Effects of elevated temperature and cadmium exposure on stress protein response in eastern oysters Crassostrea virginica (Gmelin)

Aquatic Toxicology 91 (2009) 245–254

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Effects of elevated temperature and cadmium exposure on stress protein response in eastern oysters Crassostrea virginica (Gmelin) A.V. Ivanina a , C. Taylor a,b , I.M. Sokolova a,∗ a b

Department of Biology, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC 28223, USA Johnson C. Smith University, 100 Beatties Ford Rd., Charlotte, NC 28216, USA

a r t i c l e

i n f o

Article history: Received 13 October 2008 Received in revised form 14 November 2008 Accepted 18 November 2008 Keywords: Metallothioneins Heat shock proteins Temperature Cadmium Interactions Mollusks Bivalves

a b s t r a c t Stress proteins such as heat shock proteins (HSPs) and metallothioneins (MTs) play a key role in cellular protection against environmental stress. Marine ectotherms such as eastern oysters Crassostrea virginica are commonly exposed to multiple stressors including temperature and pollution by metals such as cadmium (Cd) in estuaries and coastal zones; however, the combined effects of these stressors on their cellular protection mechanisms are poorly understood. We acclimated C. virginica from populations adapted to different thermal regimes (Washington, North Carolina and Texas) at a common temperature of 12 ◦ C, and analyzed their expression of MTs and HSPs (cytosolic HSP69, HSC72–77, HSP90 and mitochondrial HSP60) in response to the combined acute temperature stress and long-term Cd exposure. Overall, HSP and MT induction patterns were similar in oysters from the three studied geographically distant populations. HSP69 and MTs were significantly up-regulated by Cd and temperature stress implying their important role in cellular stress protection. In contrast, HSC72–77, HSP60 and HSP90 were not consistently induced by either acute heat or Cd exposure. The induction temperature for MTs was higher than for HSP69 (>28 ◦ C vs. 20 ◦ C, respectively), and MTs were more strongly induced by Cd than by temperature stress (to up to 38–94-fold compared by 3.5–7.5-fold, respectively) consistent with their predominant role in metal detoxification. Notably, heat stress did not result in an additional increase in metallothionein expression in Cd-exposed oysters suggesting a capacity limitation during the combined exposure to Cd and temperature stress. Levels of HSP69 and in some cases, HSC72–77 and HSP90 were lower in Cd-exposed oysters as compared to their control counterparts during heat stress indicating that simultaneous exposure to these two stressors may have partially suppressed the cytoprotective upregulation of molecular chaperones. These limitations of stress protein response may contribute to the reduced thermotolerance of oysters from metal-polluted environments. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Pollution of aquatic environments by trace metals including cadmium is a world-wide problem due to the persistency and continuing accumulation of metals in the environment (GESAMP, 1987; de Mora et al., 2004; Hyun et al., 2006). Cadmium (Cd) is one of the most toxic metals found in coastal zones and estuaries where it is predominantly released by human activities such as smelting, mining, battery manufacturing, and pigment and plastic production (GESAMP, 1987; Pinot et al., 2000). In ectotherms (that constitute >99% of species in aquatic realms), susceptibility to metal pollutants can be strongly modified by the environmental temperature due to its direct effects on all biochemical and physiological reactions

∗ Corresponding author. Tel.: +1 704 687 8532. E-mail address: [email protected] (I.M. Sokolova). 0166-445X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2008.11.016

(reviews in: Hochachka and Somero, 2002; Gordon, 2005; Sokolova and Lannig, 2008). Earlier studies have shown that moderately elevated temperatures exaggerate toxic effects of Cd on aquatic ectotherms through increased mitochondrial damage and oxidative stress, elevated energy demand, impaired ventilatory and/or circulatory capacities and resulting energy deficiency (review in: Sokolova and Lannig, 2008). However, it is not known whether Cd exposure can also affect the response to and/or tolerance of temperature extremes in marine ectotherms. According to the hierarchical model of the mechanisms of thermal tolerance in aquatic ectotherms (review in: Pörtner, 2002), survival under the conditions of acute heat stress is critically dependent on the molecular protective mechanisms maintaining cellular integrity such as the expression of stress proteins, and alterations of the capacity of these cytoprotective systems can have serious implications for the whole-organism survival under the extreme temperature conditions.

