Inna M. Sokolova

Impact of Ocean Acidification on Energy Metabolism of Oyster, Crassostrea gigas—Changes in Metabolic Pathways and Thermal Response

Climate change with increasing temperature and ocean acidification (OA) poses risks for marine ecosystems. According to Pörtner and Farrell [1], synergistic effects of elevated temperature and CO2-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 CO2 levels (partial pressure of CO2 in the seawater ~0.15 kPa, seawater pH ~ 7.7). Within one month of incubation at elevated Pco2 and 15 °C hemolymph pH fell (pHe = 7.1 ± 0.2 (CO2-group) vs. 7.6 ± 0.1 (control)) and Peco2 values in hemolymph increased (0.5 ± 0.2 kPa (CO2-group) vs. 0.2 ± 0.04 kPa (control)). Slightly but significantly elevated bicarbonate concentrations in the hemolymph of CO2-incubated oysters ([HCO− 3]e = 1.8 ± 0.3 mM (CO2-group) vs. 1.3 ± 0.1 mM (control)) indicate only minimal regulation of extracellular acid-base status. At the acclimation temperature of 15 °C the OA-induced decrease in pHe did not lead to metabolic depression in oysters as standard metabolism rates (SMR) of CO2-exposed oysters were similar to controls. Upon acute warming SMR rose in both groups, but displayed a stronger increase in the CO2-incubated group. Investigation in isolated gill cells revealed a similar temperaturedependence 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 1H-NMR spectroscopy revealed substantial changes in some tissues following OA exposure at 15 °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.

Cadmium-induced apoptosis in oyster hemocytes involves disturbance of cellular energy balance but no mitochondrial permeability transition.

SUMMARY Exposure to environmentally prevalent heavy metals such as cadmium can have detrimental effects on a variety of commercially and ecologically important species such as oysters. Since Cd2+ is known to induce apoptosis in immune cells of vertebrates, we have investigated the effects of this metal on isolated oyster hemocytes, the main cellular immune defense in mollusks. Enhanced apoptosis of these cells could conceivably create immunosuppressed conditions in these organisms and result in reduced disease resistance and increased opportunistic infection, resulting in decline of their populations. Cd2+ exposure induced apoptosis in oyster hemocytes in a dose-dependent manner in the range of 10-100 μmol l-1, as indicated by the translocation of phosphatidylserine to the outer leaflet of the plasma membrane. At higher concentrations (200-1000 μmol l-1), there was no further increase in apoptosis but a significant increase in the level of necrosis. In stark contrast to vertebrate immune cells, there was no decrease in the mitochondrial membrane potential or activation of caspases in response to Cd2+ in the apoptotic range. Surprisingly, Cd2+ exposure in this range did cause a significant decrease in intracellular ATP levels, indicating a severe disturbance of energy metabolism. Similarly, Cd2+ exposure of isolated mitochondria resulted in partial uncoupling of mitochondria but no difference in mitochondrial membrane potential. The results demonstrate that the important environmental pollutant Cd2+ induces apoptosis in oyster immune cells and does so through a mitochondria/caspase-independent pathway, suggesting that a novel, perhaps ancient, apoptotic pathway is active in these cells. Furthermore, it appears that the observed decrease in ATP production during apoptosis is not due to the loss of the mitochondrial proton-motive force but is more likely to be due to inhibition of the F0/F1-ATPase and/or mitochondrial ADP/ATP or substrate transport.

