Effects of cadmium exposure on the gill proteome of Cottus gobio: Modulatory effects of prior thermal acclimation

Accepted Manuscript Title: Effects of cadmium exposure on the gill proteome of Cottus gobio: Modulatory effects of prior thermal acclimation Author: Jennifer Dorts Patrick Kestemont Marie-Laetitia Th´ezenas Martine Raes Fr´ed´eric Silvestre PII: DOI: Reference:

S0166-445X(14)00159-3 http://dx.doi.org/doi:10.1016/j.aquatox.2014.04.030 AQTOX 3837

To appear in:

Aquatic Toxicology

Received date: Revised date: Accepted date:

6-3-2014 10-4-2014 29-4-2014

Please cite this article as: Dorts, J., Kestemont, P., Th´ezenas, M.-L., Raes, M., Silvestre, F.,Effects of cadmium exposure on the gill proteome of Cottus gobio: modulatory effects of prior thermal acclimation, Aquatic Toxicology (2014), http://dx.doi.org/10.1016/j.aquatox.2014.04.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Effects of cadmium exposure on the gill proteome of Cottus gobio:

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effects of prior thermal acclimation Jennifer Dortsa*, Patrick Kestemonta, Marie-Laetitia Thézenasb, Martine Raesb and Frédéric

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Silvestrea Research Unit in Environmental and Evolutionary Biology (URBE), University of Namur,

Rue de Bruxelles 61, B-5000, Namur, Belgium.

Research Unit in Cell Biology (URBC) (NARILIS), University of Namur, Rue de Bruxelles

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61, B-5000, Namur, Belgium.

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* Corresponding author: Jennifer Dorts, Research Unit in Environmental and Evolutionary Biology (URBE), University of Namur, Rue de Bruxelles 61, B-5000, Namur, Belgium. [email protected]

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Abstract Temperature and trace metals are common environmental stressors, and their importance is increasing due to global climate change and anthropogenic pollution. The aim of the present study was to investigate whether acclimation to elevated temperature affects the response of

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the European bullhead (Cottus gobio) to subsequent cadmium (Cd) exposure by using enzymatic and proteomic approaches. Fish acclimated to 15 (standard temperature), 18 or 21 °C for 28 days were exposed to 1 mg Cd/L for 4 days at the respective acclimation

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temperature. First, exposure to Cd significantly decreased the activity of the lactate dehydrogenase (LDH) in gills of fish acclimated to 15 °C or 18 °C. However, an acclimation

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to 21 °C suppressed the inhibitory effect of Cd. Second, using a proteomic analysis by 2DDIGE, we observed that thermal acclimation was the first parameter affecting the protein expression profile in gills of C. gobio, while subsequent Cd exposure seemed to attenuate

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this temperature effect. Moreover, our results showed opposite effects of these two environmental stressors at protein expression level. From the 52 protein spots displaying

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significant interaction effects of temperature and Cd exposure, a total of 28 different proteins were identified using nano LC-MS/MS and the Peptide and Protein Prophet algorithms of Scaffold software. The identified differentially expressed proteins can be categorized into

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diverse functional classes, related to protein turnover, folding and chaperoning, metabolic process, ion transport, cell signaling and cytoskeleton. Within a same functional class, we

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further reported that several proteins displayed reverse responses following sequential exposure to heat and Cd. This work provides insights into the molecular pathways potentially

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involved in heat acclimation process and the interactive effects of temperature and Cd stress in ectothermic vertebrates.

Keywords: sequential stress; proteomics; metabolic enzymes; cadmium; heat acclimation; gills

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1. Introduction The current increases in average temperatures and fluctuations in temperature extremes due to global climate change are considered as one of the new and important threats to aquatic ecosystems (Daufresne et al., 2009). Fuller et al. (2010) recognized four possible outcomes

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for a species under the influence of climate changes. Species may (1) become extinct or extirpated, (2) migrate or shift their current distribution range, (3) adapt to the changes through a change in the genetic composition of the population, or (4) employ phenotypic

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plasticity. Although one possible way of coping with climate change is migration to suitable new habitats (Parmesan and Yohe, 2003), this task is challenging or even impossible for

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some species (Fuller et al., 2010). Genetic change and phenotypic plasticity are thus the outcomes that prevent local extinction. Phenotypic plasticity (i.e., the ability of one genotype to adopt different phenotypes) is, however, likely to represent the first response of individual

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organisms under climate change (Bradshaw and Holzapfel, 2008; Somero, 2010). Such phenotypic plasticity can lead to heat acclimation which is defined as a “within lifetime”

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phenotypic adaptation involving a suite of physiological and biochemical adjustments that enhance thermotolerance and heat endurance (Horowitz, 2007). Furthermore many aquatic species are also subjected to polluted environments and

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increased temperatures may pose additional threats to survival of ectotherms by modulating their susceptibility to other stressors, such as metal contamination. Previous studies have

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shown that elevated temperatures tend to enhance toxic effects of metals on aquatic organisms that may be partially explained by the higher uptake rates of metals and a higher

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intrinsic sensitivity of organisms (reviewed in Cairns et al., 1975; Heugens et al., 2001; Sokolova and Lannig, 2008). According to Sokolova and Lannig (2008), interference with aerobic metabolism, including energy demand, oxygen supply, and mitochondrial function, forms a physiological basis for interactions between environmental temperature and metal pollution. Within this interaction context it is important to differentiate between combined exposure to temperature and metal stress, and sequential combination of these stressors (Holmstrup et al., 2010). The latter scenario may be more ecologically relevant and attenuates temperature dependence, possibly due to common defense mechanisms against heat and metal stress (Vergauwen et al., 2013). Tedengren et al. (2000), for instance, have found that pretreatment to elevated temperature increased heat shock protein (HSP70) levels in mussels Mytilus edulis and conferred greater resistance to subsequent cadmium (Cd) exposure. More recently, Vergauwen et al. (2013) have reported that heat-acclimated zebrafish Danio rerio were more able to tolerate a subsequent Cd stress. Proteomic analysis, the study of the protein complement of the cell, is increasingly used in aquatic toxicology as a powerful tool to examine potentially unforeseen responses to 3   

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environmental stress (Sanchez et al., 2011a). It is generally admitted that a detailed understanding of the formation of a phenotype requires the study of all the steps during gene regulation and their final products at the proteome level (Feder and Walser, 2005; Karr, 2008). Further, proteomics is close to physiology, gives a functional knowledge of gene expression and is often reported as an important part of the cellular phenotype (Silvestre et

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al., 2012). Currently, changes of protein expression in aquatic organisms exposed to multiple stressors simultaneously or sequentially have received little attention. Using this approach, Silvestre et al. (2010) investigated the combined effects of heat stress and micro-injected

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selenium on sturgeon larvae (Acipenser medirostris and A. transmontanus) until stage D45 (after 8 – 12 days of exposure). The authors observed that proteins involved in correct were predominantly affected by heat and/or selenium.

