Immunomodulation by the interactive effects of cadmium and hypercapnia in marine bivalves Crassostrea virginica and Mercenaria mercenaria

Fish & Shellfish Immunology 37 (2014) 299e312

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Immunomodulation by the interactive effects of cadmium and hypercapnia in marine bivalves Crassostrea virginica and Mercenaria mercenaria Anna V. Ivanina, Chelsea Hawkins, Inna M. Sokolova* Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 December 2013 Received in revised form 17 February 2014 Accepted 20 February 2014 Available online 2 March 2014

Estuarine organisms are exposed to multiple stressors including large fluctuations in partial pressure of carbon dioxide ðPCO2 Þ and concentrations of trace metals such as cadmium (Cd) that can affect their survival and fitness. Ocean acidification due to the increasing atmospheric PCO2 leads to a decrease in pH and shifts in the carbonate chemistry of seawater which can change bioavailability and toxicity of metals. We studied the interactive effects of PCO2 and Cd exposure on metal levels, metabolism and immune-related functions in hemocytes of two ecologically and economically important bivalve species, Mercenaria mercenaria (hard shell clam) and Crassostrea virginica (Eastern oyster). Clams and oysters were exposed to combinations of three PCO2 levels (w400, 800 and 2000 matm PCO2 , corresponding to the present day conditions and the projections for the years 2100 and 2250, respectively) and two Cd concentrations (0 and 50 mg l1) in seawater. Following four weeks of exposure to Cd, hemolymph of both species contained similar Cd levels (50e70 mg l1), whereas hemocytes accumulated intracellular Cd burdens up to 15e42 mg l1, regardless of the exposure PCO2 . Clam hemocytes had considerably lower Cd burdens than those of oysters (0.7e1 ng 10 6 cells vs. 4e6 ng 106 cells, respectively). Cd exposure suppressed hemocyte metabolism and increased the rates of mitochondrial proton leak in normocapnia indicating partial mitochondrial uncoupling. This Cd-induced mitochondrial uncoupling was alleviated in hypercapnia. Cd exposure suppressed immunerelated functions in hemocytes of clams and oysters, and these effects were exacerbated at elevated PCO2 . Thus, elevated PCO2 combined with Cd exposure resulted in decrease in phagocytic activity and adhesion capacity as well as lower expression of mRNA for lectin and heat shock protein (HSP70) in clam and oyster hemocytes. In oysters, combined exposure to elevated PCO2 and Cd also led to reduced activity of lysozyme in hemocytes and hemolymph. Overall, our study shows that moderately elevated PCO2 (w800e2000 matm PCO2 ) potentiates the negative effects of Cd on immunity and thus may sensitize clams and oysters to pathogens and diseases during seasonal hypercapnia and/or ocean acidification in polluted estuaries. Published by Elsevier Ltd.

Keywords: Ocean acidification Metals Immune function Hemocytes Bivalves

1. Introduction Diseases and parasites play an important role in the survival and population dynamics of marine mollusks, and are considered the major risk factor in shellfish aquaculture [6,14,21]. Human activities can negatively affect the balance of naturally coevolved hosteparasite and hostepathogen systems by introducing novel disease agents and/or leading to deterioration of the environmental quality that affects the immunity of marine organisms [14,46,87,95]. Both natural

* Corresponding author. Department of Biology, University of North Carolina at Charlotte, Charlotte, NC 28223, USA. Tel.: þ1 704 687 8532; fax: þ1 704 687 3128. E-mail address: [email protected] (I.M. Sokolova). http://dx.doi.org/10.1016/j.fsi.2014.02.016 1050-4648/Published by Elsevier Ltd.

and anthropogenic environmental stressors such as variations in temperature, salinity and pollutants can modulate the resistance of mollusks to parasites and diseases [24,30,51,52,76,95]. In recent decades, ocean acidification caused by the increasing atmospheric levels of carbon dioxide (CO2) due to the human activities has become an urgent problem in the ocean. Elevated CO2 levels in the surface waters of the ocean lead to a decrease in seawater pH, shifts in the carbonate chemistry and can affect bioavailability and toxicity of pollutants [11,83,91]. Ocean acidification has a strong impact on acidebase balance, energy metabolism and biomineralization of marine organisms [58,70,92] but its impact on the immune functions is not yet well understood [10,45,56,79] hampering our ability to predict the susceptibility of marine mollusks to parasites and diseases in the changing ocean.

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Trace metals such as cadmium (Cd) are common persistent pollutants in estuarine and coastal environments. The total input of Cd into the marine ecosystems is estimated at w8000 t year1 (about half of which results from the human activities), and the input of Cd into the ocean exceeds its output leading to the slow accumulation of Cd in water and sediments [35]. Cd is a known immunosuppressant in a variety of vertebrates and invertebrates including mollusks [18,19,22,38,85,107]. Hemocytes (blood cells) play a key role in innate immunity and wound and shell repair of mollusks [54,86,93]. These cells are sensitive to the negative effects of Cd as shown by the Cd-induced decreases in their viability, phagocytic activity and ability to kill pathogens [5,17,19,30,103,105,107]. Recent studies showed that variation of the environmental and intracellular pH impacts accumulation of Cd, modulates Cd-induced oxidative stress and mitochondrial toxicity in mollusks [61,65]. However, the interactive effects of pH changes (such as expected during ocean acidification and seasonal hypercapnia in estuaries) and Cd on the immune functions of mollusks are not well known and require further investigation. The goal of this study was to determine the interactive effects of the environmentally relevant levels of cadmium (50 mg l1) and PCO2 on the immunity of two species of estuarine bivalves, Mercenaria mercenaria (hard clams) and Crassostrea virginica (Eastern oysters). These bivalves inhabit the intertidal and upper subtidal zones in the western Atlantic, and serve as ecosystem engineers and important fishery and aquaculture resources. Clams and oysters can bioaccumulate high levels of metals in their tissues from estuarine waters and sediments [47,81,99] and are exposed to a broad range of pH and CO2 fluctuations in their environments [26,98]. Thus, these bivalves are excellent model species to investigate the interactive effects of trace metals and pH on the immune function. In order to obtain a comprehensive picture of Cd- and PCO2 -induced immunomodulation, we assessed the interactive effects of Cd and PCO2 levels on quantity, metabolic activity and key defense-related functions of hemocytes of clams and oysters. Specifically, we determined the effects of four weeks exposure to different Cd (50 mg l1 Cd) and CO2 levels (w400, 800 and 2000 matm PCO2 ) on hemocyte count as well as phagocytosis, adhesion ability and activity of lysozyme that play a key role in recognition and destruction of parasites and pathogens by bivalve hemocytes [3,33,34,54,110]. Because immune function is an energy-demanding process [39], the metabolic activity, mitochondrial capacity and proteosynthetic activity (using cellular RNA and protein content as indices) was measured in hemocytes of clams and oysters. We have also determined mRNA expression of key immune-related genes including humoral factors (C-type lectins involved in non-self-recognition, and defensin, a broad-range antimicrobial defense peptide) [101,112], and integrin, a protein which plays a key role in cell adhesion [96,109]. Expression of mRNA levels for metal binding proteins (metallothioneins) and molecular chaperones (heat shock protein 70) was measured to assess the general stress response of bivalve hemocytes exposed to different Cd and CO2 levels. This assessment of the metabolic and immunityrelated functions of hemocytes in response to elevated PCO2 and Cd stress provides insights into the potential impacts of environmental hypercapnia (such as observed during seasonal hypercapnia in estuaries and expected during ocean acidification) on immunity and health of bivalves from polluted estuaries. 2. Materials and methods 2.1. Chemicals Unless otherwise indicated, all chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA) or Fisher Scientific (Pittsburg, PA, USA) and were of analytical grade or higher.

