Combined effects of salinity and intermittent hypoxia on mitochondrial capacity and reactive oxygen species efflux in the Pacific oyster, Crassostrea gigas.

Coastal environments commonly experience fluctuations in salinity and hypoxia-reoxygenation (H/R) stress that can negatively affect mitochondrial functions of marine organisms. Although intertidal bivalves are adapted to these conditions, the mechanisms that sustain mitochondrial integrity and function are not well understood. We determined the rates of respiration and reactive oxygen species (ROS) efflux in the mitochondria of oysters, Crassostrea gigas, acclimated to high (33 psu) or low (15 psu) salinity, and exposed to either normoxic conditions (control; 21% O2) or short-term hypoxia (24 h at <0.01% O2) and subsequent reoxygenation (1.5 h at 21% O2). Further, we exposed isolated mitochondria to anoxia in vitro to assess their ability to recover from acute (∼10 min) oxygen deficiency (<0.01% O2). Our results showed that mitochondria of oysters acclimated to high or low salinity did not show severe damage and dysfunction during H/R stress, consistent with the hypoxia tolerance of C. gigas. However, acclimation to low salinity led to improved mitochondrial performance and plasticity, indicating that 15 psu might be closer to the metabolic optimum of C. gigas than 33 psu. Thus, acclimation to low salinity increased mitochondrial oxidative phosphorylation rate and coupling efficiency and stimulated mitochondrial respiration after acute H/R stress. However, elevated ROS efflux in the mitochondria of low-salinity-acclimated oysters after acute H/R stress indicates a possible trade-off of higher respiration. The high plasticity and stress tolerance of C. gigas mitochondria may contribute to the success of this invasive species and facilitate its further expansion into brackish regions such as the Baltic Sea.

Tissue- and substrate-dependent mitochondrial responses to acute hypoxia-reoxygenation stress in a marine bivalve (Crassostrea gigas).

Hypoxia is a major stressor for aquatic organisms, yet intertidal organisms such as the oyster Crassostrea gigas are adapted to frequent oxygen fluctuations by metabolically adjusting to shifts in oxygen and substrate availability during hypoxia-reoxygenation (H/R). We investigated the effects of acute H/R stress (15 min at ∼0% O2 and 10 min reoxygenation) on isolated mitochondria from the gill and the digestive gland of C. gigas respiring on different substrates (pyruvate, glutamate, succinate, palmitate and their mixtures). Gill mitochondria showed better capacity for amino acid and fatty acid oxidation compared with mitochondria from the digestive gland. Mitochondrial responses to H/R stress strongly depended on the substrate and the activity state of mitochondria. In mitochondria oxidizing NADH-linked substrates, exposure to H/R stress suppressed oxygen consumption and generation of reactive oxygen species (ROS) in the resting state, whereas in the ADP-stimulated state, ROS production increased despite little change in respiration. As a result, electron leak (measured as H2O2 to O2 ratio) increased after H/R stress in the ADP-stimulated mitochondria with NADH-linked substrates. In contrast, H/R exposure stimulated succinate-driven respiration without an increase in electron leak. Reverse electron transport (RET) did not significantly contribute to succinate-driven ROS production in oyster mitochondria except for a slight increase in the OXPHOS state during post-hypoxic recovery. A decrease in NADH-driven respiration and ROS production, enhanced capacity for succinate oxidation and resistance to RET might assist in post-hypoxic recovery of oysters mitigating oxidative stress and supporting rapid ATP re-synthesis during oxygen fluctuations, as is commonly observed in estuaries and intertidal zones.

Metabolic remodeling caused by heat hardening in the Mediterranean mussel Mytilus galloprovincialis.

