Description: Climate change with increasing temperature and ocean acidification (OA) poses risks for marine ecosystems. According to Portner and Farrell [1], synergistic effects of elevated temperature and CO(2)-induced OA on energy metabolism will narrow the thermal tolerance window of marine ectothermal animals. To test this hypothesis, we investigated the effect of an acute temperature rise on energy metabolism of the oyster, Crassostrea gigas chronically exposed to elevated CO(2) levels (partial pressure of CO(2) in the seawater similar to 0.15 kPa, seawater pH similar to 7.7). Within one month of incubation at elevated PCO(2) and 15 degrees C hemolymph pH fell (pH(e) = 7.1 +/- 0.2 (CO(2)-group) vs. 7.6 +/- 0.1 (control)) and P(e)CO(2) values in hemolymph increased (0.5 +/- 0.2 kPa (CO(2)-group) vs. 0.2 +/- 0.04 kPa (control)). Slightly but significantly elevated bicarbonate concentrations in the hemolymph of CO(2)-incubated oysters ([HCO(3)(-)](e) = 1.8 +/- 0.3 mM (CO(2)-group) vs. 1.3 +/- 0.1 mM (control)) indicate only minimal regulation of extracellular acid-base status. At the acclimation temperature of 15 degrees C the OA-induced decrease in pHe did not lead to metabolic depression in oysters as standard metabolism rates (SMR) of CO(2)-exposed oysters were similar to controls. Upon acute warming SMR rose in both groups, but displayed a stronger increase in the CO(2)-incubated group. Investigation in isolated gill cells revealed a similar temperature-dependence of respiration between groups. Furthermore, the fraction of cellular energy demand for ion regulation via Na(+)/K(+)-ATPase was not affected by chronic hypercapnia or temperature. Metabolic profiling using (1)H-NMR spectroscopy revealed substantial changes in some tissues following OA exposure at 15 degrees C. In mantle tissue alanine and ATP levels decreased significantly whereas an increase in succinate levels was observed in gill tissue. These findings suggest shifts in metabolic pathways following OA-exposure. Our study confirms that OA affects energy metabolism in oysters and suggests that climate change may affect populations of sessile coastal invertebrates such as mollusks.

Description: Highlights: • Transcriptional response to hypoxia-reoxygenation was studied in an OMZ bivalve. • Anaerobic glycolysis and protein quality control pathways were transcriptionally upregulated in hypoxia. • Hypoxia elevated mRNA levels of UCP2 but had no effect on thiol-dependent antioxidants. • No impact of hypoxia-reoxygenation was found on aerobic marker enzymes. • Responses of an OMZ bivalve show parallels to other hypoxia-tolerant bivalves. Abstract: Benthic animals inhabiting the edges of marine oxygen minimum zones (OMZ) are exposed to unpredictable large fluctuations of oxygen levels. Sessile organisms including bivalves must depend on physiological adaptations to withstand these conditions. However, as habitats are rather inaccessible, physiological adaptations of the OMZ margin inhabitants to oxygen fluctuations are not well understood. We therefore investigated the transcriptional responses of selected key genes involved in energy metabolism and stress protection in a dominant benthic species of the northern edge of the Namibian OMZ, the nuculanid clam Lembulus bicuspidatus,. We exposed clams to normoxia (~5.8 ml O2 l−1), severe hypoxia (36 h at ~0.01 ml O2 l−1) and post-hypoxic recovery (24 h of normoxia following 36 h of severe hypoxia). Using newly identified gene sequences, we determined the transcriptional responses to hypoxia and reoxygenation of the mitochondrial aerobic energy metabolism (pyruvate dehydrogenase E1 complex, cytochrome c oxidase, citrate synthase, and adenine nucleotide translocator), anaerobic glycolysis (hexokinase (HK), phosphoenolpyruvate carboxykinase (PEPCK), phosphofructokinase, and aldolase), mitochondrial antioxidants (glutaredoxin, peroxiredoxin, and uncoupling protein UCP2) and stress protection mechanisms (a molecular chaperone HSP70 and a mitochondrial quality control protein MIEAP) in the gills and the labial palps of L. bicuspidatus. Exposure to severe hypoxia transcriptionally stimulated anaerobic glycolysis (including HK and PEPCK), antioxidant protection (UCP2), and quality control mechanisms (HSP70 and MIEAP) in the gills of L. bicuspidatus. Unlike UCP2, mRNA levels of the thiol-dependent mitochondrial antioxidants were not affected by hypoxia-reoxygenation stress. Transcript levels of marker genes for aerobic energy metabolism were not responsive to oxygen fluctuations in L. bicuspidatus. Our findings highlight the probable importance of anaerobic succinate production (via PEPCK) and mitochondrial and proteome quality control mechanisms in responses to oxygen fluctuations of the OMZ bivalve L. bicuspidatus. The reaction of L. bicuspidatus to oxygen fluctuations implies parallels to that of other hypoxia-tolerant bivalves, such as intertidal species.