Metabolic plasticity and critical temperatures for aerobic scope in a eurythermal marine invertebrate (Littorina saxatilis, Gastropoda: Littorinidae) from different latitudes

SUMMARY Effects of latitudinal cold adaptation and cold acclimation on metabolic rates and aerobic scope were studied in the eurythermal marine gastropod Littorina saxatilis from temperate North Sea and sub-arctic White Sea areas. Animals were acclimated for 6-8 weeks at control temperature (13°C) or at 4°C, and their respiration rates were measured during acute temperature change (1-1.5°C h-1) in a range between 0°C and 32°C. In parallel, the accumulation of anaerobic end products and changes in energy status were monitored. Starting from 0°C, aerobic metabolic rates of L. saxatilis rose quickly with increasing temperatures up to a point at or slightly above the respective acclimation temperature. Beyond this value, thermal sensitivity of oxygen consumption rate (V̇O2) greatly decreased in a wide, 15°C range of experimental temperatures. This change in metabolic regulation was also reflected in the activation energy of aerobic metabolism (Ea), which was approximately seven times lower at temperatures above Arrhenius breakpoint temperatures (ABTs) than at temperatures below ABTs. Warming progressively led to a discrepancy between energy demand and energy production, as demonstrated by a decrease in the levels of high-energy phosphates [phosho-L-arginine (PLA) and ATP], and resulted in the onset of anaerobiosis at critically high temperatures, indicating a limitation of aerobic scope. The comparison of aerobic and anaerobic metabolic rates in L. saxatilis in air and water suggests that the heat-induced onset of anaerobiosis is due to the insufficient oxygen supply to tissues at high temperatures. Cold acclimation led to an increase in aerobic metabolic rates and a considerable downward shift of the upper critical temperature in North Sea L. saxatilis but not in White Sea L. saxatilis. Limited metabolic plasticity in response to cold acclimation in sub-arctic White Sea snails as compared with their temperate North Sea counterparts suggests that metabolic depression occurs during overwintering under the more extreme winter conditions at the White Sea.

Proteomic response to elevated PCO2 level in eastern oysters, Crassostrea virginica: evidence for oxidative stress.

SUMMARY Estuaries are characterized by extreme fluctuations in CO2 levels due to bouts of CO2 production by the resident biota that exceed its capacity of CO2 consumption and/or the rates of gas exchange with the atmosphere and open ocean waters. Elevated partial pressures of CO2 (PCO2; i.e. environmental hypercapnia) decrease the pH of estuarine waters and, ultimately, extracellular and intracellular pH levels of estuarine organisms such as mollusks that have limited capacity for pH regulation. We analyzed proteomic changes associated with exposure to elevated PCO2 in the mantle tissue of eastern oysters (Crassostrea virginica) after 2 weeks of exposure to control (∼39 Pa PCO2) and hypercapnic (∼357 Pa PCO2) conditions using two-dimensional gel electrophoresis and tandem mass spectrometry. Exposure to high PCO2 resulted in a significant proteome shift in the mantle tissue, with 12% of proteins (54 out of 456) differentially expressed under the high PCO2 compared with control conditions. Of the 54 differentially expressed proteins, we were able to identify 17. Among the identified proteins, two main functional categories were upregulated in response to hypercapnia: those associated with the cytoskeleton (e.g. several actin isoforms) and those associated with oxidative stress (e.g. superoxide dismutase and several peroxiredoxins as well as the thioredoxin-related nucleoredoxin). This indicates that exposure to high PCO2 (∼357 Pa) induces oxidative stress and suggests that the cytoskeleton is a major target of oxidative stress. We discuss how elevated CO2 levels may cause oxidative stress by increasing the production of reactive oxygen species (ROS) either indirectly by lowering organismal pH, which may enhance the Fenton reaction, and/or directly by CO2 interacting with other ROS to form more free radicals. Although estuarine species are already exposed to higher and more variable levels of CO2 than other marine species, climate change may further increase the extremes and thereby cause greater levels of oxidative stress.

