 s at 60 ◦ C. qRT-PCR was performed with four biological replicates and three technical replicates. For the mt-10, mt-20, and calreticulin assays, the thermal protocol was as presented by Dondero et al. (2005). Statistical analyses were carried out on the group mean values using a random reallocation test (Pfaffl et al., 2002). 2.8. Statistical analysis

2.6. Functional genomics analysis

2.7. qRT-PCR qRT-PCR was carried out with the same RNA extract used for microarray hybridization. Relative mRNA abundances of the mussel genes encoding metallothionein (mt10, EMBL ID AJ625847) and calreticulin (AJ624756) were evaluated with SYBR Green I chemistry (EvaGreen® dye; Bio-Rad Laboratories; Banni et al., 2011). The mRNA abundances of the genes encoding chitinase (AJ624093, AJ625569, and AJ624637) and gm2 ganglioside activator protein (AJ624405) were evaluated in multiplex Taqman assays according to Banni et al. (2011). Multiplex Taqman assays according to Negri et al. (2013) were used to assay the genes encoding the folding proteins fk506-binding protein (AJ624969), HSP90 (AJ625915), HSP70 (AJ624049), and HSP27 (AJ625244), the genes encoding the ribosomal proteins ribo-s24 (AJ626091), ribo-s28 (AJ624271), and ribo-L19 (AJ516414), and the genes encoding the proteolysisrelated proteins trypsin-like proteinase (AJ624491) and cathepsin I (AJ624869). Probes and primer pairs (Table S1) were designed using Beacon Designer v3.0 (Premier Biosoft International, Inc.). All primers and dual-labeled Taqman probes were synthesized by MWG-Biotech Gmbh. cDNA (25 ng RNA reverse transcribed to cDNA) was amplified in a CFX384 Real-Time PCR detection system (Bio-Rad Laboratories) with iQTM Multiplex Power mix (Bio-Rad Laboratories) according to the manufacturer’s instructions for the triplex protocol. All multiplex combinations accounted for the following dual fluorescence tags: 6-carboxyfluorescein/Black Hole (BH) 1, 6-carboxy-2 ,4,4 ,5 ,7,7 -hexachlorofluorescein/BH1, and Texas Red/BH2. Briefly, cDNA was amplified in the presence of 1× iQTM Multiplex Power mix, 0.3 ␮M each primer, and 0.1 ␮M each probe (Table S1) in a final volume of 10 ␮L. Relative expression data were geometrically normalized to18S rRNA (L33452),an invariant actin isotype (AJ625116), and ribosomal proteinriboL27(AJ625928) (Negri et al., 2013), which were selected from a list of genes whose expression did not vary over more than 50 conditions (including toxic treatments, stages of the life cycle, and various tissues). A specific duplex Taqman assay was developed to amplify 0.25 ng of RNA reverse transcribed to cDNA in the presence of

The results for Ni accumulation, LMS, and MDA measurements are presented as the mean ± standard deviation (SD) of 10 samples. Statistica version 6.0(Statsoft. Inc. 2002) was used for statistical analysis. The normality of the distribution was tested using the Shapiro–Wilk test. To assess multiple comparisons, a two-way ANOVA analysis of variance was performed on the data, with a student’s post test. 3. Results Ni concentrations were significantly increased in the digestive gland of animals exposed to Ni at 18 ◦ C and at 26 ◦ C compared to the control condition (18 ◦ C without Ni; Fig. 1). The effects of Ni exposure and temperature increase on LMS in the mussel digestive gland are reported in Fig. 2. We detected a pronounced effect of Ni and 26 ◦ C co-exposure on LMS compared to the control condition and to animals exposed to heat stress only (26 ◦ C). Interestingly, Ni loads in the digestive gland significantly differed between animals exposed to 18 ◦ C/Ni and to 26 ◦ C/Ni. MDA accumulation significantly increased in the digestive gland in all experimental conditions versus control conditions, and was very pronounced in animals exposed to Ni at 26 ◦ C (Fig. 3). Transcriptional profiling was performed to identify the main molecular mechanisms involved in the response of mussels to Ni exposure and heat stress. Using a 1673-feature cDNA microarray, SS Source of variation Temperature Ni Interaction Error

2

df

0.704 1 7.579 1 0.757 1 0.44 20

MS

Fs

p

0.704 319.497 7.579 3438.76 0.757 343.238 0.002

0.0001 0.0001 0.0001

* control Ni

1,6

µg/g dry weight

Functional characterization of mussel genes represented on the microarray was based on Gene Ontology (GO) annotation and was carried out with Blast2GO (Conesa et al., 2005) using default parameters. Briefly, 1673 mussel sequences with EMBL IDs were subjected to the annotation analysis; 880 sequences had no BLASTX hits (Altschul et al., 1990), while another 63 sequences did not map to GO terms. Putative annotation for 873 mussel sequences was established based on GO terms for the first 20 BLASTX hits or based on protein domains obtained from InterProScan (Quevillon et al., 2005; Banni et al., 2011). GO term enrichment was evaluated with hypergeometric statistics (p < 0.05); the distribution of GO terms in each set of interest was compared against the set reflecting the entire microarray sequence catalogue.

