No abstract

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.

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.

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 mitochondriarich cells (MRC) was distinguished from that in other cell types using Na + /K + ATPase (NKA) as a cell-speciWc marker. Caspase 3/7 activity in MRC, assessed using LSC and microplate assays, increased signiWcantly 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 reXected 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 signiWcantly 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 signiWcant 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.

The physiological response of juvenile steelhead Oncorhynchus mykiss to prolonged heat stress was examined by exposing replicated groups of fish to three different temperatures, 15, 23, and 25 • C, for 25 consecutive days followed by a 55-d recovery period at 15 • C. We found that at temperatures ≥ 25 • C, steelhead consumed significantly less food per day and had elevated feed conversion rates but experienced slower growth, reduced body size, lower body fat, and elevated heat shock protein (hsp) 70 levels relative to fish exposed to 23 • C and 15 • C. Growth decreased 24.4% and 27.1% for length and mass, respectively, between 15 • C and 23 • C, and an additional 60% and 56.5%, respectively, between 23 • C and 25 • C during exposures. While growth increments and lipid levels recovered to control levels after water temperature was reduced, body size of the fish exposed to 25 • C lagged throughout the experiment. Our results indicate a temperature threshold after which steelhead exposed to semichronic thermal stress incur a physiological debt. Heat shock protein 70 levels were detectable up to 25 d poststress in fin and liver tissues. This result provides evidence that hsp 70 is a useful metric for measuring thermal stress that can be assessed nonlethally and used to monitor thermal-habitat restoration efforts for threatened and endangered salmonids.