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Two important groups of stress proteins – heat shock proteins (HSPs) and metallothioneins (MTs) – play a key role in cellular protection against toxic metals and temperature stress (Bauman et al., 1993; Andrews, 2000; Amiard et al., 2006) and are the focus of the current study. Metallothioneins are low molecular weight cysteine-rich proteins with prominent metal-binding and redox capacities (Amiard et al., 2006; Bauman et al., 1993; Coyle et al., 2002; Palmiter, 1998). Their major function involves regulation of intracellular levels of essential and non-essential metals (including Cd) and metal detoxification (Roesijadi, 1996; Palmiter, 1998; Klaassen et al., 1999; Amiard et al., 2006), while secondary roles include antioxidant defense, protection against xenobiotics, inflammation and infection through free radical scavenging (Tamai et al., 1994; DeMoor et al., 2001; Van Cleef-Tödt et al., 2001; Coyle et al., 2002; Piano et al., 2004). HSPs also play an important role in protection against multiple stressors (including heat stress, toxic metals, ionizing and UV radiation and others) and act as molecular chaperones that assist in ATP-dependent folding and stabilization of stress-damaged proteins (Parsell and Lindquist, 1993; Somero, 1995; Hofmann et al., 2002; Boutet et al., 2003; Piano et al., 2004; Hofmann, 2005; Moraga et al., 2005). Cytosolic chaperones HSP70 and HSP90 are among the most abundant cellular proteins protecting from stress-induced damage. HSP70 is the family of universal cytosolic chaperones involved in folding of reparably damaged proteins and in degradation of those that are damaged beyond repair (Mayer and Bukau, 2005). HSP90 is another general cytosolic chaperone orchestrating the folding of many proteins; however, HSP90 alone is insufficient to assist refolding of partially denatured proteins, and requires other chaperones such as HSP70 to complete this task (Csermely et al., 1998; Mayer and Bukau, 2005). In contrast, HSP60 is predominantly found in mitochondria and chloroplasts assisting with the protein folding and stress protection in these organelles (Cechetto et al., 2000). Both metallothioneins and HSPs are essential for survival of an organism exposed to toxic metals and heat stress (Parsell and Lindquist, 1993; Somero, 1995; Roesijadi, 1996; Klaassen et al., 1999). However, it is not known how the concomitant exposure to these combined stressors affects expression of MTs and HSPs in aquatic ectotherms. The goal of this study was to analyze the induction patterns of MTs and HSPs (including cytosolic HSP69, HSC72–77, HSP90 and mitochondrial HSP60) in response to combined temperature and Cd stress in a model marine ectotherm, eastern oyster C. virginica. We tested whether Cd exposure alters stress protein expression during acute heating in oysters and compared expression patterns of MTs and HSPs in oysters from populations adapted to different climates in order to assess potential geographical variation in the studied parameters. Oysters are commonly exposed to metals including Cd and to temperature stress in their habitats and thus can serve as a useful model to address these questions. They have an ability to accumulate Cd burdens exceeding the environmental levels by orders of magnitude, making them susceptible to the toxic effects of Cd as well as important vectors of Cd transfer to the higher levels of the food chain (Roesijadi, 1996; Frew et al., 1997; Pigeot et al., 2006). Like all intertidal organisms, oysters also can experience rapid and extreme temperature fluctuations, with a change in body temperature by up to 10–20 ◦ C within a few hours during the diurnal/tidal cycles and even more dramatic changes (from 0 to 35–40 ◦ C) over the longer (seasonal) time span (Sokolova et al., 2000; Helmuth et al., 2002; Cherkasov et al., 2007). Investigations of the interactive effects of temperature and metal stress on cytoprotective mechanisms of oysters can provide a better understanding of physiological and cellular mechanisms of stress response in aquatic ectotherms, and the factors setting limits to their tolerance in the face of multiple stressors in polluted estuaries.

2. Materials and methods 2.1. Animal collection and maintenance Oysters (C. virginica) were obtained from J & B Aquafood (Jacksonville, NC, USA), Taylor Shellfish Farms (Totten Inlet, Shelton, WA, USA), and Jeri’s Seafood Inc. (Smith Point, TX, USA) in winter–early spring 2007. Oysters were shipped within 24–48 h to the University of North Carolina at Charlotte and placed in recirculated aerated tanks with artificial seawater (ASW) (Instant Ocean® , Kent Marine, Acworth, USA) at 12 ± 1 ◦ C and 30‰ which was close to the temperature and salinity of their habitats at the time of collection. It is worth noting that although our collections were performed in winter when surface water temperatures were similar in the three studied sites, their seasonal thermal regimes are different. Mean monthly surface water temperature varies between 12–15 ◦ C in winter and 28–30 ◦ C in summer near Smith Point, TX, and between 6–12 ◦ C in winter and 28–32 ◦ C in summer in Stump Sound, NC, respectively (IDARS NOAA at http://www.nodc.noaa.gov/dsdt/index.html; Cherkasov et al., 2007). The surface water temperature averages 8–10 ◦ C during the winter months and 12–13 ◦ C in summer near the study site in Totten Inlet, WA (IDARS NOAA at http://www.nodc.noaa.gov/dsdt/index.html). All three collection sites are used for commercial oyster culture for human consumption and have low background levels of metals and other pollutants. Oysters were allowed to acclimate at 12 ± 1 ◦ C and 30‰ for 2–3 weeks and fed ad libitum on alternate days with a commercial algal blend (2 mL per oyster) containing Nannochloropsis, Tetraselmis, and Isochrysis spp. with a cell size of 2–15 ␮m (PhytoPlex; Kent Marine, Acworth, GA, USA) or Nannochloropsis oculata, Phaeodactylum tricornutum and Chlorella with a cell size of 2–20 ␮m (DT’s Live Marine Phytoplankton, Premium Reef Blend, Sycamore, IL, USA). After the preliminary acclimation, half of the tanks were randomly selected, and Cd (as CdCl2 ) was added to the nominal concentration of 50 ␮g L−1 . The remaining tanks were used as controls. Oysters were exposed to Cd (50 ␮g L−1 ) or clean ASW (controls) for 45–50 days at 12 ◦ C prior to subsequent experiments. To avoid pseudoreplication, two tanks were set for each combination of Cd exposure (i.e. control and 50 ␮g L−1 ) and study population (WA, NC or TX), and oysters were haphazardly sampled from these tanks for each experiment. It is worth noting that Cd concentrations in our experimental exposures were at the upper end of Cd concentrations found in polluted estuaries (Crompton, 1997). However, our previous studies have shown that up to 60 days of exposure under these conditions result in physiologically relevant tissue burdens of Cd similar to those found in oysters from polluted estuaries (Sokolova et al., 2005; Cherkasov et al., 2006 and references therein). 2.2. Experimental exposures Because transcriptional and translational responses to environmental stress can be delayed due to the time needed to synthesize mRNAs from target genes and to translate them into proteins, we performed a pilot experiment to determine the time course of mRNA and protein expression in response to acute heating and to identify the time window of the maximum stress response for HSPs and MTs (see below Section 2.3 for the measurement techniques). Oysters were exposed to 40 ◦ C for 1 h and allowed to recover for up to 21 days at their acclimation temperature. Exposure to 40 ◦ C is an acute stressor which is strong enough to elicit significant stress response while remaining in the environmentally relevant range for intertidal organisms such as oysters (Hamdoun et al., 2003; Helmuth et al., 2002; Cherkasov et al., 2007). Analysis of expres-

