Abstract
The euryhaline fish Cyprinodon variegatus variegatus is capable of tolerating ambient salinities ranging from 0.3 to 160 PSU, but is incapable of long-term survival in freshwater (<2 mmol l(-1) Na(+)). A population isolated in several freshwater (0.4-1 mmol l(-1) Na(+)) lakes in central Florida is now designated as a subspecies (Cyprinodon variegatus hubbsi). We conducted a comparative study of Na(+) transport kinetics in these two populations when acclimated to different ambient Na(+) concentrations. Results reveal that the two subspecies have qualitatively similar low affinity Na(+) uptake kinetics (K(m)=7000-38,000 μmol l(-1)) when acclimated to 2 or 7 mmol l(-1) Na(+), but C. v. hubbsi switches to a high affinity system (K(m)=100-140 μmol l(-1)) in low-Na(+) freshwater (≤1 mmol l(-1) Na(+)). Inhibitor experiments indicate that Na(+) uptake in both subspecies is EIPA-sensitive, but sensitivity decreases with increasing external Na(+). EIPA induced a 95% inhibition of Na(+) influx in C. v. hubbsi acclimated to 0.1 mmol l(-1) Na(+), suggesting that this subspecies is utilizing a Na(+)/H(+) exchanger to take up Na(+) in low-Na(+) environments despite theoretical thermodynamic constraints. Na(+) uptake in C. v. hubbsi acclimated to 0.1 mmol l(-1) Na(+) is phenamil-sensitive but not bafilomycin-sensitive, leading to uncertainty about whether this subspecies also utilizes Na(+) channels for Na(+) uptake. Experiments with both subspecies acclimated to 7 mmol l(-1) Na(+) also indicate that a Cl(-)-dependent Na(+) uptake pathway is present. This pathway is not metolazone-sensitive (NCC inhibitor) in either species but is bumetanide-sensitive in C. v. variegatus but not C. v. hubbsi. This suggests that an apical NKCC is increasingly involved with Na(+) uptake for this subspecies as external Na(+) increases. Finally, characterization of mitochondria-rich cell (MRC) size and density in fish acclimated to different ambient Na(+) concentrations revealed significant increases in the number and size of emergent MRCs with decreasing ambient Na(+). A linear relationship between the fractional area of emergent MRCs and Na(+) uptake rate was observed for both subspecies. However, C. v. variegatus have lower Na(+) uptake rates at a given MRC fractional area compared with C. v. hubbsi, indicating that the enhanced Na(+) uptake by C. v. hubbsi at low ambient Na(+) concentrations is not strictly a result of increased MRC fractional area, and other variables, such as differential expression of proteins involved in Na(+) uptake, must provide C. v. hubbsi with the ability to osmoregulate in dilute freshwater.
