Effects of seawater salinity and pH on cellular metabolism and enzyme activities in biomineralizing tissues of marine bivalves.

Molluscan shell formation is a complex energy demanding process sensitive to the shifts in seawater CaCO3 saturation due to changes in salinity and pH. We studied the effects of salinity and pH on energy demand and enzyme activities of biomineralizing cells of the Pacific oyster (Crassostrea gigas) and the hard-shell clam (Mercenaria mercenaria). Adult animals were exposed for 14 days to high (30), intermediate (18), or low (10) salinity at either high (8.0-8.2) or low (7.8) pH. Basal metabolic cost as well as the energy cost of the biomineralization-related cellular processes were determined in isolated mantle edge cells and hemocytes. The total metabolic rates were similar in the hemocytes of the two studied species, but considerably higher in the mantle cells of C. gigas compared with those of M. mercenaria. Cellular respiration was unaffected by salinity in the clams' cells, while in oysters' cells the highest respiration rate was observed at intermediate salinity (18). In both studied species, low pH suppressed cellular respiration. Low pH led to an upregulation of Na+/K+ ATPase activity in biomineralizing cells of oysters and clams. Activities of Ca2+ ATPase and H+ ATPase, as well as the cellular energy costs of Ca2+ and H+ transport in the biomineralizing cells were insensitive to the variation in salinity and pH in the two studied species. Variability in cellular response to low salinity and pH indicates that the disturbance of shell formation under these conditions has different underlying mechanisms in the two studied species.

Potential trade-offs between biomineralization and immunity revealed by shell properties and gene expression profiles of two closely related Crassostrea species.

Species of the Ostreidae family are key ecosystem engineers and many of them - including Crassostrea gigas and Crassostreavirginica - are commercially important aquaculture species. Despite similarities in their morphology and ecology, these two species differ in their ability to defend against pathogens, potentially reflecting species-specific differential specialization of hemocytes on immune defense versus biomineralization. To test this hypothesis, we investigated the expression levels of immune- and biomineralization-related genes as well as mineralogical and mechanical properties of the shells and the calcium sequestration ability of the hemocytes of C. gigas and C. virginica The expression of biomineralization-related genes was higher in C. virginica than in C. gigas in multiple tissues including the mantle edge and hemocytes, while the expression of immune genes was higher in the hemocytes of C. gigas Hemocytes of C. virginica contained more calcium (stored intracellularly as calcium carbonate mineral) compared with those of C. gigas Analysis of the adult shells showed that the crystallinity of calcite was higher and the laths of the foliated layer of the shell were thicker in C. virginica than in C. gigas Mechanically, the shells of C. virginica were stiffer, harder and stronger than those of C. gigas Taken together, our results show that the species-specific differences in physiology (such as disease resistance and exoskeleton properties) are reflected at the cellular and molecular levels in the differential specialization of hemocytes on potentially competing functions (immunity and biomineralization) as well as different expression profiles of other tissues involved in biomineralization (such as the mantle edge).

Biomineralization-related specialization of hemocytes and mantle tissues of the Pacific oyster Crassostrea gigas.

The molluscan exoskeleton (shell) plays multiple important roles including structural support, protection from predators and stressors, and physiological homeostasis. Shell formation is a tightly regulated biological process that allows molluscs to build their shells even in environments unfavorable for mineral precipitation. Outer mantle edge epithelial cells (OME) and hemocytes were implicated in this process; however, the exact functions of these cell types in biomineralization are not clear. Pacific oysters (Crassostrea gigas) were used to study differences in the expression profiles of selected biomineralization-related genes in hemocytes and mantle cells, and the functional characteristics of hemocytes such as adhesion, motility and phagocytosis. The specialized role of OME in shell formation was supported by high expression levels of the extracellular matrix (ECM) related and cell-cell interaction genes. Density gradient separation of hemocytes revealed distinct phenotypes based on the cell morphology, gene expression patterns, motility and adhesion characteristics. These hemocyte fractions can be categorized into two functional groups, i.e. biomineralization and immune response cells. Gene expression profiles of the putative biomineralizing hemocytes indicate that in addition to their proposed role in mineral transport, hemocytes also contribute to the formation of the ECM, thus challenging the current paradigm of the mantle as the sole source of the ECM for shell formation. Our findings corroborate the specialized roles of hemocytes and the OME in biomineralization and emphasize complexity of the biological controls over shell formation in bivalves.

Effects of intermittent hypoxia on oxidative stress and protein degradation in molluscan mitochondria.

