Expression Analysis in Atlantic Salmon Liver Reveals miRNAs Associated with Smoltification and Seawater Adaptation

Abstract

Optimal smoltification is crucial for normal development, growth, and health of farmed Atlantic salmon in seawater. Here, we characterize miRNA expression in liver to reveal whether miRNAs regulate gene expression during this developmental transition. Expression changes of miRNAs and mRNAs was studied by small-RNA sequencing and microarray analysis, respectively. This revealed 62 differentially expressed guide miRNAs (gDE-miRNAs) that could be divided into three groups with characteristic dynamic expression patterns. Three of miRNA families are known as highly expressed in liver. A rare arm shift was observed during smoltification in the Atlantic salmon-specific novel-ssa-miR-16. The gDE-miRNAs were predicted to target 2804 of the genes revealing expression changes in the microarray analysis. Enrichment analysis revealed that targets were significantly enriched in smoltification-associated biological process groups. These included lipid and cholesterol synthesis, carbohydrate metabolism, protein metabolism and protein transport, immune system genes, circadian rhythm and stress response. The results indicate that gDE-miRNAs may regulate many of the changes associated with this developmental transition in liver. The results pave the way for validation of the predicted target genes and further study of gDE-miRNA and their targets by functional assays.

Keywords: Atlantic salmon; liver; microRNAs; microarray transcriptome; seawater adaptation; small-RNA sequencing; smoltification.

Figure 1

Heatmap and hierarchical clustering of the 62 guide DE-miRNAs. Each row represents a miRNA, and each column represents the expression changes at each time points relative to T1 (pre-smolt, one day before smoltification). T2–T4 and T5–T6 are relative expression changes during smoltification period and post-SWT period, respectively. The dendrogram and the row side colors on the left show the three major clusters of DE-miRNAs (Cluster 1-orange, Cluster 2-red, Cluster 3-green). The direction of expression changes in terms of log2 fold-change is illustrated by the color key above the heatmap. The annotation (1) indicates liver-specific miRNAs and (2) indicates some miRNAs with large changes from T1 to T4 (smoltified fish).

Figure 2

Significantly overrepresented biological processes in the predicted target genes of gDE-miRNAs dataset. The specific and representative subclasses of biological processes are shown here while the complete results from analysis of enriched biological processes are given in Table S8. The dot size indicates the number of DE-miRNA target genes associated with the process and the dot color indicates the significance of the enrichment (−log10 (FDR-corrected P-values)). The vertical grey dashed line represents a fold enrichment of 1.

Figure 3

Enriched gene pathways associated with predicted target genes. The specific and representative subclasses of pathways are shown on the y-axis while the complete results from the analysis of enriched Reactome pathways are given in Table S9. The enriched pathways were grouped into categories and each category were assigned a number where each number represents the following categories, 1. Cell cycle, 2. Cellular responses to stress, 3. Gene expression (Transcription), 4. Immune system, 5. Metabolism of lipids, 6. Metabolism of proteins, 7. Metabolism of RNA, 8. Signal Transduction, 9. Transport of small molecules, 10. Vesicle-mediated transport, 11. Biological oxidations, 12. Programmed cell death, 13. Metabolism of vitamins and cofactors, 14. Metabolism of nucleotides, 15. Metabolism of amino acids and derivatives, 16. Metabolism of carbohydrates, 17. Hemostasis, 18. Chromatin organization). The dot size indicates the number of DE-miRNA target genes associated with the pathway and the dot color indicates the significance of the enrichment (−log10 (FDR-corrected p-values). The vertical grey dashed line represents a fold enrichment of 1.