Effects of acid acclimation on the number of HR ionocytes and the expression of HA in the gills. The number of HR ionocytes and the fluorescence intensity of Atp6v1a signals in the gills were analyzed by immunofluorescence (IF) after acid acclimation (a). HR ionocyte number of filaments (N = 12) (b), mean fluorescence intensity of individual HR ionocytes (N = 1894–6144) (c), and average mean fluorescence intensity of HR ionocytes of filaments (N = 12) (d) were presented. The protein expression of HA in the gills acclimated to acid environments was analyzed by Western blot. The blots showed the bands with molecular weights corresponding to Atp6v1a and Gapdh (approximately 70 kDa and 35 kDa, respectively) (e). The protein expression of HA was quantified and normalized to Gapdh (N = 6) (f). Values are the mean ± SD or SEM. Student’s t-test or Mann–Whitney U test, * p < 0.05, **** p < 0.0001.