Organisms can modify and increase their thermal tolerance faster and more efficiently after a brief exposure to sublethal thermal stress. This response is called 'heat hardening' as it leads to the generation of phenotypes with increased heat tolerance. The aim of this study was to investigate the impact of heat hardening on the metabolomic profile of Mytilus galloprovincialis in order to identify the associated adjustments of biochemical pathways that might benefit the mussels' thermal tolerance. Thus, mussels were exposed sequentially to two different phases (heat hardening and acclimation phases). To gain further insight into the possible mechanisms underlying the metabolic response of the heat-hardened M. galloprovincialis, metabolomics analysis was complemented by the estimation of mRNA expression of phosphoenolpyruvate carboxykinase (PEPCK), pyruvate kinase (PK) and alternative oxidase (AOX) implicated in the metabolic pathways of gluconeogenesis, glycolysis and redox homeostasis, respectively. Heat-hardened mussels showed evidence of higher activity of the tricarboxylic acid (TCA) cycle and diversification of upregulated metabolic pathways, possibly as a mechanism to increase ATP production and extend survival under heat stress. Moreover, formate and taurine accumulation provide an antioxidant and cytoprotective role in mussels during hypoxia and thermal stress. Overall, the metabolic responses in non-heat-hardened and heat-hardened mussels underline the upper thermal limits of M. galloprovincialis, set at 26°C, and are in accordance with the OCLTT concept. The ability of heat-hardened mussels to undergo a rapid gain and slow loss of heat tolerance may be an advantageous strategy for coping with intermittent and often extreme temperatures.

Effects of variable oxygen regimes on mitochondrial bioenergetics and reactive oxygen species production in a marine bivalve, Mya arenaria.

Estuarine and coastal benthic organisms often experience fluctuations in oxygen levels that can negatively impact their mitochondrial function and aerobic metabolism. To study these impacts, we exposed a common sediment-dwelling bivalve, the soft-shell clam Mya arenaria, for 21 days to chronic hypoxia (PO2  âˆ¼4.1 kPa), cyclic hypoxia (PO2  âˆ¼12.7-1.9 kPa, mean 5.7 kPa) or normoxia (PO2  âˆ¼21.1 kPa). pH was manipulated to mimic the covariation in CO2/pH and oxygen levels in coastal hypoxic zones. Mitochondrial respiration, including proton leak, the capacity for oxidative phosphorylation (OXPHOS), the maximum activity of the electron transport system (ETS), reactive oxygen species (ROS) production, and activity and oxygen affinity of cytochrome c oxidase (CCO) were assessed. Acclimation to constant hypoxia did not affect the studied mitochondrial traits except for a modest decrease in the OXPHOS coupling efficiency. Cyclic hypoxia had no effect on OXPHOS or ETS capacity, but increased proton leak and lowered mitochondrial OXPHOS coupling efficiency. Furthermore, mitochondria of clams acclimated to cyclic hypoxia had higher rates of ROS generation compared with the clams acclimated to normoxia or chronic hypoxia. CCO activity was upregulated under cyclic hypoxia, but oxygen affinity of CCO did not change. These findings indicate that long-term cyclic hypoxia has a stronger impact on the mitochondria of M. arenaria than chronic hypoxia and might lead to impaired ATP synthesis, higher costs of mitochondrial maintenance and oxidative stress. These changes might negatively affect populations of M. arenaria in the coastal Baltic Sea under increasing hypoxia pressure.

Mitochondrial capacity and reactive oxygen species production during hypoxia and reoxygenation in the ocean quahog, Arctica islandica.

Oxygen fluctuations are common in marine waters, and hypoxia-reoxygenation (H-R) stress can negatively affect mitochondrial metabolism. The long-lived ocean quahog, Arctica islandica, is known for its hypoxia tolerance associated with metabolic rate depression, yet the mechanisms that sustain mitochondrial function during oxygen fluctuations are not well understood. We used top-down metabolic control analysis (MCA) to determine aerobic capacity and control over oxygen flux in the mitochondria of quahogs exposed to short-term hypoxia (24 h <0.01% O2) and subsequent reoxygenation (1.5 h 21% O2) compared with normoxic control animals (21% O2). We demonstrated that flux capacity of the substrate oxidation and proton leak subsystems were not affected by hypoxia, while the capacity of the phosphorylation subsystem was enhanced during hypoxia associated with a depolarization of the mitochondrial membrane. Reoxygenation decreased the oxygen flux capacity of all three mitochondrial subsystems. Control over oxidative phosphorylation (OXPHOS) respiration was mostly exerted by substrate oxidation regardless of H-R stress, whereas control by the proton leak subsystem of LEAK respiration increased during hypoxia and returned to normoxic levels during reoxygenation. During hypoxia, reactive oxygen species (ROS) efflux was elevated in the LEAK state, whereas it was suppressed in the OXPHOS state. Mitochondrial ROS efflux returned to normoxic control levels during reoxygenation. Thus, mitochondria of A. islandica appear robust to hypoxia by maintaining stable substrate oxidation and upregulating phosphorylation capacity, but remain sensitive to reoxygenation. This mitochondrial phenotype might reflect adaptation of A. islandica to environments with unpredictable oxygen fluctuations and its behavioural preference for low oxygen levels.