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sion of MT mRNA levels and protein levels for HSP70, HSP60 and HSP90 was conducted immediately following the heat stress (time 0) and after 1, 24 and 48 h and 5, 7, 14 and 21 days of recovery. Expression of MTs and HSPs was also determined in the control (non-heat-stressed) oysters. This pilot study showed that there was no significant increase in levels of MT mRNA or HSP expression at the protein level until 24 h of recovery after the heat shock, and that the strongest induction of MT mRNA and of heat shock proteins was achieved after 48 h of recovery (data not shown). This agrees with the results of earlier studies in bivalves that showed that de novo synthesis and accumulation of HSPs peak around 48 h of recovery after heat shock (Brun et al., 2008; Clegg et al., 1998; Hamdoun et al., 2003). Therefore, in all subsequent experiments mRNA and/or protein expression levels were measured after 48 h of recovery at the acclimation temperature (12 ◦ C) following exposure to a designated stress temperature. For experimental exposures, oysters acclimated to 12 ◦ C in clean ASW (controls) or in ASW with 50 ␮g L−1 Cd (Cd-exposed) were subjected to acute temperature increase (1 ◦ C h−1 ). Such acute temperature increase was intended to mimic rapid heating during low tide in the intertidal zone (e.g. Sokolova et al., 2000; Hamdoun et al., 2003; Sokolova and Boulding, 2004). Once a respective target temperature was reached (20, 24, 28, 32, 36 or 40 ◦ C), oysters were kept at this temperature for 1 h, then transferred to their acclimation temperature (12 ◦ C) and allowed to recover for 48 h. After 48 h of recovery, oysters were dissected, and their gill and hepatopancreas tissues were immediately shock frozen and stored in liquid nitrogen until further analyses. 2.3. Determination of stress protein expression Expression of metallothionein (MT) mRNA was measured by quantitative real-time PCR (QRT-PCR). There are no commercially available antibodies specific for molluscan MTs, and several commercial antibodies developed against mammalian MTs failed to cross-react with oyster MTs in our pilot studies (data not shown). Earlier it has been shown that MT expression is primarily regulated at the level of transcription (Farzana et al., 2003; Rose et al., 2006) and that Cd-induced expression of MT mRNA is correlated with the protein levels (Vasconcelos et al., 2002; Knapen et al., 2007; Vergani et al., 2007). Therefore, in this study we have used mRNA expression to estimate induction of MTs by heat stress and Cd exposure. For QRT-PCR, specific primers were designed to amplify cDNA for total MTs (MTI and MTII) and ␤-actin using known C. virginica sequences (NCBI accession numbers AY331695.1, AY331705.1 and X75894.1). Because of the high sequence similarity and the duplicated ␤-domain in MTII (Tanguy and Moraga, 2001), we were unable to design specific primers to MTII that would amplify a single product; therefore, metallothionein I and II expression could not be measured separately. Instead, we designed consensus primers that amplified a single length PCR product from either MTI or MTII and thus determined mRNA expression of both metallothioneins simultaneously. Following primers were used: for MTs I and II: MTI + II-FW 5 -ggc tgt aaa tgt ggg gag aa-3 MTI + II-RV 5 -gag aac gcc tct cat tgg tc-3 for ␤-actin: Act-Cv-F437 5 -cac agc cgc ttc ctc atc ctc c-3 Act-Cv-R571 5 -ccg gcg gat tcc ata cca agg-3 . Quantitative RT-PCR was performed using a LightCycler® 2.0 Real Time PCR System (Roche Applied Science, Indianapolis, IN,

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USA) and QuantiTect SYBR Green PCR kit (Qiagen, Valencia, CA, USA) according to the manufacturers’ instructions as described in Ivanina et al. (2008). In each run, serial dilutions of a cDNA standard were amplified to determine amplification efficiency (Pfaffl, 2001), and an internal standard was included to test for amplification variability between the runs. Amplification efficiencies (E) were 2.05 + 0.04 (N = 7), and 2.03 + 0.04 (N = 8) for MTs I & II and ␤-actin, respectively. Expression of MTs was calculated relative to the expression of ␤actin and normalized against the internal standard as proposed by Pfaffl (2001): R=

