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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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

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., (Copyright © 2018 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

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., (Copyright © 2018. Published by Elsevier B.V.)

Oxygen homeostasis is crucial for development, survival and normal function of all metazoans. A family of transcription factors called hypoxia-inducible factors (HIF) is critical in mediating the adaptive responses to reduced oxygen availability. The HIF transcription factor consists of a constitutively expressed β subunit and an oxygen-dependent α subunit; the abundance of the latter determines the activity of HIF and is regulated by a family of O(2)- and Fe(2+)-dependent enzymes prolyl hydroxylases (PHDs). Currently very little is known about the function of this important pathway and the molecular structure of its key players in hypoxia-tolerant intertidal mollusks including oysters, which are among the animal champions of anoxic and hypoxic tolerance and thus can serve as excellent models to study the role of HIF cascade in adaptations to oxygen deficiency. We have isolated transcripts of two key components of the oxygen sensing pathway - the oxygen-regulated HIF-α subunit and PHD - from an intertidal mollusk, the eastern oyster Crassostrea virginica, and determined the transcriptional responses of these two genes to anoxia, hypoxia and cadmium (Cd) stress. HIF-α and PHD homologs from eastern oysters C. virginica show significant sequence similarity and share key functional domains with the earlier described isoforms from vertebrates and invertebrates. Phylogenetic analysis shows that genetic diversification of HIF and PHD isoforms occurred within the vertebrate lineage indicating functional diversification and specialization of the oxygen-sensing pathways in this group, which parallels situation observed for many other important genes. HIF-α and PHD homologs are broadly expressed at the mRNA level in different oyster tissues and show transcriptional responses to prolonged hypoxia in the gills consistent with their putative role in oxygen sensing and the adaptive response to hypoxia. Similarity in amino acid sequence, domain structure and transcriptional responses between HIF-α and PHD homologs from oysters and other invertebrate and vertebrate species implies the highly conserved functions of these genes throughout the evolutionary history of animals, in accordance with their critical role in oxygen sensing and homeostasis., (Copyright © 2010 Elsevier Inc. All rights reserved.)

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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Marine organisms are exposed to periodical oxygen deficiency and pollution stress in estuarine and coastal zones which may strongly affect their performance and survival. We studied the combined effects of exposure to a common pollutant, cadmium (Cd), and intermittent anoxia on anaerobic metabolism, energy status and mRNA expression of 13 genes involved in and/or controlled by the hypoxia inducible factor-1 (HIF-1) pathway in hepatopancreas of an intertidal bivalve, the eastern oyster Crassostrea virginica. In control oysters, prolonged anoxia resulted in a selective suppression of nitric oxide synthase (NOS) and upregulation of cytochrome c oxidase subunit IV (COX4) while the levels of other transcripts remained unchanged. During post-anoxic recovery, mRNA expression of hypoxia inducible factor-1alpha (HIF-1alpha) was elevated, phosphoenolpyruvate carboxykinase (PEPCK), NOS and LON protease suppressed, and mRNA expression of other studied genes not changed. Notably, most of the key glycolytic genes that are stimulated by HIF-1 in mammals, either remained unchanged or were downregulated in anoxic oysters suggesting a different mechanism of molecular response to oxygen deficiency. Patterns of transcriptional response during anoxia and reoxygenation were significantly altered by Cd exposure in a gene-specific manner. Anaerobic metabolism (indicated by accumulation of l-alanine, succinate and acetate during anoxia) was also suppressed in Cd-exposed oysters. In control oysters, ATP turnover rate (M(ATP)) during anoxia was mostly sustained by anaerobic glycolysis with negligible contributions from ATP and PLA breakdown. In contrast, in Cd-exposed oysters ATP breakdown contributed significantly to anaerobic M(ATP). Thus, while control oysters could efficiently defend the ATP levels and tissue energy status during prolonged anoxia, Cd-exposed oysters experienced a disturbance in tissue energy balance indicated by the depletion of ATP, a rapid decline in adenylate energy charge and increase in ADP/ATP ratios. This energy deficiency combined with suppression of anaerobic metabolism may strongly affect performance and survival of oysters in polluted estuaries where metal pollution may co-occur with "dead zones"., (Copyright 2010 Elsevier B.V. All rights reserved.)