Oxygen fluctuations represent a common stressor in estuarine and intertidal environments and can compromise the mitochondrial integrity and function in marine organisms. We assessed the role of mitochondrial protection mechanisms (ATP-dependent and -independent mitochondrial proteases, and antioxidants) in tolerance to intermittent hypoxia or anoxia in three species of marine bivalves: hypoxia-tolerant hard clams (Mercenaria mercenaria) and oysters (Crassostrea virginica), and a hypoxia-sensitive subtidal scallop (Argopecten irradians). In clams and oysters, mitochondrial tolerance to hypoxia (18 h at 5% O2), anoxia (18 h at 0.1% O2) and subsequent reoxygenation was associated with the ability to maintain the steady-state activity of ATP-dependent and -independent mitochondrial proteases and an anticipatory upregulation of the total antioxidant capacity under the low oxygen conditions. No accumulation of end-products of lipid or protein peroxidation was found during intermittent hypoxia or anoxia in clams and oysters (except for an increase in protein carbonyl concentration after hypoxia-reoxygenation in oysters). In contrast, hypoxia/anoxia and reoxygenation strongly suppressed activity of the ATP-dependent mitochondrial proteases in hypoxia-sensitive scallops. This suppression was associated with accumulation of oxidatively damaged mitochondrial proteins (including carbonylated proteins and proteins conjugated with a lipid peroxidation product malondialdehyde) despite high total antioxidant capacity levels in scallop mitochondria. These findings highlight a key role of mitochondrial proteases in protection against hypoxia-reoxygenation stress and adaptations to frequent oxygen fluctuations in intertidal mollusks.

Intermittent hypoxia leads to functional reorganization of mitochondria and affects cellular bioenergetics in marine molluscs.

Fluctuations in oxygen (O2) concentrations represent a major challenge to aerobic organisms and can be extremely damaging to their mitochondria. Marine intertidal molluscs are well-adapted to frequent O2 fluctuations, yet it remains unknown how their mitochondrial functions are regulated to sustain energy metabolism and prevent cellular damage during hypoxia and reoxygenation (H/R). We used metabolic control analysis to investigate the mechanisms of mitochondrial responses to H/R stress (18 h at <0.1% O2 followed by 1 h of reoxygenation) using hypoxia-tolerant intertidal clams Mercenaria mercenaria and hypoxia-sensitive subtidal scallops Argopecten irradians as models. We also assessed H/R-induced changes in cellular energy balance, oxidative damage and unfolded protein response to determine the potential links between mitochondrial dysfunction and cellular injury. Mitochondrial responses to H/R in scallops strongly resembled those in other hypoxia-sensitive organisms. Exposure to hypoxia followed by reoxygenation led to a strong decrease in the substrate oxidation (SOX) and phosphorylation (PHOS) capacities as well as partial depolarization of mitochondria of scallops. Elevated mRNA expression of a reactive oxygen species-sensitive enzyme aconitase and Lon protease (responsible for degradation of oxidized mitochondrial proteins) during H/R stress was consistent with elevated levels of oxidative stress in mitochondria of scallops. In hypoxia-tolerant clams, mitochondrial SOX capacity was enhanced during hypoxia and continued rising during the first hour of reoxygenation. In both species, the mitochondrial PHOS capacity was suppressed during hypoxia, likely to prevent ATP wastage by the reverse action of FO,F1-ATPase. The PHOS capacity recovered after 1 h of reoxygenation in clams but not in scallops. Compared with scallops, clams showed a greater suppression of energy-consuming processes (such as protein turnover and ion transport) during hypoxia, indicated by inactivation of the translation initiation factor EIF-2α, suppression of 26S proteasome activity and a dramatic decrease in the activity of Na(+)/K(+)-ATPase. The steady-state levels of adenylates were preserved during H/R exposure and AMP-dependent protein kinase was not activated in either species, indicating that the H/R exposure did not lead to severe energy deficiency. Taken together, our findings suggest that mitochondrial reorganizations sustaining high oxidative phosphorylation flux during recovery, combined with the ability to suppress ATP-demanding cellular functions during hypoxia, may contribute to high resilience of clams to H/R stress and help maintain energy homeostasis during frequent H/R cycles in the intertidal zone.

Interactive effects of copper exposure and environmental hypercapnia on immune functions of marine bivalves Crassostrea virginica and Mercenaria mercenaria.

