Physiological effects of salinity on Delta Smelt, Hypomesus transpacificus.

Abiotic factors like salinity are relevant to survival of pelagic fishes of the San Francisco Bay Estuary. We tested the effects of 4 parts per thousand (ppt) salinity increases on Delta Smelt (DS) in a laboratory experiment simulating salinity increases that might occur around the low-salinity zone (LSZ) (<6 ppt). Adult DS, fed 2% body mass per day, starting at 0.5 ppt [freshwater (FW)], were exposed to weekly step-increases of 4 ppt to a maximum of 10 ppt saltwater (SW) over 19 days, and compared to FW controls. DS (n = 12/treatment per sampling) were sampled at 24, 72, and 96 h (1, 3, and 4 days) post-salinity increase for analyses of hematocrit, plasma osmolality, muscle water content, gill chloride cell (CC) Na(+)/K(+)-ATPase (NKA) and apoptosis after being weighed and measured (n = 3 tanks per treatment). No apparent increase in length or weight occurred nor did a difference in survival. Following step-increases in SW, hematocrit increased over time. Other fish responses generally showed a pattern; specifically plasma osmolality became elevated at 1 day and diminished over 4 days in SW. Percent muscle water content (%) did not show significant changes. CCs showed increased NKA, cell size and apoptosis over time in SW, indicating that CCs turnover in DS. The cell renewal process takes days, at least over 19 days. In summary, DS are affected by salinities of the LSZ and ≤10 ppt, though they employ physiological strategies to acclimate.

Rapid changes in plasma cortisol, osmolality, and respiration in response to salinity stress in tilapia (Oreochromis mossambicus).

We elucidated a time course for cortisol release in tilapia as it corresponds to changes in plasma osmolytes and respiration. Following exposure of freshwater (FW) tilapia to 25 per thousand seawater (SW), we measured plasma osmolality, [Na(+)], [K(+)], [Cl(-)], hematocrit, cortisol concentration, oxygen-consumption rate (MO2), and ventilation frequency over 5days and compared them to FW control fish. Cortisol increased rapidly by 3h and remained elevated for 3days. Plasma osmolality, [Na(+)], and [Cl(-)] were elevated at 6-8h, peaked 24h following SW exposure, and then decreased to near-FW levels by 3days. MO2 increased at 24h post-SW exposure relative to FW, while ventilation frequency increased by 3h. Overall, we interpret changes in cortisol as resulting from a change in salinity, in contrast to changes in plasma solute concentrations that could be due to adjustments resulting from the fish's cortisol response as it faces osmoregulatory distress. Increases in oxygen-consumption rate at 24h and ventilation frequency at 3h are likely as a result of the cellular stress response occurring during salinity stress. No significant changes in blood hematocrit were observed, which suggests that tilapia are capable of rapidly counteracting dehydration during acute hyperosmotic stress.

Prolonged apoptosis in mitochondria-rich cells of tilapia (Oreochromis mossambicus) exposed to elevated salinity.

The time-course of programmed cell death (apoptosis) during reorganization of gill epithelium in salinity-stressed tilapia was analyzed using a recently developed method based on laser scanning cytometry (LSC) of dissociated gill cells. Apoptosis in mitochondria-rich cells (MRC) was distinguished from that in other cell types using Na(+)/K(+) ATPase (NKA) as a cell-specific marker. Caspase 3/7 activity in MRC, assessed using LSC and microplate assays, increased significantly starting at 6 h of salinity stress and remained elevated for at least 5 days. This time-course of apoptosis in MRC during acute salinity stress was reflected in elevated apoptotic DNA fragmentation. In parallel to induction of apoptosis, MRC showed a pronounced shift to G2 phase of the cell cycle, which is indicative of G2/M cell cycle arrest, and an increase in NKA abundance per MRC. Unlike in MRC, apoptosis was not significantly increased in other gill cell types, although there was a small transient increase in DNA fragmentation at 6 h. G2 arrest was also observed. Overall, we interpret our data as evidence for a significant role of apoptosis in the extensive reorganization of MRC populations that takes place during salinity acclimation, perhaps similar to its well-established role during organismal development.

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.