Jacob Torgersen

Epithelial Cadherin Determines Resistance to Infectious Pancreatic Necrosis Virus in Atlantic Salmon

Infectious pancreatic necrosis virus (IPNV) is the cause of one of the most prevalent diseases in farmed Atlantic salmon (Salmo salar). A quantitative trait locus (QTL) has been found to be responsible for most of the genetic variation in resistance to the virus. Here we describe how a linkage disequilibrium-based test for deducing the QTL allele was developed, and how it was used to produce IPN-resistant salmon, leading to a 75% decrease in the number of IPN outbreaks in the salmon farming industry. Furthermore, we describe how whole-genome sequencing of individuals with deduced QTL genotypes was used to map the QTL down to a region containing an epithelial cadherin (cdh1) gene. In a coimmunoprecipitation assay, the Cdh1 protein was found to bind to IPNV virions, strongly indicating that the protein is part of the machinery used by the virus for internalization. Immunofluorescence revealed that the virus colocalizes with IPNV in the endosomes of homozygous susceptible individuals but not in the endosomes of homozygous resistant individuals. A putative causal single nucleotide polymorphism was found within the full-length cdh1 gene, in phase with the QTL in all observed haplotypes except one; the absence of a single, all-explaining DNA polymorphism indicates that an additional causative polymorphism may contribute to the observed QTL genotype patterns. Cdh1 has earlier been shown to be necessary for the internalization of certain bacteria and fungi, but this is the first time the protein is implicated in internalization of a virus.

In silico and in situ characterization of the zebrafish (Danio rerio) gnrh3 (sGnRH) gene

BackgroundGonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio).ResultsWe have characterized a zebrafish [Trp7, Leu8] or salmon (s) GnRH variant, gnrh 3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish.ConclusionsThe characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.

Genomic survey of early responses to viruses in Atlantic salmon, Salmo salar L.

Viral diseases are one of the main problems and risk factors in aquaculture. At present diseases are diagnosed by detection of pathogens and clinical symptoms. Identification of genes involved in early responses to viruses is important for better knowledge of antiviral defence and development of diagnostic tools. The aim of this study was to search for gene markers common for viral infections in Atlantic salmon based on microarray analyses of a wide range of samples. Gene expression profiles from fish and cell cultures infected with different viruses and treated with the synthetic double-stranded RNA poly(I:C) were compared in order to identify virus responsive genes (VRG). The list of VRG defined in this study contained 117 genes with known or unidentified functions. Several genes, including the most highly ranked one (receptor transporting protein), had not been previously reported to be involved in antiviral defence. VRG were characterized by a rapid induction and low tissue specificity, and their expression levels were related to the viral load. Immunofluorescence analyses of proteins encoded by VRG in cardiac tissue of salmon with the viral disease cardiomyopathy syndrome (CMS) revealed a common expression pattern. In head kidney leukocytes VRG showed comparable or equal responses to CpG and poly(I:C), which mimic respectively bacterial DNA and viral RNA. Most VRG showed highly correlated expression with interferon-a (IFNa). Sequence comparison of salmon VRG with those from other species gave an understanding of the evolution of these genes, which showed a remarkably rapid sequence divergence in comparison with the entire proteome. VRG emerged both before and after separation of teleosts and tetrapods, and among genes found exclusively in fish species there were members of several multigene families: tripartite motif proteins, gig1- and gig2-like proteins. Several VRG, including genes with unknown functions and orthologs to mammalian RNA helicase RIG-I and chemokine C-X-C type 10, were present in cyprinid and salmonid fish but not in the phylogenetically advanced orders, suggesting that they have been lost in the evolution of Teleostei. Apparently, a number of genes involved in antiviral responses in salmon have acquired different functional roles in higher vertebrates.

Morphological and molecular characterization of developing vertebral fusions using a teleost model

