Mucosal and systemic physiological changes underscore the welfare risks of environmental hydrogen sulphide in post-smolt Atlantic salmon (Salmo salar).

Atlantic salmon (Salmo salar) might encounter toxic hydrogen sulphide (H2S) gas during aquaculture production. Exposure to this gas can be acute or chronic, with heightened levels often linked to significant mortality rates. Despite its recognised toxicity, our understanding of the physiological implications of H2S on salmon remains limited. This report details the mucosal and systemic physiological consequences in post-smolt salmon reared in brackish water at 12 ppt after prolonged exposure to elevated H2S levels over 4 weeks. The fish were subjected to two concentrations of H2S: 1 µg/L (low group) and 5 µg/L (high group). An unexposed group at 0 µg/L served as the control. Both groups exposed to H2S exhibited incremental mortality, with cumulative mortality rates of 4.7 % and 16 % for the low and high groups, respectively. Production performance, including weight and condition factors, were reduced in the H2S-exposed groups, particularly in the high group. Mucosal response of the olfactory organ revealed higher tissue damage scores in the H2S-exposed groups, albeit only at week 4. The high group displayed pronounced features such as increased mucus cell density and oedema-like vacuoles. Transcriptome analysis of the olfactory organ unveiled that the effects of H2S were more prominent at week 4, with the high group experiencing a greater magnitude of change than the low group. Genes associated with the extracellular matrix were predominantly downregulated, while the upregulated genes primarily pertained to immune response. H2S-induced alterations in the metabolome were more substantial in plasma than skin mucus. Furthermore, the number of differentially affected circulating metabolites was higher in the low group compared to the high group. Five core pathways were significantly impacted by H2S regardless of concentration, including the phenylalanine, tyrosine, and tryptophan biosynthesis. The plasma levels of phenylalanine and tyrosine were reduced following exposure to H2S. While there was a discernible distinction in the skin mucus metabolomes among the three treatment groups, only one metabolite - 4-hydroxyproline - was significantly impacted by H2S. Furthermore, this metabolite was significantly reduced in the plasma and skin mucus of H2S-exposed fish. This study underscores that prolonged exposure to H2S, even at concentrations previously deemed sub-lethal, has discernible physiological implications that manifest across various organisational levels. Given these findings, prolonged exposure to H2S poses a welfare risk, and thus, its presence must be maintained at low levels (<1 µg/L) in salmon land-based rearing systems.

Fast and slow releasing sulphide donors engender distinct transcriptomic alterations in Atlantic salmon hepatocytes.

Hydrogen sulphide (H2S) is a naturally occurring compound generated either endogenously or exogenously and serves both as a gaseous signalling molecule and an environmental toxicant. Though it has been extensively investigated in mammalian systems, the biological function of H2S in teleost fish is poorly identified. Here we demonstrate how exogenous H2S regulates cellular and molecular processes in Atlantic salmon (Salmo salar) using a primary hepatocyte culture as a model. We employed two forms of sulphide donors: the fast-releasing salt form, sodium hydrosulphide (NaHS) and the slow-releasing organic analogue, morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate (GYY4137). Hepatocytes were exposed to either a low (LD, 20 µg/L) or high (HD, 100 µg/L) dose of the sulphide donors for 24 hrs, and the expression of key sulphide detoxification and antioxidant defence genes were quantified by qPCR. The key sulphide detoxification genes sulfite oxidase 1 (soux) and the sulfide: quinone oxidoreductase 1 and 2 (sqor) paralogs in salmon showed pronounced expression in the liver and likewise responsive to the sulphide donors in the hepatocyte culture. These genes were ubiquitously expressed in different organs of salmon as well. HD-GYY4137 upregulated the expression of antioxidant defence genes, particularly glutathione peroxidase, glutathione reductase and catalase, in the hepatocyte culture. To explore the influence of exposure duration, hepatocytes were exposed to the sulphide donors (i.e., LD versus HD) either transient (1h) or prolonged (24h). Prolonged but not transient exposure significantly reduced hepatocyte viability, and the effects were not dependent on concentration or form. The proliferative potential of the hepatocytes was only affected by prolonged NaHS exposure, and the impact was not concentration dependent. Microarray analysis revealed that GYY4137 caused more substantial transcriptomic changes than NaHS. Moreover, transcriptomic alterations were more marked following prolonged exposure. Genes involved in mitochondrial metabolism were downregulated by the sulphide donors, primarily in NaHS-exposed cells. Both sulphide donors influenced the immune functions of hepatocytes: genes involved in lymphocyte-mediated response were affected by NaHS, whereas inflammatory response was targeted by GYY4137. In summary, the two sulphide donors impacted the cellular and molecular processes of teleost hepatocytes, offering new insights into the mechanisms underlying H2S interactions in fish.