2.2. Animal maintenance The eastern oysters, C. virginica, and the hard clams, M. mercenaria, were obtained from a local shellfish supplier (Inland Seafood, Charlotte, NC). Animals were acclimated for 10 days in tanks with recirculating artificial seawater (ASW; Instant OceanÒ, Kent Marine, Acworth, GA, USA) at 22  1  C and 30  1 practical salinity units (PSU) salinity and aerated with the ambient air. After the preliminary acclimation, clams and oysters were exposed for four weeks to different metal and CO2 levels in a full 3  2 experimental design. Four weeks is considered sufficient time to achieve full acclimation to experimental conditions and physiological steady-state in mollusks [68,77]. Three CO2 levels were used that were representative of the present-day conditions (PCO2 w400 matm; normocapnia) and atmospheric CO2 concentrations predicted by one of the moderate scenarios of the Intergovernmental Panel for Climate Change [59] for the years 2100 (w800 matm PCO2 ; moderate hypercapnia) and 2250 (w2000 matm PCO2 ; extreme hypercapnia). It is worth noting that seasonal hypercapnic events in the present-day habitats of clams and oysters in the estuaries of the southeastern United States can exceed the predictions of the ocean acidification scenarios; however, such events have typically shorter duration (hours to days, rarely weeks) [20,80,98]. Target PCO2 levels were achieved by bubbling seawater with gas mixtures containing different CO2 concentrations. For normocapnia, the ambient air was used, while for hypercapnia (w800 and 2000 matm PCO2 ), the ambient air was mixed with 100% CO2 (Roberts Oxygen, NC, USA) in fixed proportions using precision mass flow controllers (Colee Parmer, IL, USA). Flow rates of air or air-CO2 mixtures were set up to maintain the target steady-state pH values during the exposures. To avoid potential variations in water chemistry, ASW for all exposures was prepared using the same batch of Instant OceanÒ salt. At each CO2 level, bivalves were maintained either in clean ASW (control) or in ASW supplemented with of 50 mg l1 Cd added as CdCl2. This Cd concentration is within the range of Cd levels found in polluted estuaries (15e80 mg l1 in water and 5e460 mg kg1 dry mass in the sediments) [40,53,57,90]. In all treatments, clams and oysters were co-exposed in the same tanks, and two replicate tanks were set for each experimental treatment. Throughout the experiments, animals were fed ad libitum every other day with a commercial algal blend (5 ml per tank) containing Nannochloropsis oculata, Phaeodactylum tricornutum and Chlorella sp. with a cell size of 2e20 mm (DT’s Live Marine Phytoplankton, Sycamore, IL, USA). Each tank contained 10e15 animals in 28 l of seawater. Water was completely changed twice a week using ASW pre-equilibrated with the respective gas mixtures to achieve the required treatment pH. In order to avoid Cd depletion, a staticrenewal design was used in Cd-exposed tanks, with Cd supplementation to the nominal concentration of 50 mg l1 during each water change. Experimental tanks were checked for mortality daily, and animals that gaped and did not respond to a mechanical stimulus were recorded as dead and immediately removed. The cumulative mortalities over the exposure period were below 5% in all experimental groups. Seawater temperature, salinity and pH were measured daily and adjusted as needed. pH was measured using a pH electrode (pH meter Model 1671, Jenco Instruments, San Diego, CA, USA) calibrated with National Institute of Standards and Technology standard pH buffer solutions (National Bureau of Standards, NBS standards, Fisher Scientific). Water samples were collected weekly to determine carbonate chemistry parameters as described elsewhere [8]. Briefly, water samples were collected in air-tight containers, poisoned with mercuric chloride and stored at þ4  C until further analysis. Total dissolved inorganic carbon (DIC) was measured by the Nutrient Analytical Services (Chesapeake Biological Laboratory,

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Solomons, MD, USA). Seawater carbonate chemistry parameters (PCO2 , total alkalinity, and the saturation state (U) for calcite and aragonite) were calculated using CO2SYS software [75] using barometric pressure values, as well as DIC, pH, temperature and salinity values for the respective samples (Table 1). For calculations, we used NBS scale for seawater pH, constants from Millero et al. [82], KSOe 4 constant from Dickson [44], and concentrations of silicate and phosphate for Instant OceanÒ seawater (0.17 mmol kg1 and 0.04 mmol kg1, respectively). 2.3. Hemolymph collection After four weeks of experimental exposures, hemolymph was extracted from the adductor muscle of clams and oysters using sterile 10-ml syringe with a 21 gauge needle containing 1 ml icecold filtered artificial seawater (ASW) to prevent aggregation of the hemocytes. For each biological replicate, hemolymph was pooled from three oysters or from two clams; a total of 5e6 biological replicates was used for each species and experimental treatment. Concentration of the cells was determined using a Brightline hemacytometer. _O Þ 2.4. Cellular oxygen consumption rates ðM 2 Hemocyte-containing samples of hemolymph (1.6e10.5  106 cells per ml) were placed immediately after withdrawal into water-jacketed chambers at 20  C (OX1LP-1 ml, Qubit Systems, Kingston, ON, Canada), _ and M O2 was determined using Clarke-type oxygen electrodes. Twopoint calibration with air-saturated seawater and saturated Na2SO3 solution was conducted prior to each measurement. We determined _ total respiration rate ðM O2 -total Þ of hemocytes and the respiration rate in the presence of 3 mg ml1 oligomycin (a specific inhibitor of mitochondrial F0,F1-ATPase). Respiration rate in the presence of oligomycin _ ðM O2 -oligo Þ is an estimate of the energy expenditure on the mitochondrial proton leak, a sum of futile proton and cation cycling that dissipates mitochondrial protonmotive force without the concomitant ATP synthesis [15,16]. The rate of the proton leak therefore can be viewed as an indicator of mitochondrial inefficiency [15,16]. Non_ mitochondrial respiration ðM O2 -KCN Þ was determined in hemocyte suspensions in the presence of a specific inhibitor of cytochrome c oxidase (100 mmol l1 KCN). In clams, 200 mmol l1 salicylhydroxamic acid (SHAM) was added to KCN to inhibit alternative oxidase (AOX) in the mitochondria; no SHAM addition was needed for oyster cells that do not exhibit AOX activity (data not shown). Cellular respiration rates were corrected for electrode drift and expressed as _ mmol O2 min1 106 cells. Total respiration ðM O2 -total Þ, mitochondrial