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protein folding, protein synthesis, protein degradation, ATP supply and structural proteins The aim of the present study was to investigate whether physiological acclimation to elevated

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temperature, in the range predicted by scientists, may modulate the response of ectotherms to subsequent environmental chemical insults. To address this question, the European

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bullhead (Cottus gobio) was chosen as an ecologically relevant organism. Bullhead is a small bottom-dwelling freshwater fish considered endangered in several parts of its distribution area as a result of pollution and habitat destruction and is known for its sensitivity to

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temperature changes (Utzinger et al. 1998; Dorts et al. 2012a). Fish were acclimated to 15 (standard temperature), 18 or 21 °C for 28 days, and then exposed to Cd at these

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temperatures for 4 days. The interactive effects of temperature and Cd exposure were investigated on gill tissue by monitoring the response of two enzymes involved in energy

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metabolism (citrate synthase CS and lactate dehydrogenase LDH). Furthermore, a proteomic approach using the two-dimensional differential in-gel electrophoresis (2D-DIGE) technique (Unlu et al., 1997) was undertaken to reveal molecular responses induced by heat acclimation that could modify the subsequent response to Cd exposure. 2. Material and methods

2.1. Fish capture and maintenance Investigations and animal care were conducted according to the guidelines for the use and care of laboratory animals and in compliance with Belgian and European regulations on animal welfare. Adult European bullhead of both genders weighing 11.3 ± 2.9 g were caught by electrofishing in the Samson River (Belgium) in May 2009. Fish were acclimatized to laboratory conditions in dechlorinated tap water at 15.3 ± 1.4 °C under a 14:10 h (light/dark) photoperiod for four weeks before the experiment. During the acclimatization period, fish were fed daily to apparent satiation with frozen chironomid (Chironomus sp.) larvae. 2.2. Experimental setup

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A total of 108 fish were randomly distributed into 9 tanks and acclimated to three temperatures: 15.1 ± 0.5 °C, 18.1 ± 0.2 °C, and 21.1 ± 0.4 °C for 28 days. There were three replicate tanks for each temperature. The temperature was increased by 3 °C per day from 15 to 18 or 21 °C. After heat acclimation, fish were distributed into 18 tanks filled with 12 L dechlorinated tap water and exposed to CdCl2 (Sigma C2544) at nominal concentrations of 0

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and 1 mg/L during 4 days at each of the three temperature (15, 18 or 21 °C). The treatments were as follows: (A) 15 °C Æ 0 mg Cd/L, (B) 15 °C Æ 1 mg Cd/L, (C) 18 °C Æ 0 mg Cd/L, (D) 18 °C Æ 1 mg Cd/L, (E) 21 °C Æ 0 mg Cd/L and (F) 21 °C Æ 1 mg Cd/L. The tested

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treatments were chosen according to our previous studies conducted with C. gobio (Dorts et al., 2012a, 2012b, 2011a). Each treatment included three replicate tanks, with 6 fish per tank.

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After 4 days of exposure, all fish were individually weighed, and gills were collected on ice, directly snap-frozen in liquid nitrogen and stored at -80 °C until homogenization. During the contamination stage, half-water was gently siphoned out and replaced every day. Animals

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were not fed during contamination while they were fed as described above during heat acclimation. No mortality was observed over the course of the experiment.

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Total Cd concentrations in the exposure water were monitored every other day using a Sector Field Inductively Coupled Plasma Mass Spectrometer (Thermo Finnigan Element 2). Actual Cd concentrations in water samples were stable over the course of the experiment; mg/L.

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the mean concentration and standard deviation in the 1 mg/L treatment were 0.89 ± 0.07

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2.3. Metabolic enzyme activities

Enzymatic activities were assessed in gills from 3 pooled fish per replicate tank. One unit of

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gill tissue was homogenized with 15 units of ice-cold phosphate buffer (100 mM, pH 7.4) containing Complete-MiniTM Protease inhibitor cocktail (Roche). The homogenates were centrifuged at 1 000 x g for 10 min at 4 °C, and the supernatants were kept at -80 °C for enzyme activity assays. Protein concentrations were measured according to Bradford (1976) using bovine serum albumin as a standard. The experimental conditions for the enzymatic activities assays were as described by Dorts et al. (2011a): Citrate synthase (CS): 100 mM Tris/HCl, 0.1 mM DTNB, 0.3 mM acetyl CoA, 0.5 mM oxaloacetate, pH 8.1. Lactate dehydrogenase (LDH): 100 mM Tris/HCl, 0.3 mM NADH, 10 mM pyruvate, pH 7.4. Reactions were assayed spectrophotometrically following the reduction of DTNB for CS (at 412 nm) and the oxidation of NADH for LDH (340 nm). Millimolar extinction coefficients used were 13.6 for DTNB and 6.22 for NADH. Enzymatic activities were performed in duplicate. They are expressed in milliunit per mg protein. One unit corresponds to the amount of the enzyme that will convert 1 µmol of substrate into product per minute.

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Results for the enzymatic activities were expressed as the mean (n = 3) ± S.D. All data were logarithm transformed to stabilize the variance and to approximate normal distribution. Differences between groups were analyzed using two-way analysis of variance (ANOVA 2) followed by a multiple comparison Fisher LSD test at a 5 % significant level. All tests were performed using the Statistica 5.5 software (StatSoft, Tulsa, OK, USA). Further, a post-hoc 2.4. Protein extraction and two-dimensional electrophoresis

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power analysis was performed with JMP 11 software (SAS Institute, Cary, NC, USA).