EtCPt CPref Eref

where Et and Eref are amplification efficiencies for the target gene and the reference (␤-actin), respectively, and CPt and CPref are differences between crosspoints for fluorescence of the sample and the internal standard for the target gene and ␤-actin, respectively. Expression of heat shock proteins (HSPs) was determined by standard immunoblotting techniques. Briefly, gill and hepatopancreas tissues from experimental oysters were homogenized in ice-cold buffer (100 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton-X, 10% glycerol, 0.1% sodium dodecylsulfate (SDS), 0.5% deoxycholate, 0.5 ␮g mL−1 leupeptin, 0.7 ␮g mL−1 pepstatin, 40 ␮g mL−1 phenylmethylsulphonyl fluoride (PMSF), and 0.5 ␮g mL−1 aprotinin). The homogenate was sonicated three times for 10 s each (output 69 Watts, Sonicator 3000, Misonix, Farmingdale, NY, USA) and centrifuged at 14,000 × g for 5 min at 4 ◦ C. Protein content was measured in the supernatant using Bio-Rad Protein Assay kit according to the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA). Bovine serum albumin (BSA) was used as a standard. 30 ␮g of sample protein per lane was loaded onto 8% polyacrylamide gels and run at 100 mA for 2 h at room temperature. The resolved proteins were transferred onto a nitrocellulose membrane in 96 mM glycine, 12 mM Tris and 20% methanol (v/v) using a TransBlot semi-dry cell (Thermo Fisher Scientific Inc, Portsmouth, NH, USA). The membranes were blocked overnight in 5% non-fat milk in Tris-buffered saline, pH 7.6 (TBST), and probed with primary monoclonal antibodies against HSP70, HSP90 and HSP60 (MA3-007, Affinity Bioreagents, Golden, CO, USA; SPA-835 & SPA-805, Stressgen Bioreagents, Ann Arbor, MI, USA, respectively). After washing off the primary antibody, membranes were probed with the respective polyclonal secondary antibodies conjugated with horseradish peroxidase (Jackson Immunoresearch, West Grove, PA, USA) and proteins detected by enhanced chemiluminescence according to the manufacturer’s instructions (Pierce, Rockford, IL, USA). In this study, anti-HSP70 antibodies produced two and three bands in the expected size range (69–78 kDa) in control and heat-stressed oysters, respectively; two larger isoforms (ca. 72–78 kDa) were constitutively expressed whereas a smaller isoform (ca. 69 kDa) was expressed in heat or Cd-stressed oysters only (see Section 3 below). This agrees with earlier studies of HSP70 family in oysters showing expression of three HSP70 isoforms, one inducible and two cognate ones (Hofmann and Somero, 1995; Hamdoun et al., 2003; Piano et al., 2004 and references therein). Thus, we have adopted the HSP70 nomenclature used in previous studies on Crassostrea (Hamdoun et al., 2003) and termed the two cognate isoforms HSC72 and HSP77, and the inducible one–HSP69. In contrast to HSP70, anti-HSP60 and -HSP90 antibodies produced single bands of the expected size, and the respective proteins were constitutively expressed. Densitometric analysis of the signal was performed by GelDoc 2000 TM System with Quantity One 1D Analysis Software (Bio-Rad Laboratories Inc., Hercules, CA, USA). To ensure that the temperature-induced differences in HSP levels are not confounded with blot-to-blot variations, different exposure temperatures were

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always run on the same blot, and each blot included a control sample as an internal standard. In some experiments, resolution of HSC72 and HSC77 was not sufficient to quantify each of the isoforms separately, and therefore, both constitutive HSP70 isoforms were quantified together and called HSC72–77 in subsequent analyses. For each experimental group (i.e. combination of the experimental temperature and Cd level), HSP and MT levels were determined in gill and hepatopancreas tissues of five individual oysters. 2.4. Cadmium determination Gill and hepatopancreas samples were freeze-dried, weighed and digested in Teflon bottles with 52.5% nitric acid (trace metal grade, Fisher Scientific, Suwanee, GA, USA) using 3–4 cycles of microwave heating and cooling until the tissues were fully digested. Cd concentrations were determined with an atomic absorption spectrometer (PerkinElmer AAnalyst 800), equipped with a graphite furnace and Zeeman background correction. National Institute of Standards and Technology (NIST) oyster tissue (1566b) was analyzed with the samples to verify the metal analyses; the percent recoveries over all batches were 94.6 + 6.6% (mean + standard deviation). Tissue levels of Cd were determined in 5–13 Cd-exposed and control oysters from each population. 2.5. Statistics Effects of cadmium exposure and temperature on expression of MTs or HSPs was analyzed for each oyster population and tissue type using linear model ANOVA or split-plot repeated measures ANOVA (for mRNA and protein expression, respectively) after testing the assumptions of normality of data distribution and homogeneity of variances. Blot number was used as a repeated measures variable for immunoblotting protein analyses in order to account for blot-to-blot signal variability, and a control sample was included on each gel as an internal standard to enable comparisons between different exposure temperatures and/or between control and Cd-exposed oysters within each population and tissue type. Because of the limited amount of sample, it was not possible to use the same internal standard on all gels, and the internal standards were different for each studied population; thus, the levels of HSP protein expression can be compared between different temperatures and between control and Cd-exposed oysters, but not across different populations. Tukey’s Honest Significant Difference (HSD) tests were used for post-hoc comparisons of sample means. Statistical analyses were performed using SAS 9.1.3 software (SAS Institute, Cary, NC, USA), and differences were considered significant if the probability for Type II error was less than 0.05.