301

_ _ _ respiration ðM O2 -mito ¼ M O2 -total  M O2 -KCN Þ and mitochondrial _ _ _ O -KCN Þ were compared in heproton leak ðM ¼ M  M O2 -leak O2 -oligo 2 mocytes of oysters and clams exposed to different CO2 and Cd levels. 2.5. Phagocytosis Phagocytosis assay was performed using a modified procedure published elsewhere [36]. Neutral Red stained, heat-stabilized zymosan (Sigma Aldrich, St. Louis, MO, USA) suspended in ASW was added to hemocytes at the final concentration of 200 zymosan particles per hemocyte. Hemocytes were incubated for 30 min at room temperature (RT), centrifuged at 1000 g for 10 min and washed once with ASW to remove extracellular zymosan. Cell-free suspensions of zymosan at known concentrations were used as standards for calibration. Acetic acid (1% in 50% ethanol) was added to hemocytes or zymosan standards to solubilize the Neutral Red and incubated for 5 min. The optical density (OD) was then read at 550 nm on a microplate spectrophotometer (Multiscan GO, Thermo Scientific, Waltham, MA, USA). This assay estimates the average phagocytic activity of the hemocyte population which depends on both the relative proportion of phagocytic cells and the number of zymosan particles ingested by each cell. Results are expressed as the number of ingested zymosan particles per 100 cells. 2.6. Adhesion capacity Isolated hemocytes (106 cells) were placed in 1 ml of ASW in the wells of a 12-well plate (Costar, Corning) and incubated for 2 h at RT. After the incubation, ASW was collected and the wells were surface-washed once with 1 ml of ASW. The ASW was pooled and centrifuged for 10 min 1000 g to collect non-adhered cells. The pellet was resuspended in 1 ml ASW, and the cells counted using a Brightline hemacytometer (Sigma Aldrich, St. Louis, MO, USA). The adhesion capacity was estimated as the percentage of adhered hemocytes in each sample. 2.7. Lysozyme activity Lysozyme activity was quantified in the hemocytes and cell-free hemolymph. Hemolymph samples were centrifuged at 1000 g for 10 min. The supernatant representing cell-free hemolymph was collected and used for determining the lysozyme activity in hemolymph. Hemocytes were washed once with the ice-cold ASW, frozen and stored at 80  C. Immediately before analysis, 500 ml of distilled water was added to the hemocytes to lyse the cells, and the cells were sonicated (1 min at output 69 W, Sonicator 3000, Misonix,

Table 1 Summary of water chemistry parameters during experimental exposures. Salinity, temperature, pHNBS and dissolved inorganic carbon (DIC) were measured in water samples collected during the exposure. Other parameters are calculated using CO2SYS. Data is represented as means  S.E.M. Ucalcite and Uaragonite e the saturations states for calcite and aragonite, respectively. The same batch of Instant OceanÒ sea salt was used throughout the course of the experiment with an average total alkalinity (TA) of 3098.40  83.13 mmol kg1 SW. N ¼ 5 for DIC and TA, and N ¼ 50e58 for other parameters. Nominal PCO2

Salinity Temperature,  C pHNBS PCO2 , matm

Ucalcite Uaragonite

Control exposure

Cadmium exposure

400 matm

800 matm

2000 matm

400 matm

800 matm

2000 matm

30.34 0.07 20.19 0.02 8.19 0.00 552.22 6.98 5.84 0.05 3.75 0.03

30.10 0.05 20.07 0.03 7.93 0.00 1075.54 6.12 3.49 0.02 2.25 0.01

30.21 0.58 20.37 0.05 7.59 0.01 2543.49 31.86 1.70 0.02 1.09 0.01

30.20 0.04 19.97 0.05 8.16 0.01 606.49 17.05 5.48 0.08 3.52 0.05

30.17 0.06 19.86 0.06 7.92 0.01 1117.47 29.66 3.43 0.06 2.20 0.04

30.24 0.05 20.05 0.13 7.60 0.02 2633.56 113.42 1.87 0.14 1.20 0.09

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Farmingdale, NY, USA). The lysate was centrifuged for 30 min at 1000 g and þ4  C and the supernatant collected for lysozyme analysis. 50 ml of hemolymph or cell lysate was incubated at RT with 950 ml of a 0.15% suspension of Micrococcus lysodeikticus (Sigma St. Louis, MO, USA) in 66 mmol l1 phosphate buffer (pH 6.2). The decrease in absorbance was continuously recorded for 5 min at 450 nm using spectrophotometer (VARIAN Cary 50 Bio UVeVis spectrophotometer, Cary, NC, USA). Standard solutions were prepared from crystalline hen egg white lysozyme (Sigma Aldrich, St. Louis, MO, USA) in 66 mmol l1 phosphate buffer (pH 6.2) and used for calibration. Protein concentrations were measured in hemolymph and cell lysates using Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA) with bovine serum albumin as a standard. Lysozyme activity was expressed as ng lysozyme mg1 protein.

primers, 1.5 ml of 10 diluted cDNA template and water to adjust to 15 ml. The reaction mixture was subjected to the following cycling: 15 min at 95  C to denature DNA and activate Taq polymerase and 40 cycles of 15 s at 94  C, 30 s at 55  C (for actin) or 60  C (for all other genes) and 30 s at 72  C. Serial dilutions of a cDNA standard were amplified in each run to determine amplification efficiency [94]. A single cDNA sample from M. mercenaria and C. virginica hemocytes was used as an internal cDNA standard and included in each run to test for run-to-run amplification variability. The target gene mRNA expression was standardized relative to b-actin mRNA as described elsewhere [61,94,102]. Our pilot studies showed that b-actin is an appropriate reference housekeeping gene in marine bivalves as its mRNA expression does not vary in response to CO2 or sublethal metal exposures [61,102]. 2.9. Cadmium determination

2.8. Quantitative real-time PCR (qRT-PCR) Hemolymph samples were centrifuged for 10 min at 1000 g. The hemocyte pellet was washed once with ice-cold ASW and stored at 80  C until RNA extraction. Total RNA was extracted from hemocytes using ZR RNA MiniPrepÔ kit (Zymo Research, Irvine, CA, USA) according to the manufacturers’ instructions. RNA concentration was measured using NanoDrop 2000 spectrophotometer (Thermo Scientific, Pittsburg, USA) and expressed as mg RNA 106 cells. Single-stranded cDNA was obtained from 0.2 or 0.3 mg of the total RNA (for clams and oysters, respectively) using 50 U ml1 SMARTScribeÔ Reverse Transcriptase (Clontech, Mountain View, CA, USA) and 20 mmol l1 of oligo(dT)18 primers. Transcript expression of target genes was determined by qRTPCR using a 7500 Fast Real-Time PCR System (Applied Biosystems/Life Technologies, Carlsbad, CA, USA) and SYBRÒ Green PCR kit (Life Technologies, Bedford, MA, USA) according to the manufacturers’ instructions. The primer sequences are given in Table 2. The qRT-PCR reaction mixture consisted of 7.5 ml of 2 SYBRÒ Green master mix, 0.3 mmol l1 of each forward and reverse gene-specific

Hemocytes were collected by centrifugation (1000 g for 10 min), washed twice in ASW to remove surface-associated Cd and re-suspended in 500 ml of distilled water. Cell suspensions and cellfree hemolymph were mixed 1:1 with 70% nitric acid and digested in a water bath at 60  C for 24 h. Cd concentrations were determined with an atomic absorption spectrometer AAnalyst 800 (Perkin Elmer, Waltham, MA, USA), 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 recovery for Cd was 102.67  9.08% (mean  standard deviation). Cd levels in hemocytes and hemolymph were expressed as ng Cd 106 cells or mg Cd l1, respectively. 2.10. Statistics For all studied traits, the effects of the factors “PCO2 ”, “Cd exposure” and their interactions were assessed using generalized linear model ANOVA after testing for the normality of data

