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.

Hot and heavy: Responses of ragworms (Hediste diversicolor) to copper-spiked sediments and elevated temperature.

Sediment contamination and seawater warming are two major stressors to macrobenthos in estuaries. However, little is known about their combined effects on infaunal organisms. Here we investigated the responses of an estuarine polychaete Hediste diversicolor to metal-contaminated sediment and increased temperature. Ragworms were exposed to sediments spiked with 10 and 20 mg kg-1 of copper at 12 and 20 °C for three weeks. No considerable changes were observed in the expression of genes related to copper homeostasis and in the accumulation of oxidative stress damage. Dicarbonyl stress was attenuated by warming exposure. Whole-body energy reserves in the form of carbohydrates, lipids and proteins were little affected, but the energy consumption rate increased with copper exposure and elevated temperature, indicating higher basal maintenance costs of ragworms. The combined effects of copper and warming exposures were mostly additive, with copper being a weak stressor and warming a more potent stressor. These results were replicable, as confirmed by two independent experiments of similar settings conducted at two different months of the year. This study suggests the higher sensitivity of energy-related biomarkers and the need to search for more conserved molecular markers of metal exposure in H. diversicolor.

Salinity variation modulates cellular stress response to ZnO nanoparticles in a sentinel marine bivalve, the blue mussel Mytilussp.

Zinc oxide nanoparticles are released into marine environments from industrial, medical and consumer uses sparking concerns about their potential ecotoxicological effects. Ecological hazard assessment of nZnO in marine ecosystems is hindered by the lack of understanding of the potential interactive effects of nZnO toxicity with other common abiotic stressors, such as salinity fluctuations, in marine organisms. To close this gap in our knowledge, we carried out a comprehensive biomarker-based assessment of the combined effects of salinity and nZnO in a sentinel marine bivalve, the blue mussels Mytilus edulis. The mussels were exposed for 21 days to clean seawater (control), an environmentally relevant concentration (100 µg Zn l-1) of nZnO or dissolved Zn (to identify the toxic effects attributable to Zn2+ toxicity) under the normal (15), low (5) and fluctuating (5-15) salinity regimes. The selected molecular and biochemical markers focused on the oxidative stress, apoptosis, detoxification system and inflammation in the gills and the digestive gland of the mussels. Biomarker analysis showed different effects of nZnO and dissolved Zn on biomarkers of oxidative stress, xenobiotic detoxification and apoptosis but similar effects of both pollutants on the levels of metallothioneins and inflammatory markers. Exposure to nZnO led to elevated levels of lipid peroxidation, upregulation of p53 and p38 stress kinases and apoptosis-related genes, most notably in the gills. Exposure to dissolved Zn led to accumulation of protein carbonyls and activated redox-sensitive detoxification enzymes (NADPH-P450 reductase and glutathione-S-transferase) in the mussels. The ambient salinity had significant effects the cellular adverse effects of nZnO in the mussels. The nZnO-induced cellular stress was detectable under the normal (15) and fluctuating (5-15) salinity conditions in the studied brackish water population of the mussels. At low salinity (5), nZnO toxicity signal was almost completely dampened. These findings indicate that chronic osmotic stress close to the tolerance limits of M. edulis prevails over the effects of the environmentally relevant nZnO and dissolved Zn concentrations in combined exposures. These stressor interactions might ameliorate the cellular toxicity of nZnO in the mussels but limit applicability of cellular stress biomarkers for detecting the toxic effects of nanopollutants in low salinity habitats.

Effects of hypoxia and reoxygenation on mitochondrial functions and transcriptional profiles of isolated brain and muscle porcine cells.

