Epigenetic regulation by Atlantic salmon miRNAs in disease and osmotic stress

Disease and stress constitute a major challenge for the aquaculture industry. 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). Incomplete smoltification and disease lead to poor fish welfare and large economic losses in Norwegian aquaculture. 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 is to provide new knowledge on A. salmon miRNAs as regulators of immune responses to pathogens as well as stress related to smoltification/SWT. We apply modern functional genomics methods like small-RNA sequencing, RT-qPCR, and in vitro assays to identify which miRNA genes are associated with immune and stress responses. Moritella viscosa and Paramoeba perurans cause winter ulcer disease and amoebic gill disease (AGD), respectively. These are our pathogen model systems, while the stress responsive miRNAs are identified by monitoring which miRNAs that change their expression during smoltification/SWT in selected organs. Our Moritella viscosa and Paramoeba perurans challenge trial results show that several miRNAs respond to bacterial and parasite disease. Many are the same miRNAs as previously identified in our challenges with viral pathogens. Furthermore, our studies in cell assays monitoring the monocyte to macrophage maturation shows that many of these pathogen-associated miRNAs are likely involved in regulation of macrophage maturation. Results from analysis of miRNA expression in head-kidney, liver and gill strongly indicate that post-transcriptional regulation of gene expression by miRNAs is important in smoltification and sea water adaptation. Gene pathway enrichment analysis shows 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 show that miRNAs responding differently to high and low fish oil diets as well as to “functional” feed designed to boost immunity vs traditional feed. This indicates that certain miRNAs are important in lipid metabolism while others are potential biomarkers for predicting whether a diet boost disease resistance or make fish more robust to stress. 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 with high quality sequences of >70.000 mRNAs (GIYK00000000.1). Following the release of this important resource, 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 groups of miRNAs associated with stress and pathogen response allows for selection of putative target genes to be validated. We are now carrying out miRCLIP-methods to validate such predicted targets. Manipulation of miRNA expression in infected cells by use of luciferase assays will further reveal miRNA’s impact on target gene expression. 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 and luciferase assays will provide final novel knowledge needed to understand how miRNA-target gene interaction affects disease and stress response. This may contribute to knowledge-based changes in today’s smoltification management (e.g. select certain miRNAs as biomarkers of SWT-ready smolt). Validation of target genes of the pathogen-responsive miRNAs opens for dedicated searches for beneficial target gene variation affecting disease response. Finally, applying gene-editing methods detailed knowledge on miRNA-target gene interaction 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.