Rethinking Nano-TiO2 Safety: Overview of Toxic Effects in Humans and Aquatic Animals.

Titanium dioxide nanoparticles (nano-TiO2 ) are widely used in consumer products, raising environmental and health concerns. An overview of the toxic effects of nano-TiO2 on human and environmental health is provided. A meta-analysis is conducted to analyze the toxicity of nano-TiO2 to the liver, circulatory system, and DNA in humans. To assess the environmental impacts of nano-TiO2 , aquatic environments that receive high nano-TiO2 inputs are focused on, and the toxicity of nano-TiO2 to aquatic organisms is discussed with regard to the present and predicted environmental concentrations. Genotoxicity, damage to membranes, inflammation and oxidative stress emerge as the main mechanisms of nano-TiO2 toxicity. Furthermore, nano-TiO2 can bind with free radicals and signal molecules, and interfere with the biochemical reactions on plasmalemma. At the higher organizational level, nano-TiO2 toxicity is manifested as the negative effects on fitness-related organismal traits including feeding, reproduction and immunity in aquatic organisms. Bibliometric analysis reveals two major research hot spots including the molecular mechanisms of toxicity of nano-TiO2 and the combined effects of nano-TiO2 and other environmental factors such as light and pH. The possible measures to reduce the harmful effects of nano-TiO2 on humans and non-target organisms has emerged as an underexplored topic requiring further investigation.

Effects of intermittent hypoxia on cell survival and inflammatory responses in the intertidal marine bivalves Mytilus edulis and Crassostrea gigas.

Hypoxia is a major stressor in estuarine and coastal habitats, leading to adverse effects in aquatic organisms. Estuarine bivalves such as blue mussels (Mytilus edulis) and Pacific oysters (Crassostrea gigas) can survive periodic oxygen deficiency but the molecular mechanisms that underlie cellular injury during hypoxia-reoxygenation are not well understood. We examined the molecular markers of autophagy, apoptosis and inflammation during short-term (1 day) and long-term (6 days) hypoxia and post-hypoxic recovery (1 h) in mussels and oysters by measuring the lysosomal membrane stability, activity of a key autophagic enzyme (cathepsin D) and mRNA expression of the genes involved in the cellular survival and inflammation, including caspase 2, 3 and 8, Bcl-2, BAX, TGF-ß-activated kinase 1 (TAK1), nuclear factor kappa B1 (NF-κB) and NF-κB activating kinases IKKα and TBK1. Crassostrea gigas exhibited higher hypoxia tolerance, as well as blunted or delayed inflammatory and apoptotic response to hypoxia and reoxygenation as shown by the later onset and/or the lack of transcriptional activation of caspases, BAX and the inflammatory effector NF-κB, compared with M. edulis Long-term hypoxia resulted in upregulation of Bcl-2 in the oysters and mussels, implying activation of anti-apoptotic mechanisms. Our findings indicate the potential importance of the cell survival pathways in hypoxia tolerance of marine bivalves, and demonstrate the utility of the molecular markers of apoptosis and autophagy for the assessment of sublethal hypoxic stress in bivalve populations.

Mitochondrial performance of a continually growing marine bivalve, Mytilus edulis, depends on body size.