Fig. 1. Cd levels in gill (A) and hepatopancreas (B) tissues of control oysters and oysters exposed for 45–50 days to 50 ␮g L−1 Cd. Populations—WA: Washington, NC: North Carolina and TX: Texas oysters. Control oysters were maintained in clean ASW, and Cd-exposed—in ASW with 50 ␮g L−1 Cd; both groups were maintained at 12 ◦ C. Vertical bars represent standard errors. Asterisks denote values significantly different from the respective controls (non-Cd-exposed oysters) (P < 0.05). Different letters denote values that significantly vary between populations; i.e. if columns do not share the same letter, the interpopulational differences within control or Cd-exposed groups are significantly different (P < 0.05). N = 5–13.

all studied tissues/populations except hepatopancreas of WA oysters where a sixfold MT induction was not statistically significant due to the high individual variation (Fig. 2A and C). Cadmium exposure resulted in strongly elevated expression of MT in tissues of oysters from all three studied populations (Fig. 2B and D and Table 1). At the control temperature (12 ◦ C), Cd-induced expression of MT was the highest in TX oysters (39–94fold compared to the oysters not exposed to Cd), followed by NC oysters (36–48-fold), and was the lowest in WA oysters (5.6–33fold). Notably, acute warming failed to induce a further increase of MT expression in Cd-exposed oysters (with the exception of hepatopancreas tissue in NC oysters, where a 3.7-fold induction was found at 40 ◦ C) (Fig. 2B and D).

3. Results 3.3. Heat shock protein expression 3.1. Cadmium accumulation Exposure to 50 ␮g L−1 Cd resulted in a significant accumulation of Cd in gill and hepatopancreas tissues (Fig. 1). Accumulated Cd burdens were similar in the gills of oysters from all three studied populations (Fig. 1A). In contrast, Cd levels in hepatopancreas were higher in NC oysters compared to their TX or WA counterparts (Fig. 1B). 3.2. Metallothionein expression In control oysters from all three studied populations, acute temperature stress induced MT expression. Notably, exposure to 28 ◦ C had no significant effect on MT expression, while heating to 40 ◦ C resulted in a 3.5–7.5-fold induction of MT which was significant in

HSP69 (an inducible isoform of HSP70 family) was not expressed in tissues of the control oysters at their acclimation temperature (12 ◦ C) but was significantly induced by elevated temperatures in all three studied populations (Figs. 3 and 4). Interactions between the effects of Cd exposure and temperature on HSP69 expression were significant in all studied populations (Table 1) indicating that the response to warming significantly differed between control and Cd-exposed oysters. In control oysters, a significant induction was seen at the temperatures at or above 24 ◦ C (in hepatopancreas tissues of WA oysters and gills of NC ones) or 20 ◦ C (all other tissues/populations) (Fig. 4). In contrast, in Cd-exposed oysters HSP69 was expressed at a considerable level already at the acclimation temperature, 12 ◦ C (Fig. 4). Exposure to elevated temperatures failed to further induce HSP69 in Cd-exposed oysters except for a

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Fig. 2. Effects of Cd exposure and heat stress on metallothionein (MTI and MTII) expression in gill (A and B) and hepatopancreas (C and D) of oysters from different populations. (A and C) Oysters maintained in clean ASW and (B and D) Cd-exposed oysters (45–50 days in ASW with 50 ␮g L−1 Cd). Control (non-heat-stressed and non-Cd-exposed) oysters are represented by 12 ◦ C columns on panes (A and C). MT levels were measured using real-time PCR and normalized to the actin mRNA levels. Vertical bars represent standard errors. Asterisks (in B and D) denote values of MT expression in Cd-exposed oysters that are significantly different from their counterparts kept in the clean ASW (shown in panes (A and C)) at the same temperature (P < 0.05). Different letters denote values that significantly vary between different exposure temperatures within the same population so that if columns do not share the same letter, the respective values are significantly different (P < 0.05). Populations—WA: Washington, NC: North Carolina and TX: Texas oysters. N = 5.