Table 2 Primer sequences and PCR product characteristics used for real time quantitative PCR (qRT-PCR). Apparent amplification efficiency (Ea), expected size of the PCR product and annealing temperature (Ta) are given.* Accession numbers from the Marine Genomics Project database, http://mgnew.clemson.edu/. All other accession numbers are from NCBI. Species

Gene

NCBI accession number

Primer name

Primer sequences

Ta ( C)

Product size (bp)

Ea

M. mercenaria

Metallothionein (MT)

GO915201

150

1.58

GO915233

50 -ATC CGT GCA ATT GTG CGT CTG AAA CGG-30 50 -TTA CAT CCG GGA CAC TTG CAG TCA-30 50 -GAC CGT CTG GGA GTT CGT AG-30 50 -AGC GTG GTT ACT CCT TCA CC-30

60

b-actin

Mmer Mmer Mmer Mmer

55

153

Defensin

GO915266

115

GO915219

55

221

1.42

Heat shock protein 70

GO915235

50 -GCA ATT ATG GCT GTC GGA AT-30 50 -GTA TGC TGG AGT TGC CGT CT-30 50 -AAT TGC TGT CCG GGT TAG AA-30 50 -GCA ATG AAC TGG TGC AGA AA-30 50 -TGA CCT CGC TAC ATT TGT CC-30 50 -AAT GAC AAA GGC CGT CTC AG-30

55

C-type lectin

Big defensin 1 F Big defensin 1 R C-type lectin 9 F C-type lectin 9 R HSP70_F HSP70_R

1.86 0.08 N¼2 1.91

55

220

1.43 0.13 N¼2

Metallothionein (MT)

MTI þ II-FW MTI þ II-RV Act-Cv-F437 Act-Cv-R571

50 -GGC TGT AAA TGT GGG GAG AA-30 50 -GAG AAC GCC TCT CAT TGG TC-30 50 -CAC AGC CGC TTC CTC ATC CTC C-30 50 -CCG GCG GAT TCC ATA CCA AGG-30

55

150

1.64

Actin

AY331695 AY331705 X75894

55

134

Integrin

MGID95214*

integrin 5 F integrin 5 R

50 -AAG GGG AAG AGA GCA ACA CA-30 50 -CCT TCC TGA GCC AAG AAC TG-30

55

130

C-type lectin

MGID93410*

131

GO915166

50 -ATT TGC TCA GCC TTG AAT GG-30 50 -GTC CCT CCC ACC CAG TAG TT-30 50 -AAT TGG GCA CCT TTG AAC TG-30 50 -CTT TGT CGT TGG TGA TGG TG-30

55

Heat shock protein 70

C-type lectin 4 F C-type lectin 4 R HSP70_F HSP70_R

1.49 0.02 N¼2 2.00 0.16 N¼2 1.17

55

159

2.17

C. virginica

MT-FW MT-Rev Act-FW Act-RV

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distribution and homogeneity of variances. All factors were treated as fixed and had three levels for PCO2 (w400, w800 and w2000 matm PCO2 ), and two levels for metal exposures (control and Cd-exposed). ANOVA results for all studied traits are given in Tables 3 and 4. Post-hoc tests (Fisher’s Least Significant Difference) were used to test the differences between the group means. Sample sizes were 5e6 for all measured traits and experimental groups, except for Cd accumulation in hemocytes and hemolymph, where a statistically significant outlier was removed in one of the groups (clams exposed to Cd at w2000 matm PCO2 ) resulting in N ¼ 4. Unless otherwise indicated, data are represented as means  standard errors of means (SEM). The differences were considered significant if the probability of Type I error was less than 0.05. 3. Results 3.1. Metal accumulation Exposure to 50 mg l1 Cd led to a significant increase of Cd levels in bivalve hemolymph, reaching 49e70 mg l1 in clams and 52e 68 mg l1 in oysters (Fig. 1). Cd levels in hemolymph of clams or oysters were not affected by exposure to elevated CO2 (Fig. 1, Tables 3 and 4). Cadmium exposure led to Cd accumulation by hemocytes, which was 4e5 times lower in clams than in oysters

Table 3 ANOVA: effects of PCO2 and Cd exposure on the hemocyte and hemolymph traits in M. mercenaria. Significant effects are highlighted in bold. Factors/interaction

Metal Hemocytes accumulation Hemolymph RNA and protein content

RNA

Cellular respiration

Total respiration

Proteins in hemocytes Proteins in hemolymph

Cd

CO2  Cd

F2,32 ¼ 0.46, P ¼ 0.634 F2,33 ¼ 0.58, P ¼ 0.564

F1,32 ¼ 9.76, P ¼ 0.004 F1,33 ¼ 68.53, P < 0.0001

F2,32 ¼ 0.13, P ¼ 0.881 F2,33 ¼ 0.62, P ¼ 0.544

F2,35 ¼ 0.14, P ¼ 0.873 F2,29 ¼ 2.03, P ¼ 0.153 F2,33 ¼ 1.59, P ¼ 0.222

F1,35 ¼ 11.74, P ¼ 0.002 F1,29 ¼ 0.02, P ¼ 0.886 F1,33 ¼ 0.04, P ¼ 0.848

F2,35 ¼ 1.17, P ¼ 0.324 F2,29 ¼ 5.26, P ¼ 0.013 F2,33 ¼ 0.29, P ¼ 0.753

F1,33 ¼ 8.27, P ¼ 0.008 F1,34 ¼ 5.95, P ¼ 0.021 F1,34 ¼ 2.54, P ¼ 0.122 F1,32 ¼ 4.76, P ¼ 0.038

F2,33 ¼ 1.07, P ¼ 0.356 F2,34 ¼ 1.60, P ¼ 0.219 F2,34 ¼ 1.74, P ¼ 0.193 F2,32 ¼ 2.05, P ¼ 0.149

F2,34 ¼ 0.17, P ¼ 0.846 F2,32 ¼ 3.67, P ¼ 0.039 F2,34 ¼ 3.13, P ¼ 0.059 F2,34 ¼ 2.46, P ¼ 0.104 F2,34 ¼ 3.66, P ¼ 0.038

F1,34 ¼ 5.56, P ¼ 0.025 F1,32 ¼ 0.19, P ¼ 0.664 F1,34 ¼ 0.35, P ¼ 0.559 F1,34 ¼ 11.77, P ¼ 0.002 F1,34 ¼ 3.74, P ¼ 0.063

F2,34 ¼ 0.14, P ¼ 0.872 F2,32 ¼ 0.21, P ¼ 0.811 F2,35 ¼ 0.52, P ¼ 0.601 F2,34 ¼ 0.04, P ¼ 0.960 F2,34 ¼ 0.64, P ¼ 0.536

F2,35 ¼ 0.23, P ¼ 0.797 F2,34 ¼ 0.61, P ¼ 0.548 F2,33 ¼ 2.32, P ¼ 0.117 F2,33 ¼ 1.47, P ¼ 0.248

F1,35 ¼ 3.01, P ¼ 0.0937 F1,34 ¼ 5.44, P ¼ 0.027 F1,33 ¼ 0.17, P ¼ 0.680 F1,33 ¼ 5.91, P ¼ 0.022

F2,35 ¼ 6.20, P ¼ 0.006 F2,34 ¼ 5.99, P ¼ 0.007 F2,33 ¼ 2.02, P ¼ 0.151 F2,33 ¼ 0.08, P ¼ 0.923

F2,33 ¼ 1.69, P ¼ 0.203 Mitochondrial F2,34 ¼ 0.64, respiration P ¼ 0.533 Proton leak F2,34 ¼ 0.16, P ¼ 0.852 Non-mitochondrial F2,32 ¼ 1.13, respiration P ¼ 0.338

