SUMMARYThe salinity tolerance of the `California' Mozambique tilapia(Oreochromis mossambicus × O. urolepis hornorum), a current inhabitant of the hypersaline Salton Sea in California, USA, was investigated to identify osmoregulatory stress indicators for possible use in developing a model of salinity tolerance. Seawater-acclimated (35 g l–1) tilapia hybrids were exposed to salinities from 35–95 g l–1, using gradual and direct transfer protocols, and physiological (plasma osmolality, [Na+],[Cl–], oxygen consumption, drinking rate, hematocrit, mean cell hemoglobin concentration, and muscle water content), biochemical(Na+, K+-ATPase) and morphological (number of mature,accessory, immature and apoptotic chloride cells) indicators of osmoregulatory stress were measured. Tilapia tolerated salinities ranging from 35 g l–1 to 65 g l–1 with little or no change in osmoregulatory status; however, in fish exposed to 75–95 g l–1 salinity, plasma osmolality, [Na+],[Cl–], Na+, K+-ATPase, and the number of apoptotic chloride cells, all showed increases. The increase in apoptotic chloride cells at salinities greater than 55 g l–1, prior to changes in physiological and biochemical parameters, indicates that it may be the most sensitive indicator of osmoregulatory stress. Oxygen consumption decreased with salinity, indicating a reduction in activity level at high salinity. Finally, `California' Mozambique tilapia have a salinity tolerance similar to that of pure Mozambique tilapia; however, cellular necrosis at 95 g l–1 indicates they may be unable to withstand extreme salinities for extended periods of time.

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did not parallel one another, suggesting the mitochondrial NADH shuttles do not function during hypoxia exposure. Large increases in the expression of PDK (PDK isoform 2) were consistent with decreased PDH activity; however, these changes in mRNA were not associated with changes in total PDK-2 protein content assessed using mammalian antibodies. No other changes in the expression of other known hypoxia-responsive genes (e.g., lactate dehydrogenase-A or -B) were observed in either muscle or liver. Gibbs free energy; pyruvate dehydrogenase kinase; energy charge; Gibbs free energy; muscle; fish THE ABILITY TO SUPPRESS CELLULAR ATP demand to match the limited capacity for oxygen-independent ATP production has emerged as the unifying adaptive strategy ensuring hypoxia survival (19). In hypoxia-tolerant animals, reductions in cellular ATP demand are achieved through the controlled arrest of processes involved in membrane ion movement (8, 40), protein synthesis (28, 56), RNA transcription, urea synthesis, gluconeogensis, and other anabolic pathways (19). The cellular signals that initiate the hypoxia-induced decrease in oxygen demand are not known. However, work in aestivating snails (3) suggests that the signal for metabolic rate depression originates from the mitochondria and is controlled to a great degree by changes in the kinetics of substrate oxidation (4,14,15,46). Mitochondria isolated from skeletal muscle of hypoxia-acclimated frogs (Rana temporaria) show reduced state III and IV respiration rates, increased mitochondrial oxygen affinity (47), and reduced electron transport chain activity (46) compared with mitochondria isolated from normoxia-exposed animals. This ability to effectively arrest mitochondrial function during hypoxia exposure is essential to limit the production of harmful reactive oxygen species (ROS) and prevent mitochondrialmediated initiation of apoptosis (32). The mechanistic basis for mitochondrial arrest is not known, but it has been suggested (49) that the mitochondrial protein complex pyruvate dehydrogenase (PDH) is involved in mediating metabolic rate depression.PDH is the rate-limiting enzyme that regulates the rate of entry of glycolytically derived pyruvate into the TCA cycle and mitochondrial oxidative metabolism (20) and is regulated by both product inhibition (NADH and acetyl-CoA) and reversible covalent modification (phosphorylation/dephosphorylation). The transformation of PDH between the active form (PDHa) and inactive form is regulated by the relative activities of pyruvate dehydrogenase kinase (PDK), which phosphorylates PDH to deactivate it, and PDH phosphatase, which activates PDH by dephosphorylation (42 (20). Expression of PDK in mammalian cell lines, in particular the PDK-1 isoform, is hypoxia responsive and regulated by the transcription factor hypoxia inducible factor-1 (HIF-1) (10). Increases in PDK-1 mRNA during hypoxia exposure has been linked to the phosphorylation of PDH, reduced mitochondrial oxygen consumption, and ROS generation (33, 43). Expression of PDK-1 in...

