Fig. 2.

The reduced cortisol levels in males from the HFL decreased their locomotor and exploratory behaviors. (A) PC1 scores representing the locomotor and exploratory behavioral responses to the novel-tank diving test (NTT) of the CTR and FLX lineages. PCA was used to reduce the dimensionality of the dataset composed of 10 behavioral metrics. PC1 accounted for 51% of the behavioral variance of which positive weights are associated with high exploratory and locomotor activities. n = 13–16 biological replicates per group in each generation. (B) Metyrapone (MET) exposure inhibited the total cortisol production in naïve adult males subjected to the NTT. STR-NTT, stressed levels following the NTT; n = 4 and 16 fish for basal and novel-tank diving test groups, respectively. Analyzed by two-way ANOVA on ranks. (C) Pharmacological cortisol reduction in naïve males with MET recapitulated the transgenerational inherited behavioral phenotype observed in the males from the HFL. n = 15 fish in each group. (D) Whole-body cortisol levels (stressed) of males from the F₀ following the NTT. n = 8–18 fish in each group. Analyzed by one-way ANOVA. (E) Cortisol (C) supplementation to the F₀ male adult fish from the HFL (HFL-C) rescued their locomotor and exploratory behaviors. n = 13–17 fish in each group. Data (A, C, and E) are presented in box plots showing the median (solid line), the mean (dashed line), the interquartile range (box), and the whiskers embracing data within the 10th and 90th percentiles; all data outside the range of the whiskers are presented as individual data points. The asterisk (*P = 0035) or P values shown above the bars represent significant differences within treatment compared with the CTR. The number sign (^#P < 0.001) identifies statistical differences between HFL and HFL-C. Behavioral data were analyzed using Student’s t test (or Mann–Whitney U test for A, HFL F₀ and LFL F₃). Whole-body cortisol levels (B and D) are expressed as mean ± SEM; P < 0.05.