Estuarine organisms such as bivalves are commonly exposed to trace metals such as copper (Cu) and hypercapnia (elevated CO2 levels) in their habitats, which may affect their physiology and immune function. This study investigated the combined effects of elevated CO2 levels (∼800-2000 µatm PCO2, such as predicted by the near-future scenarios of global climate change) and Cu (50 µg l(-1)) on immune functions of the sediment dwelling hard clams Mercenaria mercenaria and an epifaunal bivalve, the eastern oyster Crassostrea virginica. Clams and oysters were exposed for 4 weeks to different CO2 and Cu levels, and tissue Cu burdens and immune parameters were assessed to test the hypothesis that hypercapnia will enhance Cu uptake due to the higher bioavailability of free Cu(2+) and increase the immunomodulatory effects of Cu. Exposure to Cu stimulated key immune parameters of clams and oysters leading to increased number of circulating hemocytes, higher phagocytosis and adhesion ability of hemocytes, as well as enhanced antiparasitic and antibacterial properties of the hemolymph reflected in higher activities of lysozyme and inhibitors of cysteine proteases. Lysozyme activation by Cu exposure was most prominent in normocapnia (∼400 µatm PCO2) and an increase in the levels of the protease inhibitors was strongest in hypercapnia (∼800-2000 µatm PCO2), but other immunostimulatory effects of Cu were evident in all PCO2 exposures. Metabolic activity of hemocytes of clams and oysters (measured as routine and mitochondrial oxygen consumption rates) was suppressed by Cu exposure likely reflecting lower rates of ATP synthesis and/or turnover. However, this metabolic suppression had no negative effects of the studied immune functions of hemocytes such as phagocytosis or adhesion capacity. Hypercapnia (∼800-2000 µatm PCO2) slightly but significantly enhanced accumulation of Cu in hemocytes, consistent with higher Cu(2+) bioavailability in CO2-acidified water, but had little effect on cellular and humoral immune traits of clams and oysters. These findings indicate that low levels of Cu contamination may enhance immunity of estuarine bivalves while moderate hypercapnia (such as predicted by the near future scenarios of the global climate change) does not strongly affect their immune parameters.

Effects of pH and bicarbonate on mitochondrial functions of marine bivalves.

Estuarine organisms including mollusks are exposed to periodic oxygen deficiency (hypoxia) that leads to a decrease in intracellular pH and accumulation of bicarbonate (HCO3(-)). These changes can affect cellular bioenergetics; however, their effects on mitochondria of estuarine mollusks are not well understood. We determined the interactive effects of bicarbonate (0-10mM) and pH (7.2 and 6.5) on mitochondrial oxygen consumption (MO2), membrane potential (Δψ) and production of reactive oxygen species (ROS) in two common estuarine bivalves - hard clams Mercenaria mercenaria, and bay scallops Argopecten irradians. In both species, elevated HCO3(-) levels suppressed ADP-stimulated (state 3) MO2 but had little effect on the resting (state 4) respiration. These effects were not mediated by the soluble adenylyl cyclase or cyclic AMP. Effects of the low pH (6.5) on mitochondrial traits were species-specific and depended on the substrate oxidized by the mitochondria. Mild acidosis (pH6.5) had minimal effects on MO2 and Δψ of the bivalve mitochondria oxidizing pyruvate but led to increased rates of ROS production in clams (ROS production could not be measured in scallops). In succinate-respiring mitochondria of clams, mild acidosis suppressed MO2 and increased mitochondrial coupling, while in scallop mitochondria the effects of low pH were opposite. Suppression of mitochondrial oxidative phosphorylation by bicarbonate and/or acidosis may contribute to the metabolic rate depression during shell closure or environmental hypoxia/hypercapnia. These findings have implications for understanding the physiological mechanisms involved in regulation of mitochondrial bioenergetics during hypoxia exposure in estuarine bivalves.

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

Estuarine organisms are exposed to multiple stressors including large fluctuations in partial pressure of carbon dioxide (P2CO) and concentrations of trace metals such as cadmium (Cd) that can affect their survival and fitness. Ocean acidification due to the increasing atmospheric (P2CO) 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 (P2CO) 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 (P2CO) levels (∼400, 800 and 2000 µatm (P2CO), corresponding to the present day conditions and the projections for the years 2100 and 2250, respectively) and two Cd concentrations (0 and 50 µg l(-1)) in seawater. Following four weeks of exposure to Cd, hemolymph of both species contained similar Cd levels (50-70 µg l(-1)), whereas hemocytes accumulated intracellular Cd burdens up to 15-42 mg l(-1), regardless of the exposure P2CO. Clam hemocytes had considerably lower Cd burdens than those of oysters (0.7-1 ng 10(-6) cells vs. 4-6 ng 10(-6) 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 immune-related functions in hemocytes of clams and oysters, and these effects were exacerbated at elevated (P2CO). Thus, elevated (P2CO) 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 (P2CO) and Cd also led to reduced activity of lysozyme in hemocytes and hemolymph. Overall, our study shows that moderately elevated (P2CO) (∼800-2000 µatm P2CO) 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.

