Many fish encounter hypoxia on a daily cycle, but the physiological effects of intermittent hypoxia are poorly understood. We investigated whether acclimation to constant (sustained) hypoxia or to intermittent diel cycles of nocturnal hypoxia (12 h normoxia:12 h hypoxia) had distinct effects on hypoxia tolerance or on several determinants of O 2 transport and O 2 utilization in estuarine killifish. Adult killifish were acclimated to normoxia, constant hypoxia, or intermittent hypoxia for 7 or 28 days in brackish water (4 ppt). Acclimation to both hypoxia patterns led to comparable reductions in critical O 2 tension and resting O 2 consumption rate, but only constant hypoxia reduced the O 2 tension at loss of equilibrium. Constant (but not intermittent) hypoxia decreased filament length and the proportion of seawatertype mitochondrion-rich cells in the gills (which may reduce ion loss and the associated costs of active ion uptake), increased blood haemoglobin content, and reduced the abundance of oxidative fibres in the swimming muscle. In contrast, only intermittent hypoxia augmented the oxidative and gluconeogenic enzyme activities in the liver and increased the capillarity of glycolytic muscle, each of which should facilitate recovery between hypoxia bouts. Neither exposure pattern affected muscle myoglobin content or the activities of metabolic enzymes in the brain or heart, but intermittent hypoxia increased brain mass. We conclude that the pattern of hypoxia exposure has an important influence on the mechanisms of acclimation, and that the optimal strategies used to cope with intermittent hypoxia may be distinct from those for coping with constant hypoxia.

Many fish encounter hypoxia in their native environment, but the role of mitochondrial physiology in hypoxia acclimation and hypoxia tolerance is poorly understood. We investigated the effects of hypoxia acclimation on mitochondrial respiration, O 2 kinetics, emission of reactive oxygen species (ROS), and antioxidant capacity in the estuarine killifish (Fundulus heteroclitus). Killifish were acclimated to normoxia, constant hypoxia (5 kPa O 2 ) or intermittent diel cycles of nocturnal hypoxia (12 h:12 h normoxia: hypoxia) for 28-33 days and mitochondria were isolated from liver. Neither pattern of hypoxia acclimation affected the respiratory capacities for oxidative phosphorylation or electron transport, leak respiration, coupling control or phosphorylation efficiency. Hypoxia acclimation also had no effect on mitochondrial O 2 kinetics, but P 50 (the O 2 tension at which hypoxia inhibits respiration by 50%) was lower in the leak state than during maximal respiration, and killifish mitochondria endured anoxia-reoxygenation without any impact on mitochondrial respiration. However, both patterns of hypoxia acclimation reduced the rate of ROS emission from mitochondria when compared at a common O 2 tension. Hypoxia acclimation also increased the levels of protein carbonyls and the activities of superoxide dismutase and catalase in liver tissue (the latter only occurred in constant hypoxia). Our results suggest that hypoxia acclimation is associated with changes in mitochondrial physiology that decrease ROS production and may help improve hypoxia tolerance.

No abstract

Many fish experience daily cycles of hypoxia in the wild, but the physiological strategies for coping with intermittent hypoxia are poorly understood. We examined how killifish adjust O 2 supply and demand during acute hypoxia, and how these responses are altered after prolonged acclimation to constant or intermittent patterns of hypoxia exposure. We acclimated killifish to normoxia (∼20 kPa O 2), constant hypoxia (2 kPa) or intermittent cycles of nocturnal hypoxia (12 h:12 h normoxia:hypoxia) for 28 days, and then compared whole-animal O 2 consumption rates (Ṁ O2) and tissue metabolites during exposure to 12 h of hypoxia followed by reoxygenation in normoxia. Normoxiaacclimated fish experienced a pronounced 27% drop in Ṁ O2 during acute hypoxia, and modestly increased Ṁ O2 upon reoxygenation. They strongly recruited anaerobic metabolism during acute hypoxia, indicated by lactate accumulation in plasma, muscle, liver, brain, heart and digestive tract, as well as a transient drop in intracellular pH, and they increased hypoxia inducible factor (HIF)-1α protein abundance in muscle. Glycogen, glucose and glucose-6-phosphate levels suggested that glycogen supported brain metabolism in hypoxia, while the muscle used circulating glucose. Acclimation to constant hypoxia caused a stable ∼50% decrease in Ṁ O2 that persisted after reoxygenation, with minimal recruitment of anaerobic metabolism, suggestive of metabolic depression. By contrast, fish acclimated to intermittent hypoxia maintained sufficient O 2 transport to support normoxic Ṁ O2 , modestly recruited lactate metabolism and increased Ṁ O2 dramatically upon reoxygenation. Both groups of hypoxia-acclimated fish had similar glycogen, ATP, intracellular pH and HIF-1α levels as normoxic controls. We conclude that different patterns of hypoxia exposure favour distinct strategies for matching O 2 supply and O 2 demand.

