Establishment of cold resistance in zebrafish larvae after pre-exposure to cold stress. (A) Flowchart of cold exposure. Zebrafish embryos were incubated at 28°C from fertilization to 96 hpf. Larvae at 96 hpf were exposed to 16°C for 24 h and the controls were maintained at 28°C. Samples for RNA-seq were collected at 120 hpf. The pre-treated and control larvae were further exposed to 12°C for 6, 12, 24, 36 and 48 h. The time scales were shown on the left. (B) Death rates of pre-treated and control (ck) larvae exposed to 12°C for different times. Data was shown as mean ± standard deviation (n = 4). “**” above error bars indicate p < 0.01. (C) Images of pre-treated and control larvae exposed to 12°C for 36 h. Images were taken under a stereomicroscope from Zeiss with a color CCD camera. Red arrowheads indicate representative dead larvae.