Interactive effects of elevated temperature and CO(2) levels on metabolism and oxidative stress in two common marine bivalves (Crassostrea virginica and Mercenaria mercenaria).

Marine bivalves such as the hard shell clams Mercenaria mercenaria and eastern oysters Crassostrea virginica are affected by multiple stressors, including fluctuations in temperature and CO2 levels in estuaries, and these stresses are expected to be exacerbated by ongoing global climate change. Hypercapnia (elevated CO2 levels) and temperature stress can affect survival, growth and development of marine bivalves, but the cellular mechanisms of these effects are not yet fully understood. In this study, we investigated whether oxidative stress is implicated in cellular responses to elevated temperature and CO2 levels in marine bivalves. We measured the whole-organism standard metabolic rate (SMR), total antioxidant capacity (TAOC), and levels of oxidative stress biomarkers in the muscle tissues of clams and oysters exposed to different temperatures (22 and 27°C) and CO2 levels (the present day conditions of ~400ppm CO2 and 800ppm CO2 predicted by a consensus business-as-usual IPCC emission scenario for the year 2100). SMR was significantly higher and the antioxidant capacity was lower in oysters than in clams. Aerobic metabolism was largely temperature-independent in these two species in the studied temperature range (22-27°C). However, the combined exposure to elevated temperature and hypercapnia led to elevated SMR in clams indicating elevated costs of basal maintenance. No persistent oxidative stress signal (measured by the levels of protein carbonyls, and protein conjugates with malondialdehyde and 4-hydroxynonenal) was observed during the long-term exposure to moderate warming (+5°C) and hypercapnia (~800ppm CO2). This indicates that long-term exposure to moderately elevated CO2 and temperature minimally affects the cellular redox status in these bivalve species and that the earlier observed negative physiological effects of elevated CO2 and temperature must be explained by other cellular mechanisms.

Interactive effects of elevated temperature and CO2 levels on energy metabolism and biomineralization of marine bivalves Crassostrea virginica and Mercenaria mercenaria.

The continuing increase of carbon dioxide (CO2) levels in the atmosphere leads to increases in global temperatures and partial pressure of CO2 (PCO2) in surface waters, causing ocean acidification. These changes are especially pronounced in shallow coastal and estuarine waters and are expected to significantly affect marine calcifiers including bivalves that are ecosystem engineers in estuarine and coastal communities. To elucidate potential effects of higher temperatures and PCO2 on physiology and biomineralization of marine bivalves, we exposed two bivalve species, the eastern oysters Crassostrea virginica and the hard clams Mercenaria mercenaria to different combinations of PCO2 (~400 and 800µatm) and temperatures (22 and 27°C) for 15weeks. Survival, bioenergetic traits (tissue levels of lipids, glycogen, glucose and high energy phosphates) and biomineralization parameters (mechanical properties of the shells and activity of carbonic anhydrase, CA) were determined in clams and oysters under different temperature and PCO2 regimes. Our analysis showed major inter-species differences in shell mechanical traits and bioenergetics parameters. Elevated temperature led to the depletion of tissue energy reserves indicating energy deficiency in both species and resulted in higher mortality in oysters. Interestingly, while elevated PCO2 had a small effect on the physiology and metabolism of both species, it improved survival in oysters. At the same time, a combination of high temperature and elevated PCO2 lead to a significant decrease in shell hardness in both species, suggesting major changes in their biomineralization processes. Overall, these studies show that global climate change and ocean acidification might have complex interactive effects on physiology, metabolism and biomineralization in coastal and estuarine marine bivalves.

Effects of temperature and cadmium exposure on the mitochondria of oysters (Crassostrea virginica) exposed to hypoxia and subsequent reoxygenation.