Allometric decline of mass-specific metabolic rate with increasing body size in organisms is a well-documented phenomenon. Despite a long history of research, the mechanistic causes of metabolic scaling with body size remain under debate. Some hypotheses suggest that intrinsic factors such as allometry of cellular and mitochondrial metabolism may contribute to the organismal-level metabolic scaling. The aim of our present study was to determine the metabolic allometry at the mitochondrial level using a continually growing marine ectotherm, the mussel Mytilus edulis, as a model. Mussels from a single cohort that considerably differed in body size were selected, implying faster growth in the larger specimens. We determined the body mass-dependent scaling of the mitochondrial proton leak respiration, respiration in the presence of ADP indicative of the oxidative phosphorylation (OXPHOS), and maximum activity of the mitochondrial electron transport system (ETS) and cytochrome c oxidase (COX). Respiration was measured at normal (15°C), and elevated (27°C) temperatures. The results demonstrated a pronounced allometric increase in both proton leak respiration and OXPHOS activity of mussel mitochondria. Mussels with faster growth (larger body size) showed an increase in OXPHOS rate, proton leak respiration rate, and ETS and COX activity (indicating an overall improved mitochondrial performance) and higher respiratory control ratio (indicating better mitochondrial coupling and potentially lower costs of mitochondrial maintenance at the same OXPHOS capacity) compared with slower growing (smaller) individuals. Our data show that the metabolic allometry at the organismal level cannot be directly explained by mitochondrial functioning.

Effects of prolonged food limitation on energy metabolism and burrowing activity of an infaunal marine bivalve, Mya arenaria.

Benthic organisms are subject to prolonged seasonal food limitation in the temperate shallow coastal waters that can cause energetic stress and affect their performance. Sediment-dwelling marine bivalves cope with prolonged food limitation by adjusting different physiological processes that might cause trade-offs between maintenance and other fitness-related functions. We investigated the effects of prolonged (42 days) food deprivation on bioenergetics, burrowing performance and amino acid profiles in a common marine bivalve, Mya arenaria collected in winter and spring. Food limitation of >15 days decreased respiration of the clams by 80%. Total tissue energy content was higher in spring-collected clams (reflecting higher lipid content) than in their winter counterparts. Prolonged food deprivation decreased the tissue energy content of clams, especially in winter. The levels of free amino acids transiently increased during the early phase of food deprivation possibly reflecting suppression of the protein synthesis or enhanced protein degradation. The levels of amino acids considered essential for bivalves were more tightly conserved than those of non-essential amino acids during starvation. The burrowing capacity of clams was negatively affected by food deprivation so that the time required for a burial cycle increased by 35-50% after 22-42 days of starvation. During the early phase of starvation, clams preferentially used lipids as fuel for burrowing, whereas carbohydrates were used at the later phase. These findings suggest that although M. arenaria can withstand prolonged food deprivation by lowering their basal maintenance costs and switching their fuel usage, their ecological functions (e.g. bioturbation and the energy transferable to the next trophic level) could be negatively impacted by starvation.

Effects of hypoxia and reoxygenation on intermediary metabolite homeostasis of marine bivalves Mytilus edulis and Crassostrea gigas.

Marine benthic invertebrates are frequently exposed to fluctuating oxygen levels resulting in hypoxia-reoxygenation (H/R) stress in the intertidal, estuarine and shallow coastal habitats. H/R stress can strongly affect the organisms' physiological performance due to the negative shifts in bioenergetics and redox balance. H/R stress commonly leads to the depletion of energy substrates and accumulation of anaerobic end products, but the effects of H/R stress on the homeostasis of the intermediate nitrogenous compounds are not well understood. We studied the effects of the short-term and long-term hypoxia (1 and 6 days, respectively) and subsequent reoxygenation on the metabolite profiles of free amino acids (FAAs), as well as the intermediates of the urea cycle and purine metabolism in two species of hypoxia-tolerant intertidal bivalves, the blue mussels Mytilus edulis and the Pacific oysters Crassostrea gigas. Accumulation of succinate was assessed to determine the role of anaerobiosis in the metabolic responses to H/R stress. Our study showed that the more hypoxia-tolerant of the two studied species (C. gigas) had lower rate of succinate accumulation during hypoxia (indicating stronger metabolic rate suppression) and was better able to maintain the homeostasis of nitrogenous intermediates during H/R stress compared with the less hypoxia-tolerant M. edulis. Furthermore, analysis of the metabolite profiles indicate that the oysters maintain high levels of cytoprotective compounds (such as taurine and GABA), accumulate lower levels of potential prooxidants (such as succinate and hypoxanthine) and experience less damage to oxidation-prone thiol-containing amino acids such as cysteine, homocysteine and methionine during hypoxia and reoxygenation compared with the blue mussels. This study indicates a potentially important role of intermediate metabolite homeostasis in the tolerance to prolonged hypoxia and H/R stress in marine organisms and opens avenue for further testing of this hypothesis in a broader comparative framework.

