The effects of acclimation temperature, salinity, and behavior on the thermal tolerance of Mozambique tilapia (Oreochromis mossambicus).

Mozambique tilapia have been shown to be incredibly stress tolerant with respect to environmental salinity, hypoxia, and ammonia concentrations. Temperature challenges to this species have shown that they have difficulty with cold acclimation. The purpose of this study was to measure the effects of acclimation temperature and salinity on the thermal tolerance of Mozambique tilapia as assessed by critical thermal maxima (CTMax) and critical thermal minima (CTMin). We also monitored fish behavior and quantified ventilation rate. To our knowledge, this study was the first to investigate upper and lower thermal tolerances, and the effect of environmental salinity in this physiologically impressive species. Using predictive regression analyses of the thermal limits, thermal tolerance polygons were constructed and total areas were calculated 678.9°C2 for freshwater (FW)-acclimated tilapia, and 739.4°C2 seawater (SW)-acclimated tilapia. During the thermal challenges, we observed two novel behaviors in response to thermal challenge, ventilation cessation behavior (VCB) and aquatic surface respiration (ASR), and we conclude that the use of these behaviors extended the thermal limits of these fish in both FW and two-thirds SW by limiting the exposure of the gill epithelium to the changing environment.

Hagfish: Champions of CO2 tolerance question the origins of vertebrate gill function.

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.

The physiological responses of green sturgeon (Acipenser medirostris) to potential global climate change stressors.

The green sturgeon (Acipenser medirostris) is an anadromous species with a distinct population segment in the San Francisco Bay-Sacramento River Delta that is currently listed as threatened. Although this species is able to tolerate salinity challenges as soon as 6 mo posthatch, its ability to deal with unpredictable salinity fluctuations remains unknown. Global climate change is predicted to result in large freshwater (FW) flushes through the estuary during winter and greater tidal influence during the summer. We exposed green sturgeon acclimated to 15 (EST) or 24 (BAY) g/L salinity to a rapid FW influx, where salinity was reduced to 0 g/L in 3 h in order to simulate the effect of the "winter" scenario. Both groups survived, enduring a 10% plasma osmolality reduction after 3 h. BAY-acclimated sturgeon upregulated both Na(+), K(+)-ATPase (NKA) activity and caspase 3/7 activity, but no changes were observed in the EST-acclimated fish. In addition, we exposed FW-acclimated sturgeon to a dual 12-h salinity fluctuation cycle (0-24-0 g/L) in order to simulate the effect of greater tidal influence. At 6 h, the sturgeon showed a significant increase in plasma osmolality, and branchial NKA and caspase 3/7 activities were increased, indicating an acclimation response. There was no acclimation at 18 h, and plasma osmolality was higher than the peak observed at 6 h. The second fluctuation elicited an upregulation of the stress proteins ubiquitin and heat shock 70-kDa protein (HSP 70). Sturgeon can acclimate to changes in salinity; however, salinity fluctuations resulted in substantial cellular stress.

Salinity stress results in rapid cell cycle changes of tilapia (Oreochromis mossambicus) gill epithelial cells.

We have developed a technique for immunocytochemistry of fish gill cells that we used to quantify tilapia (Oreochromis mossambicus) mitochondria-rich cells (MRC) and other gill cells (non-MRC) within different cell cycle phases by laser scanning cytometry. Gill cells fixed on coverslips were triple stained with propidium iodide to distinguish G1 vs. G2 phases, Ser10-phosphorylated histone H3 antibody to label mitotic cells, and Na(+)/K(+) ATPase antibody to label MRC. These parameters were measured at 0 (control), 4, 8, 16, 24, 48, 72, and 168 hr (1 week) following exposure of freshwater (FW) acclimated fish to 2/3 seawater (SW). MRC increased mitotic activity very rapidly peaking at 8 hr following SW exposure. This change in mitotic MRC is indicative of epithelial reorganization during SW acclimation. In contrast to MRC, the proportion of non-MRC (likely pavement cells (PVC)) in mitosis did not change significantly in response to SW exposure. Moreover, twice as many MRC were in mitosis compared with non-MRC, suggesting that MRC turn over faster than other cell types during SW acclimation. Following the mitosis peak, MRC accumulated in G2 phase over a period of 16-72 hr post-SW exposure. We also observed G2 arrest with similar kinetics following SW exposure in tilapia non-MRC (likely PVC). We interpret the G2 arrest that occurs after an initial wave of transient increase in MRC mitosis as a means for conserving energy for dealing with the osmotic stress imposed during the exposure of FW fish to SW.

Osmo- and ionoregulatory responses of green sturgeon (Acipenser medirostris) to salinity acclimation.

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.

The acute temperature tolerance of green sturgeon (Acipenser medirostris) and the effect of environmental salinity.

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 effect of elevated salinity on 'California' Mozambique tilapia (Oreochromis mossambicus x O. urolepis hornorum) metabolism.