1,2

* 0,8 0,4 0 18°C

26°C

Fig. 1. Ni accumulation in Mytilus galloprovincialis digestive gland in animals exposed for 4 days to Ni (137 ␮g/L) along with an 8 ◦ C temperature increase gradient (18 ◦ C and 26 ◦ C) Data, expressed in ␮g/g dry weight (n = 10), were analyzed by twoway ANOVA + Student’s post test. * : Statistically significant differences (p < 0.01) in comparison with control condition. Shown are also the Two-way ANOVA results (above).

B. Mohamed et al. / Aquatic Toxicology 148 (2014) 104–112

Table 1 Number of DEGS depicted in animals exposed to heat stress and nickel against 18 ◦ C without Ni supply.

SS df MS Fs p Source of variation Temperature 236.819 1 236.819 31.206 0.0001 490.782 1 490.782 64.670 0.0001 Ni 4.361 1 4.361 0.575 0.457 Interaction 151.780 20 7.589 Error

45

Destabilisation time (mn)

40

*

30

*

25 20 15 10 5 0 18°C

26°c

Fig. 2. Effects of exposure to Ni and temperature on lysosomal membrane stability in Mytilus galloprovincialis digestive gland. Mussels were exposed for 4 days to Ni (137 ␮g/L) along with 8 ◦ C temperature increase (18 ◦ C and 26 ◦ C). Data, expressed as labialization period (Moore, 1976) (n = 10), were analyzed by twoway ANOVA + Student’s post test. * : Statistically significant differences (p < 0.01) in comparison with control condition. Shown are also the Two-way ANOVA results (above).

we generated transcriptome profiles for the female digestive gland exposed to 26 ◦ C, 18 ◦ C/Ni, and 26 ◦ C/Ni for 4 days in comparison to control animals maintained at 18 ◦ C. Microarray analysis revealed distinct patterns for 385 differentially expressed genes (DEGs; differential expression under at least one condition; Fig. 4, Table 1, Tables S2 and S3). Of the 385 DEGs, only 68 genes were shared among the three experimental conditions datasets, as detailed in the Venn diagram in Fig. 4. To further characterize the major patterns of gene expression following exposure to an 8 ◦ C increase in temperature (to 26 ◦ C) for 4 days, we performed GO term enrichment analysis of the 171 DEGs

SS Source of variation Temperature Ni Interaction Error

df

MS

1176 1 1176. 630.785 1 630.785 6.869 1 6.869 84.267 20 4.213

Fs

p

279.14 149.712 1.630

0.0001 0.0001 0.216

50 45

nmole/mg proteins

Condition

26 ◦ C

18 ◦ C/Ni

26 ◦ C/Ni

DEGs Up-regulated Down-regulated

171 92 79

188 95 93

262 129 133

Control Ni

35

40

107

*

control Ni

35 30

*

25 20 15

obtained in this condition (Table 2, Table S3). Our data highlighted the following contributing biological processes: cellular component assembly, chitin metabolic process, cell migration, response to unfolded protein, post-embryonic organ development, oocyte development, and epithelial morphogenesis. Chitin metabolic process and response to unfolded protein were represented by six (the five chitinase variantsAJ623376, AJ624637, AJ625051, AJ516900, AJ625778, AJ625569, and AJ624093, as well as the chitinase b precursor AJ624087) and three (small HSP26, AJ624926; small HSP27,AJ625244; and calreticulin, AJ624756) up-regulated DEGs, respectively. RNA was extracted from the digestive gland of animals exposed to Ni along with the two tested temperatures. Dual-color microarray hybridizations revealed 188 DEGs (49.46% down-regulated) and 262 DEGs (50.76% down-regulated) for animals exposed to 18 ◦ C and 26 ◦ C, respectively, between Ni-exposed animals and controls (Table 1, Table S4). GO analysis of the 188 DEGs in animals exposed to Ni at 18 ◦ C highlighted seven biological processes, mainly characterized by the up-regulation of genes involved in “translation,” “response to unfolded proteins,” and “chitin catabolic process.” Down-regulated patterns were displayed by genes involved in cell development, cellular catabolic process, anatomical structure formation involved in morphogenesis, and response to chemical stimulus (Table 2, Table S4). The transcriptional response of mussels exposed to Ni at 26 ◦ C was different from the response of control animals maintained at 26 ◦ C, as well as from those exposed to Ni and a temperature of 18 ◦ C. The main pattern for mussels exposed to Ni at 26 ◦ C was characterized by the up-regulation of 10/12 genes involved in the GO process of “translation,” 5/5 in “response to unfolded proteins,”8/18 in “regulation of cellular metabolic process,” and 6/11 in “proteolysis.” Down-regulation occurred in11/13 genes associated with “organ development,” 9/11 involved in “positive regulation of biological process,” and 5/6 related to “catabolic process” (Table 2, Table S4). The only consistent pattern was that related to the chitin metabolic process. We also carried out qRT-PCR to confirm and refine the relative expression levels of 14 homologous genes belonging to the most important biological processes, including the genes encoding metallothionein (mt10, EMBL ID AJ625847), calreticulin (AJ624756), fk506-binding protein (AJ624969),HSP90 (AJ625915), HSP27 (AJ625244), HSP70 (AJ624049), gm2 ganglioside activator protein (AJ624405), trypsin-like proteinase (AJ624491), cathepsin l (AJ624869), three chitinase variants (AJ624093, AJ625569, and AJ624637), and the ribosomal proteins ribo-s24 (AJ626091), ribos28 (AJ624271), and ribo-L19 (AJ516414). Microarray and qPCR data showed a positive relationship in all cases (Fig. S1).

