Epigenetic regulation by Atlantic salmon miRNAs in disease and osmotic stress

Disease and stress due to incomplete smoltification constitute a major challenge for the aquaculture industry. This leads to poor fish welfare and large economic losses. More knowledge is necessary to improve health and reduce the negative effect of bacterial and parasite diseases as well as stress related to smoltification and sea-water transfer (SWT). miRNAs are gene expression regulators in vertebrates that are pivotal in pathogen response and in adaptation to new environmental conditions. We have previously characterized all known miRNA genes in A. salmon. Our aim in this project was to provide new knowledge on A. salmon miRNAs as regulators of immune responses as well as regulators of smoltification and stress related to SWT. We have applied modern functional genomics methods like small-RNA sequencing, RT-qPCR, and in vitro assays to identify which of the more than 400 miRNA genes in Atlantic salmon that are associated with immune and stress responses. Differential expression of miRNAs and protein coding genes in challenge materials from Moritella viscosa (winter ulcer) and Paramoeba perurans (gill disease, AGD) have revealed immune response associated miRNAs (IR-miRNAs) while stress responsive miRNAs were identified by differential expression analysis of miRNAs in in selected organs during smoltification/SWT. Our results from pathogene challenge trials showed that a small group of miRNAs respond to bacterial and parasite disease. Many of these were previously identified as IR-miRNAs in our challenge studies with viral pathogens (SAV, IPNV). Furthermore, our studies in cell assays monitoring the monocyte to macrophage maturation showed that several of these IR-miRNAs are likely involved in regulation of macrophage maturation. Results from analysis of miRNA expression in head-kidney, liver and gill have strongly indicated that post-transcriptional regulation of gene expression by miRNAs is important in smoltification and sea water adaptation. Aiming to identify the genes regulated by these miRNAs (target genes) and better understand what gene pathways are affected, we have carried out the first full-length sequencing of the salmon transcriptome. Following the release of this important resource (>70.000 high quality, full-length sequenced mRNAs (GIYK00000000.1)), we generated a database (MicroSalmon) with information on any A. salmon transcript that may be the target gene of any of the Atlantic salmon miRNAs (http://github.com/AndreassenLab/MicroSalmon/). Together, the prediction of the target genes of miRNAs associated with stress and pathogen response have allowed for enrichment analysis’ indicating what gene networks which is likely regulated by these miRNAs. Gene pathway enrichment analysis showed that the miRNAs expressed differently during smoltification/SWT may regulate important biological processes like hormone biosynthesis, stress management, immune response and ion transport. Our feeding trials showed that some IR-miRNAs responded differently to high and low fish oil diets as well as to “functional” feed designed to boost immunity. This indicated that certain miRNAs were important in lipid metabolism while others were potential biomarkers for predicting whether a diet boost disease resistance or makes fish more robust to stress. Enrichment analysis of all the IR-miRNAs revealed they were associated with several immune response gene networks commonly activated in response to viral and bacterial pathogens. As the first research group on salmon genomics we have now established miRCLIP-methods for in vivo identification of the target genes in Atlantic salmon. Furthermore, we are also manipulating the expression of IR-miRNAs in infected cells by use of luciferase assays. This will will further reveal miRNA’s impact on the protein translation of the in vivo identified target genes from the miRCLIP methods. In summary; we have disclosed molecular details of disease and stress mechanisms that involve gene regulation by certain pathogen- and smoltification-responsive miRNAs. Functional studies by miRCLIP have revealed the target genes of each of these miRNAs. The luciferase assays will provide knowledge on the effect (magnitude) these miRNAs have on the immune and stress responses by directly measuring the negative effect they have on target gene protein translation. The identification of the target genes of the IR-miRNAs opens for dedicated searches for beneficial target gene variation at their miRNA binding sequences. Certain miRNAs involved in the regulation of smoltification may also be applied as additional biomarkers to identify SWT-ready smolt. Finally, applying gene-editing methods the detailed knowledge on miRNA-target gene interaction revealed in this project may be utilized to efficiently introduce beneficial variation into breeding stocks to help improve fish health and decrease losses.

Disease constitute a major challenge for the aquaculture industry and cause large economic losses. In order to reduce the negative effect of disease and improve fish health, more knowledge on host-pathogen interaction is necessary. Fish health could also be improved by making knowledge based changes in management regimes. In the current management of smoltification and sea water transfer (SWT) the average mortality is relative high indicating poor fish welfare. MiRNAs are epigenetic regulators of gene expression that are pivotal in pathogen response and in adaption to new environmental conditions in vertebrates, but there are few studies in A. salmon. Our aim is to provide new knowledge on gene regulation by A. salmon miRNAs using modern functional genomics methods like small-RNA sequencing, RT-qPCR, and in vitro assays to study miRNA expression and miRNA-target gene interactions. Studies of host-pathogen interactions following challenge with Moritella viscosa and Paramoeba perurans, two pathogens causing large economic losses in Norwegian aquaculture will reveal miRNAs responding to diseases caused by bacteria and parasites. We will also characterize the miRNAs associated with smoltification and SWT. The characterization of pathogen and stress responsive miRNAs along with the genes regulated by miRNAs will point out gene networks involved in disease response and adaption to osmotic stress. Comparing groups that are resistant/susceptible to disease (P.perurans), any miRNAs and gene networks associated with disease resistance may be identified. Finally, the disease responsive miRNAs will be used in feeding trials with different diets to identify diet responsive miRNAs that can be used as biomarkers to predict whether a diet boost disease resistance. In addition to disclose molecular details of disease mechanisms the results may also contribute knowledge that can help make changes in management (smoltification and SWT) that improve fish health and decrease losses.