Recurrent oxidant treatment induces dysregulation in the brain transcriptome of Atlantic salmon (Salmo salar) smolts.

Peracetic acid (PAA) is an organic peroxide that produces free radicals, which contribute to its potent disinfection power. At therapeutic doses, PAA is considered a mild stressor that can trigger transient local and systemic oxidative stress in fish, but the resulting consequences in the brain have yet to be identified. Therefore, we report the brain transcriptome of Atlantic salmon (Salmo salar) smolts that have been periodically exposed to PAA. Fish were treated three times (every 15 days) with PAA with either short (15 min) or long (30 min) exposure periods. After the third treatment, the whole brain was collected and subjected to biochemical and transcriptomic analyses. The level of reactive oxygen species in the brain was not significantly affected by recurrent PAA treatments. Microarray analysis was performed on the whole brain and revealed 205 differentially expressed genes (DEGs), regardless of the duration of the treatment. The short exposure duration had a more considerable impact on the brain transcriptome, correlating with 70% more DEGs than the long exposure. Strikingly, the brain transcriptome was characterised by the downregulation of gene expression, especially in the short exposure group, and around 82% of the identified DEGs were downregulated. Some of the highly affected genes were key molecules of the vasotocinergic and isotocinergic systems and the corticotropin-releasing factor signalling system, indicating interference of the stress axis but could also suggest an anxiolytic effect. In addition, there were alterations in genes involved in cellular metabolism and processing, signalling and trafficking, and innate immunity, which underscores the physiological changes in the brain following recurrent PAA treatment. Overall, the transcriptomic data reveal that recurrent oxidant treatment could influence brain functions, and although the magnitude was marginal, the alterations suggested neurological adaptations of fish to PAA as a potential chemical stressor. The results identify the risks of PAA, which would be valuable in drafting a framework for its empirically driven use in fish farming.

Regulation of the molecular repertoires of oxidative stress response in the gills and olfactory organ of Atlantic salmon following infection and treatment of the parasite Neoparameoba perurans.

The present study investigated the involvement of key molecular regulators of oxidative stress in amoebic gill disease (AGD), a parasitic infestation in Atlantic salmon. In addition, the study evaluated how these molecular biomarkers responded when AGD-affected fish were exposed to a candidate chemotherapeutic peracetic acid (PAA). Atlantic salmon were experimentally infected with the parasite Neoparameoba perurans, the causative agent of AGD, by bath exposure and after 2 weeks, the fish were treated with three commercial PAA products (i.e., Perfectoxid, AquaDes and ADDIAqua) at a dose of 5 ppm. Two exposure durations were evaluated - 30 min and 60 min. Sampling was performed 24 h and 2 weeks after PAA treatment (equivalent to 2- and 4-weeks post infection). At each sampling point, the following parameters were evaluated: gross gill pathology, gill parasitic load, plasma reactive oxygen species (ROS) and total antioxidant capacity (TAC), histopathology and gene expression profiling of genes with key involvement in oxidative stress in the gills and olfactory organ. AGD did not result in systemic oxidative stress as ROS and TAC levels remained unchanged. There were no clear patterns of AGD-mediated regulation of the oxidative stress biomarkers in both the gills and olfactory organ; significant changes in the expression were mostly related to time rather than infection status. However, the expression profiles of the oxidative stress biomarkers in AGD-affected salmon, following treatment with PAA, revealed that gills and olfactory organ responded differently - upregulation was prominent in the gills while downregulation was more frequent in the olfactory organ. The expression of catalase, glutathione S-transferase and thioredoxin reductase 2 was significantly affected by the treatments, both in the gills and olfactory organ, and these alterations were influenced by the duration of exposure and PAA product type. Parasitic load in the gills did significantly increase after treatment regardless of the product and exposure duration; the parasite was undetectable in some fish treated with AquaDes for 30 mins. However, PAA treated groups for 30 min showed lower macroscopic gill scores than the infected-untreated fish. Histology disclosed the classic pathological findings such as multifocal hyperplasia and increased number of mucous cells in AGD-affected fish. Microscopic scoring of gill injuries showed that AGD-infected-PAA-treated fish had lower scores, however, an overall trend could not be established. The morphology and structural integrity of the olfactory organ were not significantly altered by parasitism or PAA treatment. Collectively, the results indicate that AGD did not affect the systemic and mucosal oxidative status of Atlantic salmon. However, such a striking profile was changed when AGD-affected fish were exposed to oxidative chemotherapeutics. Moreover, the gills and olfactory organ demonstrated distinct patterns of gene expression of oxidative stress biomarkers in AGD-infected-PAA-treated fish. Lastly, PAA treatment did not fully resolve the infection, but appeared not to worsen the mucosal health either.

