Laser scanning cytometry and tissue microarray analysis of salinity effects on killifish chloride cells

Abstract

SUMMARYThe effects of salinity on chloride cells (CC) and Na+/K+-ATPase content in gill epithelium of euryhaline killifish Fundulus heteroclitus were analyzed using laser scanning cytometry (LSC) and tissue microarrays (TMAs). Salinity acclimations consisted of acute transfer from freshwater (FW) to 1× seawater (SW) and gradual transfer from FW to 2.4× SW. Suspensions of dissociated gill epithelial cells were stained with DASPMI and evaluated using LSC. CC number and volume are proportional to external salinity, being lower in FW(0.5±0.2×105 and 405±32 μm3,respectively) and higher after 5 weeks in 2.4× SW(3.7±0.9×105 and 2697±146 μm3,respectively). TMAs were constructed from fixed gill tissues and developed using antibody for Na+/K+-ATPase to visualize CCs in situ and compare their characteristics with isolated CCs. Na+/K+-ATPase content per CC increases transiently (from 2.2±0.5×106 to 4.8±1.1×106relative fluorescence units, RFU) after 1 week of acute acclimation to 1× SW but returns to baseline values(2.4±0.5×106 RFU) within 5 weeks. In contrast, gradual acclimation to 2.4× SW permanently increases Na+/K+-ATPase content per CC (from 2.0±0.8×106 to 6.7±2.7×106 RFU after 5 weeks). CC size in situ did not correlate well to salinity because of basolateral membrane infoldings. Taken together, these data suggest that euryhaline fishes are capable of sensing environmental salinity to utilize transient short-term and permanent long-term adaptations for coping with salinity changes. These results also demonstrate the power of LSC and TMA for comparative biology.