We used confocal laser scanning microscopy to validate a new and fast co-labelling method to study the distribution of mitochondria-rich (MR) cells in gill filaments and to differentiate between MR cells that are in contact with the water (cells labelled with both DASPMI and Concanavalin-A) and those that are not (DASPMI-positive only). This method was used to describe differences in MR cell density that occur in the gills of tilapia Oreochromis mossambicus adapted to fresh water or sea water. In fresh water, the total MR cell density was 6233 cells mm-2 and the density of the subpopulation of MR cells that are in contact with the water was 3458 mm-2. After seawater adaptation, cell density decreased to 3061 cells mm-2 for all MR cells of which 2445 cells mm-2 were in contact with water. The percentage of double-labelled MR cells in the total MR cell population had increased from 55 to 80 %. MR cell size (measured as the maximal cross-sectional area) increased from 87 µm2 in fresh water to 217 µm2 in sea water. Biochemical determination of specific and total Na+/K+-ATPase activity in gill homogenates showed no difference between freshwater- and seawater-adapted fish. Quantification of 'mature' chloride cell density in fixed gill filaments using scanning electron microscopy resulted in an overestimate of chloride cell density due to shrinkage of the sample.

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