Table 1 ANOVA: effects of temperature and Cd exposure on expression of HSP69 protein and metallothionein mRNA (MT) in eastern oysters Crassostrea virginica from three geographically distant populations (Washington (WA), North Carolina (NC) and Texas (TX) states). ANOVA factor effects Cd exposure

Temperature

Cd × temperature

Gills HP Gills HP Gills HP

F1,20 = 3.28, P = 0.086 F1,22 = 5.42, P = 0.029 F1,21 = 8.20, P = 0.009 F1,11 = 18.83, P = 0.001 F1,15 = 3.06, P = 0.101 F1,23 = 1.32, P = 0.262

F6,54 = 20.13, P < 0.0001 F6,50 = 2.28, P = 0.051 F6,46 = 16.90, P < 0.0001 F6,46 = 11.23, P < 0.0001 F6,46 = 13.20, P < 0.0001 F6,46 = 5.33, P = 0.0003

F6,54 = 18.99, P < 0.0001 F6,50 = 8.29, P < 0.0001 F6,46 = 9.81, P < 0.0001 F6,46 = 14.00, P < 0.0001 F6,46 = 6.51, P < 0.0001 F6,46 = 3.03, P = 0.014

Gills HP Gills HP Gills HP

F1,24 = 41.6, P < 0.0001 F1,24 = 1.39, P = 0.250 F1,24 = 19.30, P = 0.002 F1,24 = 13.70, P = 0.001 F1,24 = 54.5, P < 0.0001 F1,24 = 27.5, P < 0.0001

F2,24 = 0.74, P = 0.488 F2,24 = 2.09, P = 0.146 F2,24 = 2.65, P = 0.091 F2,24 = 4.87, P = 0.017 F2,24 = 0.80, P = 0.462 F2,24 = 1.57, P = 0.227

F2,24 = 0.88, P = 0.429 F2,24 = 0.51, P = 0.606 F2,24 = 2.14, P = 0.135 F2,24 = 4.34, P = 0.024 F2,24 = 3.28, P = 0.055 F2,24 = 1.92, P = 0.168

HSP69 WA Population

NC TX

MT WA Population

NC TX

Significant effects are highlighted in bold.

slight increase at 40 ◦ C in gills from WA and NC oysters (Fig. 4). Notably, in most cases HSP69 expression at elevated temperatures was considerably lower in Cd-exposed oysters compared to their control counterparts (Table 1 and Fig. 4). Expression of cognate isoforms of HSP70 family (HSC72 and HSC77), HSP90 and HSP60 did not show a consistent pattern of upregulation in response to Cd exposure or temperature stress. For these HSPs, effects of Cd exposure were not statistically significant in most of the studied populations (P > 0.05), with the exception of HSC72–77 in WA and TX oysters and HSP90 in TX oysters, which showed significantly lower levels in Cd-exposed oysters as com-

pared to their control counterparts (Fig. 5). Temperature had no significant effect on the expression of HSC72–77, HSP90 or HSP60 in most of the studied populations (P > 0.05; data not shown) with the exception of HSP60 from gill tissues of WA oysters and HP tissues of TX oysters where a slight but significant increase in the mid-temperature range was observed (Fig. 6). 4. Discussion Our study showed that exposure to heat or Cd resulted in a strong upregulation of the inducible isoform of the HSP70 fam-

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Fig. 3. Representative immunoblots of HSPs in eastern oysters. (A) HSP70 family (top band: HSC72–77, bottom band: HSP69), (B) HSP90 and (C) HSP60. Representative immunoblots from gill tissues of non-Cd-exposed oysters are given.

ily (HSP69) and metallothioneins (MTs) in all studied populations of C. virginica, and that the heat-induced expression patterns of these two stress proteins differed between control and Cd-exposed oysters. MT expression was considerably elevated in gill and hepatopancreas of Cd-exposed oysters as compared to their control counterparts that agrees with the results of earlier studies showing Cd-induced MT accumulation in mollusks (review in: Roesijadi, 1996). Interestingly, MTs were also noticeably upregulated in response to acute heat stress (40 ◦ C) in control C. virginica. A similar modest but significant increase in MT expression in response to heat stress was earlier shown in other aquatic ectotherms including fish and mollusks (Carpené et al., 1992; Van Cleef-Tödt et al., 2001; Piano et al., 2004). Although both stressors resulted in elevated MT levels, MTs were more strongly induced by Cd than by temperature stress (to up to 38–94-fold compared with 3.5–7.5-fold, respectively) consistent with their predominant role in metal detoxification. While the mechanisms of MT induction by Cd via transcriptional activation by the metal-responsive transcription factor MTF-1 are well known (Andrews, 2000; Saydam et al., 2003 and references therein), the mechanisms and physiological significance of the heat-induced upregulation of MTs are not fully understood. Overexpression of MTs during the heat shock may result from a direct activation of the MT promoter by heat shock transcription factor (HSF) as shown in mammalian models (Tamai et al., 1994; Liu and Thiele, 1996) or from the indirect effects of the heat-induced oxidative stress (Abele et al., 2002, 2007) via the activation of an antioxidant response element in MT promoter or through the redox-mediated changes in intracellular free metal pools activating MTF-1 (Andrews, 2000; Saydam et al., 2003). Irrespective of the exact mechanisms of the MT induction by heat stress, our data show that MTs are an integral part of the general stress response in aquatic ectotherms and cannot be viewed as specific biomarkers of environmental metal exposure. Moreover, potential modulation of MTs expression by environmental factors such as