Proteins from gill tissue were extracted from 3 pooled fish per replicate tank. The

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homogenization of frozen gill tissue was performed using a Potter-Elvehjem apparatus in homogenization buffer containing 250 mM sucrose, 10 mM Tris-HCl, 1 mM EDTA, pH 7. The

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homogenate was centrifuged at 1 000 x g for 8 min at 4 °C. The supernatant was centrifuged at 7 700 x g for 10 min at 4 °C; the pellet was dissolved in homogenization buffer and centrifuged again at 10 000 x g for 10 min at 4 °C. The final pellet was dissolved in DLA

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buffer (7 M urea, 2 M thiourea, 4 % CHAPS, 30 mM Tris/HCl, pH 8.5) and 2 % ASB-14. Protein concentrations were measured according to Bradford (1976) using bovine serum

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albumin as a standard.

The experimental procedure for 2D-DIGE was adapted from Dorts et al. (2011a) with small modifications. In brief, protein extracts were labeled prior to electrophoresis with fluorescent

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amine reactive Cyanine dyes following the manufacturer’s recommended protocols (GE Healthcare). Immobiline DryStrips (24 cm, pH 4-7; GE Healthcare) were rehydrated overnight

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before cup-loading of proteins and isoelectric focusing (IEF). IEF was performed with an Ettan IPGphor II isoelectric focusing unit (GE Healthcare). Focused IPG strips were

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equilibrated twice in equilibration buffer supplemented (1) with DTT and then (2) with iodoacetamide. The second dimension was carried out on 10 % 24 cm, 1 mm thick acrylamide gel and casted in an Ettan DALTsix system gelcaster (GE Healthcare). The SDSPAGE step was performed in Ettan DALTsix separation unit (GE Healthcare). Gels were scanned using a Typhoon 9400 scanner (GE Healthcare). Image analysis and statistics were performed using the DeCyder 5.0 software (GE Healthcare). Briefly, the differential in-gel analysis (DIA) module co-detected and differentially quantified the protein spot intensity in each image using the internal standard sample as a reference to normalize the data. At a second step, the Biological Variation Analysis (BVA) was used to calculate ratios between samples and internal standard abundances by performing a gel-to-gel matching of the internal standard spot maps for each gel. The log standard abundance was used in the statistical analyses. Initially, a one-way ANOVA among the six experimental groups first allowed the selection of spots whose abundance was significantly modified at p < 0.05. On this basis, a principal component analysis (PCA) was performed within the ade4 package for the R statistical environment (Dray et al., 2007), including proteins present in 6   

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100 % of the gels. PCA reduces the large number of dimensions of a dataset into a smaller number of dimensions in such a way that most of the variance of the dataset is described by the first principal component (PC). Second, effects of temperature, Cd exposure and their interaction on protein expression were analyzed using a two-way ANOVA. Protein spots displaying significant (p < 0.05) interaction effects of temperature and Cd exposure were of

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interest for identification. 2.5. Mass spectrometry and protein identification

Peptide sequencing and protein identification were carried out as previously described (Dorts

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et al., 2011b). Briefly, spots were excised from preparative gels using the Ettan Spot Picker (GE Healthcare), and proteins were digested with trypsin by in-gel digestion. Peptides were

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analyzed by using nano-LC-ESI-MS/MS HCT-Ultra ETDII coupled with a nano-LC Dionex UltiMate 3000 (Bruker, Bremen, Germany). Peak lists were created using DataAnalysis 4.0 (Bruker) and saved as XML file for use with ProteinScape 2.0 (Bruker) with Mascot 2.2 as

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search engine (Matrix Science). Enzyme specificity was set to trypsin, and the maximum number of missed cleavages per peptide was set at one. Carbamidomethylation was allowed

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as fixed modification and oxidation of methionine as variable modification. Mass tolerance for monoisotopic peptide window was 0.4 Da and MS/MS tolerance window was set to 0.4 Da. The peak lists were searched against the full NCBInr database (9694989 sequences

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downloaded on September the 15th 2009). Scaffold (version Scaffold-2_06_01, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein

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identifications. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.2) and X! Tandem (The GPM, thegpm.org; version 2007.01.01.). Peptide

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identifications were accepted if they could be established at greater than 95% probability as specified by the Peptide Prophet algorithm (Keller et al., 2002). Protein identifications were accepted if they could be established at greater than 99% probability and contained at least 1 identified peptide. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii et al., 2003). A hierarchical cluster analysis (PermutMatrix; Caraux and Pinloche, 2005) of identified protein using an algorithm resulting from the combination of Ward’s aggregation method and the Euclidean-based distance metrics (Meunier et al., 2007) was performed. This method groups proteins according to the similarity of their expression pattern. 3. Results 3.1. Metabolic enzyme activities The activities of metabolic enzymes assayed in gill tissue of bullhead in response to thermal acclimation and subsequent Cd exposure are depicted in Table 1. First, CS activity was significantly (p < 0.01) influenced by temperature, independently of Cd exposure. Acclimation to 21 °C weakly increased the activity of CS by 12 %. Nevertheless, we did not observe any 7   

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interaction between temperature and Cd exposure for its activity. This result should be interpreted with caution as according to the post-hoc power calculation, the power of the ANOVA was 0.20 for the interaction effect. The power was 0.87 and 0.47 for effects of temperature and Cd exposure, respectively. Second, LDH activity displayed significant effects of temperature (p < 0.01), Cd exposure (p < 0.001) and their interaction (p < 0.01).

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We observed that exposure to Cd significantly decreased the activity of LDH by 58 % in gills of fish acclimated to 15 °C (p < 0.001) or 18 °C (p < 0.05) compared to unexposed animals. In contrast, no significant changes occurred in fish acclimated to 21 °C and then exposed to

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Cd, with activities reaching 101.3 mU/mg proteins. According to the post-hoc power analysis, the power of the ANOVA was 0.95, 0.99 and 0.90 for effects of temperature, Cd exposure

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and their interaction, respectively. 3.2. Proteomic analysis

A representative 2D gel is illustrated in Fig. 1. The average number of spots detected on

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gels was 1496 ± 116. The one-way ANOVA test among the six experimental groups revealed that 151 protein spots (10.1 % of all detected spots) were differentially expressed at p < 0.05.