Intertidal bivalves are commonly exposed to multiple stressors including periodic hypoxia, temperature fluctuations and pollution, which can strongly affect energy metabolism. We used top-down control and elasticity analyses to determine the interactive effects of intermittent hypoxia, cadmium (Cd) exposure and acute temperature stress on mitochondria of the eastern oyster Crassostrea virginica. Oysters were acclimated at 20°C for 30 days in the absence or presence of 50 µg l(-1) Cd and then subjected to a long-term hypoxia (6 days at <0.5% O(2) in seawater) followed by normoxic recovery. Mitochondrial function was assessed at the acclimation temperature (20°C), or at elevated temperature (30°C) mimicking acute temperature stress in the intertidal zone. In the absence of Cd or temperature stress, mitochondria of oysters showed high resilience to transient hypoxia. In control oysters at 20°C, hypoxia/reoxygenation induced elevated flux capacity of all three studied mitochondrial subsystems (substrate oxidation, phosphorylation and proton leak) and resulted in a mild depolarization of resting mitochondria. Elevated proton conductance and enhanced capacity of phosphorylation and substrate oxidation subsystems may confer resistance to hypoxia/reoxygenation stress in oyster mitochondria by alleviating production of reactive oxygen species and maintaining high aerobic capacity and ATP synthesis rates during recovery. Exposure to environmental stressors such as Cd and elevated temperatures abolished the putative adaptive responses of the substrate oxidation and phosphorylation subsystems, and strongly enhanced proton leak in mitochondria of oysters subjected to hypoxia/reoxygenation stress. Our findings suggest that Cd exposure and acute temperature stress may lead to the loss of mitochondrial resistance to hypoxia and reoxygenation and thus potentially affect the ability of oysters to survive periodic oxygen deprivation in coastal and estuarine habitats.

Interactive effects of salinity and elevated CO2 levels on juvenile eastern oysters, Crassostrea virginica.

Rising levels of atmospheric CO(2) lead to acidification of the ocean and alter seawater carbonate chemistry, which can negatively impact calcifying organisms, including mollusks. In estuaries, exposure to elevated CO(2) levels often co-occurs with other stressors, such as reduced salinity, which enhances the acidification trend, affects ion and acid-base regulation of estuarine calcifiers and modifies their response to ocean acidification. We studied the interactive effects of salinity and partial pressure of CO(2) (P(CO2)) on biomineralization and energy homeostasis in juveniles of the eastern oyster, Crassostrea virginica, a common estuarine bivalve. Juveniles were exposed for 11 weeks to one of two environmentally relevant salinities (30 or 15 PSU) either at current atmospheric P(CO2) (∼400 µatm, normocapnia) or P(CO2) projected by moderate IPCC scenarios for the year 2100 (∼700-800 µatm, hypercapnia). Exposure of the juvenile oysters to elevated P(CO2) and/or low salinity led to a significant increase in mortality, reduction of tissue energy stores (glycogen and lipid) and negative soft tissue growth, indicating energy deficiency. Interestingly, tissue ATP levels were not affected by exposure to changing salinity and P(CO2), suggesting that juvenile oysters maintain their cellular energy status at the expense of lipid and glycogen stores. At the same time, no compensatory upregulation of carbonic anhydrase activity was found under the conditions of low salinity and high P(CO2). Metabolic profiling using magnetic resonance spectroscopy revealed altered metabolite status following low salinity exposure; specifically, acetate levels were lower in hypercapnic than in normocapnic individuals at low salinity. Combined exposure to hypercapnia and low salinity negatively affected mechanical properties of shells of the juveniles, resulting in reduced hardness and fracture resistance. Thus, our data suggest that the combined effects of elevated P(CO2) and fluctuating salinity may jeopardize the survival of eastern oysters because of weakening of their shells and increased energy consumption.

Proteomic response to elevated PCO2 level in eastern oysters, Crassostrea virginica: evidence for oxidative stress.