temperature, salinity or oxidative stress (Carpené et al., 1992; Van Cleef-Tödt et al., 2001; Piano et al., 2004; Lucu et al., 2008; this study) must be taken into account before elevated MT levels can be interpreted as a sign of metal exposure in aquatic ectotherms. Interestingly, the expression patterns of MTs in response to heat stress were different in control and Cd-exposed oysters. In contrast to the control oysters, in which the exposure to extreme temperatures (40 ◦ C) resulted in a significant upregulation of MTs, MT levels did not change in response to temperature in their Cd-exposed counterparts. This may indicate that in Cd-exposed oysters, the transcriptional activation of MTs has reached its maximum capacity and no further increase was possible in response to heat stress. Alternatively, this may suggest that high MT levels in Cd-exposed oysters were sufficient to counteract the negative effects of elevated temperature on the cellular redox balance and to effectively bind metals released from the intracellular storage sites by the heatinduced oxidative stress (Andrews, 2000; Saydam et al., 2003). It is worth noting that our earlier studies in C. virginica showed that combined exposure to Cd and elevated temperature (28 ◦ C) resulted in elevated oxidative damage in oyster tissues (Lannig et al., 2006) suggesting that high MT levels cannot fully prevent disturbances to the cellular redox status during this combined stress exposure; however, whether or not it is associated with elevated levels of intracellular free metals, is not known. Our current study does not allow distinguishing between these two alternative explanations, and further research is needed to determine whether the absence of heat-induced MT upregulation in Cd-exposed oysters reflects a capacity limitation of the transcriptional response or the sufficient level of protection rendered by elevated MT levels. HSP69 expression was strongly upregulated in response to a long-term exposure to 50 ␮g L−1 Cd in oysters from all three studied populations indicating that Cd exposure induces damage to intracellular proteins. Other HSPs analyzed in this study (including cytosolic HSC72–77, HSP90 and mitochondrial HSP60) were not consistently upregulated by Cd stress. These findings agree with

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Fig. 4. Effects of Cd exposure and heat stress on the protein expression of inducible HSP69 isoform in gill (A, C and E) and hepatopancreas (B, D and F) of oysters from different populations. ASW: oysters maintained in clean ASW without Cd addition, Cd: oysters exposed to 45–50 days to 50 ␮g L−1 Cd in ASW. HSP69 levels were below the detection limit in the control oysters (non-Cd-exposed and non-heat-stressed) oysters at 12 ◦ C. Populations—WA: Washington, NC: North Carolina and TX: Texas oysters. Vertical bars represent standard errors. Asterisks and daggers denote values of HSP69 expression that are significantly different from the respective values at 12 ◦ C for non-Cd-exposed or Cd-exposed oysters, respectively (P < 0.05). Optical density of the signal is reported in arbitrary units (A.U.). N = 5.

the results of earlier studies in bivalves showing a major role of inducible HSP70 isoforms in protecting against toxicity of metals including Cd (Piano et al., 2004; Franzellitti and Fabbri, 2005, 2006; Ivanina et al., 2008). In contrast to chronic low-level exposures such as used in the present study, acute exposures to high Cd concentrations may also result in upregulation of HSP90 and/or HSP60 in addition to HSP69 (Sanders and Martin, 1991; Sanders et al., 1994; Choi et al., 2008; Ivanina et al., 2008) suggesting that these chaperones may play ancillary roles in cell protection of mollusks when the capacity of the inducible HSP70 isoforms is overwhelmed. As expected, HSP69 was also strongly upregulated in response to the heat stress in control oysters; this agrees with earlier studies in aquatic ectotherms including bivalves (Roberts et al., 1997; Chapple et al., 1998; Hofmann et al., 2002; Kregel, 2002; Hamdoun et al., 2003; Piano et al., 2004; Hofmann, 2005; Brun et al., 2008). Interestingly, in those bivalves in which both inducible and constitutive isoforms of HSP70 are found, heat exposure typically leads to overexpression of the inducible but not cognate isoforms (Clegg et al., 1998; Hamdoun et al., 2003; Franzellitti and Fabbri, 2005).

This study supports this pattern in that only the inducible HSP69 but not the constitutive HSP72–77, HSP90 or HSP60 showed consistent upregulation in response to heat stress in oysters. Induction temperature (Ton ) for HSP69 was about 8 ◦ C above the acclimation temperature, with a significant induction of HSP69 at 20 ◦ C in oysters from all three studied populations (note that this is a conservative estimate because lower temperatures have not been tested). This value is within the range of induction temperatures for HSP70 in aquatic ectotherms that typically vary from 7 to 12 ◦ C above the acclimation temperature (Tirard et al., 1995; Roberts et al., 1997; Tomanek and Somero, 1999; Buckley et al., 2001; Hamdoun et al., 2003; Fangue et al., 2006). Notably, in our study, the maximum expression of HSPs was often observed at intermediate temperatures and declined at the extremely high temperature; such curtailed protein synthesis was found in other bivalves at extreme temperatures and likely reflects damage to proteosynthetic machinery (Hofmann and Somero, 1996; Hofmann, 2005). There were no significant interpopulational differences in Ton for HSP69 induction or the temperatures at which HSP69 expression was