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On this basis, a principal component analysis (PCA) was performed as a useful tool allowing the representation of the original dataset in a new reference system formed by new variables called factors or principal components (PC) (Fig. 2). Out of the potential 151 variables, we

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found two relevant PCs that explained 67 % of the total variance. PC1 accounts for 44 % of total variability and allowed the graphical discrimination of the three different acclimation

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temperatures, while PC2 allowed the effective separation of the Cd-exposed groups in the positive part and the unexposed groups in the negative part of the graph. Regarding the

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unexposed groups, we observed a very clear separation between the three acclimation temperatures: PC1 represents the groups acclimated to 15 °C and 18 °C in the left side and the 21 °C-acclimated group in the right side. This graphical discrimination is less apparent among the Cd-exposed groups. Thus, changes in the protein expression profile induced by heat acclimation seemed to be attenuated by subsequent exposure to Cd. Using a two-way ANOVA test among the six experimental groups, we attempted to reveal the molecular mechanisms induced by heat acclimation that could modify the subsequent Cd response. In fact, a significant interaction effect implied that a heat acclimation affects the subsequent response to Cd exposure, highlighting their joint modes of action. According to the two-way ANOVA test, 9 and 43 protein spots displayed significant interaction effects of temperature and Cd exposure at p < 0.01 and p < 0.05, respectively. All these spots were excised and submitted to MS/MS identification. A total of 28 different proteins were identified using nano LC-MS/MS and searches in the NCBI nr databases. Peptide and Protein Prophet Algorithms were used to validate MS/MS based peptide and protein identifications. The identified differentially expressed proteins represented a heterogeneous group and could be 8   

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categorized into diverse functional classes (Table 2). Analysis of the differentially expressed proteins suggested a stress response, as six chaperones were identified, namely protein disulfide isomerase associated 3 (PDIA3) (spot 733), heat shock protein 4 (HSPA4) (spot 222), calreticulin (spot 904), T-complex protein 1 subunit alpha (TCP1) (spot 707), chaperonin containing TCP1, subunit 6A (CCT6A) (spot 677), and stress protein HSC70-2

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(HSC70-2) (spots 501 and 506). Next to this stress response, alterations in the expression of several proteins involved in the proteasome machinery were detected. These were Cdc48 (spots 303 and 304) and 26S proteasome non-ATPase regulatory subunit 2 (PSMD2) (spot

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333). Besides, three proteins involved in protein biosynthesis were identified, namely elongation factor Tu (EF-Tu) (spot 1009), eukaryotic translation elongation factor 2, like (EF-

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G) (spots 297 and 302), and eukaryotic translation initiation factor 3 subunit K (EIF3K) (spot 1626). Alterations in the expression of several proteins involved at different levels of metabolic pathways were detected. These were NADH-ubiquinone oxidoreductase 75 kDa

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subunit (NDUS1) (spot 453), ATP synthase subunit beta (ATP5B) (spot 892), aldehyde dehydrogenase family 7 member A1 homolog (AL7A1) (spot 760), glucose-6-phosphate 1-

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dehydrogenase (G6PD) (spot 746), and Hibadhb (also known as 3-hydroxyisobutyrate dehydrogenase b) (spot 1416). The abundance of some cytoskeleton-associated proteins was also affected. This was the case for tubulin beta-1 chain (TBB1) (spot 775), tubulin

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alpha-1C chain (TBA1C) (spot 732), type II cytokeratin (KRT5) (spot 697), beta-actin (spots 1425, 1501, 1521, 1553 and 1562), and actin-related protein 2/3 complex, subunit 2

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(ARPC2) (spot 1462). Three signaling related proteins were identified, namely 14-3-3 protein beta/alpha B (spot 1468), serine/threonine-protein phosphatase (PPIG) (spot 1262) and GDP

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dissociation inhibitor 2 (GDI2) (spot 920). Spots 753 and 1410 were respectively identified as vacuolar-type H+ transporting ATPase B1 subunit (VAOD1) and voltage-dependent anionselective channel protein 1 (VDAC1), which are involved in ion transport. Lastly, spots 608 and 1273 were identified as apoptosis-inducing factor, mitochondrion-associated 1 (AIFM1), which has a dual role in controlling cellular life and death, and annexin, a calcium-dependent phospholipid-binding protein involved in diverse cellular processes. Hierarchical cluster analysis was further applied to group proteins according to the similarity of their expression pattern. This analysis yielded five protein groups with 9, 6, 6, 3 and 3 different proteins, respectively. A sixth group included one protein (NDUS1) (Fig. 3). We approached the interaction between temperature and Cd exposure by discussing the fold change 1 mg Cd/L versus control for each temperature (bottom graphs in Fig. 3). Within group 1, Cd exposure increased protein abundance at 15 °C and 18 °C, while a decrease was observed at 21 °C (negative fold change of -1.32 ± 0.16). The reverse trend was recorded in group 2. The abundance of proteins in group 3 was increased due to Cd exposure at 15 °C, while a decrease was observed at higher temperatures (18 °C and 21 9   

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°C). Within group 4, Cd exposure increased protein abundance at all temperatures. We further observed increasing fold change with increasing temperature (from 1.06 ± 0.06 at 15 °C to 1.21 ± 0.12 at 18 °C to 1.84 ± 0.48 at 21 °C). NDUS1 exhibited a reverse trend. Finally, Cd exposure increased protein abundance within group 5 at 15 °C and 21 °C, while a decrease was observed at the intermediate temperature 18 °C. Further, based on the protein

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expression profiles within unexposed groups (top graphs in Fig. 3) we observed that protein abundance increased with increasing temperature in groups 1, 3 and NDUS1. The reverse trend was recorded for protein groups 2, 4 and 5. Taken together, these results showed that

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for all protein groups (except group 5), exposure to Cd clearly interfered with the changes of protein expression profile induced by heat acclimation. This opposite effect was mainly

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observed at the highest temperature 21 °C. In addition, we observed differing responses of proteins belonging to the same functional class, e.g., calreticulin (protein group 2) versus the

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other molecular chaperones (protein group 1). 4. Discussion

Given the increasing multiplicity of environmental stressors associated with global change,

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there is a need to develop a better understanding of the interactive effects of multiple stressors on ecosystems. To the best of our knowledge, the present study was the first attempt to evaluate the interactive effects of temperature and Cd exposure in sequential

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combination, i.e., fish acclimated to elevated temperature and subsequently exposed to Cd, by using both enzymatic and proteomic approaches. The temperatures applied in the present

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study were far below the upper limit of normal feeding or death for C. gobio, which was found to be 27.7 °C (Elliott and Elliott, 1995). Further, our previous study showed that exposure to

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temperatures from 4 to 8 °C higher than temperature C. gobio would have experienced in the wild during the recrudescence and mating periods significantly influenced its reproduction (Dorts et al., 2012a).

