Embryonic incubation temperature has a long-term effect on the immune response of adult zebrafish

Zebrafish eggs were collected at 28 °C, and split into three embryonic incubation temperature groups (24 °C, 28 °C, 32 °C). When larvae reaching first feeding, the incubation temperature of larvae from 24 °C and 32 °C was gradually changed to 28 °C, and all fish were kept at 28 °C for later development until adulthood. At 100 d post-fertilization, 7 replicates from each temperature group were intraperitoneally (i.p.) injected with 2 µl of 50 mg/ml lipopolysaccharide (LPS) from Pseudomonas aeruginosa 10, while another 7 replicates were i.p. injected with 2 µl phosphate-buffered saline as control. At 12 h post-injection, the spleen was dissected, and total RNA was extracted. Five LPS-treated replicates and five controls were used for building RNA libraries and sequencing. The differential gene expression analysis were performed between fish from different embryonic incubation temperatures (24 °C vs 28 °C; 32 °C vs 28 °C), and between LPS treatment and control within each temperature group (24 °C LPS vs control; 28 °C LPS vs control; 32 °C LPS vs control). Totally, 251 differentially expressed genes (DEGs, 71 up-/180 down-regulated) were identified in fish from 24 °C embryonic incubation temperature compared to fish kept at constant 28 °C (DESeq2, adjusted p-value < 0.05, |fold change| > 1.5); and 660 DEGs (385 up-/275 down-regulated) were identified in fish from 32 °C embryonic incubation temperature compared to fish kept at 28 °C. By comparing LPS-treated fish to control, 567 DEGs (271 up-/296 down-regulated) were identified in fish from embryonic incubation temperature of 24 °C, 140 DEGs (80 up-/60 down-regulated) were identified in fish kept at constant 28 °C; and 49 DEGs (11 up-/38 down-regulated) were identified in fish from the 32 °C embryonic incubation temperature group. Overall design: Thirty samples were analyzed, including five LPS-treated replicates and five controls from each of three embryonic incubation temperatures (24 °C, 28 °C, 32 °C).