Description: Increased anthropogenic emission of CO2 changes the carbonate chemistry and decreases the pH of the ocean. This can affect the speciation and the bioavailability of metals in polluted habitats such as estuaries. However, the effects of acidification on metal accumulation and stress response in estuarine organisms including bivalves are poorly understood. We studied the interactive effects of CO2 and two common metal pollutants, copper (Cu) and cadmium (Cd), on metal accumulation, intracellular ATP/ubiquitin-dependent protein degradation, stress response and energy metabolism in two common estuarine bivalves—Crassostrea virginica (eastern oyster) and Mercenaria mercenaria (hard shell clam). Bivalves were exposed for 4–5 weeks to clean seawater (control) and to either 50 μg L−1 Cu or 50 μg L−1 Cd at one of three partial pressures of CO2 (PCO2PCO2 ∼395, ∼800 and ∼1500 μatm) representative of the present-day conditions and projections of the Intergovernmental Panel for Climate Change (IPCC) for the years 2100 and 2250, respectively. Clams accumulated lower metal burdens than oysters, and elevated PCO2PCO2 enhanced the Cd and Cu accumulation in mantle tissues in both species. Higher Cd and Cu burdens were associated with elevated mRNA expression of metal binding proteins metallothionein and ferritin. In the absence of added metals, proteasome activities of clams and oysters were robust to elevated PCO2PCO2, but PCO2PCO2 modulated the proteasome response to metals. Cd exposure stimulated the chymotrypsin-like activity of the oyster proteasome at all CO2 levels. In contrast, trypsin- and caspase-like activities of the oyster proteasome were slightly inhibited by Cd exposure in normocapnia but this inhibition was reversed at elevated PCO2PCO2. Cu exposure inhibited the chymotrypsin-like activity of the oyster proteasome regardless of the exposure PCO2PCO2. The effects of metal exposure on the proteasome activity were less pronounced in clams, likely due to the lower metal accumulation. However, the general trends (i.e. an increase during Cd exposure, inhibition during exposure to Cu, and overall stimulatory effects of elevated PCO2PCO2) were similar to those found in oysters. Levels of mRNA for ubiquitin and tumor suppressor p53 were suppressed by metal exposures in normocapnia in both species but this effect was alleviated or reversed at elevated PCO2PCO2. Cellular energy status of oysters was maintained at all metal and CO2 exposures, while in clams the simultaneous exposure to Cu and moderate hypercapnia (∼800 μatm PCO2PCO2) led to a decline in glycogen, ATP and ADP levels and an increase in AMP indicating energy deficiency. These data suggest that environmental CO2 levels can modulate accumulation and physiological effects of metals in bivalves in a species-specific manner which can affect their fitness and survival during the global change in estuaries.

Description: Ectotherms are exposed to a range of environmental temperatures and may face extremes beyond their upper thermal limits. Such temperature extremes can stimulate aerobic metabolism toward its maximum, a decline in aerobic substrate oxidation, and a parallel increase of anaerobic metabolism, combined with ROS generation and oxidative stress. Under these stressful conditions, marine organisms recruit several defensive strategies for their maintenance and survival. However, thermal tolerance of ectothermic organisms may be increased after a brief exposure to sub- lethal temperatures, a process known as "hardening". In our study, we examined the ability of M. galloprovincialis to increase its thermal tolerance under the effect of elevated temperatures (24, 26 and 28 °C) through the "hardening" process. Our results demonstrate that this process can increase the heat tolerance and antioxidant defense of heat hardened mussels through more efficient ETS activity when exposed to temperatures beyond 24 °C, compared to non-hardened individuals. Enhanced cell protection is reflected in better adaptive strategies of heat hardened mussels, and thus decreased mortality. Although hardening seems a promising process for the maintenance of aquacultured populations under increased seasonal temperatures, further investigation of the molecular and cellular mechanisms regulating mussels’ heat resistance is required.

Description: 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.

Description: Life on tidal coasts presents physiological major challenges for sessile species. Fluctuations in oxygen and temperature can affect bioenergetics and modulate metabolism and redox balance, but their combined effects are not well understood. We investigated the effects of intermittent hypoxia (12h/12h) in combination with different temperature regimes (normal (15 °C), elevated (30 °C) and fluctuating (15 °C water/30 °C air)) on the Pacific oyster Crassostrea (Magallana) gigas. Fluctuating temperature led to energetic costly metabolic rearrangements and accumulation of proteins in oyster tissues. Elevated temperature led to high (60%) mortality and oxidative damage in survivors. Normal temperature had no major negative effects but caused metabolic shifts. Our study shows high plasticity of oyster metabolism in response to oxygen and temperature fluctuations and indicates that metabolic adjustments to oxygen deficiency are strongly modulated by the ambient temperature. Co-exposure to constant elevated temperature and intermittent hypoxia demonstrates the limits of this adaptive metabolic plasticity.

Description: 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. Our 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.

Description: 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.

Description: Benthic organisms, such as infaunal bivalve, can experience and withstand prolonged food limitation in the temperate shallow coastal area. These organisms withstand the long food limitation by adjusting physiological processes which might cause trade-off between maintenance and other functions (e.g. burrowing). We have investigated the effects of prolonged food limitation (42 days without added food) on the tissue energy content, metabolites, RNA to DNA ratio, and burrowing activity of a common bioturbator, Mya arenaria. Mya arenaria decrease their respiration by 80% over 15 days of food limitation, and increase the time required for one burial cycle by 30-50% after 22 days of food limitation. We noticed a preferential switch in the fuel usage for burrowing depending on the stage of food limitation. The tissue energy content and free amino acid concentration decreases as a result of food limitation, however, there is a seasonal pattern. Our study suggest, although M. arenaria can withstand prolonged food limitation by lowering their basal maintenance cost and switching their fuel usage, their ecological function (bioturbation) could still be negatively affected.