Mucosal immune and stress responses of Neoparamoeba perurans-infected Atlantic salmon (Salmo salar) treated with peracetic acid shed light on the host-parasite-oxidant interactions.

Treatment development for parasitic infestation is often limited to disease resolution as an endpoint response, and physiological and immunological consequences are not thoroughly considered. Here, we report the impact of exposing Atlantic salmon affected with amoebic gill disease (AGD) to peracetic acid (PAA), an oxidative chemotherapeutic. AGD-affected fish were treated with PAA either by exposing them to 5 ppm for 30 min or 10 ppm for 15 min. Unexposed fish from both infected and uninfected groups were also included. Samples for molecular, biochemical, and histological evaluations were collected at 24 h, 2 weeks, and 4 weeks post-treatment. Behavioral changes were observed during PAA exposure, and post-treatment mortality was higher in the infected and PAA treated groups, especially in 10 ppm for 15 min. Plasma indicators showed that liver health was affected by AGD, though PAA treatment did not exacerbate the infection-related changes. Transcriptome profiling in the gills showed significant changes, triggered by AGD and PAA treatments, and the effects of PAA were more notable 24 h after treatment. Genes related to immune pathways of B- and T- cells and protein synthesis and metabolism were downregulated, where the magnitude was more remarkable in 10 ppm for 15 min group. Even though treatment did not fully resolve the pathologies associated with AGD, 5 ppm for 30 min group showed lower parasite load at 4 weeks post-treatment. Mucous cell parameters (i.e., size and density) increased within 24 h post-treatment and were significantly higher at termination, especially in AGD-affected fish, with some treatment effects influenced by the dose of PAA. Infection and treatments resulted in oxidative stress-in the early phase in the gill mucosa, while systemic reactive oxygen species (ROS) dysregulation was evident at the later stage. Infected fish responded to elevated circulating ROS by increasing antioxidant production. Exposing the fish to a crowding stress revealed the interference in the post-stress responses. Lower cortisol response was displayed by AGD-affected groups. Collectively, the study established that PAA, within the evaluated treatment protocols, could not provide a convincing treatment resolution and, thus, requires further optimization. Nonetheless, PAA treatment altered the mucosal immune and stress responses of AGD-affected Atlantic salmon, shedding light on the host-parasite-treatment interactions. .

Multiomics Provide Insights into the Key Molecules and Pathways Involved in the Physiological Adaptation of Atlantic Salmon (Salmo salar) to Chemotherapeutic-Induced Oxidative Stress.

Although chemotherapeutics are used to treat infections in farmed fish, knowledge on how they alter host physiology is limited. Here, we elucidated the physiological consequences of repeated exposure to the potent oxidative chemotherapeutic peracetic acid (PAA) in Atlantic salmon (Salmo salar) smolts. Fish were exposed to the oxidant for 15 (short exposure) or 30 (long exposure) minutes every 15 days over 45 days. Unexposed fish served as the control. Thereafter, the ability of the remaining fish to handle a secondary stressor was investigated. Periodic chemotherapeutic exposure did not affect production performance, though survival was lower in the PAA-treated groups than in the control. Increased ventilation, erratic swimming, and a loss of balance were common behavioural manifestations during the oxidant exposure. The plasma reactive oxygen species levels increased in the PAA-treated groups, particularly after the third exposure, suggesting an alteration in the systemic oxidative stress status. Plasma indicators for internal organ health were affected to a certain degree, with the changes mainly observed after the second and third exposures. Metabolomics disclosed that the oxidant altered several circulating metabolites. Inosine and guanosine were the two metabolites significantly affected by the oxidative stressor, regardless of exposure time. A microarray analysis revealed that the gills and liver were more responsive to the oxidant than the skin, with the gills being the most sensitive. Moreover, the magnitude of the transcriptomic modifications depended on the exposure duration. A functional analysis showed that genes involved in immunity and ribosomal functions were significantly affected in the gills. In contrast, genes crucial for the oxidation-reduction process were mainly targeted in the liver. Skin mucus proteomics uncovered that the changes in the mucosal proteome were dependent on exposure duration and that the oxidant interfered with ribosome-related processes. Mucosal mapping revealed gill mucous cell hypertrophy after the second and third exposures, although the skin morphological parameters remained unaltered. Lastly, repeated oxidant exposures did not impede the ability of the fish to mount a response to a secondary stressor. This study provides insights into how a chemical oxidative stressor alters salmon physiology at both the systemic and mucosal levels. This knowledge will be pivotal in developing an evidence-driven approach to the use of oxidative therapeutics in fish, with some of the molecules and pathways identified as potential biomarkers and targets for assessing the physiological cost of these treatments.