Oxygen fluctuations might occur in mammalian tissues under physiological (e.g. at high altitudes) or pathological (e.g. ischemia-reperfusion) conditions. Mitochondria are the key target and potential amplifiers of hypoxia-reoxygenation (H-R) stress. Understanding the mitochondrial responses to H-R stress is important for identifying adaptive mechanisms and potential therapeutic solutions for pathologies associated with oxygen fluctuations. We explored metabolic response to H-R stress in two tissue types (muscle and brain) with different degrees of hypoxia tolerance in a domestic pig Sus scrofa focusing on the cellular responses independent of the systemic regulatory mechanisms. Isolated cells from the skeletal muscle (masseter) and brain (thalamus) were exposed to acute short-term (15 min) hypoxia followed by reoxygenation. The mitochondrial oxygen consumption, reactive oxygen species (ROS) production rates and transcriptional profiles of hypoxia-responsive mRNA and miRNA were determined. Mitochondria of the porcine brain cells showed a decrease in the resting respiration and ATP synthesis capacity whereas the mitochondria from the muscle cells showed robust respiration and less susceptibility to H-R stress. ROS production was not affected by the short-term H-R stress in the brain or muscle cells. Transcriptionally, prolyl hydroxylase domain protein EGLN3 was upregulated during hypoxia and suppressed during reoxygenation in porcine muscle cells. The decline in EGLN3 mRNA during reoxygenation was accompanied by an upregulation of hypoxia-inducible factor subunit α (HIF1A) transcripts in the muscle cells. However, in the brain cells, HIF1A mRNA levels were suppressed during reoxygenation. Other functionally important transcripts and miRNAs involved in antioxidant response, apoptosis, inflammation, and substrate oxidation were also differentially expressed between the muscle and brain cells. Suppression of miRNA levels during acute intermittent hypoxia was stronger in the brain cells affecting ~ 55% of all studied miRNA transcripts than in the muscle cells (~ 25% of miRNA) signifying transcriptional derepression of the respective mRNA targets. Our study provides insights into the potential molecular and physiological mechanisms contributing to different hypoxia sensitivity of the studied tissues and can serve as a starting point to better understand the biological processes associated with hypoxia stress, e.g. during ischemia and reperfusion.

Gone with sunscreens: Responses of blue mussels (Mytilus edulis) to a wide concentration range of a UV filter ensulizole.

Organic UV filters have emerged as a new threat to marine organisms, but ecotoxicological studies have so far focused on only a few substances despite the chemical diversity of these synthetic sunscreen agents. Here we examined the responses of blue mussels Mytilus edulis to ensulizole, a non-lipophilic UV filter commonly found in the Baltic Sea. Mussels were exposed for three weeks to five ensulizole concentrations of 10, 102, 103, 104, and 105 ng/L. Stress on stress response was evaluated by subjecting mussels to air exposure. A battery of biomarkers related to detoxification and antioxidant defense, oxidative stress damage, energy reserves and metabolism, autophagy, apoptosis, inflammation, and DNA damage was measured in the gills and the digestive gland. In general, ensulizole affected the antioxidant response, energy storage, and cell death-related processes in mussel tissues. Mussels exposed to low, environmentally relevant concentrations of ensulizole had a shorter air survival time than the control. Ensulizole often showed the non-monotonic concentration-response curves, suggesting the complex effects of this UV filter at molecular, biochemical, and organismal levels.

Interactive effects of ZnO nanoparticles and temperature on molecular and cellular stress responses of the blue mussel Mytilus edulis.

