Behavioral and physiological response of zebrafish to the alarm substances (AS). Side-view video of swimming behavior of individual zebrafish before (A) and after (B) treatment with alarm substances. (C–F) Graphs of behavioral parameters of fright reaction, including the time in upper half of the test tank (C), the number of freezing bouts (D), the amount of time spent frozen (E), and the number of erratic movements (F). Alarm substances increased the whole body cortisol levels (G). All p values between control and test groups were lower than 0.001.

Gene expression patterns of zebrafish after fright reaction. (A) Principal component analysis (PCA) plot shows clustering of samples based on treatments and tissues. (B) Heatmap of cross correlations of all expression datasets showing different datasets being hierarchically clustered. (C and D) Pairwise comparisons of gene expression abundances between test group and control group in olfactory mucosae (C) and brain (D). (E) Venn diagram shows shared genes identified as differentially expressed across tissues. (F) RT-qPCR and RNA-seq fold change values are highly correlated. T, Test group; C, Control group.

Selected GO term enrichment for the genes identified as differentially expressed in the olfactory mucosae and brain of zebrafish. The enrichment values are indicated in colored scale and p values are indicated with different size.

Divergence estimates for the differentially expressed and unbiased genes. Pairwise dN (A), dS (B), and dN/dS (C) values were estimated by comparing orthologs between zebrafish and grass carp. Zebrafish lineage-specific dN (D), dS (E), and dN/dS (F) values were estimated using six-species alignments among zebrafish, grass carp, cave fish, medaka, stickleback, and spotted gar. The relative frequency distribution of dN/dS ratios for each category of genes were calculated from zebrafish–grass carp comparison (G) and zebrafish lineage only (H). Outliers were removed from the boxplot. Significant differences are indicated by the asterisks, based on Wilcoxon rank sum test, *P < 0.05, **P < 0.001.

Adaptive evolution of fright reaction genes in zebrafish. Comparison of the proportions of genes showing evidence of positive selection between differentially expressed and unbiased genes. The percentage of positively selected genes was identified using site mode (A) and branch site model (B) using codeml. (C, D) Codon usage bias in differentially expressed versus unbiased genes, as measured by frequency of optimal codons (Fop) (C) and effective number of codons (D). (E) Breadth of expression between differentially expressed and unbiased genes measured by the specificity index value (τ). Significant differences were indicated by the asterisks, based on Chi-squared test, *P < 0.05, **P < 0.001. NS means non significant.