Galanin deficiency results in higher mortality and bacterial burden after yolk infection. (A,B) Survival rate in mock-injected larvae (non-inf +/+), infected wild-types (inf +/+), infected gal−/− mutants (inf -/-) and infected gal−/− mutants treated with NAX 5055 (inf -/- NAX) after (A) M. marinum or (B) S. aureus yolk infection. (C,D) Bacterial burden in infected wild-type larvae, infected gal−/− mutants and infected gal−/− mutants treated with NAX 5055 after (C) M. marinum or (D) S. aureus yolk infection. Data are combined from three biological replicates (n = 10 larvae/group). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, non-significant.

Galanin deficiency results in higher mortality and bacterial burden after vein infection. (A,B) Survival rate in mock injected larvae (non-inf +/+), infected wild-types (inf +/+), infected gal−/− mutants (inf -/-) and infected gal−/− mutants treated with NAX 5055 (inf -/- NAX) after (A) M. marinum or (B) S. aureus vein infection. (C,D) Bacterial burden in infected wild-type larvae, infected gal−/− mutants and infected gal−/− mutants treated with NAX 5055 after (C) M. marinum or (D) S. aureus vein infection. Data are combined from three biological replicates (n = 10 larvae/group). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, non-significant.

Expression levels of immune-related genes determined by RT-qPCR. irg1l, il1b, tnfa and cxcl8a levels after (A) M. marinum or (B) S. aureus infection. Data are mean ± s.e.m. of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (determined using ANOVA with a Fisher’s LSD post hoc). ns, non-significant.

Transcriptome analysis by RNA-seq showing modulation of infection-induced gene regulation by galanin mutation. (A) Venn diagram showing overlaps between clusters of genes significantly upregulated or downregulated by infection of gal+/+ and gal−/− mutant larvae; (B) heat map displaying the normalized fold changes of transcripts that reached statistical significance as differentially expressed in infected gal+/+ and infected gal−/− zebrafish larvae; (C) scatter plot showing the effect of galanin deficiency on gene expression. For all genes showing significant regulation upon infection (blue and gray dots) or the combined infection in the mutant (gray dots). Gray dots represent the overlap between control infection and mutant infection. The gray line indicates the point at which galanin knockout treatment does not alter infection-induced gene regulation; (D) GO analysis for upregulated genes in biological processes. Paired analysis was per-formed using DESeq2 by comparing each group to each non-infected group. Significantly regulated genes were selected by using a p.adj < 0.05 and |FoldChange| > 1.5 cutoff.

Pathway analysis. Comparison of regulated genes between gal+/+ and gal−/− larvae after M. marinum infection in KEGG Pathways: (A) Cytokine-cytokine receptor interaction and (B) JAK-STAT signaling pathway. Blue stars indicate genes regulated in gal+/+, while green in gal−/−.