10

4. Discussion

5 0 18°C

26°C

Fig. 3. Malondialdehyde (MDA) accumulation in Mytilus galloprovincialis digestive gland in animals exposed for 4 days to 137 ␮g/L Ni along with an 8 ◦ C temperature increase gradient (18 ◦ C and 26 ◦ C). Data, expressed in nmole/mg proteins (n = 10), were analyzed by two-way ANOVA + Student’s post test. * : Statistically significant differences (p < 0.01) in comparison with control condition. Shown are also the Twoway ANOVA results (above).

M. galloprivincialis is among the most economically important species inhabiting tidal zones in southern Mediterranean coasts. An increasing body of evidence indicates that tidal zones are experiencing severe contamination episodes, such as the metal hotspots associated with global climate change (Gracey et al., 2008; Tomanek and Zuzow, 2010). A number of physiological processes studied at the metabolomic, proteomic, and transcriptomic levels

108

B. Mohamed et al. / Aquatic Toxicology 148 (2014) 104–112

Fig. 4. Mytilus galloprovincialis gene expression profiles of digestive gland tissue in animals exposed to 26 ◦ C, 18 ◦ C/Ni and 26 ◦ C/Ni. The heat map (A) (Pearson correlation, complete linkage algorithm) reports the log2 relative expression level with respect to the selected reference condition (18 ◦ C without Ni supply). 385 differentially expressed genes were generated in at least one condition. Microarray data were analyzed using the Linear Mode for Microarray Analysis (LIMMA) software as described in Banni et al. (2011). (B) statistics with adjusted p value, 0.05 and B.0 were used as threshold for rejection of the null hypothesis (no variation). Supporting information to Fig. 4 is present on Tables S2 and S3. Venn diagram representation of gene expression patterns (Panel B) clearly depicted that only 68 DEGs are shared between the three experimental conditions and 101 DEGs between 26 ◦ C and 26 ◦ C plus Ni. All DEGs are obtained respect to the control condition 18 ◦ C without Ni supply. Data used to generate the Venn-diagram were obtained from microarray analysis (Table S3).

have been proposed to underlie the response of mussels to thermal and multi-factorial environmental stressors (Connor and Gracey, 2012; Kamel et al., 2012; Lockwood et al., 2010; Negri et al., 2013; Tomanek and Zuzow, 2010; Gracey et al., 2008). Recent studies indicate that metal accumulation in mollusks is dependent on concentration and time (Negri et al., 2013; Attig et al., 2010; Das and Jana, 1999). In the present study, Ni accumulation in the digestive gland, the most metabolically active organ in mussels, was found to be affected by seawater temperature. Our data showed that bioaccumulation was higher in animals exposed to the physiological temperature (18 ◦ C). The observed reduction of Ni uptake at high temperature could be the results of physiological changes that would counterbalance a higher impact at 26 ◦ C. In this study, LMS analysis revealed a toxic effect following exposure to Ni, an effect that became pronounced when associated with an 8 ◦ C increase in temperature (Fig. 2). LMS is the main lysosomal response to a wide range of environmental stressors (Moore and Viarengo, 1987; Viarengo et al., 2007). The direct relationship between LMS and scope for growth allows LMS to be linked to potential effects at the organismal and population levels, as proposed by Allen and Moore (2004).

