2461 Environmental Toxicology and Chemistry, Vol. 25, No. 9, pp. 2461–2469, 2006  2006 SETAC Printed in the USA 0730-7268/06 $12.00 + .00 COMBINED EFFECTS OF TEMPERATURE ACCLIMATION AND CADMIUM EXPOSURE ON MITOCHONDRIAL FUNCTION IN EASTERN OYSTERS CRASSOSTREA VIRGINICA GMELIN (BIVALVIA: OSTREIDAE) ANTON S. CHERKASOV,AMY H. RINGWOOD, and INNA M. SOKOLOVA* Biology Department, University of North Carolina at Charlotte, 9201 University City Boulevard, Charlotte, North Carolina 28223, USA ( Received 10 October 2005; Accepted 27 February 2006) Abstract—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 g/L) on mitochondrial function were studied in oysters (Crassostrea virginica) acclimated to 12 and 20°C in winter and to 20 and 28°C in fall. Cadmium exposure had different effects on mitochondrial bioenergetics of oysters depending on the acclimation temperature. In oysters acclimated to 12°C, Cd exposure resulted in elevated intrinsic rates of mitochondrial oxidation, whereas at 28°C, a rapid and pronounced decrease of mitochondrial oxidative capacity was found in Cd-exposed oysters. At the intermediate acclimation temperature (20°C), effects of Cd exposure on intrinsic rates of mitochondrial oxidation were negligible. Degree of coupling significantly decreased in mitochondria from 28°C-acclimated oysters but not in that from 12°C- or 20°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. Keywords—Mitochondrial respiration Mitochondrial membrane potential Cadmium Temperature Bivalves INTRODUCTION Metabolic regulation plays a key role in environmental stress tolerance, because matching energy demand with suf- ficient energy supply is crucial for survival. Environmental stressors may shift this finely tuned balance, leading to energy deficiency, which in turn will negatively affect performance and survival of the organisms. Temperature and heavy metals, including Cd, are important stressors in estuarine habitats [1,2]. Earlier research has shown that temperature directly affects oxidation rates as well as the balance between different mi- tochondrial functions (e.g., phosphorylation, production of re- active oxygen species, proton leak) in poikilotherm mito- chondria, resulting in mitochondrial dysfunction at extreme temperatures [3,4]. Moreover, acclimation or seasonal accli- matization to ambient temperatures is accompanied by con- siderable changes in the intracellular milieu, such as shifts in intracellular pH (pH i ), changes in substrate catabolism, and levels of expression of antioxidants and heat shock proteins, which in turn may strongly affect mitochondrial function [4]. Studies in plant, mammalian, and invertebrate models also have shown that mitochondria are highly sensitive to Cd. Mi- tochondrial response to Cd typically involves decreased phos- phorylation efficiency and progressive uncoupling at increas- ing Cd levels, resulting in impaired aerobic capacity for aden- osine triphosphate (ATP) production [5–9]. High sensitivity of mitochondrial function to Cd and tem- perature in poikilotherms implies that mitochondrial energy metabolism is a key intracellular target, on which the effects of these two important environmental stressors converge. Our * To whom correspondence may be addressed (insokolo@uncc.edu). earlier study has shown that acute rise in temperature, such as may be expected during summer low tides in the intertidal zone, resulted in a strongly increased sensitivity of oyster mi- tochondria to Cd [8]. However, the effects of elevated tem- perature on Cd sensitivity may differ when environmental tem- perature changes slowly, such as during seasonal acclimati- zation on a short-term basis or global climate change on a long-term scale, and an organism has time to adjust its phys- iology to the temperature shift. Under these conditions, chang- es in intracellular milieu resulting from temperature accli- mation may modulate sensitivity to Cd in a way that is car- dinally different from the response to an acute rise in tem- perature. However, to our knowledge, the effects of temperature acclimation and seasonal acclimatization on the sensitivity of mitochondrial bioenergetics to Cd have not been studied, and their role in modulating the toxic effects of Cd on poikilotherm mitochondria is not fully understood. The aim of the present study was to analyze the role of temperature acclimation and seasonal acclimatization in mod- ulating Cd effects on mitochondrial capacity in a model es- tuarine bivalve, eastern oyster (Crassostrea virginica). The hypothesis tested was that the adjustments in mitochondrial structure and function associated with prolonged temperature acclimation or seasonal acclimatization may affect mitochon- drial sensitivity to Cd in a different way and/or to a different extent than an acute rise in temperature that does not allow time for acclimatory adjustments. To test this hypothesis, phos- phorylation capacity, proton leak, coupling, and membrane potential were studied in mitochondria from control and Cd- exposed oysters acclimated to different temperatures within the environmentally relevant range (12, 20, and 28°C) in fall and winter.