Uncoupling proteins (UCPs) belong to the mitochondrial anion carrier gene family which has been implicated in diverse physiological functions ranging from thermoregulation to antioxidant defense. In mammals, the UCP family is well characterized and contains five members (UCP1-5). In contrast, invertebrate homologues of uncoupling proteins are much less studied both from the viewpoints of structure and function. In this study we report nucleotide and predicted protein structure of an important member of UCP family, UCP5 from eastern oysters Crassostrea virginica. UCP5 from oysters appears to be a close homolog of the mammalian brain mitochondrial carrier protein (BMCP1, or UCP5) and is the first full-length UCP described from a Lophotrochozoan invertebrate. Evolutionary analysis of UCP sequences indicates at least three monophyletic UCP branches (UCP1-3, UCP4 and UCP5) that have diverged early in the evolution, prior to the divergence of vertebrates and invertebrates. In oysters, two forms of UCP5 transcript are found (UCP5S and UCP5L) that differ by 152 bp in length due to the presence of an intron in UCP5L. UCP5 was expressed in all studied oyster tissues, unlike mammals, where UCP5 is predominantly expressed in brains and male gonads. Hypoxia-reoxygenation stress, sublethal Cd exposure (50 ?g L(?1) Cd for 56 days) and acclimation to different temperatures (12 and 20 °C) had no significant effect on UCP5 mRNA expression in oysters indicative of its relative unimportance in antioxidant defense and temperature adaptation of oyster mitochondria. These data suggest that despite the relatively high degree of evolutionary conservation of the UCP5 amino acid sequence, its functional significance in mitochondria changed in the course of evolution of mollusks and vertebrates.

Temperature and heavy metals such as cadmium (Cd) are important stressors which can strongly affect physiology of marine ectotherms in polluted estuaries. Mitochondria are among the key intracellular targets for these stressors, but the mechanisms of Cd-induced mitochondrial damage are not fully understood. In this study we determined the effects of acclimation temperature (12, 20 and 28 degrees C) and Cd exposure (0 or 50 microg L(-1) Cd) in vivo on activity and mRNA expression of a key mitochondrial enzyme, aconitase, which is known as a sensitive marker of oxidative stress, and on mRNA expression of LON protease involved in the degradation of oxidatively damaged mitochondrial proteins, in eastern oysters Crassostrea virginica. Sensitivity of mitochondrial aconitase to exposure to Cd in vitro (0 or 50 microM) was also determined in oysters acclimated to different temperatures and Cd levels. Acclimation at 28 degrees C resulted in a strong decrease in activity of mitochondrial aconitase as well as mRNA expression of aconitase and LON protease suggesting mitochondrial dysfunction at elevated temperatures. Exposure of isolated mitochondria to 50 microM Cd in vitro resulted in a 20-25% inhibition of mitochondrial aconitase reflecting oxidative damage of this enzyme. However, long-term (3-6 weeks) exposure of whole oysters to Cd had no effect on mitochondrial aconitase activity suggesting that this enzyme is well protected against Cd-induced oxidative stress in vivo. Aconitase mRNA expression was positively correlated with the enzyme activity within control and Cd-exposed groups; however, this correlation was strikingly different when compared between control and Cd-exposed oysters. The level of aconitase transcript was considerably lower (3-13-fold) in Cd-exposed oysters while the specific aconitase activities were similar in control and Cd-exposed oysters indicating regulation at the post-transcriptional level. LON protease expression was upregulated by 2-4-fold in Cd-exposed oysters suggesting an increase in mitochondrial protein degradation as a novel protective mechanism against Cd-induced mitochondrial stress. Our data indicate that mitochondrial aconitase is not a good biomarker for Cd-induced oxidative stress in oysters in vivo, because of its complex regulation at transcriptional and post-transcriptional levels, low sensitivity to Cd effects in vivo but high sensitivity to acclimation temperature that can potentially mask effects of other stressors under the field conditions.

Temperature and heavy metals such as cadmium (Cd) are important environmental stressors that can strongly affect mitochondrial function of marine poikilotherms. In this study, we investigated the combined effects of temperature (20 degrees C and 30 degrees C) and Cd stress on production of reactive oxygen species (ROS) and oxidative stress in a marine poikilotherm Crassostrea virginica (the eastern oyster) using mitochondrial aconitase as a sensitive biomarker of oxidative damage. We also assessed potential involvement of mitochondrial uncoupling proteins (UCPs) in antioxidant protection in oyster mitochondria using purine nucleotides (GDP, ATP and ADP) as specific inhibitors, and free fatty acids as stimulators, of UCPs. Our results show that exposure to Cd results in elevated ROS production and oxidative damage as indicated by aconitase inactivation which is particularly pronounced at elevated temperature. Unexpectedly, oyster mitochondrial aconitase was inhibited by physiologically relevant levels of ATP (IC(50)=1.93 and 3.04 mmol l(-1) at 20 degrees C and 30 degrees C, respectively), suggesting that allosteric regulation of aconitase by this nucleotide may be involved in regulation of the tricarboxylic acid flux in oysters. Aconitase was less sensitive to ATP inhibition at 30 degrees C than at 20 degrees C, consistent with the elevated metabolic flux at higher temperatures. ADP and GDP also inhibited mitochondrial aconitase but at the levels well above the physiological concentrations of these nucleotides (6-11 mmol l(-1)). Our study shows expression of at least three UCP isoforms in C. virginica gill tissues but provides no indication that UCPs protect mitochondrial aconitase from oxidative inactivation in oysters. Overall, the results of this study indicate that temperature stress exaggerates toxicity of Cd leading to elevated oxidative stress in mitochondria, which may have important implications for survival of poikilotherms in polluted environments during seasonal warming and/or global climate change, and suggest a novel temperature-dependent mechanism of allosteric regulation of TCA flux in oyster mitochondria.