Experimental evidence indicates that an increase in environmental temperature results in elevated mortality rates in metal-exposed ectotherms (reviewed in Cairns et al., 1975; Heugens et al., 2001; Sokolova and Lannig, 2008). In a previous study we observed that 4 days exposure to elevated temperature (21 °C) and high Cd concentration (1 mg Cd/L) resulted in a high mortality rate of C. gobio (Dorts et al., 2012b). Interestingly, no mortality was observed over the course of the present experiment in which fish were acclimated to 15, 18 or 21 °C for 28 days prior to 4 days Cd exposure. An inseparable outcome of heat acclimation is the cross-tolerance phenomenon, in which adjusting to one environmental stressor can, in addition to the primary adaptation, confer protection against an additional type of stress (reviewed in Horowitz, 2007). From our observations, we can deduce that acclimation to elevated temperature conferred increased resistance of bullhead to subsequent Cd exposure. Similarly, previous studies have shown that heat acclimation 10   

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enhanced the resistance of several aquatic species to a subsequent Cd stress (Moller et al., 1994; Roch and Maly, 1979; Vergauwen et al., 2013). Possibly, either the warm acclimation provoked a general stress response that protected organisms against future severe stress situations, or resulted in specific defense mechanisms that also provided protection against Cd exposure (Kultz, 2005; Vergauwen et al., 2013).

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Metabolic regulation plays a key role in environmental stress tolerance, because matching energy demand with sufficient energy supply is crucial for survival. Elevated temperature and trace metals, including Cd, are known to strongly impact the metabolic physiology of

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ectothermic organisms (Cherkasov et al., 2006; Sokolova and Lannig, 2008). To assess tissue metabolic capacities, we measured the activity of citrate synthase (CS), the first

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enzyme of the Krebs cycle located within the mitochondria, and lactate dehydrogenase (LDH), the terminal enzyme of anaerobic glycolysis located in the cytoplasm. While no interaction between temperature and Cd exposure was observed for the CS activity, we

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showed an interactive effect of both stressors for the LDH activity. Exposure to Cd decreased its activity at low acclimation temperatures, while no change occurred at an increased

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temperature of 6 °C above standard temperature. We further illustrated how a stressor, i.e., a heat acclimation, that does by itself not affect the LDH activity may do so in sequential combination with another stressor, such as trace metal. Pelletier et al. (1993) have shown

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that thermal acclimation and seasonal changes had relatively slight effect on glycolytic enzyme activities in muscle of the cod Gadus morhua, while Guderley et al. (2001) did not

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observe any change in the activity of CS and LDH in muscle of the threespine stickleback Gasterosteus aculeatus acclimated to elevated temperature. In contrast, several studies

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have reported changes in the activity of metabolic enzymes following warm acclimation in various fish species (e.g., Das et al., 2006; Stobel et al., 2013; Verma et al., 2007). To further our understanding with respect to the interactive effects of both stressors, a proteomic analysis without a priori assumptions was performed. The proteomic approach developed in the present study is innovative in this field of research (i.e., sequential stress) and provides new answers about the effects of multiple stressors at the cellular phenotype level. We believe that it is of great importance to be aware that the phenotype, studied at its finest level, can change under thermal acclimation and that these changes can modify how organisms respond to additional stress. Changes in protein expression associated with longterm acclimation have received little attention in aquatic organisms. Fields et al. (2012) assessed the proteomic response in gill tissue of mussels (Mytilus galloprovincialis and M. trossulus) to chronic (4 weeks) temperature acclimation. The authors showed higher abundances of several cytoskeletal proteins (tubulin isoforms) and metabolic proteins (ubiquinol cytochrome c reductase, succinate dehydrogenase and ATP synthase β-subunit), as well as increases in abundances of several molecular chaperones (78-kDa glucose11   

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related proteins, HSP70, and small HSP25) and an oxidative stress protein (DyP-type peroxidase) during warm acclimation. Furthermore, there is only one proteomic study which has addressed the interactive effects of multiple stressors (such as temperature and metal pollution) in aquatic ectotherms. Silvestre et al. (2010) have highlighted mechanisms of action of heat stress and selenium exposure, along with their combined effects, on sturgeon

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larvae (Acipenser medirostris and A. transmontanus) by using a proteomic analysis. Nonetheless, the authors have used micro-injection of selenium while heat stress was much higher than the one expected by global warming. Moreover, there was no acclimation of

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organisms and no subsequent exposure that could have allowed analysis of phenotypic plasticity during long-term stress.

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Using a combination of univariate and multivariate methods, we firstly reported that an acclimation to elevated temperature was the first parameter affecting the protein expression profile in gills of C. gobio, while the subsequent response to Cd was clearly affected by heat

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acclimation. In the PCA, a clear separation between the three acclimation temperatures was observed within the groups unexposed to Cd, while this graphical discrimination was less

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apparent among the Cd-exposed groups. Changes in the branchial protein expression profile induced by heat acclimation were attenuated, or alternatively, masked by subsequent exposure to Cd. Second, subsequent identification of proteins by mass spectrometry and

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bioinformatics allowed exploring the potential molecular pathways induced by heat acclimation that could modify the subsequent response to Cd. Many affected proteins are

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associated with different central aspects of the evolutionary conserved cellular stress response (Kultz, 2005; 2003), such as molecular chaperones, proteolysis and redox

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regulation (mainly dehydrogenases). Other differentially expressed proteins that were identified are related to protein synthesis, ion transport, metabolic process, cell signaling, and cytoskeletal reorganization.