California Mozambique tilapia (Oreochromis mossambicus x O. urolepis hornorum) are extremely saline tolerant and have been previously shown to reduce whole-animal oxygen consumption rate (MO(2)) upon exposures to salinities greater than that of seawater (SW). In this study tilapia were acclimated to 15, 30, 45, 60 and 75 g/L salinity for 1, 5, 14, or 28 days. There was little change in plasma osmolality or muscle water content in salinities below 60 g/L, and branchial Na(+), K(+)-ATPase (NKA) activity was low in 15 and 30 g/L relative to 60 and 75 g/L. When tilapia were exposed to 75 g/L, plasma osmolality and NKA activity were significantly increased within 5 days of exposure relative to those in 15 and 30 g/L, and remained elevated over the entire 28 days acclimation, indicating that short term salinity challenges (i.e., 5 days) are predictive of longer exposure durations in this species. MO(2) following transfer to 15 and 30 g/L was elevated, reflecting the high energy demand required for switching from a hyper- to a hypo-osmoregulatory strategy. The MO(2) of 60 g/L-exposed fish was significantly reduced at 1, 5, and 14 days, relative to 30 g/L-exposed fish; however by 28 days there were no significant differences. We investigated the potential for a metabolic basis for the salinity-induced MO(2) reduction, using forward stepwise linear regression to correlate enzyme activities of brain, liver, and kidney with MO(2). Brain NKA was correlated with MO(2) after 5 days (p<0.01, r(2)=0.944) and both brain NKA and hepatic total ATPase were correlated with the reduced MO(2) at 14 days (p=0.027, r(2)=0.980 and p=0.025, r(2)=0.780, respectively). These results may indicate a tissue-level metabolic suppression, which has not been previously described as a response to hypersaline exposure in fishes.

Regulation of pyruvate dehydrogenase in the common killifish, Fundulus heteroclitus, during hypoxia exposure.

We examined the metabolic responses of the hypoxia-tolerant killifish (Fundulus heteroclitus) to 15 h of severe hypoxia and recovery with emphasis on muscle substrate usage and the regulation of the mitochondrial protein pyruvate dehydrogenase (PDH), which controls carbohydrate oxidation. Hypoxia survival involved a transient activation of substrate-level phosphorylation in muscle (decreases in [creatine phospate] and increases in [lactate]) during which time mechanisms to reduce overall ATP consumption were initiated. This metabolic transition did not affect total cellular [ATP], but had an impact on cellular energy status as indicated by large decreases in [ATP]/[ADP(free)] and [ATP]/[AMP(free)] and a significant loss of phosphorylation potential and Gibbs free energy of ATP hydrolysis (DeltafG'). The activity of PDH was rapidly (within 3 h) decreased by approximately 50% upon hypoxia exposure and remained depressed relative to normoxic samples throughout. Inactivation of PDH was primarily mediated via posttranslational modification following the accumulation of acetyl-CoA and subsequent activation of pyruvate dehydrogenase kinase (PDK). Estimated changes in cytoplasmic and mitochondrial [NAD(+)]/[NADH] 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.

Salinity-dependent changes in Na(+)/K (+)-ATPase content of mitochondria-rich cells contribute to differences in thermal tolerance of Mozambique tilapia.

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.

The effect of temperature on juvenile Mozambique tilapia hybrids (Oreochromis mossambicus x O. urolepis hornorum) exposed to full-strength and hypersaline seawater.

The effects of temperature on the salinity tolerance of Mozambique-Wami tilapia hybrids (Oreochromis mossambicus x O. urolepis hornorum) were investigated by transferring 35 g/l, 25 degrees C-acclimated fish to 35, 43, 51 or 60 g/l salinity at 15, 25 or 35 degrees C for 24 h, and by assaying gill tissue for branchial Na(+), K(+)-ATPase activity at the three temperatures after acclimating the fish to 15, 25 or 35 degrees C for 2 weeks. Tilapia survived all salinities at 25 and 35 degrees C; however, at 15 degrees C, mortality was 85.7% and 100% in the 51 g/l and 60 g/l groups, respectively. There was a significant interaction between temperature and salinity, as plasma osmolality, [Na(+)] and [Cl(-)] were significantly increased at 51 and 60 g/l salinity in 35 degrees C water (P<0.001). Additionally, muscle water content was significantly reduced at 43 g/l, 15 degrees C relative to pre-transfer values (P<0.001). Branchial Na(+), K(+)-ATPase activity was reduced at 15 degrees C regardless of acclimation temperature, and 25 degrees C-acclimated gill tissue did not show an increase in activity when assayed at 35 degrees C. Results indicate that the effects of a combined temperature-salinity transfer on plasma osmolality and ion concentrations, as well as muscle water content, are greater than when either challenge is given alone. Additionally, branchial Na(+), K(+)-ATPase activity is altered when assayed at varying temperatures; in the case of 15 degrees C, regardless of acclimation temperature. Our enzyme activity data may indicate the presence of a high temperature isoform of branchial Na(+), K(+)-ATPase enzyme.

Physiological, biochemical and morphological indicators of osmoregulatory stress in 'California' Mozambique tilapia (Oreochromis mossambicus x O. urolepis hornorum) exposed to hypersaline water.

The salinity tolerance of the 'California' Mozambique tilapia (Oreochromis mossambicus x 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.