BackgroundSpinal disorders are a major cause of disability for humans and an important health problem for intensively farmed animals. Experiments have shown that vertebral deformities present a complex but comparable etiology across species. However, the underlying molecular mechanisms involved in bone deformities are still far from understood. To further explicate the mechanisms involved, we have examined the fundamental aspects of bone metabolism and pathogenesis of vertebral fusions in Atlantic salmon (Salmo salar).ResultsExperimentally, juvenile salmon were subjected to hyperthermic conditions where more than 28% developed fused vertebral bodies. To characterize the fusion process we analyzed an intermediate and a terminal stage of the pathology by using x-ray, histology, immunohistochemistry, real-time quantitative PCR and in situ hybridization. At early stage in the fusion process, disorganized and proliferating osteoblasts were prominent at the growth zones of the vertebral body endplates. PCNA positive cells further extended along the rims of fusing vertebral bodies. During the developing pathology, the marked border between the osteoblast growth zones and the chondrocytic areas connected to the arches became less distinct, as proliferating cells and chondrocytes blended through an intermediate zone. This cell proliferation appeared to be closely linked to fusion of opposing arch centra. During the fusion process a metaplastic shift appeared in the arch centra where cells in the intermediate zone between osteoblasts and chondrocytes co-expressed mixed signals of chondrogenic and osteogenic markers. A similar shift also occurred in the notochord where proliferating chordoblasts changed transcription profile from chondrogenic to also include osteogenic marker genes. In progressed fusions, arch centra and intervertebral space mineralized.ConclusionLoss of cell integrity through cell proliferation and metaplastic shifts seem to be key events in the fusion process. The fusion process involves molecular regulation and cellular changes similar to those found in mammalian deformities, indicating that salmon is suitable for studying general bone development and to be a comparative model for spinal deformities.

Metabolism, health and fillet nutritional quality in Atlantic salmon (Salmo salar) fed diets containing n-3-rich microalgae.

Microalgae, as primary producers of EPA and DHA, are among the most prominent alternative sources to fish oil for n-3 long-chain PUFA in animal and human nutrition. The present study aimed to assess technical, nutritional and fish health aspects of producing n-3-rich Atlantic salmon (Salmo salar) fish fillets by dietary supplementation of increasing levels of a DHA-producing Schizochytrium sp. and reduced or without use of supplemental fish oil. Atlantic salmon smolt were fed diets with graded levels of microalgae for 12 weeks, during which all fish showed high feed intake rates with postprandial plasma leptin levels inversely correlating with final mean fish body weights. Fish performance was optimal in all experimental treatments (thermal growth coefficient about 4·0 and feed conversion ratio 0·8–0·9), protein digestibility was equal in all diets, whereas dietary lipid digestibility inversely correlated with the dietary levels of the SFA 16 : 0. Fillet quality was good and similar to the control in all treatments in terms of n-3 long-chain PUFA content, gaping, texture and liquid losses during thawing. Histological fluorescence staining and immunofluorescence analysis of salmon intestines (midgut: base of intestine and villi) revealed significant effects on slime, goblet cell production and inducible nitric oxide synthase (iNOS) activity with increasing levels of dietary Schizochytrium sp. supplementation. Microarray analysis did not reveal any signs of toxicity, stress, inflammation or any other negative effects from Schizochytrium sp. supplementation in diets for Atlantic salmon.

In vivo and in vitro insulin and fasting control of the transmembrane fatty acid transport proteins in Atlantic salmon (Salmo salar)

We have examined the nutritional and insulin regulation of the mRNA expression of transmembrane fatty acid (FA) transporters [FA transport protein-1 (FATP1) and CD36] together with the lipoprotein lipase (LPL), the cytosolic FA carrier FA binding protein (FABP3), and mitochondrial FA-CoA and -carnitine palmitoyl transferase carriers (CPT)1 and -2 in Atlantic salmon tissues and myocyte cell culture. Two weeks of fasting diminished FATP1, CD36, and LPL in adipose tissue, suggesting a reduction in FA uptake, while FABP3 increased in liver, probably enhancing the transport of FA to the mitochondria. Insulin injection decreased FATP1 and CD36 in white and red muscles, while both transporters were upregulated in the adipose tissue in agreement with the role of insulin-inhibiting muscle FA oxidation and stimulating adipose fat stores. Serum deprivation of 48 h in Atlantic salmon myotubes increased FATP1, FABP3, and CPT-2, while CPT-1 was diminished. In myotubes, insulin induced FATP1 expression but decreased CD36, FABP3, and LPL, suggesting that FATP1 could be more involved in the insulin-stimulated FA uptake. Insulin increased the FA uptake in myotubes mediated, at least in part, through the relocation of FATP1 protein to the plasma membrane. Overall, Atlantic salmon FA transporters are regulated by fasting and insulin on in vivo and in vitro models.

Soft texture of atlantic salmon fillets is associated with glycogen accumulation.

Atlantic salmon (Salmo salar L.) with soft fillets are not suited for manufacturing high quality products. Therefore fillets with insufficient firmness are downgraded, leading to severe economic losses to the farming and processing industries. In the current study, morphological characteristics of salmon fillets ranging from soft to hard were analysed. Different microscopic techniques were applied, including novel methods in this field of research: morphometric image analysis, periodic acid Schiff staining, immunofluorescence microscopy, transmission electron microscopy and fourier transform infrared microscopy. The results showed that the myocytes of soft muscle had detached cells with mitochondrial dysfunctions, large glycogen aggregates and enlarged inter cellular areas, void of extracellular matrix proteins, including lower amounts of sulfated glycoproteins. Myofibre-myofibre detachment and disappearance of the endomysium in soft muscles coincided with deterioration of important connective tissue constituents such as Collagen type I (Col I), Perlecan and Aggrecan. In summary our investigations show for the first time an association between soft flesh of Atlantic salmon and massive intracellular glycogen accumulation coinciding with degenerated mitochondria, myocyte detachment and altered extracellular matrix protein distribution. The results are important for further understanding the etiology of soft salmon.

