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First published online April 8, 2004
Journal of Experimental Biology 207, 1729-1739 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00930

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

Raquel N. Lima and Dietmar Kültz^*

University of California, Davis, One Shields Avenue, Davis, CA 95616, USA

^* Author for correspondence (e-mail: dkueltz{at}ucdavis.edu)

Accepted 10 February 2004

The 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 1x seawater (SW) and gradual transfer from FW to 2.4x 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±0.2x10⁵ and 405±32 µm³, respectively) and higher after 5 weeks in 2.4x SW (3.7±0.9x10⁵ 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±0.5x10⁶ to 4.8±1.1x10⁶ relative fluorescence units, RFU) after 1 week of acute acclimation to 1x SW but returns to baseline values (2.4±0.5x10⁶ RFU) within 5 weeks. In contrast, gradual acclimation to 2.4x SW permanently increases Na⁺/K⁺-ATPase content per CC (from 2.0±0.8x10⁶ to 6.7±2.7x10⁶ 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.

Key words: chloride cell, salinity adaptation, killifish, Fundulus heteroclitus, gill epithelium, osmoregulation, tissue microarray, laser scanning cytometry, Na⁺/K⁺-ATPase

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