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Fig. 5. Effects of Cd exposure and heat stress on the protein expression of constitutive HSP 90 and HSC72–77 isoforms in oysters. ASW: oysters maintained in clean ASW without Cd addition, Cd: oysters exposed to 45–50 days to 50 ␮g L−1 Cd in ASW. Control (non-Cd-exposed and non-heat-stressed) oysters are represented by the white12 ◦ C column in each graph. Populations—WA: Washington, NC: North Carolina and TX: Texas oysters. (A and B) HSP90 in gill and hepatopancreas tissues of TX oysters, respectively; (C and D) HSC72–77 in gill and hepatopancreas tissues of WA oysters, respectively; (E) HSC72–77 in hepatopancreas tissues of TX oysters. P values for the effects of Cd exposure (as determined by the split-plot repeated measures ANOVA) are given; in all other studied populations and tissue types, Cd effects on HSC72–77 and HSP90 expression were not significant (P > 0.05; data not shown). Temperature had no significant effect on HSC72–77 or HSP90 expression in oysters (ANOVA, P > 0.05). Optical density of the signal is reported in arbitrary units (A.U.). Vertical bars represent standard errors, N = 5.

Fig. 6. Effects of Cd exposure and heat stress on the protein expression of HSP60 in oysters. ASW: oysters maintained in clean ASW without Cd addition, Cd: oysters exposed to 45–50 days to 50 ␮g L−1 Cd in ASW. Control (non-Cd-exposed and non-heat-stressed) oysters are represented by the white12 ◦ C column in each graph. Populations—WA: Washington, NC: North Carolina and TX: Texas oysters. (A) Gill tissues of WA oysters; (B) hepatopancreas tissues of TX oysters. P values for the effects of temperature (as determined by the split-plot repeated measures ANOVA) are given. In all other studied populations and tissue types, temperature had no significant effect on HSP60 expression (P > 0.05; data not shown). Cd exposure had no significant effect on HSP60 expression in oysters (ANOVA, P > 0.05). Optical density of the signal is reported in arbitrary units (A.U.). Vertical bars represent standard errors. N = 5.

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maximal suggesting that laboratory acclimation at the common temperature (12 ◦ C) could override potential differences (if any) in the HSP69 expression patterns that might have arisen due to the thermal adaptations of oyster populations from different climates. It is worth noting that Cd exposure apparently affected the ability of oysters to mount adequate HSP response to acute warming. Indeed, in most populations, HSP69 levels were lower in Cd-exposed oysters compared to their control counterparts at all tested temperatures except the acclimation temperature, 12 ◦ C. In some populations, levels of other molecular chaperones such as HSC72–77 and HSP90 were also lower in oysters exposed to the combined Cd and heat stress as compared to their control counterparts at the same temperatures. Since it is unlikely that Cd exposure reduces the degree of protein damage thus counteracting the effects of heat stress (in fact, elevated HSP69 levels in Cd-exposed oysters at 12 ◦ C suggest the opposite), the most parsimonious explanation of this decrease in HSP levels is an impaired ability of HSP synthesis in oysters during the combined exposure to heat and Cd. The mechanisms responsible for this impairment of stress protein response are currently unknown. One of the possible explanations is the negative effect of Cd on cellular biosynthetic capacity such as transcription and/or translation processes (Pytharopoulou et al., 2006). Indeed, a decrease in synthesis of HSP70 isoforms along with an overall inhibition of de novo protein synthesis was reported during acute exposures to high metal concentrations in bivalves (Pacific oysters Crassostrea gigas and blue mussels Mytilus edulis), earthworms Lumbricus terrestris and a green algae Enteromorpha intestinalis (Steinert and Pickwell, 1988; Lewis et al., 2001; Nadeau et al., 2001). However, our earlier studies in C. virginica showed that exposure to 50 ␮g L−1 Cd for up to 40 days resulted in an increased rate of cellular protein synthesis (Cherkasov et al., 2006) suggesting that Cd-induced inhibition of the global protein synthesis is unlikely to fully account for the observed decrease in HSP levels. Other possible (not mutually exclusive) explanations are cellular energy deficiency due to the mitochondrial dysfunction in oysters exposed to the combined temperature and Cd stress that can limit the amount of ATP available for HSP synthesis and/or function (Sokolova, 2004; Cherkasov et al., 2006; Lannig et al., 2006, 2008; Sokolova and Lannig, 2008), or specific interference of Cd with transcriptional or translational pathways for HSP production. Irrespective of the exact mechanisms, an observed decrease in HSP levels may negatively affect cellular function under the acute heat stress in Cd-exposed oysters thus contributing to the reduced thermotolerance in Cd-exposed oysters. As a corollary, the current study demonstrates that the concomitant exposure to acute heat and Cd stress can result in an overload and/or partial impairment of cellular protective mechanisms in oysters that can be expected to negatively affect their tolerance of temperature extremes (e.g. such as occurs during the summer low tide in the intertidal zone). Earlier studies also showed a reduced tolerance to a long-term exposure to moderately elevated temperatures in Cd-exposed oysters due to the reduced aerobic scope and the resultant shift of the thermal tolerance limits to the lower values (review in: Sokolova and Lannig, 2008). Taken together, this decrease in tolerance to both acute and long-term exposure to elevated temperatures may have important implications for survival of oysters in polluted estuaries during tidal and seasonal temperature fluctuations in the intertidal zone and/or during the global climate change.

Acknowledgements The authors wish to thank John R. Schwarz (Seafood Safety Laboratory, Texas A&M University at Galveston, TX) and James

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