Temperature is an important abiotic factor that modulates all aspects of ectotherm physiology, including sensitivity to pollutants. Nanoparticles are emerging pollutants in coastal environments, and their potential to cause toxicity in marine organisms is a cause for concern. Here we studied the interactive effects of temperature (including seasonal and experimental warming) on sublethal toxicity of ZnO nanoparticles (nano-ZnO) in a model marine bivalve, the blue mussel Mytilus edulis. Molecular markers were used to assess the pollutant-induced cellular stress responses in the gills and the digestive gland of mussels exposed for 21 days to 10 µg l-1 and 100 µg l-1 of nano-ZnO or dissolved Zn under different temperature regimes including ambient temperature (10 °C and 15 °C in winter and summer, respectively) or experimental warming (+5 °C). Exposure to high concentration (100 µg l-1) of nano-ZnO caused oxidative injury to proteins and lipids and induced a marked apoptotic response indicated by increased transcript levels of apoptosis-related genes p53, caspase 3 and the MAPK pathway (JNK and p38) and decreased mRNA expression of anti-apoptotic Bcl-2. No significant induction of inflammatory cytokine-related response (TGF-ß and NF-κB) of tissues was observed in nano-ZnO exposed-mussels. Furthermore, the oxidative injury and apoptotic response could differentiate the effects of nano-ZnO from those of dissolved Zn in the mussels. This study revealed that oxidative stress and stress-related transcriptional responses to nano-ZnO were strongly modified by warming and season in the mussels. No single biomarker could be shown to consistently respond to nano-ZnO in all experimental groups, which implies that multiple biomarkers are needed to assess nano-ZnO toxicity to marine organisms under the variable environmental conditions of coastal habitats.

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.

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.

Biomarker-based assessment of sublethal toxicity of organic UV filters (ensulizole and octocrylene) in a sentinel marine bivalve Mytilus edulis.

The global occurrence of organic UV filters in the marine environment is of increasing ecotoxicological concern. Here we assessed the toxicity of UV filters ensulizole and octocrylene in the blue mussels Mytilus edulis exposed to 10 or 100 µg l-1 of octocrylene and ensulizole for two weeks. An integrated battery of biochemical and molecular biomarkers related to xenobiotics metabolism and cellular toxicity (including oxidative stress, DNA damage, apoptosis, autophagy and inflammation) was used to assess the toxicity of these UV filters in the mussels. Octocrylene (but not ensulizole) accumulated in the mussel tissues during the waterborne exposures. Both studied UV filters induced sublethal toxic effects in M. edulis at the investigated concentrations. These effects involved induction of oxidative stress, genotoxicity (indicated by upregulation of DNA damage sensing and repair markers), upregulation of apoptosis and inflammation, and dysregulation of the xenobiotic biotransformation system. Octocrylene induced cellular stress in a concentration-dependent manner, whereas ensulizole appeared to be more toxic at the lower (10 µg l-1) studied concentration than at 100 µg l-1. The different concentration-dependence of sublethal effects and distinct toxicological profiles of ensulizole and octocrylene show that the environmental toxicity is not directly related to lipophilicity and bioaccumulation potential of these UV filters and demonstrate the importance of using bioassays for toxicity assessment of emerging pollutants in coastal marine ecosystems.

Salinity-dependent effects of ZnO nanoparticles on bioenergetics and intermediate metabolite homeostasis in a euryhaline marine bivalve, Mytilus edulis.

Engineered nanoparticles including ZnO nanoparticles (nZnO) are important emerging pollutants in aquatic ecosystems creating potential risks to coastal ecosystems and associated biota. The toxicity of nanoparticles and its interaction with the important environmental stressors (such as salinity variation) are not well understood in coastal organisms and require further investigation. Here, we examined the interactive effects of 100 µg l-1 nZnO or dissolved Zn (as a positive control for Zn2+ release) and salinity (normal 15, low 5, and fluctuating 5-15) on bioenergetics and intermediate metabolite homeostasis of a keystone marine bivalve, the blue mussel Mytilus edulis from the Baltic Sea. nZnO exposures did not lead to strong disturbances in energy or intermediate metabolite homeostasis regardless of the salinity regime. Dissolved Zn exposures suppressed the mitochondrial ATP synthesis capacity and coupling as well as anaerobic metabolism and modified the free amino acid profiles in the mussels indicating that dissolved Zn is metabolically more damaging than nZnO. The environmental salinity regime strongly affected metabolic homeostasis and altered physiological and biochemical responses to nZnO or dissolved Zn in the mussels. Exposure to low (5) or fluctuating (5-15) salinity affected the physiological condition, energy metabolism and homeostasis, as well as amino acid metabolism in M. edulis. Generally, fluctuating salinity (5-15) appeared bioenergetically less stressful than constantly hypoosmotic stress (salinity 5) in M. edulis indicating that even short (24 h) periods of recovery might be sufficient to restore the metabolic homeostasis in this euryhaline species. Notably, the biological effects of nZnO and dissolved Zn became progressively less detectable as the salinity stress increased. These findings demonstrate that habitat salinity must be considered in the biomarker-based assessment of the toxic effects of nanopollutants on coastal organisms.