Both high temperature and metal exposure, including Cd, are known to cause damage and misfolding of proteins (De Smet and Blust, 2001; Medicherla and Goldberg, 2008; Parsell and Lindquist, 1993). Damaged proteins are generally either rescued by chaperones, or degraded by proteases, or they form insoluble aggregates, in particular when the chaperone/protease machinery is overwhelmed (Yerbury et al., 2005). Interestingly, a number of molecular chaperones (HSC70, HSPA4, TCP1, CCT6A and PDIA3) as well as proteins involved in the proteasome machinery (Cdc48 and PSMD2) were differentially expressed in response to temperature and Cd exposure. These proteins clustered in protein groups 1 and 3 showed similar expression profiles (except at the intermediate temperature 18 °C). First, within unexposed groups we observed over-expression of the aforementioned proteins with increasing temperature, suggesting a possible increase of protein damage at high temperature with an attempt to maintain protein folding. Second, while Cd exposure 12   

Page 12 of 27

increased their expression at the standard temperature, a decrease was observed at the highest temperature compared to unexposed fish. In contrast, calreticulin, a molecular calcium-binding chaperone promoting folding, oligomeric assembly and quality control of glycoproteins in the endoplasmic reticulum, was under-expressed with increasing temperature in unexposed groups, while Cd exposure increased its expression at 21 °C

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(protein group 2). Although the mechanisms underlying the observed effects need further studies, one would think that acclimation to elevated temperature of 6 °C modulates the Cd response through the branchial chaperone/protease machinery of C. gobio. Besides,

cr

alterations in the expression of several proteins involved in protein biosynthesis have been detected. EIF3K was under-expressed with increasing temperature (protein group 4), while

us

we observed a reverse trend for EF-Tu and EF-G encompassed in protein group 3. EIF3K is a component of the eukaryotic translation initiation factor 3 (EIF3) complex, which plays a central role in initiating translation of mRNAs in multiple steps, including generation of

an

ribosomal subunits and formation of preinitiation complexes (Dong and Zhang, 2006). Next, during polypeptide lengthening, EF-Tu promotes the GTP-dependent binding of aminoacyl-

M

tRNA to the ribosomal A site and EF-G catalyzes the GTP-dependent ribosomal translocation steps of tRNA (Kulczycka et al., 2011). Previously, Logan and Somero (2010) reported increased expression of genes involved in translation in gills of Gillichthys mirabilis

d

following warm acclimation. Further, Sanchez et al. (2011b) observed that 4 days exposure to Cd decreased the expression of genes involved in protein biosynthesis in liver of the

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largemouth bass (Micropterus salmoides). Protein synthesis can account for up to ninety percent of the total energy consumption in fish cell (Smith and Houlihan, 1995). Additionally,

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gill tissue is known to be among the most active tissues in terms of protein turnover, second only to liver (Lyndon and Houlihan, 1998). It is therefore noteworthy that a substantial increase in protein turnover and protein repair might entail an energetic cost in gills of C. gobio. In the current study, two proteins (NDUS1 and ATP5B) involved in ATP generation through the mitochondrial respiratory chain were identified and showed reverse responses (protein group 6 and 4, respectively). Experimental evidence indicates that mitochondria, and hence energy metabolism, are likely to be a common intracellular target for increased temperature and Cd (Dorta et al., 2003; Portner, 2001; Sokolova and Lannig, 2008). Cytoskeleton is involved in the maintenance of cell shape, locomotion, intracellular organization, and transport. Thus, alterations in the expression of any of the three major protein filaments, i.e., microfilaments (actin), microtubules (tubulin) and intermediate filaments (keratin), can cause adverse effects to the cells (Nawaz et al., 2005). The present study reported a number of cytoskeleton-associated proteins differentially expressed following heat acclimation and Cd exposure in gills of C. gobio. Tubulin isoforms and type II cytokeratin were over-expressed with increasing temperature in unexposed groups, while Cd 13   

Page 13 of 27

exposure decreased their expression at the highest temperature (protein group 1). The reverse pattern was recorded for beta-actin and ARPC2 (protein group 2). Recently, Fields et al. (2012) reported increased expression of cytoskeletal proteins, e.g., actin and tubulin, in gill tissue of mussels (Mytilus galloprovincialis and M. trossulus) following warm acclimation. In a previous study, alterations in the expression of type II keratin proteins were also

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detected in gills of C. gobio exposed for 4 days to Cd (Dorts et al., 2011a). Interestingly, cytoskeleton (re)organization has not been recognized as a main aspect of the stress response. The components of the cytoskeleton, such as actin, are also evolutionarily highly

cr

conserved, showing their importance for the cell survival (Schmidt and Hall, 1998). In that requires further investigation (Wang et al., 2009).

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sense, this group of proteins could be included in the general stress response, but this Several oxidoreductases, mainly dehydrogenases, which are involved in metabolic pathways and/or influence the cell redox balance, were also variably expressed in response to

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temperature and Cd exposure. This was the case for Hibadhb, a mitochondrial enzyme involved in valine metabolism, G6PD, an enzyme involved in the pentose phosphate

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pathway, and AL7A1, a multifunctional enzyme mediating important protective effects. Within unexposed groups, we observed under-expression of Hibadhb and G6PD with increasing temperature (protein group 5), while AL7A1 was over-expressed following heat acclimation

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(protein group 1). Besides, we previously observed significant interactive effects of temperature and Cd on the LDH activity. Thus, the changes in enzyme activity assays and in

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protein expression profiles suggest that acclimation to elevated temperature modulates the Cd response through metabolic readjustments and a possible cell redox imbalance in gills of Another protein was identified as AIFM1, a ubiquitous mitochondrial

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C. gobio.

oxidoreductase involved in apoptosis. During apoptosis, it is translocated from the mitochondria to the nucleus to function as a pro-apoptotic factor in a caspase-independent pathway, while in normal mitochondria it functions as an anti-apoptotic factor via its oxidoreductase activity.

Numerous proteins identified in the present study are considered as members of the cellular stress response and/or are also commonly detected in proteomic studies, as for instance ATP synthase subunit beta, EF-G and beta-actin (Wang et al., 2009). Nonetheless this work provides for the first time insights into the molecular pathways potentially involved in heat acclimation process and interfering with a subsequent Cd stress. The interaction response was more complex than expected. We clearly demonstrated opposite effects of these two environmental stressors at protein expression level. The mechanisms underlying these unexpected effects need further studies in aquatic ectotherms. We also observed that proteins within a same functional class exhibited reverse responses following sequential exposure to heat and Cd. What this means for the actual function of the cell and/or the 14   

Page 14 of 27

individual remains unknown to date. Earlier studies have revealed significant interactions of heat and metal stress, but results are not consistent (Holmstup et al., 2010). Indeed, concurrent or sequential exposure of biological systems to multiple stressors induces complex (additive, antagonistic, or synergistic) interactive effects that are difficult to model and predict from single-stressor effects (Altshuler et al., 2011; Folt et al., 1999). Furthermore,

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these types of investigations are still in their infancy at protein expression level. Cairns et al. (1975) previously stated that similar physiological processes can be affected by a toxicant and temperature, complicating the outcome of combined or sequential exposure, depending

cr

on the species (Cairns et al., 1978), toxicant (Bao et al., 2008) and endpoint (Smit and Van Gestel, 1997) considered.