Cadmium effects on mitochondrial function are enhanced by elevated temperatures in a marine poikilotherm, Crassostrea virginica Gmelin (Bivalvia: Ostreidae)

SUMMARY Marine intertidal mollusks, such as oysters, are exposed to multiple stressors in estuaries, including varying environmental temperature and levels of trace metals, which may interactively affect their physiology. In order to understand the combined effects of cadmium and elevated temperature on mitochondrial bioenergetics of marine mollusks, respiration rates and mitochondrial volume changes were studied in response to different cadmium levels (0–1000 μmol l–1) and temperatures (15, 25 and 35°C) in isolated mitochondria from the eastern oyster Crassostrea virginica acclimated at 15°C. It was found that both cadmium and temperature significantly affect mitochondrial function in oysters. Elevated temperature had a rate-enhancing effect on state 3 (ADP-stimulated) and states 4 and 4+ (representative of proton leak) respiration, and the rate of temperature-dependent increase was higher for states 4 and 4+ than for state 3 respiration. Exposure of oyster mitochondria to 35°C resulted in a decreased respiratory control and phosphorylation efficiency (P/O ratio) compared to that of the acclimation temperature (15°C), while an intermediate temperature (25°C) had no effect. Cadmium exposure did not lead to a significant volume change in oyster mitochondria in vitro. Low levels of cadmium (1–5 μmol l–1) stimulated the rate of proton leak in oyster mitochondria, while not affecting ADP-stimulated state 3 respiration. In contrast, higher cadmium levels (10–50 μmol l–1) had little or no effect on proton leak, but significantly inhibited state 3 respiration by 40–80% of the control rates. Elevated temperature increased sensitivity of oyster mitochondria to cadmium leading to an early inhibition of ADP-stimulated respiration and an onset of complete mitochondrial uncoupling at progressively lower cadmium concentrations with increasing temperature. Enhancement of cadmium effects by elevated temperatures suggests that oyster populations subjected to elevated temperatures due to seasonal warming or global climate change may become more susceptible to trace metal pollution, and vice versa.

Interactive effects of salinity and elevated CO2 levels on juvenile eastern oysters, Crassostrea virginica

SUMMARY Rising levels of atmospheric CO2 lead to acidification of the ocean and alter seawater carbonate chemistry, which can negatively impact calcifying organisms, including mollusks. In estuaries, exposure to elevated CO2 levels often co-occurs with other stressors, such as reduced salinity, which enhances the acidification trend, affects ion and acid–base regulation of estuarine calcifiers and modifies their response to ocean acidification. We studied the interactive effects of salinity and partial pressure of CO2 (PCO2) on biomineralization and energy homeostasis in juveniles of the eastern oyster, Crassostrea virginica, a common estuarine bivalve. Juveniles were exposed for 11 weeks to one of two environmentally relevant salinities (30 or 15 PSU) either at current atmospheric PCO2 (∼400 μatm, normocapnia) or PCO2 projected by moderate IPCC scenarios for the year 2100 (∼700–800 μatm, hypercapnia). Exposure of the juvenile oysters to elevated PCO2 and/or low salinity led to a significant increase in mortality, reduction of tissue energy stores (glycogen and lipid) and negative soft tissue growth, indicating energy deficiency. Interestingly, tissue ATP levels were not affected by exposure to changing salinity and PCO2, suggesting that juvenile oysters maintain their cellular energy status at the expense of lipid and glycogen stores. At the same time, no compensatory upregulation of carbonic anhydrase activity was found under the conditions of low salinity and high PCO2. Metabolic profiling using magnetic resonance spectroscopy revealed altered metabolite status following low salinity exposure; specifically, acetate levels were lower in hypercapnic than in normocapnic individuals at low salinity. Combined exposure to hypercapnia and low salinity negatively affected mechanical properties of shells of the juveniles, resulting in reduced hardness and fracture resistance. Thus, our data suggest that the combined effects of elevated PCO2 and fluctuating salinity may jeopardize the survival of eastern oysters because of weakening of their shells and increased energy consumption.

Effects of elevated temperature and cadmium exposure on stress protein response in eastern oysters Crassostrea virginica (Gmelin).

