Embryonic incubation temperature has a long-term effect on the microRNA transcriptome of adult zebrafish

Zebrafish eggs were collected at 28 °C, and split into three temperature groups (24 °C, 28 °C, 32 °C) for incubation. When larvae reaching first feeding, the incubation temperature of larvae from 24 °C and 32 °C was gadually changed to 28 °C, and all fish were kept at 28 °C for later development until adulthood. At 100 d post-fertilization, seven replicates from each group were intraperitoneally (i.p.) injected with 2 μl of 50 mg/ml lipopolysaccharide (LPS) from Pseudomonas aeruginosa 10, while another seven 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 isolated. Five LPS-treated replicates and five controls were used for building small RNA libraries and sequencing. A total of 112 novel miRNAs were identified using miRDeep2. By comparing fish from different embryonic incubation tmeperatures, 32 miRNAs (29 up-/3 down-regulated) were identified differentially expressed (DEmiRs, adjusted p-value < 0.05, |fold change| > 2.0, DESeq2) in fish from embryonic incubation temperature of 32 °C compared to fish kept at constant 28 °C, 3 DEmiRs (2 up-/1 down-regulated) were identified in response to LPS in fish kept at 28 °C. A total of 9,116 target genes were predicted using miRanda (v3.3a), including 8,224 genes targeted by 32 DE miRNAs and 892 genes targeted by 3 DE miRNAs. 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).