Transcriptome Responses of Atlantic Salmon (Salmo salar L.) to Viral and Bacterial Pathogens, Inflammation, and Stress.

Transcriptomics provides valuable data for functional annotations of genes, the discovery of biomarkers, and quantitative assessment of responses to challenges. Meta-analysis of Nofima's Atlantic salmon microarray database was performed for the selection of genes that have shown strong and reproducible expression changes. Using data from 127 experiments including 6440 microarrays, four transcription modules (TM) were identified with a total of 902 annotated genes: 161 virus responsive genes - VRG (activated with five viruses and poly I:C), genes that responded to three pathogenic bacteria (523 up and 33 down-regulated genes), inflammation not caused by infections - wounds, melanized foci in skeletal muscle and exposure to PAMP (180 up and 72 down-regulated genes), and stress by exercise, crowding and cortisol implants (33 genes). To assist the selection of gene markers, genes in each TM were ranked according to the scale of expression changes. In terms of functional annotations, association with diseases and stress was unknown or not reflected in public databases for a large part of genes, including several genes with the highest ranks. A set of multifunctional genes was discovered. Cholesterol 25-hydroxylase was present in all TM and 22 genes, including most differentially expressed matrix metalloproteinases 9 and 13 were assigned to three TMs. The meta-analysis has improved understanding of the defense strategies in Atlantic salmon. VRG have demonstrated equal or similar responses to RNA (SAV, IPNV, PRV, and ISAV), and DNA (gill pox) viruses, injection of bacterial DNA (plasmid) and exposure of cells to PAMP (CpG and gardiquimod) and relatively low sensitivity to inflammation and bacteria. Genes of the highest rank show preferential expression in erythrocytes. This group includes multigene families (gig and several trim families) and many paralogs. Of pathogen recognition receptors, only RNA helicases have shown strong expression changes. Most VRG (82%) are effectors with a preponderance of ubiquitin-related genes, GTPases, and genes of nucleotide metabolism. Many VRG have unknown roles. The identification of TMs makes possible quantification of responses and assessment of their interactions. Based on this, we are able to separate pathogen-specific responses from general inflammation and stress.

Responses to Mineral Supplementation and Salmon Lice (Lepeophtheirus salmonis) Infestation in Skin Layers of Atlantic Salmon (Salmo salar L.).

The crustacean ectoparasite salmon louse (Lepeophtheirus salmonis), which severely affects Atlantic salmon health and welfare is one of the main problems of commercial aquaculture. In the present study, fish were fed a diet supplemented with extra minerals through the inclusion of a commercial additive (Biofeed Forte Salmon), substituting wheat in the control diet, before experimental infestation with salmon lice. Lice counts reduced with time but with no apparent effect of the diets. Further, fish fed the mineral diet had an overall higher number of blue (acidic) mucous cells, while the ratio of purple mucous cells was higher in the mineral diet. The transcriptional response in skin was enhanced at 7 dpc (copepodite life stage) in fish fed the mineral diet including immune and stress responses, while at 21 dpc (pre-adult life stage), the difference disappeared, or reversed with stronger induction in the control diet. Overall, 9.3% of the genes affected with lice also responded to the feed, with marked differences in outer (scale + epidermis) and inner (dermis) skin layers. A comparison of transcriptome data with five datasets from previous trials revealed common features and gene markers of responses to lice, stress, and mechanically induced wounds. Results suggested a prevalence of generic responses in wounded skin and lice-infected salmon.

Piscine Orthoreovirus-1 Isolates Differ in Their Ability to Induce Heart and Skeletal Muscle Inflammation in Atlantic Salmon (Salmo salar).