Stress proteins such as heat shock proteins (HSPs) and metallothioneins (MTs) play a key role in cellular protection against environmental stress. Marine ectotherms such as eastern oysters Crassostrea virginica are commonly exposed to multiple stressors including temperature and pollution by metals such as cadmium (Cd) in estuaries and coastal zones; however, the combined effects of these stressors on their cellular protection mechanisms are poorly understood. We acclimated C. virginica from populations adapted to different thermal regimes (Washington, North Carolina and Texas) at a common temperature of 12 degrees C, and analyzed their expression of MTs and HSPs (cytosolic HSP69, HSC72-77, HSP90 and mitochondrial HSP60) in response to the combined acute temperature stress and long-term Cd exposure. Overall, HSP and MT induction patterns were similar in oysters from the three studied geographically distant populations. HSP69 and MTs were significantly up-regulated by Cd and temperature stress implying their important role in cellular stress protection. In contrast, HSC72-77, HSP60 and HSP90 were not consistently induced by either acute heat or Cd exposure. The induction temperature for MTs was higher than for HSP69 (>28 degrees C vs. 20 degrees C, respectively), and MTs were more strongly induced by Cd than by temperature stress (to up to 38-94-fold compared by 3.5-7.5-fold, respectively) consistent with their predominant role in metal detoxification. Notably, heat stress did not result in an additional increase in metallothionein expression in Cd-exposed oysters suggesting a capacity limitation during the combined exposure to Cd and temperature stress. Levels of HSP69 and in some cases, HSC72-77 and HSP90 were lower in Cd-exposed oysters as compared to their control counterparts during heat stress indicating that simultaneous exposure to these two stressors may have partially suppressed the cytoprotective upregulation of molecular chaperones. These limitations of stress protein response may contribute to the reduced thermotolerance of oysters from metal-polluted environments.

The transcriptomic responses of the eastern oyster, Crassostrea virginica, to environmental conditions

Understanding the mechanisms by which organisms adapt to environmental conditions is a fundamental question for ecology and evolution. In this study, we evaluate changes in gene expression of a marine mollusc, the eastern oyster Crassostrea virginica, associated with the physico‐chemical conditions and the levels of metals and other contaminants in their environment. The results indicate that transcript signatures can effectively disentangle the complex interactive gene expression responses to the environment and are also capable of disentangling the complex dynamic effects of environmental factors on gene expression. In this context, the mapping of environment to gene and gene to environment is reciprocal and mutually reinforcing. In general, the response of transcripts to the environment is driven by major factors known to affect oyster physiology such as temperature, pH, salinity, and dissolved oxygen, with pollutant levels playing a relatively small role, at least within the range of concentrations found in the studied oyster habitats. Further, the two environmental factors that dominate these effects (temperature and pH) interact in a dynamic and nonlinear fashion to impact gene expression. Transcriptomic data obtained in our study provide insights into the mechanisms of physiological responses to temperature and pH in oysters that are consistent with the known effects of these factors on physiological functions of ectotherms and indicate important linkages between transcriptomics and physiological outcomes. Should these linkages hold in further studies and in other organisms, they may provide a novel integrated approach for assessing the impacts of climate change, ocean acidification and anthropogenic contaminants on aquatic organisms via relatively inexpensive microarray platforms.

Effects of cadmium on cellular protein and glutathione synthesis and expression of stress proteins in eastern oysters, Crassostrea virginica Gmelin.

