Introduction
Smoltification is a complex developmental process that leads to changes in the fish physiology, morphology and behaviour including remodelling of the gill from an ion-absorbing epithelium to an ion-secreting epithelium
[1-3]
. Optimal smoltification is, therefore, crucial for normal development, growth, and health of farmed Atlantic salmon in seawater phase
. The aim of this study was to explore whether miRNAs are involved in regulation of gene expression during this important developmental transition by characterizing miRNAs differentially expressed during smoltification and the following seawater adaptation (SWA) in gill of Atlantic salmon .
Material and methods
Total RNA from Atlantic salmon gill collected before smoltification (T1), halfway through smoltification (T2), three quarters into smoltification (T3), one day prior to seawater transfer (SWT) (T4), one week after SWT (T5) and one month after SWT (T6) were used to study
expression changes of miRNAs and mRNAs . Small-RNA sequencing and microarray analysis were applied to identify miRNAs and mRNAs that showed significant differences in expression when groups from the different time points were compared.
In silico
prediction of miRNA-target-genes were carried out applying the MicroSalmon GitHub repository
(http://github.com/AndreassenLab/MicroSalmon/)
with differentially expressed miRNAs (DE-miRNAs)
and differentially expressed mRNAs (DE-mRNAs)
as input . Finally,
gene ontology and pathway enrichment analysis of predicted DE-miRNA targets were conducted using PANTHER Overrepresentation Test (http://www.pantherdb.org/ ) to reveal
significantly enriched biological processes and gene pathways associated with smoltification and SWA .
Results
In total, 32
mature miRNAs were differentially expressed, and 18 of these mature miRNAs were characterized as
the biologically important guide miRNAs (gDE-miRNAs) by examining the relative abundance of the 5p and 3p miRNAs originating from same precursors
. Hierarchical clustering analysis of gDE-miRNAs showed two major clusters, one with a decreasing and one with an increasing expression during smoltification and seawater adaptation . The gDE-miRNAs were predicted to target 747 of the genes that showed significant differences in their expression in the microarray analysis. Enrichment analysis of these predicted target genes revealed that they were enriched in several biological processes including processes associated with immune system (e.g., regulation of macrophage derived foam cell differentiation, regulation of dendritic cell antigen processing and presentation, viral entry into host cell and T cell activation),
regulation of cell growth and lipid metabolic process (e.g., tissue regeneration and steroid biosynthetic process) , reactive oxygen species metabolic process and response to stress . Additional pathway enrichment also showed that pathways associated with immune system (e.g., c ytokine signaling in Immune system , neutrophil degranulation and adaptive immune system), extracellular matrix organization, signal transduction and metabolism of lipids, vitamins and cofactors as enriched.
Discussion and conclusion
The finding that some enriched biological processes and pathways
were associated with immune system,
were as expected as gills are the main mucosal surfaces and immune barriers where several immune related cells including the coordinated expression of cytokines are presented
. T he gill mucus cell population has also been reported to increase
in number in response to increased salinity
. Regulation of cell growth and steroid synthesis may play a role in
smoltification-associated osmoregulatory changes as growth hormone and cortisol have been suggested to stimulate salinity tolerance and NKA activity by promoting the formation of the seawater mitochondria-rich cells or chloride cells
[9,10]
. Gene ontology group r esponse to stress was also enriched, which is in agreement with previous study that reported that stress response genes w ere upregulated in the gill of Atlantic salmon in response to smoltification and SW A
. The findings that all transcripts enriched in these biological processes were predicted targets of the gDE-miRNAs, indicate that miRNAs are involved in post-transcriptional regulation of gene expression changes that are crucial for remodelling of the gill during this developmental transition. Furthermore, t his study points out
pairs of gDE-miRNAs and their predicted targets that may be validated and further studied by functional assays to explore their particular role in the smoltification process.
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