A. hydrophila-induced increased mortality in copper-stressed larvae. (A)A. hydrophila used in this study induced increased expression of immune-related genes in zebrafish larvae at 6 hpi (hours post infection), with increased expression observed in il-6, TLR signaling genes, MAPK signaling genes, stat1a, c3a, defb1, and hsp70.3 after only immersion infection with A. hydrophila. (B) Larvae were immersed with a different dosage of A. hydrophila at 68 hpf (hours post fertilization), and cumulative mortalities of the no-copper-stressed control and the copper-stressed larvae were calculated separately at 6, 12, 24, 36, 48, 60, and 72 hpi. (C) Larvae were immersed only or immersed after injury with 10⁷A. hydrophila at 68 hpf, and cumulative mortalities of the no-copper-stressed control and copper-stressed larvae were calculated separately at 6, 12, 24, 36, 48, 60, and 72 hpi. Three biological replicates were performed. ANOVA post-hoc Tukey's test. Data are presented as mean ± SD. ***P < 0.001; **P < 0.01; *P < 0.05.

Percentages of coro1a promotor-driven GFP-positive cells in copper-stressed and the no-copper-stressed control larvae before and after A. hydrophila infection. FACS (flow cytometry) plots (A1–A8) (red boxes indicating GFP-positive cells) and percentages (B) of coro1a promotor-driven GFP-positive macrophages and neutrophils in copper-stressed and control larvae with or without A. hydrophila infection. Three biological replicates were performed. ANOVA post-hoc Tukey's test. Data are presented as mean ± SD. ***P < 0.001; **P < 0.01; and ns, no significance.

Copper-stressed macrophages and neutrophils respond more quickly to A. hydrophila infection. (A) Schematic view of tail injury, A. hydrophila infection, and GFP-positive cell counting, with immersion after injury being used in the assays. (B) The recruitment of GFP-positive macrophages and neutrophils around injury locus (in the fixed red boxes) were analyzed at 0, 2, 4, and 6 hpi, using Tg (coro1a:eGFP) transgenic fish. (C–E) Quantification analysis showed a significant increase in the number of GFP-positive cells in the area near the injury locus in the copper-stressed Tg(coro1a:eGFP) larvae compared with the no-copper-stressed control at 0, 2, 4, and 6 hpi (C), in the copper-stressed Tg (mpx:eGFP) larvae (D), and in the copper-stressed Tg (lyz:eGFP) larvae (E). Three biological replicates were performed. Analysis with hypergeometric distribution in R-console software for bi-modal distribution to exhibit individual variations in each group in one experiment. Data are presented as mean ± SD. ***P < 0.001; **P < 0.01; *P < 0.05; and ns, no significance.

DEGs in copper-stressed coro1a, mpx, or lyz promotor-driven GFP-positive cells. (A) Schematic view of copper-stressed coro1a, mpx, or lyz promotor-driven GFP-positive cells as measured separately by FACS for RNA-Seq and comparison. (B,C) KEGG pathways over-represented for up-regulated (B) and down-regulated DEGs (C) of copper-stressed coro1a, mpx, and lyz promotor-driven GFP-positive cells, respectively, and the over-representation analysis was performed for all DEGs (adjusted P < 0.05) at 68 hpf.

Phagocytosis of macrophages and neutrophils in copper-stressed and control larvae. (A) FACS plots (A1,A2,A4,A5) and percentages of red E. coli-positive cells in the total coro1a promotor-driven GFP-positive macrophage and neutrophil cells (A3) and in the total lyz promotor-driven GFP-positive neutrophil cells (A6), respectively, following copper stimulation, red boxes indicating GFP and red positive cells by FACS. (B) Schematic view of E. coli phagocytosis activity tests for macrophages or neutrophils in copper-stressed or control larvae. (C) Mean (±SD) number of red E. coli cells per macrophage or neutrophil in the no-copper-stressed control or copper-stressed larvae. Macrophage with phagocytic red E. coli(C1,C3) or neutrophil cells (C2,C4) from the control (C1,C2) and copper-stressed larvae (C3,C4), and mean (±SD) number of red E. coli cells per macrophage or neutrophil in coro1a-GFP cells (C5) or lyz-GFP cells (C6) was calculated. Two biological replicates were performed. Analysis with hypergeometric distribution in R-console software for bi-modal distribution to exhibit individual variations in each group in one experiment. ***P < 0.001; **P < 0.01.

Increased apoptosis of macrophages and neutrophils in copper-stressed larvae. Annexin-V [(A2–A10), in red boxes] and PI (B1–B4) labeling of apoptotic macrophages and neutrophils, and GFP-positive cells were selected for the analysis [(A1), in blue box]. FACS panels (A2,A3) and percentage of apoptotic macrophages and neutrophils (A4) in copper-stressed coro1a promotor-driven GFP transgenic larvae; FACS panels (A5,A6,A8,A9) and percentages of apoptotic neutrophils in copper-stressed mpx transgenic larvae (A7) or lyz transgenic larvae (A10). Three biological replicates were performed. ANOVA post-hoc Tukey's test. Data are presented as mean ± SD. ***P < 0.001; and ns, no significance.

ROS- and mitochondrial ROS (mROS)-mediated apoptosis signaling in copper-stressed macrophages and neutrophils. ROS red labeling [(A1–A4), in red boxes] and relative ROS fluorescence (A5) in macrophages and neutrophils in copper-stressed coro1a-driven GFP transgenic larvae. (B) Schematic view for testing the mROS-mediated apoptosis gene expression in copper-stressed macrophages and neutrophils via cell direct qRT-PCR. (C) Expression of different caspase genes in macrophages and neutrophils from copper-stressed coro1a-driven GFP transgenic larvae before and after A. hydrophila infection. (D) Expression of mROS-mediated apoptotic genes in aforementioned cells. Three biological replicates were performed. ANOVA post-hoc Tukey's test. Data are presented as mean ± SD. ***P < 0.001; **P < 0.01; *P < 0.05; and ns, no significance.

Expression of immune-related genes in copper-stressed macrophages and neutrophils under no A. hydrophila infection conditions. Expression of immune-related genes, including TLR signaling genes, MAPK signaling genes, il1b, stat1a, and c3a, in copper-stressed coro1a-GFP-positive cells (A), mpx-GFP-positive cells (B), and lyz-GFP-positive cells (C). Three biological replicates were performed. ANOVA post-hoc Tukey's test. Data are presented as mean ± SD.***P < 0.001; **P < 0.01; *P < 0.05; and ns, no significance.

Expression of immune-related genes in copper-stressed macrophages and neutrophils after A. hydrophila infection. Expression of immune-related genes in copper-stressed coro1a-GFP-positive cells (A), mpx-GFP-positive cells (B), and lyz-GFP-positive cells (C) at 6 hpi (A1–C1) and 24 hpi (A2–C2), respectively. Three biological replicates were performed. ANOVA post-hoc Tukey's test. Data are presented as mean ± SD. ***P < 0.001; **P < 0.01; *P < 0.05; and ns, no significance.