Measurements of MDA accumulation, a biomarker indicative of lipid peroxidation, suggested a significant increase in MDA accumulation in mussels exposed to Ni and higher temperature compared to control animals and to animals challenged with a temperature increase alone (Fig. 3). This finding is in accordance with recent reports of marked oxidative stress damage in mussels exposed to metals and increasing temperatures (Negri et al., 2013). Since the ability of mussels to respond physiologically to environmental stressors is supposed to be based in large measure on the capacity for broad-scale, transcriptome-level changes in gene expression (Banni et al., 2011; Dondero et al., 2011; Gracey et al., 2008; Negri et al., 2013), and in view of the negative effect of Ni exposure in combination with a temperature increase on LMS and MDA accumulation, we sought to examine how the transcriptome of M. galloprovincialis was affected by an acute increase in temperature and exposure to sublethal Ni levels. However, it is important to note that even if transcriptomic data may provide an overview of the cell response to stress, changes in the protein genes products or consequent biochemical and physiological modifications are also required in describing the biological effect of stressors on living organisms.

B. Mohamed et al. / Aquatic Toxicology 148 (2014) 104–112

109

Table 2 GO term over-representation analysis of DEGs in the digestive gland tissue of mussels exposed to nickel and heat stress. Condition

Go term

26 ◦ C

Cellular component assembly

8

0

Chitin metabolic process

6

6

Cell migration Response to unfolded protein Post-embryonic organ development Oocyte development Morphogenesis of an epithelium Response to chemical stimulus

5 3 3 3 3 8

5 3 0 0 0 3

12

10

Cell development

8

1

Cellular catabolic process

7

1

Chitin metabolic process

6

6

Anatomical structure formation involved in morphogenesis Response to unfolded protein Regulation of cellular metabolic process

5

0

4 18

4 8

Proteolysis

12

7

Organ development

13

2

Chitin metabolic process

10

7

Ribosome biogenesis

12

10

6

1

5 11

5 2

18 ◦ C/Ni

Ribosome biogenesis

26 ◦ C/Ni

Catabolic process Response to unfolded protein Positive regulation of biological process

N

(Up)

Gene ID AJ624597, AJ516735, AJ625595, AJ516886, AJ626032, AJ623967, AJ625866, AJ625862 AJ625051, AJ624637, AJ624093, AJ625778, AJ624087, AJ625569 AJ624597, AJ516735, AJ626032, AJ625862, AJ624502 AJ624756, AJ625244, AJ624926 AJ516735, AJ516886, AJ624502 AJ624597, AJ625236, AJ626032 AJ624597, AJ516735, AJ516886 AJ625244, AJ624592, AJ625847, AJ625915, AJ624383, AJ623967, AJ625116, AJ624502 AJ626091, AJ626403, AJ623956, AJ625370, AJ624271, AJ623890, AJ516414, AJ516444, AJ624503, AJ624921, AJ624785, AJ624109 AJ516838, AJ624597, AJ626032, AJ626097, AJ624383, AJ623967, AJ625116, AJ624502 AJ624056, AJ624597, AJ624495, AJ625525, AJ624916, AJ624405, AJ623552 AJ625051, AJ624637, AJ624093, AJ625778, AJ624087, AJ625569 AJ624597, AJ624383, AJ623967, AJ625116, AJ624502 AJ624756, AJ625915, AJ624969, AJ625244 AJ624260, AJ625521, AJ625915, AJ625912, AJ623779, AJ516537, AJ624597, AJ625244, AJ623737, AJ623890, AJ624756, AJ516444, AJ516838, AJ625370, AJ626032, AJ624130, AJ625132, AJ624383 AJ625525, AJ625475, AJ516473, AJ624363, AJ625769, AJ624898, AJ624491, AJ624522, AJ623459, AJ624869, AJ625490, AJ624465 AJ625488, AJ516735, AJ516886, AJ624383, AJ516473, AJ626467, AJ625236, AJ625425, AJ623925, AJ625960, AJ624499, AJ624502, AJ625490 AJ623428, AJ625276, AJ623376, AJ624637, AJ625051, AJ516900, AJ625778, AJ625569, AJ624093, AJ624087 AJ626091, AJ626403, AJ623956, AJ625370, AJ624271, AJ623890, AJ516414, AJ516444, AJ624503, AJ624921, AJ624785, AJ624109 AJ624260, AJ625525, AJ624597, AJ623552, AJ624405, AJ516599 AJ624926, AJ624756, AJ625915, AJ624969, AJ625244 AJ625488, AJ625915, AJ516895, AJ516886, AJ624383, AJ623737, AJ626032, AJ624499, AJ626403, AJ516600, AJ625490

Gene ontology terms enrichment analysis was carried out comparing the GO term frequency distribution into each condition against that in the whole microarray set (hypergeometric statistics, p, 0.05). Only the lowest node per branch of the hierarchical structure of the Gene Ontology that fulfils the filter condition-cut off three sequenceswas reported. Showed are: experimental condition; Level, level in the GO tee of biological processes; GO Term, over-represented feature; N, number of mussel sequences associated to each GO term; up, Number of up-regulated genes; Gene ID, EMBL accession number of each sequence found. The over-represented GO terms in heat stresses animals versus 18 ◦ C without Ni supply (hypergeometric statistics, p, 0.05).