No abstract

Hypoxia is a pervasive stressor in aquatic environments, and both phenotypic plasticity and evolutionary adaptation could shape the ability to cope with hypoxia. We investigated evolved variation in hypoxia tolerance and the hypoxia acclimation response across fundulid killifishes that naturally experience different patterns of hypoxia exposure. We compared resting O2 consumption rate (MO2), and various indices of hypoxia tolerance (critical O2 tension [Pcrit], regulation index [RI], O2 tension [PO2] at loss of equilibrium [PLOE], and time to LOE [tLOE] at 0.6 kPa O2) in Fundulus confluentus, F. diaphanus, F. heteroclitus, F. rathbuni, Lucania goodei, and L. parva. We examined the effects of chronic (28 d) exposure to constant hypoxia (2 kPa) or nocturnal intermittent hypoxia (12 h normoxia: 12 h hypoxia) in a subset of species. Some species exhibited a two-breakpoint model in MO2 caused by early, modest declines in MO2 in moderate hypoxia. We found that hypoxia tolerance varied appreciably across species: F. confluentus was the most tolerant (lowest PLOE and Pcrit, longest tLOE), whereas F. rathbuni and F. diaphanus were the least tolerant. However, there was not a consistent pattern of interspecific variation for different indices of hypoxia tolerance, with or without taking phylogenetic relatedness into account, likely because these different indices are underlaid by partially distinct mechanisms. Hypoxia acclimation generally improved hypoxia tolerance, but the magnitude of plasticity and responsiveness to different hypoxia patterns varied interspecifically. Our results therefore suggest that hypoxia tolerance is a complex trait that is best appreciated by considering multiple indices of tolerance.

No abstract

The aim of the present study was to determine the roles of externally versus internally oriented CO/H-sensitive chemoreceptors in promoting cardiorespiratory responses to environmental hypercarbia in the facultative air-breathing fish, Chitala ornata (the clown knifefish). Fish were exposed to environmental acidosis (pH ~ 6.0) or hypercarbia (≈ 30 torr PCO) that produced changes in water pH equal to the pH levels of the acidotic water to distinguish the relative roles of CO versus H. We also injected acetazolamide to elevate arterial levels of PCO and [H] in fish in normocarbic water to distinguish between internal and external stimuli. We measured changes in gill ventilation frequency, air breathing frequency, heart rate and arterial blood pressure in response to each treatment as well as the changes produced in arterial PCO and pH. Exposure to normocarbic water of pH 6.0 for 1 h did not produce significant changes in any measured variable. Exposure to hypercarbic water dramatically increased air breathing frequency, but had no effect on gill ventilation. Hypercarbia also produced a modest bradycardia and fall in arterial blood pressure. Injection of acetazolamide produced similar effects. Both hypercarbia and acetazolamide led to increases in arterial PCO and falls in arterial pH although the changes in arterial PCO/pH were more modest following acetazolamide injection as were the increases in air breathing frequency. The acetazolamide results suggest that the stimulation of air breathing was due, at least in part, to stimulation of internally oriented CO/H chemoreceptors monitoring blood gas changes.