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.

Season-dependent effects of ZnO nanoparticles and elevated temperature on bioenergetics of the blue mussel Mytilus edulis.

Input of ZnO nanoparticles (nZnO) from multiple sources have raised concerns about the potential toxic effects on estuarine and coastal organisms. The toxicity of nZnO and its interaction with common abiotic stressors (such as elevated temperature) are not well understood in these organisms. Here, we examined the bioenergetics responses of the blue mussel Mytilus edulis exposed for 21 days to different concentrations of nZnO or dissolved zinc (Zn2+) (0, 10, 100 µg l-1) and two temperatures (ambient and 5 °C warmer) in winter and summer. Exposure to nZnO had little effect on the protein and lipid levels, but led to a significant depletion of carbohydrates and a decrease in the electron transport system (ETS) activity. Qualitatively similar but weaker effects were found for dissolved Zn. In winter mussels, elevated temperature (15 °C) led to elevated protein and lipid levels increasing the total energy content of the tissues. In contrast, elevated temperature (20 °C) resulted in a decrease in the lipid and carbohydrate levels and suppressed ETS in summer mussels. These data indicate that moderate warming in winter (but not in summer) might partially compensate for the bioenergetics stress caused by nZnO toxicity in M. edulis from temperate areas such as the Baltic Sea.

Mitochondrial Mechanisms Underlying Tolerance to Fluctuating Oxygen Conditions: Lessons from Hypoxia-Tolerant Organisms.

Oxygen (O2) is essential for most metazoan life due to its central role in mitochondrial oxidative phosphorylation (OXPHOS), which generates >90% of the cellular adenosine triphosphate. O2 fluctuations are an ultimate mitochondrial stressor resulting in mitochondrial damage, energy deficiency, and cell death. This work provides an overview of the known and putative mechanisms involved in mitochondrial tolerance to fluctuating O2 conditions in hypoxia-tolerant organisms including aquatic and terrestrial vertebrates and invertebrates. Mechanisms of regulation of the mitochondrial OXPHOS and electron transport system (ETS) (including alternative oxidases), sulphide tolerance, regulation of redox status and mitochondrial quality control, and the potential role of hypoxia-inducible factor (HIF) in mitochondrial tolerance to hypoxia are discussed. Mitochondrial phenotypes of distantly related animal species reveal common features including conservation and/or anticipatory upregulation of ETS capacity, suppression of reactive oxygen species (ROS)-producing electron flux through ubiquinone, reversible suppression of OXPHOS activity, and investment into the mitochondrial quality control mechanisms. Despite the putative importance of oxidative stress in adaptations to hypoxia, establishing the link between hypoxia tolerance and mitochondrial redox mechanisms is complicated by the difficulties of establishing the species-specific concentration thresholds above which the damaging effects of ROS outweigh their potentially adaptive signaling function. The key gaps in our knowledge about the potential mechanisms of mitochondrial tolerance to hypoxia include regulation of mitochondrial biogenesis and fusion/fission dynamics, and HIF-dependent metabolic regulation that require further investigation in hypoxia-tolerant species. Future physiological, molecular and genetic studies of mitochondrial responses to hypoxia, and reoxygenation in phylogenetically diverse hypoxia-tolerant species could reveal novel solutions to the ubiquitous and metabolically severe problem of O2 deficiency and would have important implications for understanding the evolution of hypoxia tolerance and the potential mitigation of pathological states caused by O2 fluctuations.