Estuaries are characterized by extreme fluctuations in CO(2) levels due to bouts of CO(2) production by the resident biota that exceed its capacity of CO(2) consumption and/or the rates of gas exchange with the atmosphere and open ocean waters. Elevated partial pressures of CO(2) (P(CO(2)); i.e. environmental hypercapnia) decrease the pH of estuarine waters and, ultimately, extracellular and intracellular pH levels of estuarine organisms such as mollusks that have limited capacity for pH regulation. We analyzed proteomic changes associated with exposure to elevated P(CO(2)) in the mantle tissue of eastern oysters (Crassostrea virginica) after 2 weeks of exposure to control (∼39 Pa P(CO(2))) and hypercapnic (∼357 Pa P(CO(2))) conditions using two-dimensional gel electrophoresis and tandem mass spectrometry. Exposure to high P(CO(2)) resulted in a significant proteome shift in the mantle tissue, with 12% of proteins (54 out of 456) differentially expressed under the high P(CO(2)) compared with control conditions. Of the 54 differentially expressed proteins, we were able to identify 17. Among the identified proteins, two main functional categories were upregulated in response to hypercapnia: those associated with the cytoskeleton (e.g. several actin isoforms) and those associated with oxidative stress (e.g. superoxide dismutase and several peroxiredoxins as well as the thioredoxin-related nucleoredoxin). This indicates that exposure to high P(CO(2)) (∼357 Pa) induces oxidative stress and suggests that the cytoskeleton is a major target of oxidative stress. We discuss how elevated CO(2) levels may cause oxidative stress by increasing the production of reactive oxygen species (ROS) either indirectly by lowering organismal pH, which may enhance the Fenton reaction, and/or directly by CO(2) interacting with other ROS to form more free radicals. Although estuarine species are already exposed to higher and more variable levels of CO(2) than other marine species, climate change may further increase the extremes and thereby cause greater levels of oxidative stress.

Physiological Variation in Response to Vibrio and Hypoxia by Aquacultured Eastern Oysters in the Southeastern United States.

Eastern oysters (Crassostrea virginica) have long been recognized as model organisms of extreme environmental tolerance, showing resilience to variation in temperature, salinity, hypoxia, and microbial pathogens. These phenotypic responses, however, show variability between geographic locations or habitats (e.g., tidal). Physiological, morphological, and genetic differences occur in populations throughout a species' geographical range, which may have been shaped by regional abiotic and biotic variations. Few studies of C. virginica have explored the combined factors of physiological mechanisms of divergent phenotypes between locations and the genetic relationships of individuals between these locations. To characterize genetic relationships of four locations with aquacultured oysters along the North Carolina and Virginia coast, we sequenced a portion of cytochrome oxidase subunit I (COI) that revealed significant variation in haplotype distribution between locations. We then measured mitochondrial physiology and expression of the innate immunity response of hemocytes to lab acclimation and combined stress conditions to compare basal expression and stress response in oysters between these locations. For stress sensing genes, toll-like receptors had the strongest location-specific response to hypoxia and Vibrio, whereas mannose receptor and a stress-receptor were specific to hypoxia and bacteria, respectively. The expression of stress response genes also showed location-specific and stressor-specific changes in expression, particularly for big defensin and the complement gene Cq3. Our results further suggested that genetic similarity of oysters from different locations was not clearly related to physiological and molecular responses. These results are informative for understanding the range of physiological plasticity for stress responses in this commercially important oyster species. They also have implications in the oyster farming industry as well as conservation efforts to restore endangered native oyster beds.

Food-type may jeopardize biomarker interpretation in mussels used in aquatic toxicological experimentation.

To assess the influence of food type on biomarkers, mussels (Mytilus galloprovincialis) were maintained under laboratory conditions and fed using 4 different microalgae diets ad libitum for 1 week: (a) Isochrysis galbana; (b) Tetraselmis chuii; (c) a mixture of I. galbana and T. chuii; and (d) a commercial food (Microalgae Composed Diet, Acuinuga). Different microalgae were shown to present different distribution and fate in the midgut. I. galbana (≈4 µm Ø) readily reached digestive cells to be intracellularly digested. T. chuii (≈10 µm Ø and hardly digestible) was retained in stomach and digestive ducts for long times and extracellularly digested. Based on these findings, it appeared likely that the presence of large amounts of microalgal enzymes and metabolites might interfere with biochemical determinations of mussel's biomarkers and/or that the diet-induced alterations of mussels' digestion could modulate lysosomal and tissue-level biomarkers. To test these hypotheses, a battery of common biochemical, cytological and tissue-level biomarkers were determined in the gills (including activities of pyruvate kinase, phosphoenolpyruvate carboxykinase and cytochrome c oxidase) and the digestive gland of the mussels (including protein, lipid, free glucose and glycogen total content, lysosomal structural changes and membrane stability, intracellular accumulation of neutral lipids and lipofuscins, changes in cell type composition and epithelial thinning, as well as altered tissue integrity). The type of food was concluded to be a major factor influencing biomarkers in short-term experiments though not all the microalgae affected biomarkers and their responsiveness in the same way. T. chuii seemed to alter the nutritional status, oxidative stress and digestion processes, thus interfering with a variety of biomarkers. On the other hand, the massive presence of I. galbana within digestive cells hampered the measurement of cytochemical biomarkers and rendered less reliable the results of biochemical biomarkers (as these could be attributed to both the mussel and the microalgae). Research to optimize dietary food type, composition, regime and rations for toxicological experimentation is urgently needed. Meanwhile, a detailed description of the food type and feeding conditions should be always provided when reporting aquatic toxicological experiments with mussels, as a necessary prerequisite to compare and interpret the biological responses elicited by pollutants.

