Pre-acclimation to hypoxia increased the cold resistance of zebrafish larvae. (A) Flowchart of stressor exposure. Zebrafish embryos were incubated in air at 28°C from fertilization to 96 hpf and then pre-acclimated to hypoxia or cold for 24 h. Sample collection for RNA-seq and lethal hypoxia and cold exposure were performed at 120 hpf. (B and C) Survival rates of zebrafish larvae after lethal cold (B) and hypoxia (C). Data was shown as mean ± standard deviation (n = 5). Different letters above the error bars indicate significant difference (p < 0.05) between treatment groups. (D and E) Photographs of zebrafish larvae after lethal cold (D) and hypoxia (E) challenge. Dead fish displayed an obvious body curvature.

Genes co-induced by hypoxia and cold. The expression of genes in zebrafish larvae after hypoxia and cold pre-acclimation was characterized using qPCR. Data was shown as mean ± standard deviation (n = 3). Significant differences between control and hypoxia or cold exposed samples were demonstrated using asterisks. “*” p < 0.05, “**” p < 0.01 and “***” p < 0.001.

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