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.

SUMMARYCadmium (Cd) is an important toxicant in estuarine and coastal environments that can strongly affect energy balance of aquatic organisms by increasing the organismʼs basal energy demand and reducing its aerobic capacity. Mechanisms of cadmiuminduced 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·mol·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.

Marine ectotherms, including oysters are exposed to variable environmental conditions in coastal shallow waters and estuaries. In the light of global climate change, additional stressors like pollution might pose higher risk to populations. On the basis of the concept of oxygen- and capacity-limited thermal tolerance in aquatic ectotherms (40), we show that a persistent pollutant, cadmium, can have detrimental effects on oysters (Crassostrea virginica). During acute warming from 20 to 28 degrees C (4 degrees C/48 h) standard metabolic rate (SMR) rose in control and cadmium-exposed (50 microg Cd2+/l) animals, with a consistently higher SMR in Cd-exposed oysters. Additionally, Cd-exposed oysters showed a stronger temperature-dependent decrease in hemolymph oxygen partial pressures. This observation indicates that the effect of temperature on aerobic metabolism was exacerbated due to the additional Cd stress. The oxygen delivery systems could not provide enough oxygen to cover Cd-induced elevated metabolic demands at high temperatures. Interestingly, cardiac performance (measured as the heart rate and hemolymph supply to tissues) rose to a similar extent in control and Cd-exposed oysters with warming indicating that cardiac output was unable to compensate for elevated energy demand in Cd-exposed oysters. Together with the literature data on metal-induced reduction of ventilatory capacity, these findings suggest that synergistic effects of elevated temperatures and cadmium exposure led to oxygen limitation by impaired performance in oxygen supply through ventilation and circulation. Overall, cadmium exposure resulted in progressive hypoxemia in oysters at high temperatures, suggesting that the thermal tolerance window is narrowed in marine ectotherms inhabiting polluted areas compared with pristine environments.

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Cadmium and temperature have strong impacts on the metabolic physiology of aquatic organisms. To analyze the combined impact of these two stressors on aerobic capacity, effects of Cd exposure (50 microg/L) on mitochondrial function were studied in oysters (Crassostrea virginica) acclimated to 12 and 20 degrees C in winter and to 20 and 28 degrees C in fall. Cadmium exposure had different effects on mitochondrial bioenergetics of oysters depending on the acclimation temperature. In oysters acclimated to 12 degrees C, Cd exposure resulted in elevated intrinsic rates of mitochondrial oxidation, whereas at 28 degrees C, a rapid and pronounced decrease of mitochondrial oxidative capacity was found in Cd-exposed oysters. At the intermediate acclimation temperature (20 degrees C), effects of Cd exposure on intrinsic rates of mitochondrial oxidation were negligible. Degree of coupling significantly decreased in mitochondria from 28 degrees C-acclimated oysters but not in that from 12 degrees C- or 20 degrees C-acclimated oysters. Acclimation at elevated temperatures also increased sensitivity of oyster mitochondria to extramitochondrial Cd. Variation in mitochondrial membrane potential explained 41% of the observed variation in mitochondrial adenosine triphosphate synthesis and proton leak between different acclimation groups of oysters. Temperature-dependent sensitivity of metabolic physiology to Cd has significant implications for toxicity testing and for extrapolation of laboratory studies to field populations of aquatic poikilotherms, indicating the importance of taking into account the thermal regime of the environment.

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Cadmium (Cd) is an important toxicant in estuarine and coastal environments which can strongly affect energy balance of aquatic organisms by increasing the organism's basal energy demand and reducing its aerobic capacity. Mechanisms of Cd‐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 (GSH). We studied the effects of Cd exposure on protein and GSH synthesis and expression of stress protein (heat shock proteins HSP60, HSP70 and HSP90) and metallothioneins in isolated gill and hepatopancreas (HP) cells of the eastern oysters Crassostrea virginica. Our study showed that exposure to Cd (10–2000 ìM for 4 h) resulted in dose‐dependent increase in the rate of the protein synthesis in oyster cells. GSH synthesis was inhibited by the highest Cd concentration (2 mM) especially in HP cells. Elevated protein synthesis was associated with the increased expression of metallothioneins and heat shock proteins. Stress protein response considerably differed between gill and HP cells. In HP, there was an increase in expression of MT mRNA, but no change in HSP protein level in response to Cd exposure. In gill cells no significant increase in MT expression was found and level of HSP60 and 70 significantly increased in Cd‐exposed cells.