Early Embryonic Gene Expression Profiling of Zebrafish Prion Protein (Prp2) Morphants

Background The Prion protein (PRNP/Prp) plays a crucial role in transmissible spongiform encephalopathies (TSEs) like Creutzfeldt-Jakob disease (CJD), scrapie and mad cow disease. Notwithstanding the importance in human and animal disease, fundamental aspects of PRNP/Prp function and transmission remains unaccounted for. Methodology/Principal Findings The zebrafish (Danio rerio) genome contains three Prp encoding genes assigned prp1, prp2 and prp3. Currently, the second paralogue is believed to be the most similar to the mammalian PRNP gene in structure and function. Functional studies of the PRNP gene ortholog was addressed by prp2 morpholino (MO) knockdown experiments. Investigation of Prp2 depleted embryos revealed high mortality and apoptosis at 24 hours post fertilization (hpf) as well as impaired brain and neuronal development. In order to elucidate the underlying mechanisms, a genome-wide transcriptome analysis was carried out in viable 24 hpf morphants. The resulting changes in gene expression profiles revealed 249 differently expressed genes linked to biological processes like cell death, neurogenesis and embryonic development. Conclusions/Significance The current study contributes to the understanding of basic Prp functions and demonstrates that the zebrafish is an excellent model to address the role of Prp in vertebrates. The gene knockdown of prp2 indicates an essential biological function for the zebrafish ortholog with a morphant phenotype that suggests a neurodegenerative action and gene expression effects which are apoptosis related and effects gene networks controlling neurogenesis and embryo development.

Localization of mRNAs and Proteins in Methyl Methacrylate–embedded Tissues

Precise localization of proteins and mRNA in histological sections is necessary for evaluating spatial gene expression patterns. Here we report sensitive detection of the gene products in fish tissues by immunohistochemistry (IHC) and in situ hybridization (ISH) assays on sections of whole specimens and vertebra embedded in methyl methacrylate (MMA) resin. This plastic resin favors easy preparation of various specimen types and enables preparation of large sections with well-preserved cell morphology. IHC analysis of the muscle regulatory factor MyoD in transverse sections of juvenile cod revealed MyoD-positive cells in the dorsolateral parts of the adaxial muscle. ISH revealed less spatially restricted signals of the bone morphogenic protein bmp4 in muscle and brain. To assess the applicability of ISH on sections of bony tissue, col1a1 and col2a1 expression was investigated in non-decalcified vertebra sections of Atlantic salmon. The former was identified in both chondrocytes and osteoblasts, whereas the latter was mostly evident in chondrocytes. We conclude that MMA resin offers easy preparation of large and problematic tissues and the possibility of carrying out both IHC and ISH analyses using standard protocols.

Exercise induced mechano-sensing and Substance P mediated bone modeling in Atlantic salmon

Mechanical stress plays a vital role in maintaining bone architecture. The process by which osteogenic cells convert the mechanical signal into a biochemical response governing bone modeling is not clear, however. In this study, we investigated how Atlantic salmon (Salmo salar) vertebra responds to exercise-induced mechanical loading. Bone formation in the vertebrae was favored through increased expression of genes involved in osteoid production. Fourier transform infrared spectroscopy (FT-IR) showed that bone matrix secreted both before and during sustained swimming had different properties after increased load compared to control, suggesting that both new and old bones are affected. Concomitantly, both osteoblasts and osteocytes in exercised salmon showed increased expression of the receptor nk-1 and its ligand substance P (SP), both known to be involved in osteogenesis. Moreover, in situ hybridization disclosed SP mRNA in osteoblasts and osteocytes, supporting an autocrine function. The functional role of SP was investigated in vitro using osteoblasts depleted for SP. The cells showed severely reduced transcription of genes involved in mineralization, demonstrating a regulatory role for SP in salmon osteoblasts. Investigation of α-tubulin stained osteocytes revealed cilia-like structures. Together with SP, cilia may link mechanical responses to osteogenic processes in the absence of a canaliculi network. Our results imply that salmon vertebral bone responds to mechanical load through a highly interconnected and complex signal and detection system, with SP as a key factor for initializing mechanically-induced bone formation in bone lacking the canaliculi system.