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.

Interactive effects of osmotic stress and burrowing activity on protein metabolism and muscle capacity in the soft shell clam Mya arenaria.

Bioturbators such as sediment-dwelling marine bivalves are ecosystem engineers that enhance sediment-water exchange and benthic-pelagic coupling. In shallow coastal areas, bivalves are exposed to frequent disturbance and salinity stress that might negatively affect their activity and physiological performance; however, the mechanisms underlying these effects are not fully understood. We investigated the effects of osmotic stress (low and fluctuating salinity) and repeated burrowing on aerobic and contractile capacity of the foot muscle (assessed by the activity of succinate dehydrogenase and myosin ATPase) as well as the levels of organic osmolytes (free amino acids) and biochemical markers of protein synthesis and proteolysis in key osmoregulatory and energy storing tissues (gills and hepatopancreas, respectively) in a common bioturbator, the soft shell clam Mya arenaria. Osmotic stress and exhaustive exercise altered the foot muscle capacity of soft shell clams and had a strong impact on protein and amino acid homeostasis in tissues not directly involved in locomotion. Acclimation to constant low salinity (5 practical salinity units) depleted the whole-body free amino acid pool and affected protein synthesis but not protein breakdown in the gill. In contrast, fluctuating (5-15) salinity increased protein breakdown rate, suppressed protein synthesis, caused oxidative damage to proteins in the gill and selectively depleted whole-body glycine pool. Clams acclimated to normal salinity (15) increased the aerobic capacity of the foot muscle upon repeated burrowing, whereas acclimation to low and fluctuating salinity reduced this adaptive muscle plasticity. Under the normal and low salinity conditions, exhaustive exercise induced protein conservation pathways (indicated by suppression of protein synthesis and catabolism), but this effect was disrupted by fluctuating salinity. These findings indicate that exhaustive exercise and osmotic stress interactively affect whole-body protein homeostasis and functional capacity of the foot muscle in soft shell clams which might contribute to reduced burrowing activity of bivalve bioturbators in osmotically challenging environments such as estuaries and shallow coastal zones.

Effects of mechanical disturbance and salinity stress on bioenergetics and burrowing behavior of the soft-shell clam Mya arenaria.

Bioturbation of sediments by burrowing organisms plays a key role in the functioning of coastal ecosystems. Burrowing is considered an energetically expensive activity, yet the energy costs of burrowing and the potential impacts of multiple stressors (such as salinity stress and wave action) on bioenergetics and burrowing performance of marine bioturbators are not well understood. We investigated the effects of mechanical disturbance and salinity stress on the burrowing behavior, aerobic capacity and energy expense of digging in a common marine bioturbator, the soft-shell clam Mya arenaria from the Baltic Sea (control salinity 15). Mya arenaria showed large individual variability in the burrowing efficiency, with an average of ∼7% of the body energy reserves used per burial. Clams with higher mitochondrial capacity and lower energy expenditure per burial showed higher endurance. Acclimation for 3-4 weeks to low (5) or fluctuating (5-15) salinity reduced the burrowing speed and the number of times the clams can rebury but did not affect the mitochondrial capacity of the whole body or the gill. Acclimation to the fluctuating salinity shifted the predominant fuel use for burrowing from proteins to lipids. Our data indicate that the reduced burrowing performance of clams under the salinity stress is not due to the limitations of energy availability or aerobic capacity but must involve other mechanisms (such as impaired muscle performance). The reduction in the burrowing capacity of clams due to salinity stress may have important implications for survival, activity and ecological functions of the clams in shallow coastal ecosystems.

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.