Immune-related Hemocyte count functions Zymosan uptake Adhesion Lysozyme activity in hemocytes Lysozyme activity in hemolymph mRNA expression

CO2

MT HSP 70 Lectin Defensin

303

Table 4 ANOVA: effects of PCO2 and Cd exposure on the hemocyte and hemolymph traits in C. virginica. Significant effects are highlighted in bold. Factors/interaction CO2 F2,34 ¼ 0.38, P ¼ 0.686 F2,33 ¼ 0.63, P ¼ 0.542 RNA F2,35 ¼ 0.35, RNA and protein P ¼ 0.708 content Proteins in F2,29 ¼ 0.73, hemocytes P ¼ 0.491 Proteins in F2,35 ¼ 1.57, hemolymph P ¼ 0.225 Cellular Total respiration F2,34 ¼ 0.31, P ¼ 0.738 respiration Mitochondrial F2,35 ¼ 0.05, P ¼ 0.954 respiration Proton leak F2,34 ¼ 0.14, P ¼ 0.872 Non-mitochondrial F2,33 ¼ 1.74, respiration P ¼ 0.195 Immune-related Total hemocytes F2,35 ¼ 0.26, functions numbers P ¼ 0.771 Zymosan uptake F2,31 ¼ 2.63, P ¼ 0.091 Adhesion F2,34 ¼ 6.15, P ¼ 0.006 Lysozyme activity F2,35 ¼ 1.60, in hemocytes P ¼ 0.218 Lysozyme activity F2,32 ¼ 0.86, in hemolymph P ¼ 0.436 mRNA MT F2,34 ¼ 1.29, expression P ¼ 0.291 HSP 70 F2,32 ¼ 0.25, P ¼ 0.781 Lectin F2,34 ¼ 0.72, P ¼ 0.494 Integrin F2,35 ¼ 3.12, P ¼ 0.059

Metal Hemocytes accumulation Hemolymph

Cd

CO2  Cd

F1,32 ¼ 42.42, P < 0.0001 F1,33 ¼ 48.37, P < 0.0001 F1,35 ¼ 0.70, P ¼ 0.409 F1,29 ¼ 2.85, P ¼ 0.105 F1,35 ¼ 29.09, P < 0.0001 F1,34 ¼ 3.76, P ¼ 0.062 F1,35 ¼ 1.16, P ¼ 0.290 F1,34 ¼ 4.49, P ¼ 0.043 F1,33 ¼ 9.38, P ¼ 0.005 F1,35 ¼ 11.99, P ¼ 0.002 F1,31 ¼ 0.04, P ¼ 0.842 F1,34 ¼ 2.71, P ¼ 0.111 F1,35 ¼ 13.77, P ¼ 0.001 F1,32 ¼ 21.57, P < 0.0001 F1,34 ¼ 67.21, P < 0.0001 F1,32 ¼ 4.29, P ¼ 0.048 F1,34 ¼ 0.00, P ¼ 0.947 F1,35 ¼ 1.71, P ¼ 0.201

F2,32 ¼ 0.46, P ¼ 0.639 F2,33 ¼ 0.48, P ¼ 0.623 F2,35 ¼ 1.58, P ¼ 0.222 F2,29 ¼ 2.47, P ¼ 0.106 F2,35 ¼ 1.08, P ¼ 0.353 F2,34 ¼ 2.73, P ¼ 0.082 F2,35 ¼ 3.85, P ¼ 0.033 F2,34 ¼ 4.14, P ¼ 0.026 F2,33 ¼ 0.49, P ¼ 0.616 F2,35 ¼ 0.40, P ¼ 0.675 F2,31 ¼ 0.31, P ¼ 0.739 F2,34 ¼ 3.60, P ¼ 0.040 F2,35 ¼ 1.32, P ¼ 0.282 F2,32 ¼ 1.18, P ¼ 0.324 F2,34 ¼ 1.34, P ¼ 0.279 F2,32 ¼ 2.73, P ¼ 0.084 F2,34 ¼ 1.14, P ¼ 0.334 F2,35 ¼ 0.83, P ¼ 0.447

(Fig. 1A, B). In clam hemocytes, significant Cd accumulation was observed during Cd exposure in normocapnia but not in hypercapnia (Fig. 1A). In oyster hemocytes, Cd accumulation was independent of CO2 levels (Fig. 1B). 3.2. Respiration of hemocytes In the control group of clams, oxygen consumption of hemocytes was high in clams maintained in normocapnia and extreme hypercapnia (PCO2 w400 and w2000 matm, respectively) but considerably lower in those exposed to moderate hypercapnia (PCO2 w800 matm) (Table 3; Fig. 2A). A similar, albeit non-significant trend was found for the mitochondrial respiration of clam hemocytes (Fig. 2C). In contrast, proton leak in hemocytes of control clams was not affected by exposure PCO2 (Fig. 2E). Exposure to Cd suppressed total and mitochondrial respiration in hemocytes of clams maintained in normo- and hypercapnia (Fig. 2A, C). Proton leak of hemocytes from Cd-exposed clams maintained in normocapnia did not significantly differ from that of their control counterparts. In extreme hypercapnia, Cd exposure led to a decrease of the proton leak rates in clam hemocytes (Fig. 2E). Overall, mitochondrial respiration accounted for 82e97% of the total respiration in clam hemocytes, and mitochondrial proton leak accounted for 40e46% of the total respiration. These percentages did not significantly differ between control and Cd-exposed clams (t-test: P > 0.05, N ¼ 3). Respiration rates of hemocytes from control oysters were insensitive to elevated exposure PCO2 (Table 4; Fig. 2B, D, E). In

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Fig. 1. Effects of PCO2 and metal exposure on accumulation of Cd in hemocytes and hemolymph of clams (M. mercenaria) and oysters (C. virginica). A, B e Cadmium accumulation in hemocytes, C, D e cadmium concentration in hemolymph of clams (A, C) and oysters (B, D). Columns that share letters represent values that do not significantly differ among different CO2 levels within the same Cd treatment group (P > 0.05). Asterisks indicate values that are significantly different from the control (non-Cd exposed) group at the respective PCO2 (P < 0.05). Vertical lines represent the SEM. N ¼ 4e6.

