Effects of incubation time and oPRL on ncc (A), nhe3b (B), ecac (C), prlra (D), prlrb (E) and β-actin (F) gene expression in cultured gill filaments. Solid line, control; dashed line, oPRL (1.0 μg/ml). Gene expression is presented as fold-change from 0 h. Means ± SEM (n = 6). Filaments were dissected from anesthetized fish and pre-incubated for 3 h prior to treatment with oPRL-supplemented medium. Effects of hormone treatment and time were evaluated by two-way ANOVA. For clarity, only the results of follow-up analyses for a significant main effect of hormone treatment are presented. ^*, **, ***Significantly different from the time-matched controls at P < 0.05, 0.01 and 0.001, respectively, by Student’s t-test. Whole-mount in situ hybridization showing ncc expression in gill filaments cultured for 4 h in the presence or absence of oPRL as compared with pre-incubated and 0 h control filaments (G). Scale bar = 50 μm.