Copper effects on key metabolic enzymes and mitochondrial membrane potential in gills of the estuarine crab Neohelice granulata at different salinities

Abstract

The estuarine crab Neohelice granulata was exposed (96 h) to a sublethal copper concentration under two different physiological conditions (hyperosmoregulating crabs: 2 ppt salinity, 1 mg Cu/L; isosmotic crabs: 30 ppt salinity, 5 mg Cu/L). After exposure, gills (anterior and posterior) were dissected and activities of enzymes involved in glycolysis (hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase), Krebs cycle (citrate synthase), and mitochondrial electron transport chain (cytochrome c oxidase) were analyzed. Membrane potential of mitochondria isolated from anterior and posterior gill cells was also evaluated. In anterior gills of crabs acclimated to 2 ppt salinity, copper exposure inhibited hexokinase, phosphofructokinase, pyruvate kinase, and citrate synthase activity, increased lactate dehydrogenase activity, and reduced the mitochondrial membrane potential. In posterior gills, copper inhibited hexokinase and pyruvate kinase activity, and increased citrate synthase activity. In anterior gills of crabs acclimated to 30 ppt salinity, copper exposure inhibited phosphofructokinase and citrate synthase activity, and increased hexokinase activity. In posterior gills, copper inhibited phosphofructokinase and pyruvate kinase activity, and increased hexokinase and lactate dehydrogenase activity. Copper did not affect cytochrome c oxidase activity in either anterior or posterior gills of crabs acclimated to 2 and 30 ppt salinity. These findings indicate that exposure to a sublethal copper concentration affects the activity of enzymes involved in glycolysis and Krebs cycle, especially in anterior (respiratory) gills of hyperosmoregulating crabs. Changes observed indicate a switch from aerobic to anaerobic metabolism, characterizing a situation of functional hypoxia. In this case, reduced mitochondrial membrane potential would suggest a decrease in ATP production. Although gills of isosmotic crabs were also affected by copper exposure, changes observed suggest no impact in the overall tissue ATP production. Also, findings suggest that copper exposure would stimulate the pentose phosphate pathway to support the antioxidant system requirements. Although N. granulata is very tolerant to copper, acute exposure to this metal can disrupt the energy balance by affecting biochemical systems involved in carbohydrate metabolism.

Transcriptomic responses of the endangered freshwater mussel Margaritifera margaritifera to trace metal contamination in the Dronne River, France

“…Amongst the different physiological and biochemical processes negatively correlated with Cu in aquatic organisms, we can cite glycolysis, Krebs cycle, ionic and osmotic regulation, acid-base balance, ammonia excretion, oxygen consumption, and growth. Most of these effects can be directly or indirectly associated with an insufficient aerobic production of energy to maintain cell metabolism and homeostasis (for references, see Lauer et al 2012). Due to the high reactivity of ionic (Bfree^) Cu, cells have developed complex and elegant mechanisms for shuttling Cu into, through, and out of cells as needed for its many critical reactions, without allowing free Cu to exert cytotoxic effects.…”

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“…The high expression of PEPCK in muscle during 12-24 h could be a result of gluconeogenesis in the muscle during this time period. In a previous study, Wang (et al 2012) found reduction of free amino acids in the muscle of Eriocheir sinensis after salinity stress for 12 h. Free amino acid and glycerol can be used to maintain the glucose level in the tissue of crab Neohelice granulata through gluconeogenesis under hypo-osmotic stress (Lauer et al, 2012;Nery and Santos, 1993). As muscle is a pool of amino acids in crustaceans, it can function as a source of amino acid supply to other tissues (Wang et al, 2012) and the amino acid can be further converted to glycogen through gluconeogenesis (Ramamurthi et al, 1968;Rosas et al, 2001;Wang and Scheer, 1963).…”

Contrasting Effects of Predation Risk and Copper on Copepod Respiration Rates

“…However, Cu is toxic at elevated concentrations, potentially causing oxidative stress through reactive oxygen species (ROS) generation and inhibiting enzymes by blocking other cofactor sites (Grosell 2011). Adverse effects of Cu exposure in marine invertebrates include metabolic disruption, cytotoxicity, and neurotoxicity (Brown et al 2004;Lauer et al 2012). More specifically in copepods, Cu exposure may delay development and thus increase age at maturity (Sullivan et al 1983;Kwok et al 2008), reduce reproductive output (Reeve et al 1977;Fitzer et al 2013), and reduce foraging activity (Sharp and Stearns 1997).…”