Bioenergetic responses of freshwater mussels Unio tumidus to the combined effects of nano-ZnO and temperature regime.

Bivalves from the cooling reservoirs of electrical power plants (PP) are exposed to the chronic heating and chemical pollution making them a suitable model to study the combined effects of these stressors. We investigated the effect of in situ exposures to chemical and thermal pollution in the PP cooling ponds on the metabolic responses of unionid bivalves (Unio tumidus) to a novel widespread pollutant, ZnO nanoparticles (nZnO). Male U. tumidus from the reservoirs of Dobrotvir and Burshtyn PPs (DPP and BPP) were maintained in clean water at 18 °C, or exposed for 14 days to one of the following conditions: nZnO (3.1 µM) or Zn2+ (3.1 µM, a positive control for Zn impacts) at 18 °C, elevated temperature (T, 25 °C), or nZnO at 25 °C (nZnO + T). Baseline levels of glycogen, lipids and ATP were similar in the two studied populations, whereas the levels of proteins, lactate/pyruvate ratio (L/P) and extralysosomal cathepsin D level were higher in the tissues of BPP mussels. The levels of glycogen and glucose declined in most experimental exposures indicating elevated energy demand except for a slight increase in the digestive gland of warming-exposed BPP mussels and in the gills of the nZnO + T-exposed DPP-mussels. Experimental exposures stimulated cathepsin D activity likely reflecting onset of autophagic processes to compensate for stress-induced energy demand. No depletion of ATP in Zn-containing exposures was observed indicating that the cellular metabolic adjustments were sufficient for such compensation. Unexpectedly, experimental warming mitigated most metabolic responses to nZnO in co-exposures. Our data thus indicate that metabolic effects of nZnO strongly depend on the environmental context of the mussels (such as temperature and acclimation history) which must be taken into account for the molecular and cellular biomarker-based assessment of the nanoparticle effects in the field.

Oxidative stress and expression of chaperones in aging mollusks.

The mechanisms of aging are not well understood in animals with continuous growth such as fish, reptiles, amphibians and numerous invertebrates, including mollusks. We studied the effects of age on oxidative stress, cellular defense mechanisms (including two major antioxidant enzymes, superoxide dismutase (SOD) and catalase), and molecular chaperones in two mollusks--eastern oysters Crassostrea virginica and hard clams Mercenaria mercenaria. In order to detect the age-related changes in these parameters, correction for the effects of size was performed where appropriate to account for growth-related dilution. Fluorescent age pigments accumulated with age in both species. Protein carbonyls did not change with age or size indicating that they are not a good marker of aging in mollusks possibly due to the fast turnover and degradation of oxidized proteins in growing tissues. SOD did not show a compensatory increase with aging in either species, while catalase significantly decreased with age. Mitochondrial heat shock protein (HSP60) decreased with age in mollusks suggesting an age-related decline in mitochondrial chaperone protection. In contrast, changes in cytosolic chaperones were species-specific. HSP70 increased and HSP90 declined with age in clams, whereas in oysters HSP70 expression did not change, and HSP90 increased with aging.

Effects of cadmium exposure on expression and activity of P-glycoprotein in eastern oysters, Crassostrea virginica Gmelin.