Effects of a common pharmaceutical, atorvastatin, on energy metabolism and detoxification mechanisms of a marine bivalve Mytilus edulis.

Biologically active compounds from pharmaceuticals cause concern due to their common occurrence in water and sediments of urbanized coasts and potential threat to marine organisms. Atorvastatin (ATO), a globally prescribed drug, is environmentally stable and bioavailable to marine organisms; however, the physiological and toxic effects of this drug on ecologically important coastal species are yet to be elucidated. We studied the effect of ATO (˜1.2 µg L-1) on bioenergetics (including whole-organism and mitochondrial respiration, as well as tissue energy reserves and mRNA expression of genes involved in mitochondrial biogenesis and fatty acid metabolism in the gills and the digestive gland) of a keystone bivalve Mytulis edulis (the blue mussel) from the Baltic Sea. Xenobiotic detoxification systems including activity and mRNA expression of P-glycoprotein, and Phase I and II biotransformation enzymes (cytochrome P450 monooxygenase CYP1A and glutathione transferase, GST) were also assessed in the gill and digestive gland of the mussels. Exposure to ATO caused rapid uptake and biotransformation of the drug by the mussels. Standard metabolic rate of ATO-exposed mussels increased by 56% indicating higher maintenance costs, yet no changes were detected in the respiratory capacity of isolated mitochondria. ATO exposure led to ˜60% decrease in the lysosomal membrane stability of hemocytes and ˜3-fold decrease in the whole-organism P-glycoprotein-driven and diffusional efflux of xenobiotics indicating altered membrane properties. The digestive gland was a major target of ATO toxicity in the mussels. Exposure of mussels to ATO led to depletion of lipid, carbohydrate and protein pools, and suppressed transcription of key enzymes involved in mitochondrial biogenesis (peroxisome proliferator-activated receptor gamma coactivator 1-alpha PGC-1α) and fatty acid metabolism (acetyl-CoA carboxylase and CYP4Y1) in the digestive gland. No bioenergetic disturbances were observed in the gills of ATO-exposed mussels, and elevated GST activity indicated enhanced ATO detoxification in this tissue. These data demonstrate that ATO can act as a metabolic disruptor and chemosensitizer in keystone marine bivalves and warrant further investigations of statins as emerging pollutants of concern in coastal marine ecosystems.

Compatible osmolytes modulate mitochondrial function in a marine osmoconformer Crassostrea gigas (Thunberg, 1793).

Salinity is an important environmental factor affecting physiology of marine organisms. Osmoconformers such as marine mollusks maintain metabolic function despite changes of the osmolarity and composition of the cytosol during salinity shifts. Currently, metabolic responses to the salinity-induced changes of the intracellular milieu are not well understood. We studied the effects of osmolarity (450 vs. 900 mOsm) and compatible osmolytes (70-590 mM of taurine or betaine) on isolated gill mitochondria of a marine osmoconformer, the Pacific oyster Crassostrea gigas. Physiological concentrations of taurine enhanced mitochondrial ATP synthesis and electron transport system (ETS) capacity, increased mitochondrial coupling and stimulated the forward flux through the Complex I. Notably, the stimulatory effects of taurine were more pronounced at 900 mOsm compared to 450 mOsm. In contrast, betaine proportionally increased the rates of the mitochondrial proton leak, oxidative phosphorylation and ETS flux (with no net effect on the mitochondrial coupling) and suppressed the activity of cytochrome c oxidase in oyster mitochondria. However, the effective concentration of betaine (590 mM) was higher than typically found in bivalves, and thus betaine is not likely to affect oyster mitochondria under the physiological conditions in vivo. Our findings indicate that taurine may support the mitochondrial bioenergetics during hyperosmotic stress in oysters. Compatibility of taurine with the metabolic functions and its beneficial effects on mitochondria may have contributed to its broad distribution as an osmolyte in marine osmoconformers and might explain the earlier reports of the positive effects of taurine supplementation on energy metabolism of other organisms, including mammals.