normocapnia, Cd exposure did not affect the total or mitochondrial respiration of oyster hemocytes, but led to a significant increase of the mitochondrial proton leak (Fig. 2B, D, E). Exposure to Cd at elevated PCO2 led to the suppression of the total and mitochondrial respiration which was significant at w800 matm PCO2 . Elevated PCO2 also abolished the Cd-induced increase of the proton leak in oyster hemocytes (Fig. 2B, D, E). Mitochondrial respiration accounted for w82% of the total oxygen consumption of hemocytes of control and Cd-exposed oysters, but proportion of oxygen consumption devoted to counteracting the proton leak was significantly higher in hemocytes from Cd-exposed oysters compared to their control counterparts (63% vs. 30%; P < 0.01, N ¼ 3). 3.3. Immune-related functions of hemocytes The total concentration of circulating hemocytes in hemolymph of clams and oysters was not affected by PCO2 but significantly affected by Cd exposure (Table 3). Hemocyte counts tended to increase in response to Cd exposure in both studied species of mollusks (Fig. 3A, B). Elevated CO2 levels led to a decrease of the phagocytic activity of clam hemocytes, which was especially pronounced in Cd-exposed clams (Table 3; Fig. 3C). In control oysters, phagocytic activity was not affected by PCO2 , while in Cd-exposed ones a significant decrease in phagocytosis was observed at w800 matm PCO2 (Table 4; Fig. 3D). Adhesion capacity was suppressed in hemocytes of clams and oysters exposed to elevated CO2 (Tables 3 and 4; Fig. 3E, F). Cd exposure had no significant effect on adhesion capacity of clam hemocytes (Table 3; Fig. 3E), while in oysters, the lowest adhesion capacity of hemocytes was found during the combined exposure to Cd and extreme hypercapnia (w2000 matm PCO2 ) (Fig. 3F). Lysozyme activity in clam hemocytes was not affected by increased CO2 levels but was suppressed by Cd exposure (Table 3;

Fig. 4A). In cell-free clam hemolymph, the Cd-induced suppression of the lysozyme activity was only marginally significant (P ¼ 0.06; Table 3) while elevated CO2 increased lysozyme activity (Table 3, Fig. 4C). In oysters, Cd exposure suppressed lysozyme activity in hemocytes and hemolymph (Table 4; Fig. 4B, D). Elevated PCO2 led to a significant increase in lysozyme activity of hemocytes from control but not Cd-exposed oysters (Fig. 4B). Lysozyme activity of oyster hemolymph was not affected by PCO2 (Fig. 4D). 3.4. Protein and RNA content of hemocytes and hemolymph In clam hemocytes, exposure to elevated CO2 had no effect on total RNA concentration, while Cd exposure led to a significant decrease in RNA content (Table 3; Fig. 5A). Total protein content of hemocytes significantly increased at w2000 matm PCO2 in control clams and at w800 matm PCO2 in Cd-exposed ones (Fig. 5C). The protein content of clam hemolymph was low (<0.5 mg ml1) and not affected by PCO2 or Cd exposure (Table 3; Fig. 5E). In oyster hemocytes, RNA content was not affected by PCO2 or Cd exposure (Table 4; Fig. 5B). Protein content of hemocytes and hemolymph of oysters tended to be higher during the Cd exposure; however, this effect was only significant at w400 matm PCO2 in hemocytes and at w400 and w2000 matm PCO2 in hemolymph (Fig. 5D, F). 3.5. mRNA expression of stress- and immune-related genes In clams, mRNA expression of metallothionein (MT) was significantly affected by the interactions of Cd and CO2 exposure (Table 3) indicating that the transcriptional response of this gene to Cd exposure was modulated by PCO2 . Elevated CO2 levels led to increased expression of MT mRNA in clam hemocytes in the absence of Cd exposure (Fig. 6A). In clams maintained in normocapnia, Cd exposure led to a notable (w2-fold) albeit statistically

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Fig. 2. Effects of PCO2 and metal exposure on cellular respiration of clams (M. mercenaria) and oysters (C. virginica). A, B e Total MO2, C, D e Mitochondrial MO2, E, F e Mitochondrial proton leak of clams (A, C, E) and oysters (B, D, F). Columns that do not share letters represent values that significantly differ among different CO2 treatments within the same Cd treatment group (P < 0.05). Asterisks indicate values that are significantly different from the control (non-Cd exposed) group at the respective PCO2 (P < 0.05). Vertical lines represent the SEM. N ¼ 5e6.

non-significant increase in MT mRNA levels (Fig. 6A). This Cdinduced increase in MT expression was significantly suppressed at elevated PCO2 (Fig. 6A). In oysters, Cd exposure led to a large (w7fold) and significant increase in MT mRNA levels, and MT expression was not affected by PCO2 (Table 4; Fig. 6B). Expression of HSP70 mRNA in clam hemocytes did not change in response to elevated PCO2 (Table 3; Fig. 6C). At w400 and w800 matm PCO2 , Cd had no effect on HSP70 expression in clam hemocytes while at w2000 matm PCO2 Cd exposure led to a significant decrease in HSP70 expression (Fig. 6C). In oyster hemocytes, HSP70 mRNA levels were suppressed by exposure to extreme hypercapnia (w2000 matm PCO2 ) in the absence of Cd, and by Cd exposure at w800 matm PCO2 (Fig. 6D). Elevated CO2 levels had no effect on lectin mRNA levels in clam hemocytes in the absence of Cd (Fig. 6E). However, in hemocytes of Cd-exposed clams lectin expression declined with increasing CO2 levels (Fig. 6E). In oyster hemocytes, lectin mRNA expression was unaffected by Cd or CO2 exposure (Fig. 6F). Elevated CO2 levels had no effect on mRNA expression of defensin in clams (Table 3), while Cd exposure significantly affected defensin expression (Table 3). Overall, Cd exposure led to a w2-fold decrease in expression of this gene in clam hemocytes (Fig. 6G).

Defensin mRNA levels could not be measured in oyster hemocytes due to the lack of specific primers. In oyster hemocytes, expression of integrin mRNA was significantly suppressed by exposure to hypercapnia in the absence of Cd (Table 3; Fig. 6H). Integrin mRNA levels were not affected by Cd exposure (Table 4; Fig. 6H). Integrin mRNA levels could not be measured in clams due to the lack of specific primers. 4. Discussion 4.1. Cd accumulation in hemolymph and hemocytes Cd concentration in the hemolymph of Cd-exposed clams and oysters (w50e70 mg l1) was similar to that in the ambient water (w50 mg l1 Cd). This agrees with an earlier study showing similar concentrations of Cd in the hemolymph and the environment of oysters [30]. The absence of hyperaccumulation of Cd in the blood plasma of clams and oysters suggests that Cd in the plasma is either free and/or loosely bound to low affinity ligands. Studies on marine bivalves (including hard clams M. mercenaria and blue mussels Mytilus edulis) support this conclusion and show that Cd in the blood plasma is predominantly non-specifically bound to low-

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Fig. 3. Effects of PCO2 and metal exposure on immune-related traits of hemocytes of clams (M.mercenaria) and oysters (C. virginica). A, B e Total blood cell count, C, D e average rate of hemocyte phagocytosis, E, F e cell adhesion rates of clams (A, C, E) and oysters (B, D, F). Columns that do not share letters represent values that significantly differ among different CO2 treatments within the same Cd treatment group (P < 0.05). Asterisks indicate values that are significantly different from the control (non-Cd exposed) group at the respective PCO2 (P < 0.05). Vertical lines represent the SEM. N ¼ 5e6.

