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Coastal environments commonly experience strong oxygen fluctuations. Resulting hypoxia/reoxygenation stress can negatively affect mitochondrial functions, since oxygen deficiency impairs ATP generation, whereas a surge of oxygen causes mitochondrial damage by oxidative stress. Marine intertidal bivalves are adapted to fluctuating oxygen conditions, yet the underlying molecular mechanisms that sustain mitochondrial integrity and function during oxygen fluctuations are not yet well understood. We used targeted mRNA expression analysis to determine the potential involvement of the mitochondrial quality control mechanisms in responses to short-term hypoxia (24 h at <0.01% O2) and subsequent reoxygenation (1.5 h at 21% O2) in two hypoxia-tolerant marine bivalves, the Pacific oysters Crassostrea gigas and the blue mussels Mytilus edulis. We hypothesized that the genes involved in the mitochondrial quality control will be upregulated during hypoxia, and the less hypoxia-tolerant of the two studied species (M. edulis) will show a stronger dependence on transcriptional upregulation of these pathways than C. gigas. To test these hypotheses, mRNA expression of 17 (C. gigas) and 11 (M. edulis) marker genes involved in mitochondrial fusion, fission, proteolysis and mitophagy was analyzed in the digestive gland of M. edulis and C. gigas in normoxia and during hypoxia-reoxygenation (H/R) stress. In the mussels, the mRNA expression of the transcripts related to mitochondrial dynamics and quality control was strongly altered during H/R stress showing a shift toward fission, suppression of fusion, an increase in mitochondrial proteolysis and onset of mitophagy. These changes indicate that H/R stress induces mitochondrial injury in M. edulis requiring upregulation of the protective mechanisms to segregate the dysfunctional mitochondria by fission and degrade the oxidative damaged proteins and/or organelles. Unlike mussels, the transcript levels of all studied genes in the oysters remained at the baseline (normoxic) levels during H/R stress. This muted transcriptional response of C. gigas is in agreement with earlier findings showing better ability to maintain cellular homeostasis and higher resistance to apoptosis during H/R stress in the oysters compared with the mussels. The revealed species-specific differences in the expression of the mitochondrial quality control pathways shed light on the potentially important mechanisms of mitochondrial protection against H/R-induced damage that might contribute to hypoxia tolerance in marine bivalves.

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 to normoxic control animals (21% O­2). We demonstrated that flux capacities 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 oxygen flux capacities of all three mitochondrial subsystems. Control over oxidative phosphorylation (OXPHOS) respiration was mostly exerted by substrate oxidation regardless of H/R stress, whereas the control of the proton leak subsystem over LEAK respiration increased during hypoxia and returned to normoxic level during reoxygenation. During hypoxia, reactive oxygen species (ROS) efflux was elevated in the LEAK state, while 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.

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 the oysters acclimated to high (33) or low (15) salinity, and exposed to either normoxic conditions (control; 21% O2) or to 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 salinity 15 might be closer to the metabolic optimum of C. gigas than salinity 33. 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 indicate 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 to brackish regions such as the Baltic Sea.

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