The Mozambique tilapia (Oreochromis mossambicus) is prone to osmoregulatory disturbances when faced with fluctuating ambient temperatures. To investigate the underlying causes of this phenomenon, freshwater (FW)- and seawater (SW)-acclimated tilapia were transferred to 15, 25, or 35 degrees C for 2 weeks, and along with typically used indicators of osmoregulatory status [plasma osmolality and branchial and intestinal specific Na(+), K(+)-ATPase (NKA) activity], we used tissue microarrays (TMA) and laser-scanning cytometry (LSC) to characterize the effects of temperature acclimation. Tissue microarrays were stained with fluorescently labeled anti-Na(+), K(+)-ATPase antibodies that allowed for the quantification of NKA abundance per unit area within individual branchial mitochondria-rich cells (MRCs) as well as sections of renal tissue. Mitochondria-rich cell counts and estimates of size were carried out for each treatment by the detection of DASPMI fluorescence. The combined analyses showed that SW fish have larger but fewer MRCs that contain more NKA per unit area. After a 2-week acclimation to 15 degrees C tilapia experienced osmotic imbalances in both FW and SW that were likely due to low NKA activity. SW-acclimated fish compensated for the low activity by increasing MRC size and subsequently the concentration of NKA within MRCs. Although there were no signs of osmotic stress in FW-acclimated tilapia at 25 degrees C, there was an increased NKA capacity that was most likely mediated by a higher MRC count. We conclude on the basis of the different responses to temperature acclimation that salinity-induced changes in the NKA concentration of MRCs alter thermal tolerance limits of tilapia.

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The green sturgeon is a long-lived, highly migratory species with populations that are currently listed as threatened. Their anadromous life history requires that they make osmo- and ionoregulatory adjustments in order to maintain a consistent internal milieu as they move between fresh-, brackish-, and seawater. We acclimated juvenile green sturgeon (121 +/- 10.0 g) to 0 (freshwater; FW), 15 (estuarine; EST), and 24 g/l (SF Bay water; BAY) at 18 degrees C for 2 weeks and measured the physiological and biochemical responses with respect to osmo- and ionoregulatory mechanisms. Plasma osmolality in EST- and BAY-acclimated sturgeon was elevated relative to FW-acclimated sturgeon (P < 0.01), but there was no difference in muscle water content or abundance of stress proteins. Branchial Na(+), K(+)-ATPase (NKA) activity was also unchanged, but abundance within mitochondrion-rich cells (MRC) was greater in BAY-acclimated sturgeon (P < 0.01). FW-acclimated sturgeon had the greatest NKA abundance when assessed at the level of the entire tissue (P < 0.01), but there were no differences in v-type H(+)ATPase (VHA) activity or abundance between salinities. The Na(+), K(+), 2Cl(-) co-transporter (NKCC) was present in FW-acclimated sturgeon gills, but the overall abundance was lower relative to sturgeon in EST or BAY water (P < 0.01) where this enzyme is crucial to hypoosmoregulation. Branchial caspase 3/7 activity was significantly affected by acclimation salinity (P < 0.05) where the overall trend was for activity to increase with salinity as has been commonly observed in teleosts. Sturgeon of this age/size class were able to survive and acclimate following a salinity transfer with minimal signs of osmotic stress. The presence of the NKCC in FW-acclimated sturgeon may indicate the development of SW-readiness at this age/size.

We investigated the effect of environmental salinity on the upper thermal tolerance of green sturgeon (Acipenser medirostris), a threatened species whose natural habitat is vulnerable to temperature and salinity variation as a result of global climate change. Freshwater (FW)-reared sturgeon were gradually acclimated to salinities representing FW, estuary water (EST), or San Francisco Bay water (BAY) at 18 degrees C, and their critical thermal maximum (CTMax) was measured by increasing temperature 0.3 degrees C/min until branchial ventilation ceased. CTMax was 34.2+/-0.09 degrees C in EST-acclimated fish, with FW- and BAY-acclimated fish CTMax at 33.7+/-0.08 and 33.7+/-0.1 degrees C, respectively. Despite the higher CTMax in EST-acclimated fish, FW-acclimated sturgeon ventilation rate reached a peak that was 2 degrees C higher than EST- and BAY-acclimated groups and had a greater range of temperatures within which they exhibited normal ventilatory function as assessed by Q10 calculation. The osmoregulatory consequences of exposure to near-lethal temperatures were assessed by measuring plasma osmolality and hematocrit, as well as white muscle, brain, and heart tissue water contents. Hematocrit was increased following CTMax exposure, most likely owing to the elevated metabolic demands of temperature increase, and plasma osmolality was significantly increased in EST- and BAY-acclimated fish, which was likely the result of a greater osmotic gradient across the gill as metabolism increased. To our knowledge, this represents the first evidence for an effect of salinity on the upper thermal tolerance of sturgeon, as well as the first investigation of the osmoregulatory consequences of exposure to near-lethal temperatures.

The gill is widely accepted to have played a key role in the adaptive radiation of early vertebrates by supplanting the skin as the dominant site of gas exchange. However, in the most basal extant craniates, the hagfishes, gills play only a minor role in gas exchange. In contrast, we found hagfish gills to be associated with a tremendous capacity for acid-base regulation. Indeed, Pacific hagfish exposed acutely to severe sustained hypercarbia tolerated among the most severe blood acidoses ever reported (1.2 pH unit reduction) and subsequently exhibited the greatest degree of acid-base compensation ever observed in an aquatic chordate. This was accomplished through an unprecedented increase in plasma [HCO3−] (>75 mM) in exchange for [Cl−]. We thus propose that the first physiological function of the ancestral gill was acid-base regulation, and that the gill was later co-opted for its central role in gas exchange in more derived aquatic vertebrates.