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As a corollary, the current study highlighted complex interaction patterns of elevated temperature and Cd exposure on the LDH activity and protein expression profiles in gill tissue of an ecologically relevant species with few genomic sequences available in

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databases. Nonetheless, a combination of enzyme activity assays and protein expression profiles clearly demonstrated that increases in water temperature consistent with climate

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change predictions may modulate the response of ectotherms to chemical insults. We showed that the phenotype, studied at its finest level, can change during a heat acclimation and that these changes can modify the ability of fish to respond to an additional Cd stress.

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The differentially expressed proteins that were identified in gills of C. gobio provide a good starting point for a comprehensive understanding of the heat acclimation process and of the

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interactive effects of multiple stressors, such as temperature and metal pollution on fish species. Additional investigations are now needed to link the cellular phenotype as observed

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herein to changes at higher levels of biological organization in order to inform on the health of organisms in their natural environments, an issue that is particularly relevant for environmental scientists examining the consequences of global change.

Acknowledgments

The authors thank A. Evrad and M.-C. Forget from URBE, E. Delaive and C. Demazy from URBC, P. Cambier from URBV, University of Namur (Namur, Belgium) for valuable help during animal husbandry, biochemical, proteomic, and chemical analysis respectively. This study was supported by a FNRS PhD fellowship to J. Dorts. The proteomic platform of the URBC is supported by the FNRS, Fonds National de la Recherche Scientifique (Belgium).

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Bao, V.W.W., Koutsaftis, A., Leung, K.M.Y., 2008. Temperature-dependent toxicities of chlorothalonil and copper pyrithione to the marine copepod Tigripopus japonicus and dinoflagellate Pyrocystis lunula. Aust. J. Ecotoxicol. 14, 45–54. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-

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Caraux, G., Pinloche, S., 2005. PermutMatrix: a graphical environment to arrange gene expression profiles in optimal linear order. Bioinformatics, 21, 1280-1281.

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Dorts, J., Bauwin, A., Kestemont, P., Jolly, S., Sanchez, W., Silvestre, F., 2012. Proteasome and antioxidant responses in Cottus gobio during a combined exposure to heat stress and cadmium. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 155, 318–324. Dray, S., Dufour, A.B., 2007. The ade4 package: Implementing the duality diagram for ecologists. J. Stat. Softw. 22, 1-20. 3 populations in North-West England. Freshwater Biol. 33, 411-418.

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Horowitz, M., 2007. Heat acclimation and cross-tolerance against novel stressors: genomicphysiological linkage. Prog. Brain Res. 162, 373-392. Karr, T.L., 2008. Application of proteomics to ecology and population biology. Heredity 100, 200-206. Keller, A., Nesvizhskii, A.I., Kolker, E., Aebersold, R., 2002. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem. 74, 5383-5392. Kulczycka, K., Dlugosz, M., Trylska, J., 2011. Molecular dynamics of ribosomal elongation factors G and Tu. Eur. Biophys. J. 40, 289-303.

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Kultz, D., 2003. Evolution of the cellular stress proteome: from monophyletic origin to ubiquitous function. J. Exp. Biol. 206, 3119-3124. Kultz, D., 2005. Molecular and evolutionary basis of the cellular stress response. Annu. Rev. Physiol. 67, 225-257. Logan, C.A., Somero, G.N., 2010. Transcriptional responses to thermal acclimation in the

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Vergauwen, L., Knapen, D., Hagenaars, A., Blust, R., 2013. Hypothermal and hyperthermal acclimation differentially modulate cadmium accumulation and toxicity in the zebrafish. Chemosphere, 91, 521–529. Verma, A.K., Pal, A.K., Manush, S.M., Das, T., Dalvi, R.S., Chandrachoodan, P.P., Ravi, P.M., Apte, S.K., 2007. Persistent sub-lethal chlorine exposure elicits the temperature

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space.

EMBO

Reports,

us

in

6,

1131-1136.

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te

d

M

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folding

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Figure captions Fig. 1. Representative 2D gel showing the protein expression profile obtained from gills of C. gobio acclimated to 15, 18 or 21 °C temperatures for 28 days, and then exposed to 1 mg Cd/L for 4 days. Proteins of the samples obtained for the different experimental conditions were differentially labeled with Cy3 and Cy5. An internal standard composed of equal

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amounts of each sample and labeled with Cy2 was added. Labeled samples (25 µg of each of the Cy3 and Cy5 labeled samples and of the Cy2 labeled internal standard) were loaded on 24 cm pH 4-7 IPG strips and subjected to IEF. Proteins were further separated by SDS-

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PAGE (10 %) in the second dimension. Numbers allocated by the DeCyder software indicate spots with significant changes (temperature-Cd interaction effect; p < 0.05) in intensity (n =

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3).

Fig. 2. Representation of the main principal components found after PCA. There are six experimental groups in triplicates: (A) 15 °C – 0 mg Cd/L, (B) 15 °C – 1 mg Cd/L, (C) 18 °C –

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0 mg Cd/L, (D) 18 °C – 1 mg Cd/L, (E) 21 °C – 0 mg Cd/L and (F) 21 °C – 1 mg Cd/L. Ellipses have been drawn to illustrate the clustering of the five different conditions (15 °C, 18

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°C, 21 °C, 0 mg Cd/L, and 1 mg Cd/L). The proteins that participated in the PCA were present in 100 % of the gels and passed the filter of the one-way ANOVA (p < 0.05) test. Fig. 3. Protein groups with similar expression profile, comprising all identified proteins

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displaying significant (p < 0.05) interaction effects of temperature and Cd exposure in gills of C. gobio. Mean ± S.D. is shown. Top graphs: protein standard abundance at 15, 18 or 21 °C

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acclimation temperature.

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within unexposed groups. Bottom graphs: fold change 1 mg Cd/L versus control for each

22   

Page 21 of 27

Table 1. Activities (mU/mg proteins) of citrate synthase (CS) and lactate dehydrogenase (LDH) measured in gill tissue of C. gobio in response to thermal acclimation followed by Cd exposure. 21 °C

0 mg Cd/L

98.3 ± 15.5

95.3 ± 8.3

120.8 ± 8.3

1 mg Cd/L

97.8 ± 8.9

84.9 ± 5.4

0 mg Cd/L

82.3 ± 8.0

a

110.0 ± 33.9

1 mg Cd/L

34.7 ± 3.2 b

46.3 ± 10.5 b

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LDH

18 °C

102.7 ± 6.3 a

94.0 ± 15.2 a

101.3 ± 28.3 a

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CS

15 °C

Data are presented as mean ± S.D. (n = 3). If a significant interaction effect of temperature

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and Cd exposure was observed, different letters (a and b) mean significant (p < 0.05)

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te

d

M

an

differences.