affinity ligands such as histidine-rich proteins [1,43,88,89,99,100]. In contrast, the intracellular concentrations of Cd in hemocytes were far above the ambient Cd levels. Thus, assuming cellular water content of 75% and the average volume of a hemocyte of 1.1  1013 l in clams and 1.8  1013 l in oysters [2,84], Cd concentrations were 9e15 mg l1 and 31e42 mg l1 in Cd-exposed clams and oysters, respectively, which is 180e840 times higher than ambient levels. Hyperaccumulation of Cd and other trace metals in hemocytes compared to the surrounding plasma is commonly found in mollusks [30,42,51,78]. Most of the intracellular Cd is likely bound to cysteine-rich ligands (such as glutathione and metallothioneins) as well as structural proteins and enzymes [12,37,48,108] accounting for the ability of hemocytes to accumulate high Cd levels. Despite similar Cd levels in the blood plasma of the two studied species, Cd accumulation in hemocytes was significantly higher in oysters than in clams. A four weeks exposure to Cd led to a 17e26fold increase of Cd burdens in oyster hemocytes above the background levels compared to a 4e7-fold increase in the hemocytes of clams. Lower Cd accumulation in hemocytes of clams compared to

oysters may reflect better metal handling abilities of clams. Earlier studies in hard clams showed active elimination of Cd by diapedesis of hemocytes and by specialized brown cells which accumulate and excrete metals [81,114,115]; such mechanisms have not been described in oysters. Moreover, lower metabolic and filtration rates in clams compared to oysters may also affect the rates of uptake of the waterborne Cd which mostly occurs in the gills [67,69,77]. Earlier studies also demonstrated significantly lower Cd accumulation in soft tissues of clams compared to oysters under the same Cd exposure regime indicating that the differences in metal handling abilities affect the overall body metal burdens in these species [55]. These differences in metal-handling capacities between the two studied species may reflect adaptations of the sediment-dwelling clams to higher metal concentrations (that are typically orders of magnitude higher in the sediments than the water [35,57]); compared to an epifaunal species such as oysters [81,114,115]. Elevated PCO2 did not affect Cd levels in the hemolymph and had a minor impact on Cd accumulation in hemocytes of clams and oysters. In both species, there was a slight trend of lower Cd

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Fig. 4. Effects of PCO2 and metal exposure on lysozyme activity of clams (M.mercenaria) and oysters (C. virginica). A, B e Lysozyme activity in hemocytes, C, D e Lysozyme activity in cell-free hemolymph of clams (A, C) and oysters (B, D). Columns that do not share letters represent values that significantly differ among different CO2 treatments within the same Cd treatment group (P < 0.05). Asterisks indicate values that are significantly different from the control (non-Cd exposed) group at the respective PCO2 (P < 0.05). Vertical lines represent the standard error of the mean (SEM). N ¼ 5e6.

accumulation in hemocytes at elevated CO2 levels; this trend was significant in clams but not in oysters. An earlier study using acute hypercapnic exposures (w15,000e30,000 matm) also showed that hypercapnia suppressed Cd uptake by isolated mantle cells of hard clams [61]. In contrast, during the whole-organism exposures to Cd, Cd accumulation in the mantle tissue was enhanced by moderate hypercapnia (w800 and 1500 matm in oysters and hard clams, respectively) [55]. These data indicate that effects of PCO2 on Cd accumulation in bivalves are tissue-specific and may depend on the mode of exposure and degree of hypercapnia.

w800 matm PCO2 but not at w400 or 2000 matm PCO2 . RNA content of the hemocytes was suppressed in Cd-exposed clams at w400 or 2000 matm PCO2 indicating that a decrease in the proteosynthetic activity may be responsible for the lack of the protein accumulation in clam hemocytes under these conditions. Overall, our data indicate that low, sublethal levels of Cd used in this study do not significantly impede the ability of clams and oysters to maintain normal protein levels in hemolymph and hemocytes and may under some conditions lead to protein accumulation, possibly reflecting elevated expression of stress proteins [9,29,60,62].

4.2. Interactive effects of Cd and PCO2 on protein content of hemocytes and hemolymph

4.3. Interactive effects of Cd and PCO2 on hemocyte metabolism and immune functions

Exposure to Cd led to elevated protein content of both hemocytes and hemolymph of oysters reflecting a change in the protein synthesis and/or degradation. Earlier studies showed that exposure to low, sublethal concentrations Cd stimulate protein synthesis in oyster tissues, most likely due to the elevated synthesis of stress proteins (such as metallothioneins and HSPs) [29,60,62]. In gill tissues of oysters, the elevated proteosynthetic activity in response to Cd was also reflected in higher RNA/DNA ratios [73]. A similar increase in RNA content was not seen in oyster hemocytes indicating that the increase in the protein synthesis and secretion by hemocytes occurs without a considerable change of the ribosomal abundance. Alternatively, elevated protein content in the blood cells and plasma of oysters may reflect increased half-life of the proteins due to the suppressed protein degradation [50,66,113]. The latter explanation appears less likely in oysters, where long-term exposure to sub-lethal Cd levels (50 mg l1) led to elevated activity of the proteasome [55]. In clams, no effect of Cd on the protein levels in the blood plasma was detected, and Cd-induced changes in the protein content of the hemocytes were less consistent. Clam hemocytes showed elevated protein levels during Cd exposure at

Exposure to Cd led to an increase of the total concentrations of circulating hemocytes in clams and oysters. This is consistent with earlier findings in Cd-exposed oysters C. virginica [27,28], and mussels Mytilus edulis and Mytella falcata [36,41]. The Cd-induced increase in the numbers of circulating hemocytes may be explained by cell proliferation and/or by redistribution of the resident hemocytes between the tissue and hemolymph [23]. Earlier studies showed that Cd stimulates hematopoiesis in C. virginica [27] as well as in other invertebrates (such as insects) [13]. This may explain higher hemocyte numbers in Cd-exposed bivalves found in our present study. Migration of hemocytes from tissues into the hemolymph appears a less likely explanation for the observed increase in the concentrations of circulating hemocytes, because Cd is known to induce inflammation and increase infiltration of hemocytes from free circulation into the tissues [41,105]. Elevated CO2 levels had no effect on hemocyte concentrations in hemolymph of oysters or hard clams. This is similar to earlier findings in the blue mussels M. edulis showing no change in the hemocyte counts when maintained for a month under the hypercapnic conditions ranging from 1160 to 3316 matm PCO2 (pH w7.7e

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Fig. 5. Effects of PCO2 and metal exposure on proteosynthetic responses of hemocytes and hemolymph of clams (M. mercenaria) and oysters (C. virginica). A, B e Total RNA concentration, C, D e Total protein concentration in hemocytes, E, F e total proteins concentration in hemolymph of clams (A, C, E) and oysters (B, D, F). Columns that do not share letters represent values that significantly differ among different CO2 treatments within the same Cd treatment group (P < 0.05). Asterisks indicate values that are significantly different from the control (non-Cd exposed) group at the respective PCO2 (P < 0.05). Vertical lines represent the SEM. N ¼ 5e6.