In this study, our microarray data identified 92 up-regulated DEGs out of a total of 171DEGs at 26 ◦ C; our GO analyses revealed distinct biological processes probably involved in the adaptation of M. galloprovincialis to higher temperatures. The response of mussels exposed to 26 ◦ C was mainly characterized by the up-regulation of protein folding genes (three genes) and the down-regulation of cellular component assembly-related genes (eight genes). Among the genes represented on our microarray and involved with protein folding, genes encoding calreticulin and the small heat shock proteins HSP27 and HSP26 seem to play an important role in the response to heat stress (increase of 8 ◦ C). Calreticulin is a calciumbinding protein that regulates Ca2+ homeostasis and acts as a misfolding chaperone for protein quality control in the endoplasmic reticulum, preventing the export of misfolded proteins to the Golgi apparatus (Rizvi et al., 2004). Up-regulation of the genes encoding calreticulin, HSP26, and HSP27 was recently reported to be highly involved in the mechanism of resistance to acute heat stress (Negri et al., 2013; Lockwood et al., 2010). Our data provide evidence of the marked down-regulation of eight genes involved in the cellular component assembly

process in mussels exposed to 26 ◦ C. Among these genes, six are involved in the microtubule-based movements sub-process (genes encoding two alpha 2 isoforms, AJ625595 and AJ625866; integrin beta 4, AJ516735; thymosin B actin, AJ625862; dynein light chain 2, AJ516886;beta-actin, AJ623967), and two transcripts are associated with translation (genes encoding heterogeneous nuclear ribonucleoprotein, AJ624597; RNA binding homolog 2, AJ626032). Cytoskeletal protection was previously proposed as a potential mechanism of thermotolerance in mussels (Tomanek, 2010); dynein light chain 2, integrin beta 4, thymosin B actin, and beta-actin interfere with microtubule integrity (Izidoro-Toledo et al., 2013; Dong et al., 2005; Wu et al., 1995). However, and in contrast to our data, Tomanek and Zuzow (2010) reported a crucial up-regulation of genes involved in cytoskeletal protection, such as those encoding actin and tubulin, in heat-stressed mussels. This difference may be explained by dissimilar experimental conditions (exposure period and temperature). The toxic effects of Ni on numerous aquatic organisms have attracted interest during the last decade (Attig et al., 2010; Banni et al., 2011; Dondero et al., 2011; Vijayavel et al., 2009). To our

110

B. Mohamed et al. / Aquatic Toxicology 148 (2014) 104–112

Fig. 5. The k-means algorithm was used for the computation of gene expression trends of 29 unique genes (those involved in ribosome biogenesis, protein folding and proteolysis) whose expression was modulated in at least one condition. k-Means is an iterative procedure aimed to reduce the variance to a minimum within each cluster (Sturn et al., 2002). Shown are also the log2 relative expression level of the 29 DEGs in the three experimental conditions (26 ◦ C, 18 ◦ C/Ni and 26 ◦ C/Ni) with respect to the selected reference condition (18 ◦ C without Ni supply).

knowledge, this is the first report of the transcriptional changes in mussels exposed to Ni along with a temperature increase. Our data indicate a differential modulation of mRNA abundance in mussels co-exposed to Ni at 18 ◦ C and at 26 ◦ C. Ni exposure at 18 ◦ C resulted in 188 DEGs; GO analysis revealed seven biological processes mainly characterized by the up-regulation of genes involved in “ribosome biogenesis” (10/12 genes) and “response to unfolded proteins” (4/4 genes). Among genes represented on the array and encoding proteins involved in the ribosome biogenesis process, 10 ribosomal protein subunit-related genes were up-regulated (Fig. 5, Table S5). Compared to the effect of Ni at 18 ◦ C, the mussels’ response to exposure to Ni and 26 ◦ C was mainly characterized by a clear increase in the up-regulation of genes involved in “ribosome biogenesis” and “response to unfolded proteins,” and the involvement of “proteolysis” as a new process. Furthermore, exposure to 26 ◦ C and Ni was marked by the down-regulation of genes involved in “regulation of cellular metabolic process,” “organ development,” “catabolic process,” and “positive regulation of biological process” (Table 2). The involvement of ribosomal transcripts in the response to Ni exposure is possibly due to an increase in the ribosome synthesis rate to support cellular activities such as protein translation, transcriptional activation, and mRNA stabilization during exposure to sublethal Ni levels. Similar data were recently reported by

