Epigenetic regulation by Atlantic salmon miRNAs in disease and osmotic stress

Disease constitute a major challenge for the aquaculture industry and cause large economic losses. In order to improve health and reduce the negative effect of bacterial and parasite disease as well as stress related to smoltification and sea-water transfer (SWT), more knowledge is necessary. miRNAs are regulators of gene expression that are pivotal in pathogen response and in adaption to new environmental conditions in vertebrates. 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 sea-water transfer (SWT). We apply modern functional genomics methods like small-RNA sequencing, RT-qPCR, and in vitro assays developed by our group to identify which miRNA genes are associated with immune and stress responses. Moritella viscosa and Paramoeba perurans, that cause large economic losses in Norwegian aquaculture, are our pathogen model systems while the stress responsive miRNAs are identified by monitoring which miRNAs that respond with changes in expression during smoltification and after SWT in selected organs. Our Moritella viscosa challenge trial results shows that several miRNAs respond to bacterial disease. Many are the same miRNAs 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 as well as selectively secreted in extracellular vesicles from monocytes/macrophages. 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 adaption. 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 have 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 in functional assays currently under development in our project. In summary; we have disclosed molecular details of disease and stress mechanisms that involve gene regulation by certain pathogen- and smoltification-responsive miRNAs. The final validation of target genes will provide novel understanding of miRNA-target gene interaction that may to contribute to knowledge based changes in today’s smoltification management e.g. by applying the smoltification-responsive miRNAs as biomarkers of SWT-ready smolt. Detailed knowledge about the pathogen-responsive miRNAs and their target genes may also be applied for selection of beneficial variation in breeding stocks (marker-assisted breeding). Finally, the pathogen-responsive miRNAs may, in the future, also be applied as novel therapeutic means by manipulation of their expression/interaction by e.g. gene-editing methods 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.

Publications from Cristin

Expression analysis of Moritella viscosa-challenged Atlantic salmon identifies disease-responding Genes, MicroRNAs and their predicted target genes and pathways

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

Characterization of miRNAs in Embryonic, Larval, and Adult Lumpfish Provides a Reference miRNAome for Cyclopterus lumpus

Differential Expression of miRNAs and Their Predicted Target Genes Indicates That Gene Expression in Atlantic Salmon Gill Is Post-Transcriptionally Regulated by miRNAs in the Parr-Smolt Transformation and Adaptation to Sea Water

Modulation of hepatic miRNA expression in Atlantic salmon (Salmo salar) by family background and dietary fatty acid composition

Transcriptome Profiling of Atlantic Salmon Adherent Head Kidney Leukocytes Reveals That Macrophages Are Selectively Enriched During Culture

MicroSalmon: A Comprehensive, Searchable Resource of Predicted MicroRNA Targets and 3’UTR Cis-Regulatory Elements in the Full-Length Sequenced Atlantic Salmon Transcriptome

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The tiny secret to healthier fish

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A transcriptome generated from hybrid-corrected PacBio long-reads in Atlantic Salmon, which provides 71000 high-quality full-length sequenced mRNAs, and identifies thousands of novel splice variants

Differential Expression of miRNAs and Their Predicted Target Genes Indicates That Gene expression in Atlantic Salmon Gill is Post-Transcriptionally Regulated by miRNAs in the Smoltification and Adaptation to Seawater

IDENTIFICATION OF NOVEL AND CONSERVED miRNAS IN PIKEPERCH (SANDER LUCIOPERCA)

microRNAs, ikke-kodende, regulatoriske RNA molekyler assosiert med infekssjonsykdom, og smoltifisering og tilpasning til sjøvann

Differential Expression of miRNAs and Their Predicted Target Genes Indicates That Gene Expression in Atlantic Salmon Gill Is Post‐Transcriptionally Regulated by miRNAs in the Parr‐Smolt Transformation and Adaptation to Sea Water

A de novo full-length mRNA transcriptome for Lepeophtheirus salmonis

A de novo full-length mRNA transcriptome generated from hybrid-corrected PacBio long-reads improves the transcript annotation and identifies thousands of novel splice variants in Atlantic Salmon

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MicroSalmon: A Comprehensive, Searchable Resource of Predicted MicroRNA Targets and 3’UTR Cis‐Regulatory Elements in the Full‐Length Sequenced Atlantic Salmon Transcriptome

Atlantic salmon Full-Length mRNA Transcriptome Generated From Hybrid-Corrected PacBio Long-Reads

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