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 hypoxia-reoxygenation stress on mitochondrial proteome and bioenergetics of the hypoxia-tolerant marine bivalve Crassostrea gigas.

Mitochondria are key intracellular targets of hypoxia-reoxygenation (H/R) stress due to their central role in generation of ATP and reactive oxygen species (ROS). Intertidal oysters Crassostrea gigas are adapted to frequent H/R cycles and maintain aerobic function despite frequent oxygen fluctuations. To gain insight into the molecular mechanisms of H/R tolerance, we assessed changes in mitochondrial respiration and (phospho)proteome of C. gigas during hypoxia and recovery. Oyster mitochondria maintained OXPHOS capacity despite a decline in cytochrome c oxidase activity during H/R stress. Rearrangements of the mitochondrial proteome during H/R stress involved upregulation of mitochondrial electron transport system and iron-binding proteins, and suppression of the pathways that channel electrons to ubiquinone, possibly as a mechanism to limit ROS production. H/R stress led to upregulation of a mitophagic activator PGAM5 and dephosphorylation of metalloendopeptidase OMA1, indicating stimulation of mitochondrial quality control mechanisms. Changes in abundance and phosphorylation levels of key proteins involved in mitochondrial protein homeostasis indicate suppression of protein synthesis during hypoxia, likely as an energy-saving mechanism, and its subsequent reactivation during reoxygenation. Thus, shifts in the mitochondrial (phospho-)proteome might play an important role in H/R stress resistance of oysters ensuring mitochondrial integrity and function during oxygen fluctuations. SIGNIFICANCE: Hypoxia-reoxygenation (H/R) stress elicits shifts in proteome and phosphoproteome of mitochondria in a hypoxia-tolerant model bivalve, oyster Crassostrea gigas, upregulating electron transport system, limiting electron flow to ubiquinone and activating mitochondrial quality control and protein homeostasis mechanisms. These findings provide insights into the potential role of proteomic shifts in adaptive response to H/R stress and serve as an important benchmark to understand the mechanisms of mitochondrial sensitivity to hypoxia and reoxygenation.

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.

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 cadmium and hypoxia on metabolic responses and bacterial loads of eastern oysters Crassostrea virginica Gmelin.

Pollution by toxic metals including cadmium (Cd) and hypoxia are important stressors in estuaries and coastal waters which may interactively affect sessile benthic organisms, such as oysters. We studied metabolic responses to prolonged hypoxic acclimation (2 weeks at 5% O2) in control and Cd-exposed (30 d at 50 µg L(-1) Cd) oysters Crassostrea virginica, and analyzed the effects of these stressors on abundance of Vibrio spp. in oysters. Hypoxia-acclimated oysters retained normal standard metabolic rates (SMR) at 5% O2, in contrast to a decline of SMR observed during acute hypoxia. However, oysters spent more time actively ventilating in hypoxia than normoxia resulting in enhanced Cd uptake and 2.7-fold higher tissue Cd burdens in hypoxia. Cd exposure led to a significant decrease in tissue glycogen stores, increase in free glucose levels and elevated activity of glycolytic enzymes (hexokinase and aldolase) indicating a greater dependence on carbohydrate catabolism. A compensatory increase in activities of two key mitochondrial enzymes (citrate synthase and cytochrome c oxidase) was found during prolonged hypoxia in control oysters but suppressed in Cd-exposed ones. Cd exposure also resulted in a significant increase in abundance of Vibrio parahaemolyticus and Vibrio vulnificus levels during normoxia and hypoxia, respectively. Overall, Cd- and hypoxia-induced changes in metabolic profile, Cd accumulation and bacterial flora of oysters indicate that these stressors can synergistically impact energy homeostasis, performance and survival of oysters in polluted estuaries and have significant consequences for transfer of Cd and bacterial pathogens to the higher levels of the food chain.