Negri et al. (2013) in mussels exposed to copper along a heat-stress gradient. This transcriptional up-regulation was found to be very important in the presence of the highest temperature. Our data may suggest that mussels adopt a compensatory strategy in response to the effects of exposure to sublethal Ni levels; as also observed in the response to copper (Negri et al., 2013), this strategy is mainly characterized by the up-regulation of genes contributing to protein synthesis. During acute heat stress, protein homeostasis depends in part on the degradation of irreversibly denatured proteins, and thus on proteolysis (Tomanek and Zuzow, 2010). Here, proteolysis-related genes were up-regulated in mussels exposed to Ni at 18 ◦ C, and the abundance of these transcripts was increased in mussels exposed to Ni at 26 ◦ C (Table 2, Fig. 5, Table S5). The involvement of proteolysis indicated by our data is consistent with previous reports of higher levels of protein ubiquitination, a signal of proteolysis in M. galloprovincialis under heat stress conditions (Tomanek and Zuzow, 2010; Lockwood et al., 2010). However, this study is the first report of the involvement of transcription of proteolysis-related genes in the response to Ni exposure at physiological temperature, probably due to a protein-altering effect of Ni. The LMS measurements from the present investigation (Fig. 2) are consistent with the increase in catabolic processes suggested by the transcriptomic analysis.

B. Mohamed et al. / Aquatic Toxicology 148 (2014) 104–112

Interestingly, the increase in the expression of proteolysisrelated genes in mussels exposed to Ni at 18 ◦ C and 26 ◦ C was associated with similar trends in the expression of five genes encoding chaperones (Fig. 5). However, the expression data for the genes encoding misfolding protein calreticulin and the unfolding protein small HSP27 clearly indicated a negative effect of Ni exposure on these genes versus the effect of elevated temperature alone (26 ◦ C; Fig. 5). Lockwood et al. (2010) suggested that compared to other mussel species, the response of M. galloprovincialis to acute heat stress is primarily distinguished by higher induction of genes encoding small molecular chaperones. These genes have been shown to be important elements of the stress response and stress resistance in other organisms, and therefore could be key factors that increase thermo tolerance in M. galloprovincialis (Negri et al., 2013; Tomanek and Zuzow, 2010). The observed relative decrease in the gene expression of calreticulin and HSP27 in mussels exposed to Ni at the two tested temperatures maybe due to Ni toxicity. Several studies have characterized the oxidative stress responses of Mytilus congeners to heat stress and Ni exposure (Kamel et al., 2012; Lockwood et al., 2010; Attig et al., 2010), revealing alterations in crucial cellular components. Consistent with these observations, the MDA accumulation observed in this work (Fig. 3) suggests the occurrence of marked oxidative stress damage in mussels exposed to Ni and 26 ◦ C. However, our transcriptional data did not reveal differential expression in any oxidative stressrelated genes. This observation maybe due to the microarray used in this study, which only contained 1673 sequences, increasing the probability of failing to detect transcriptional changes involved in the oxidative-stress response. Specifically, our microarray lacked probes for the genes encoding superoxide dismutase, catalase, and glutathione S-transferase, which are typically involved in the response to oxidative stress. Mussels exposed to heat stress alone or in combination with Ni consistently up-regulated genes involved in the chitin metabolic process. The expression pattern of genes encoding three chitinase variants (AJ624093, AJ625569, and AJ624637) was confirmed via qRT-PCR (Fig. S1). In chitin-containing organisms, chitinases are involved in many biological processes such as morphogenesis, cell division, and immunity (Banni et al., 2011; Kuranda and Robbins, 1991; Merzendorfer and Zimoch, 2003). Furthermore, chitinases play a role in digestion and in the control of growth and remodeling processes in mussels; the activities of the chitinases have been reported to be highly correlated with food availability. In this study, sustained up-regulation of these genes in Ni-and/or heat-stressed mussels should not be considered a response to stressors, but is more likely associated with the normal rhythm of the expression of some genes that depend on the time of the year, as previously reported by Gracey et al. (2008), Banni et al. (2011), and Connor and Gracey (2012).

5. Conclusions In summary, our study has provided insight into how an increase of 8 ◦ C associated with sublethal Ni exposure may affect the mussel’s transcriptomic and physiological responses. Temperature and Ni exerted additive effects on LMS and MDA accumulation, exacerbating the toxic effects of metal cations present at sublethal concentrations. Our transcriptomic data confirmed the crucial contribution of heat shock proteins in the response to heat stress and, for the first time, highlight the important involvement of biological processes such as ribosome biogenesis and proteolysis in the response to Ni exposure at physiological and stressing temperatures.

111

Acknowledgments This work was supported by funds from Theme 6 of the EC Seventh Framework Program through the Marine Ecosystem Evolution in a Changing Environment Collaborative Project (Grant no. MEECE 212085) and by funds from the Ministry of Scientific Research and Technology, Tunisia (Unité de Recherche en Biochimie et Toxicologie Environnementale), ISA Chott-Mariem.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aquatox. 2014.01.004.