Heavy metal pollution is a worldwide problem, and cadmium (Cd) is one of the most noxious pollutants in aquatic environments. We studied P-glycoprotein (P-gp) expression and function in control and Cd exposed (50microgL(-1) Cd, 30-40 days) oysters Crassostrea virginica as a possible mechanism of cell protection against Cd. Our data show that P-gp is expressed on cell membrane and in mitochondria of oyster gills and hepatopancreas. Inhibitor studies with verapamil, cyclosporine A and JS-2190 suggest that in the gills, mitochondrial P-gp pumps substrates from cytosol into the mitochondria, while cell membrane P-gp pumps substrates from cytosol out of the cell. Cd exposure resulted in a 2-2.5-fold increase in P-gp protein expression in cell membranes and a 3.5-7-fold increase in transport activity measured as the inhibitor-sensitive rhodamine B extrusion rate. In contrast, p-gp mRNA levels were similar in control and Cd-exposed oysters. No difference in P-gp protein expression was observed between mitochondria of control and Cd-exposed oysters but the apparent transport activity was higher in mitochondria from Cd-exposed oysters. Overall, a stronger increase in substrate transport activity in Cd-exposed oysters compared to a relatively weaker change in P-gp protein levels suggests that P-gp activity is post-translationally regulated. Our data show that direct determination of P-gp transport activity may be the best measure of the xenobiotic-resistant phenotype, whereas p-gp mRNA levels are not a good marker due to the likely involvement of multiple post-transcriptional regulatory steps. Cd exposure resulted in a significantly elevated rate of oxygen consumption of isolated oyster gills by 46%. Specific inhibitors of ATPase function of P-gp (cyclosporine A and JS-2190) had no significant effect on tissue oxygen consumption indicating that P-gp contribution to energy budget is negligible and supporting indirect estimates based on the ATP stoichiometry of substrate transport that also suggest low energy demand for P-gp function.

Detoxification and cellular stress responses of unionid mussels Unio tumidus from two cooling ponds to combined nano-ZnO and temperature stress.

Bivalve mollusks from the cooling reservoirs of fuel power plants (PP) are acclimated to the chronic heating and chemical pollution. We investigated stress responses of the mussels from these ponds to determine their tolerance to novel environmental pollutant, zinc oxide nanoparticles (nZnO). Male Unio tumidus from the reservoirs of Dobrotvir and Burschtyn PPs (DPP and BPP), Ukraine were exposed for 14 days to nZnO (3.1 µM), Zn2+ (3.1 µM) at 18 °C, elevated temperature (T, 25 °C), or nZnO at 25 °C (nZnO + T). Control groups were held at 18 °C. Zn-containing exposures resulted in the elevated concentrations of total and Zn-bound metallothionein (MT and Zn-MT) in the digestive gland, an increase in the levels of non-metalated MT (up to 5 times) and alkali-labile phosphates and lysosomal membrane destabilization in hemocytes. A common signature of nZnO exposures was modulation of the multixenobiotic-resistance protein activity (a decrease in the digestive gland and increase in the gills). The origin of population strongly affected the cellular stress responses of mussels. DPP-mussels showed depletion of caspase-3 in the digestive gland and up-regulation of HSP70, HSP72 and HSP60 levels in the gill during most exposures, whereas in the BPP-mussels caspase-3 was up-regulated and HSPs either downregulated or maintained stable. BPP-mussels were less adapted to heating shown by a glutathione depletion at elevated temperature (25 °C). Comparison with the earlier studies on mussels from pristine habitats show that an integrative 'eco-exposome'-based approach is useful for the forecast of the biological responses to novel adverse effects on aquatic organisms.

Effects of environmental hypercapnia and metal (Cd and Cu) exposure on acid-base and metal homeostasis of marine bivalves.

Elevated CO2 levels reduce seawater pH and may affect bioavailability of trace metals in estuaries. We studied the interactive effects of common metal pollutants (50 µg l(-1) Cd or Cu) and PCO2 (~395, 800 and 2000 µatm) on metal levels, intracellular pH, expression of metal binding proteins and stress biomarkers in estuarine bivalves Crassostrea virginica (oysters) and Mercenaria mercenaria (hard clams). Cd (but not Cu or hypercapnia) exposure affected the acid-base balance of hemocytes resulting in elevated intracellular pH. Cd and Cu exposure led to the increase in the tissue metal burdens, and metal accumulation was reduced by elevated PCO2 in the mantle but not hemocytes. No change was found in the intracellular free Cd(2+), Cu(2+) or Fe(2+) during Cu or Cd exposure indicating that these metals are bound to intracellular ligands. Free Zn(2+) content in oyster hemocytes was suppressed by Cd and Cu exposure and below the detection limits in clam hemocytes, which went hand-in-hand with the elevated mRNA expression of metallothioneins and ferritin in Cd- and Cu-exposed bivalves, enhanced by hypercapnia. The metal-binding and antioxidant mechanisms of oysters and clams were sufficient to effectively maintain intracellular redox status, even though metal exposure combined with moderate hypercapnia (~800 µatm PCO2) led to the elevated production of reactive oxygen species in hemocytes. Overall, while hypercapnia modulates metal accumulation, binding capacity and oxidative stress in estuarine bivalves, the physiological effects of elevated CO2 are mild compared to the effects of other common stressors.