23   

Page 22 of 27

Table 2. Detailed list of proteins differentially expressed in gill tissue of C. gobio in response to thermal acclimation followed by Cd exposure and identified by nano LC-MS/MS. Spot

Accession

No.

No.a

Protein name

Species

Matching

Theorical

Protein group

peptides

pI/Mw (kDa)

No.b

D0QEL0

Protein disulfide isomerase associated 3

Salmo salar

2

222

Q6NZU0

Heat shock protein 4

Danio rerio

5

904

A3QT58

Calreticulin

Paralichthys olivaceus

2

707

Q9W791

T-complex protein 1 subunit alpha

Xenopus laevis

5

677

Q7ZYX4

Chaperonin containing TCP1, subunit 6A

Danio rerio

501

B6F134

Stress protein HSC70-2

Seriola quinqueradiata

506

B6F134

Stress protein HSC70-2

Seriola quinqueradiata

1

5.1/96

1

4.4/49

2

5.9/60

3

4

6.7/58

4

5.3/71

3

15

5.3/71

3

us

Protein degradation

5.4/55

cr

733

ip t

Protein folding

3

333

C0H8V2

26S proteasome non-ATPase regulatory subunit 2

Salmo salar

8

5.2/100

1

304

B8XQT3

Cdc48

Larimichthys crocea

19

5.2/89

1

303

B8XQT3

Cdc48

Larimichthys crocea

25

5.2/89

1

Tetraodon nigroviridis

2

6.2/49

3

Danio rerio

2

6.3/95

3

Danio rerio

4

6.3/95

3

Danio rerio

2

4.9/25

4

Salmo salar

4

5.7/80

6

Q4S9H0

Elongation factor Tu

297

Q7ZVM3

Eukaryotic translation elongation factor 2, like

302

Q7ZVM3

Eukaryotic translation elongation factor 2, like

1626

Q567V6

Eukaryotic translation initiation factor 3 subunit K

Metabolic process 453

B5X3P9

NADH-ubiquinone

oxidoreductase

75

M

1009

an

Protein biosynthesis

kDa

mitochondrial

subunit,

C1J0J0

ATP synthase subunit beta

Gillichthys seta

9

5.2/54

4

760

B5X0S9

Aldehyde dehydrogenase family 7 member A1 homolog

Salmo salar

5

7/59

1

746

B5X1I3

Glucose-6-phosphate 1-dehydrogenase

Salmo salar

4

6.7/59

5

1416

A8DSV9

Hibadhb

Haplochromis burtoni

4

6.8/35

5

Salmo salar

17

4.8/50

1

Salmo salar

5

5/48

1

Danio rerio

3

5.3/59

1

te

Cytoskeleton

d

892

C0H808

Tubulin beta-1 chain

732

C0PU76

Tubulin alpha-1C chain

697

Q9PUB5

Type II cytokeratin

1553

A9QUS4

Beta-actin

Rachycentron canadum

4

5.3/42

2

1501

A9QUS4

Beta-actin

Rachycentron canadum

2

5.3/42

2

1425

A9QUS4

Beta-actin

Rachycentron canadum

9

5.3/42

2

1521

A9QUS4

Beta-actin

Rachycentron canadum

6

5.3/42

2

1562

A9QUS4

Beta-actin

Rachycentron canadum

5

5.3/42

2

1462

Q6P2T5

Actin related protein 2/3 complex, subunit 2

Danio rerio

2

6.6/34

2

Cell signaling

Ac ce p

775

1468

Q7T356

14-3-3 protein beta/alpha-B

Danio rerio

2

4.7/27

2

1262

B5X0W0

Serine/threonine-protein phosphatase

Salmo salar

2

5.9/47

4

920

Q6TNT9

GDP dissociation inhibitor 2

Danio rerio

2

5.6/51

1

Ion transport 753

Q9PUK7

Vacuolar-type H+ transporting ATPase B1 subunit

Anguilla anguilla

10

5.3/56

2

1410

B5X4Q7

Voltage-dependent anion-selective channel protein 1

Salmo salar

7

6.1/31

5

Other functions 608

A2BGE5

Apoptosis-inducing factor, mitochondrion-associated 1

Danio rerio

4

5.1/81

2

1273

C3KJS6

Annexin

Anoplopoma fimbria

7

5.5/30

3

a

Accession number in UniProt/TrEMBL

b

Proteins were clustered in groups according to the similarity of their expression profile

24   

Page 23 of 27

Ac ce p

te

d

M

an

us

cr

ip t

Figure 1

25   

Page 24 of 27

Ac ce p

te

d

M

an

us

cr

ip t

Figure 2

26   

Page 25 of 27

Figure 3 Protein group 2 (β‐actin, ARPC2, calreticulin,  14‐3‐3 protein beta/alpha B, VAOD1, AIFM1) 

an

us

cr

ip t

Protein group 1 (TBB1, TBA1C, KRT5, PDIA3,  HSPA4, Cdc48, PSMD2, GDI2, AL7A1) 

Protein group 3 (TCP1, CCT6A, HSC70‐2, EF‐ Tu, EF‐G, annexin) 

Ac ce p

te

d

M

Protein group 4 (ATP5B, EIF3K, PPIG) 

Protein group 5 (G6PD, Hibadhb, VDAC1)

NDUS1

27   

Page 26 of 27

Highlights of the revised manuscript reference number AQTOX-D-14-00142 entitled “Effects of cadmium exposure on the gill proteome of Cottus gobio: modulatory effects of prior thermal acclimation”. Fish acclimated to elevated temperature were subsequently exposed to cadmium.

‐ 

Interaction of both stressors on LDH activity and protein expression were complex.

‐ 

Both stressors have opposite effects at branchial protein expression level.

‐ 

Proteins belonging to the same functional class exhibited differing responses.

‐ 

Prior acclimation to elevated temperature modulated the effects of cadmium exposure.

Ac ce p

te

d

M

an

us

cr

ip t

‐ 

28   

Page 27 of 27