References Allen, J.I., Moore, M.N., 2004. Environmental prognostics: is the current use of biomarkers appropriate for environmental risk evaluation? Marine Environmental Research 58 (25), 227–232. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. Journal of Molecular Biology 215, 403–410. Attig, H., Dagnino, A., Negri, A., Jebali, J., Boussetta, H., Viarengo, A., Dondero, F., Banni, M., 2010. Uptake and biochemical responses of mussels Mytilus galloprovincialis exposed to sublethal nickel concentrations. Ecotoxicology and Environmental Safety 73 (7), 1712–1719. Banni, M., Dondero, F., Jebali, J., Guerbej, H., Boussetta, H., Viarengo, A., 2007. Assessment of heavy metal contamination using real-time PCR analysis of mussel metallothionein mt10 and mt20 expression: a validation along the Tunisian coast. Biomarkers 12 (4), 369–383. Banni, M., Negri, A., Mignone, F., Boussetta, H., Viarengo, A., Dondero, F., 2011. Gene expression rhythms in the mussel Mytilus galloprovincialis (Lam.) across an annual cycle. PLoS ONE 6 (5), e18904. Canesi, L., Negri, A., Barmo, C., Banni, M., Gallo, G., Viarengo, A., Dondero, F., 2011. The organophosphate chlorpyrifos interferes with the responses to 17b-estradiol in the digestive gland of the marine mussel Mytilus galloprovinciali. PLos One 6 (5), e19803. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Analytical Biochemistry 162, 156–169. Conesa, A., Gotz, S., Garcıa-Gomez, J.M., Terol, J., Talon, M., Robles, M., 2005. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21, 3674–3686. Connor, K.M., Gracey, A.Y., 2012. High-resolution analysis of metabolic cycles in the intertidal mussel Mytilus californianus. American Journal of Physiology—Regulatory, Integrative and Comparative Physiology 302 (1), R103–R111. D’haeseleer, P., 2005. How does gene expression clustering work? Nature Biotechnology 23, 1499–1501. Das, S., Jana, B.B., 1999. Dose-dependent uptake and Eichhornia-induced elimination of cadmium in various organs of the freshwater mussel, Lamellidens marginalis (Linn.). Ecological Engineering 12, 207–229. Dondero, F., Piacentini, L., Banni, M., Rebelo, M., Burlando, B., Viarengo, A., 2005. Quantitative PCR analysis of two molluscan metallothionein genes unveils differential expression and regulation. Gene 345, 259–270. Dondero, F., Banni, M., Negri, A., Boatti, L., Dagnino, A., Viarengo, A., 2011. Interactions of a pesticide/heavy metal mixture in marine bivalves: a transcriptomic assessment. BMC Genomics 12, 195. Dong, J.H., Ying, G.X., Liu, X., Wang, W.Y., Wang, Y., Ni, Z.M., Zhou, C.F., 2005. Expression de thymosin beta4 ARNm par les microglies activées dans l’hippocampe dénervé. Neuroreport 16 (15), 1629–1633. Farcy, E., Voiseux, C., Lebel, J.M., Fiévet, B., 2009. Transcriptional expression levels of cell stress marker genes in the Pacific oyster Crassostrea gigas exposed to acute thermal stress. Cell Stress and Chaperones 14, 371–380. Gérard-Monnier, D., Erdelmeier, I., Régnard, K., Moze-Henry, N., Yadan, J.C., Chaudière, J., 1998. Reactions of 1-methyl-2-phenylindole with malondialdehyde and 4-hydroxyalkenals. Analytical applications to a colorimetric assay of lipid peroxidation. Chemical Research in Toxicology 11 (10), 1176–1183. Gracey, A.Y., Chaney, M.L., Boomhower, J.P., Tyburczy, W.R., Connor, K., Somero, G.N., 2008. Rhythms of gene expression in a fluctuating intertidal environment. Current Biology 18, 1501–1507. Izidoro-Toledo, T.C., Borges, A.C., Araújo, D.D., Mazzi, D.P., Nascimento Jr., F.O., Sousa, J.F., Alves, C.P., Paiva, A.P., Trindade, D.M., Patussi, E.V., Peixoto, P.M., Kinnally, K.W., Espreafico, E.M., 2013. A myosin-Va tail fragment sequesters dynein light chains leading to apoptosis in melanoma cells. Cell Death and Disease 21, 4–547. Kamel, N., Attig, H., Dagnino, A., Boussetta, H., Banni, M., 2012. Increased temperatures affect oxidative stress markers and detoxification response to benzo[a]pyrene exposure in mussel Mytilus galloprovincialis. Archives of Environmental Contamination and Toxicology 63 (4), 534–543.