SUMMARY Cadmium (Cd) is an important toxicant in estuarine and coastal environments that can strongly affect energy balance of aquatic organisms by increasing the organisms basal energy demand and reducing its aerobic capacity. Mechanisms of cadmium-induced increase in basal metabolic costs are not well understood and may involve elevated detoxification costs due to the synthesis of cellular protective proteins and glutathione. We studied the short-term effects of cadmium exposure (4 h) on protein and glutathione (GSH) synthesis and expression of stress proteins (heat shock proteins HSP60, HSP70 and HSP90) and metallothioneins in isolated gill and hepatopancreas cells of the eastern oyster, Crassostrea virginica. Our study showed that exposure to cadmium resulted in a dose-dependent increase in the rate of protein synthesis in oyster cells, which reached 150% of the control at the highest tested Cd level (2000 μmol l–1). GSH synthesis was significantly inhibited by the highest Cd concentrations, especially in hepatopancreas, which resulted in a slight but significant decrease in the total GSH concentrations. Elevated protein synthesis was associated with the increased expression of metallothioneins and heat shock proteins. Interestingly, stress protein response differed considerably between gill and hepatopancreas cells. In hepatopancreas, expression of metallothionein mRNA (measured by real-time PCR) increased 2–8-fold in response to Cd exposure, whereas no significant increase in metallothionein expression was found in Cd-exposed gill cells. By contrast, HSP60 and HSP70 protein levels increased significantly in Cd-exposed gill cells (by 1.5–2-fold) but not in hepatopancreas. No change in HSP90 expression was detected in response to Cd exposure in oyster cells. These data indicate that metallothionein expression may provide sufficient protection against Cd-induced damage to intracellular proteins in hepatopancreas, alleviating the need for overexpression of molecular chaperones. By contrast, Cd detoxification mechanisms such as inducible metallothioneins and GSH appear to be insufficient to fully prevent protein damage in gill cells, thus necessitating induction of HSPs as a secondary line of cellular defense. Therefore, gills are likely to be among the most Cd-sensitive tissues in oysters, which may have important implications for impaired oxygen uptake contributing to energy misbalance and reduced aerobic scope in Cd-exposed oysters.

Effects of acclimation temperature and cadmium exposure on cellular energy budgets in the marine mollusk Crassostrea virginica : linking cellular and mitochondrial responses

SUMMARY In order to understand the role of metabolic regulation in environmental stress tolerance, a comprehensive analysis of demand-side effects (i.e. changes in energy demands for basal maintenance) and supply-side effects (i.e. metabolic capacity to provide ATP to cover the energy demand) of environmental stressors is required. We have studied the effects of temperature (12, 20 and 28°C) and exposure to a trace metal, cadmium (50 μg l–1), on the cellular energy budget of a model marine poikilotherm, Crassostrea virginica (eastern oysters), using oxygen demand for ATP turnover, protein synthesis, mitochondrial proton leak and non-mitochondrial respiration in isolated gill and hepatopancreas cells as demand-side endpoints and mitochondrial oxidation capacity, abundance and fractional volume as supply-side endpoints. Cadmium exposure and high acclimation temperatures resulted in a strong increase of oxygen demand in gill and hepatopancreas cells of oysters. Cd-induced increases in cellular energy demand were significant at 12 and 20°C but not at 28°C, possibly indicating a metabolic capacity limitation at the highest temperature. Elevated cellular demand in cells from Cd-exposed oysters was associated with a 2–6-fold increase in protein synthesis and, at cold acclimation temperatures, with a 1.5-fold elevated mitochondrial proton leak. Cellular aerobic capacity, as indicated by mitochondrial oxidation capacity, abundance and volume, did not increase in parallel to compensate for the elevated energy demand. Mitochondrial oxidation capacity was reduced in 28°C-acclimated oysters, and mitochondrial abundance decreased in Cd-exposed oysters, with a stronger decrease (by 20–24%) in warm-acclimated oysters compared with cold-acclimated ones (by 8–13%). These data provide a mechanistic basis for synergism between temperature and cadmium stress on metabolism of marine poikilotherms. Exposure to combined temperature and cadmium stress may result in a strong energy deficiency due to the elevated energy demand on one hand and a reduced mitochondrial capacity to cover this demand on the other hand, which may have important implications for surviving seasonally and/or globally elevated temperatures in polluted estuaries.