6.7) [10]. In contrast, in the Mediterranean mussel Mytilus galloprovincialis and a clam Chamelea gallina, one-week exposure to elevated CO2 (pH w7.7e7.4) altered the numbers of circulating hemocytes, with the direction and magnitude of the change dependent on the salinity and temperature of exposure [79]. Albeit limited, the current data appear to indicate that long-term (w4 weeks) exposure to hypercapnia does not alter the hemocyte numbers in bivalves, although shorter exposures to elevated CO2 may have variable effects. Metabolic activity of clam and oyster hemocytes was suppressed by Cd exposure, although this effect was significant only at some PCO2 levels. The inhibitory effect of Cd exposure on the metabolic activity of clam hemocytes were significant at the two extreme PCO2 levels e w400 and w2000 matm but not at w800 matm PCO2 . In contrast, Cd exposure affected the metabolic activity of oyster hemocytes most strongly at w400 and 800 matm PCO2 , while the metabolic effects of Cd were negligible at w2000 matm PCO2 . Unlike the present finding for hemocytes, long-term exposure to 50 mg l1 Cd resulted in elevated standard metabolic rates in the whole oysters and their isolated gill cells [29,64,72,73]. This indicates that tissues may differ in the metabolic responses to Cd potentially

leading to the reallocation of energy to maintenance of other tissues at the expense of immune system. Metabolic responses of hemocytes to Cd exposure in normocapnia also show a partial loss of mitochondrial coupling as the proton leak constitutes a higher proportion of the mitochondrial respiration in Cd-exposed hemocytes. This is consistent with the known effects of Cd as an uncoupler of molluscan mitochondria [31,63,65,71,106]. Interestingly, the uncoupling effects of Cd on mitochondrial respiration were alleviated by hypercapnia in hemocytes of clams but not in oysters, possibly reflecting reduced sensitivity of clam mitochondria to Cd at low intracellular pH [65]. Lower metabolic activity and impaired mitochondrial function go hand-in-hand with suppression of immunity-related functions in hemocytes of Cd-exposed clams and oysters. Negative effects of metals including Cd on innate immunity have been earlier described in mollusks [4,5 17,22,25,38,85], and other invertebrates as well as in vertebrates [19,74,103,104]. Suppression of the innate immunity by Cd is likely mediated by the negative effects of this metal on fundamental cellular functions such as cell adhesion, stability of cytoskeleton, energy metabolism and Ca2þ homeostasis that play a key role in the motility, attachment and phagocytosis

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Fig. 6. Effects of PCO2 and metal exposure on mRNA expression of stress-response and immune-response genes in hemocytes of clams (M. mercenaria) and oysters (C. virginica). A, B e mRNA expression of MT relative to b-actin mRNA, C, D e mRNA expression of HSP70 relative to b-actin mRNA, E, F e mRNA expression of lectin relative to b-actin mRNA of clams (A, C, E) and oysters (B, D, F). Columns that do not share letters represent values that significantly differ among different CO2 treatments within the same Cd treatment group (P < 0.05). Asterisks indicate values that are significantly different from the control (non-Cd exposed) group at the respective PCO2 (P < 0.05). Vertical lines represent the SEM. N ¼ 5e6.

[7,32,49,71,97,111]. This is consistent with the findings of the present study showing the negative effects of Cd on adhesion and phagocytosis of bivalve hemocytes. The immunomodulatory effects of Cd were PCO2 -dependent in both studied species and most pronounced at elevated PCO2 (w2000 matm in clams and w800e2000 matm in oysters). In clams, Cd exposure had no effect on immunity-related parameters in normocapnia. At elevated PCO2 levels, Cd inhibited lysozyme activity of clam hemocytes. Furthermore, phagocytosis, as well as expression of lectin, defensin and HSP70 mRNA decreased with increasing PCO2 in hemocytes of Cd-exposed clams. Similarly, in oysters, the immunosuppressive effects of Cd were more strongly pronounced at elevated PCO2 . In normocapnia, Cd exposure led to the reduced lysozyme activity in oyster hemolymph, while other immune-related parameters remained unchanged. In contrast, at

elevated PCO2 , Cd exposure led to the reduced lysozyme activities in both hemolymph and hemocytes, suppressed hemocyte adhesion and phagocytosis, and led to reduced mRNA expression of lectin and HSP70 in oysters. This indicates that elevated PCO2 such as expected during ocean acidification and/or seasonal hypercapnia in estuaries may enhance the immunosuppressive effects of Cd in bivalves. Effects of elevated PCO2 on the immune functions of clams and oysters were mild in the absence of Cd stress, and the direction of these effects differed among different immune-related traits. Thus, in control (non-Cd-exposed) clams, hemocyte adhesion was suppressed by elevated CO2 levels (w2000 matm) while lysozyme activity in hemolymph increased. In control oysters, hypercapnia enhanced the lysozyme activity of hemocytes but suppressed mRNA expression of integrin and HSP70. Although the relative importance

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of different immune-related functions of hemocytes and hemolymph in the overall immune protection of mollusks is not well understood (and likely varies depending on the pathogen and/or immune stimulus), these data indicate that immune function in the two studied bivalves displays resilience to environmental hypercapnia, but enhanced sensitivity to Cd exposure when it co-occurs with elevated PCO2 . 4.4. Conclusions and perspectives Our study shows that Cd is a potent immunosuppressor in marine bivalves which negatively affects metabolism and immunerelated functions of hemocytes of clams and oysters at low, sublethal levels (50 mg l1). Moderate environmental hypercapnia (w800e2000 matm PCO2 ) potentiates the negative effects of Cd on immune functions of hemocytes and thus may sensitize clams and oysters from polluted estuaries to pathogens and diseases. Earlier studies also showed that moderately elevated PCO2 can suppress immunostimulatory effects of other factors [10] or modify the normal response of the immune system to changes in environmental temperature and salinity [79]. Taken together, these studies suggest that environmental hypercapnia e such as occurs in summer in many eutrophicated estuaries of the eastern United States and is predicted to occur globally during ocean acidification e can have an impact on the health status of bivalve populations. The degree and direction of the impact of hypercapnia on immunity and thus health of estuarine mollusks is strongly dependent on the environmental context (such as pollution levels, temperature or salinity regime). Moreover, immune responses to hypercapnia may be further modified by evolutionary adaptations to ocean acidification over the course of decades and centuries, which was not captured in the relatively short-term exposures in this study. Given the complexity of the stressor interactions, further studies are urgently needed to assess and predict the effects of ocean acidification on the immunity and health of marine organisms in the environmentally relevant context of the multiple stressors. Acknowledgments This work was supported by funds provided by the National Science Foundation award IOS-095107, Charlotte Research Institute and UNC Charlotte’s Faculty Research Grant to I.M.S. The authors thank Bushra Khan and Amy Ringwood for assistance with the metal analyses. References [1] Abebe AT, Devoid SJ, Sugumaran M, Etter R, Robinson WE. Identification and quantification of histidine-rich glycoprotein (HRG) in the blood plasma of six marine bivalves. Comp Biochem Physiol Part B Biochem Mol Biol 2007;147: 74e81. [2] Allam B, Ashton-Alcox KA, Ford SE. Flow cytometric comparison of haemocytes from three species of bivalve molluscs. Fish Shellfish Immunol 2002;13:141e58. [3] An MI, Choi CY. Activity of antioxidant enzymes and physiological responses in ark shell, Scapharca broughtonii, exposed to thermal and osmotic stress: effects on hemolymph and biochemical parameters. Comp Biochem Physiol Part B Biochem Mol Biol 2010;155:34e42. [4] Anderson RS, Oliver LM, Jacobs D. Immunotoxicity of cadmium for the eastern oyster (Crassostrea virginica [Gmelin, 1791]): effects on hemocyte chemiluminescence. J Shellfish Res 1992;11:31. [5] Auffret M, Mujdzic N, Corporeau C, Moraga D. Xenobiotic-induced immunomodulation in the European flat oyster, Ostrea edulis. Mar Environ Res 2002;54:585e9. [6] Barber BJ, McGladdery S. Current status of shellfish and broodstock movement and disease transfer risks in the Gulf of Maine region. Gulf of Maine Council on the Marine environment; 2001. [7] Belyaeva EA, Dymkowska D, Wieckowski MR, Wojtczak L. Reactive oxygen species produced by the mitochondrial respiratory chain are involved in

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