112

B. Mohamed et al. / Aquatic Toxicology 148 (2014) 104–112

Kienle, C., Kohler, H.R., Gerhardt, A., 2009. Behavioural and developmental toxicity of chlorpyrifos and nickel chloride to zebra fish (Danio rerio) embryos and larvae. Ecotoxicology and Environmental Safety 72, 1740–1747. Kuranda, M.J., Robbins, P.W., 1991. Chitinase is required for cell separation during growth of Saccharomyces cerevisiae. Journal of Biological Chemistry 266 (29), 19758–19767. Lockwood, B.L., Sanders, J.G., Somero, G.N., 2010. Transcriptomic responses to heat stress in invasive and native blue mussels (genus Mytilus): molecular correlates of invasive success. Journal of Experimental Biology 213, 3548–3558. Meehl, G.A., Stocker, T.F., Collins, W.D., Friedlingstein, P., Gaye, A.T., Gregory, J.M., Kitoh, A., Knutti, R., Murphy, J.M., Noda, A., Raper, S.C.B., Watterson, I.G., Weaver, A.J., Zhao, Z.C., 2007. Global climate projections. In: Solomon, S., Qin, D., Manning, M. (Eds.), Climate Change 2007: The Physical Science Basis. Cambridge University Press, Cambridge, pp. 686–688. Merzendorfer, H., Zimoch, L., 2003. Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases. Journal of Experimental Biology 206, 4393–4412. Moore, M.N., 1976. Cytochemical demonstration of latency of lysosomal hydrolases in digestive gland cells of the common mussel Mytilus edulis, and changes induced by thermal stress. Cell Tissue Research 175, 279–287. Moore, M.N., Viarengo, A., 1987. Lysosomal membrane fragility and catabolism of cytosolic proteins: evidence for a direct relationship. Experientia 43 (3), 320–323. Negri, A., Oliveri, C., Sforzini, S., Mignione, F., Viarengo, A., Banni, M., 2013. Transcriptional response of the mussel Mytilus galloprovincialis (Lam.) following exposure to heat stress and copper. Plos One, http://dx.doi.org/10.1371/ journal.pone.0066802. Park, H., Ahn, I.Y., Lee, H.E., 2007. Expression of heat shock protein 70 in the thermally stressed antarctic clam Laternula elliptica. Cell Stress and Chaperones 12 (3), 275–282. Pfaffl, M.W., Horgan, G.W., Dempfle, L., 2002. Relative expression software tool (REST) for groupwise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Research 30, e36. Quevillon, E., Silventoinen, V., Pillai, S., Harte, N., Mulder, N., Apweiler, R., Lopez, R., 2005. InterProScan: protein domains identifier. Nucleic Acids Research 33, W116–W120.

Rizvi, S.M., Mancino, L., Thammavongsa, V., Cantley, R.L., Raghavan, M., 2004. A polypeptide binding conformation of calreticulin is induced by heat shock, calcium depletion, or by deletion of the C-terminal acidic region. Molecular Cell 15 (6), 913–923. Silvestre, F., Linares-Casenave, J., Doroshov, S.I., Kültz, D., 2010. A proteomic analysis of green and white sturgeon larvae exposed to heat stress and selenium. Science of the Total Environment 408 (16), 3176–3188. Smyth, G.K., 2004. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Statistical Application on Genetics and Molecular Biology 3, Article 3. Sturn, A., Quackenbush, J., Trajanoski, Z., 2002. Genesis: cluster analysis of microarray data. Bioinformatics 18, 207–218. Tomanek, L., 2010. Variation in the heat shock response and its implication for predicting the effect of global climate change on species’ bioegographic distribution ranges and metabolic costs. Journal of Experimental Biology 213, 971–979. Tomanek, L., Zuzow, M.J., 2010. The proteomic response of the mussel congeners Mytilus galloprovincialis and M. trossulus to acute heat stress: implications for thermal tolerance limits and metabolic costs of thermal stress. Journal of Experimental Biology 213 (Pt 20), 3559–3574. Venier, P., De Pitta‘, C., Pallavicini, A., Marsano, F., Varotto, L., Romualdi, C., Dondero, F., Viarengo, A., Lanfranchi, G., 2006. Development of mussel mRNA profiling: can gene expression trends reveal coastal water pollution? Mutation Research 602, 121–134. Verlecar, X.N., Jena, K.B., Chainy, G.B., 2007. Biochemical markers of oxidative stress in Perna viridis exposed to mercury and temperature. Chemico-Biological Interactions 167 (3), 219–226. Viarengo, A., Lowe, D., Bolognesi, C., Fabbri, E., Koehler, A., 2007. The use of biomarkers in biomonitoring: a 2-tier approach assessing the level of pollutant-induced stress syndrome in sentinel organisms. Comparative Biochemistry and Physiology C: Toxicology & Pharmacology 146 (3), 281–300. Vijayavel, K., Gopalakrishnan, S., Thiagarajan, B., Thilagam, H., 2009. Immunotoxic effects of nickel in the mud crab Scylla serrata. Fish and Shellfish Immunology 26, 133–139. Wu, C., Fields, A.J., Kapteijn, B.A.E., McDonald, J.A., 1995. The role of a4b1 integrin in cell motility and fibronectin matrix assembly. Journal of Cell Science 108, 821–829.