Fig. 3. (A) The effect of exposure to hypercarbia (filled bars), for periods ranging from 1 h to 24 h, on trout cytoplasmic carbonic anhydrase (tCAc) mRNA expression in the gills of rainbow trout (Oncorhynchus mykiss) relative to the expression of ß-actin mRNA (relative tCAc mRNA expression), as determined by real-time PCR. Values are means ± 1 s.e.m. and N=6 for each period of exposure to hypercarbia. For statistical analysis, values were compared to corresponding relative tCAc mRNA expression values for fish (N=6 in each case) held under control (normocarbic) conditions for the corresponding period of time; a single group of control fish sampled at 3 h served as the control for 1, 2 and 3 h of exposure to hypercarbia. In each case, mRNA expression in the control group was given a relative value of 1 (open bar), and an asterisk therefore indicates relative tCAc mRNA expression in the hypercarbic fish that was significantly different from 1 (one-sample Student's t-test, P<0.05). (B,C) Representative images of tCAc mRNA localization by in situ hybridization in the gills of (B) control and (C) hypercarbic (24 h at a nominal PW_CO2 of 6 Torr; 1 Torr=133.3 Pa) trout. Scale bars, 20 µm.