Piscine orthoreovirus 1 (PRV-1) is the causative agent of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). The virus is widespread in Atlantic salmon and was present in Norway long before the first description of HSMI in 1999. Furthermore, in Canada the virus is prevalent in farmed Atlantic salmon but HSMI is not and Canadian isolates have failed to reproduce HSMI experimentally. This has led to the hypothesis that there are virulence differences between PRV-1 isolates. In this study we performed a dose standardized challenge trial, comparing six PRV-1 isolates, including two Norwegian field isolates from 2018, three historical Norwegian isolates predating the first report of HSMI and one Canadian isolate. The Norwegian 2018 isolates induced lower viral protein load in blood cells but higher plasma viremia. Following peak replication in blood, the two Norwegian 2018 isolates induced histopathological lesions in the heart consistent with HSMI, whereas all three historical Norwegian and the Canadian isolates induced only mild cardiac lesions. This is the first demonstration of virulence differences between PRV-1 isolates and the phenotypic differences are linked to viral proteins encoded by segment S1, M2, L1, L2 and S4.

The Atlantic Salmon Gill Transcriptome Response in a Natural Outbreak of Salmon Gill Pox Virus Infection Reveals New Biomarkers of Gill Pathology and Suppression of Mucosal Defense.

The salmon gill poxvirus (SGPV) is a large DNA virus that infects gill epithelial cells in Atlantic salmon and is associated with acute high mortality disease outbreaks in aquaculture. The pathological effects of SGPV infection include gill epithelial apoptosis in the acute phase of the disease and hyperplasia of gill epithelial cells in surviving fish, causing damage to the gill respiratory surface. In this study, we sampled gills from Atlantic salmon presmolts during a natural outbreak of SGPV disease (SGPVD). Samples covered the early phase of infection, the acute mortality phase, the resolving phase of the disease and control fish from the same group and facility. Mortality, the presence and level of SGPV and gill epithelial apoptosis were clearly associated. The gene expression pattern in the acute phase of SGPVD was in tune with the pathological findings and revealed novel transcript-based disease biomarkers, including pro-apoptotic and proliferative genes, along with changes in expression of ion channels and mucins. The innate antiviral response was strongly upregulated in infected gills and chemokine expression was altered. The regenerating phase did not reveal adaptive immune activity within the study period, but several immune effector genes involved in mucosal protection were downregulated into the late phase, indicating that SGPV infection could compromise mucosal defense. These data provide novel insight into the infection mechanisms and host interaction of SGPV.

Oxidant-induced modifications in the mucosal transcriptome and circulating metabolome of Atlantic salmon.

Here we report the molecular networks associated with the mucosal and systemic responses to peracetic acid (PAA), a candidate oxidative chemotherapeutic in Atlantic salmon (Salmo salar). Smolts were exposed to different therapeutic doses (0, 0.6 and 2.4 mg/L) of PAA for 5 min, followed by a re-exposure to the same concentrations for 30 min 2 weeks later. PAA-exposed groups have higher external welfare score alterations, especially 2 weeks after the re-exposure. Cases of fin damage and scale loss were prevalent in the PAA-exposed groups. Transcriptomic profiling of mucosal tissues revealed that the skin had 12.5 % more differentially regulated genes (DEGs) than the gills following PAA exposure. The largest cluster of DEGs, both in the skin and gills, were involved in tissue extracellular matrix and metabolism. There were 22 DEGs common to both mucosal tissues, which were represented primarily by genes involved in the biophysical integrity of the mucosal barrier, including cadherin, collagen I α 2 chain, mucin-2 and spondin 1a. The absence of significant clustering in the plasma metabolomes amongst the three treatment groups indicates that PAA treatment did not induce any global metabolomic disturbances. Nonetheless, five metabolites with known functions during oxidative stress were remarkably affected by PAA treatments such as citrulline, histidine, tryptophan, methionine and trans-4-hydroxyproline. Collectively, these results indicate that salmon were able to mount mucosal and systemic adaptive responses to therapeutic doses of PAA and that the molecules identified are potential markers for assessing the health and welfare consequences of oxidant exposure.

Wound healing in post-smolt Atlantic salmon (Salmo salar L.).

Skin biopsies (5 mm) taken from behind the dorsal fin on Atlantic salmon post-smolts were followed over a 2 month period. The healing process was dominated by hemostasis, acute inflammation, and epidermal repair the first 14 days post wounding (dpw), as shown through imaging, histological evaluation, and transcriptomics. Most of the immune genes showed decreased expression after two weeks, approaching the levels of intact skin, as also reflected in sections where reduced inflammation in the wound bed was observed. Transcriptional events suggest recruitment of lymphocytes to the wound site during the acute phase, with activation of humoral responses from 14 dpw and onward. From the histology, a more adherent mucus was observed that correlated with altered transcription of glycosyltransferases. This may indicate different properties and functions of the mucus during the wound healing process. Wound contraction started between 14 and 36 dpw. The occurrence of these events was concurrent with granulation tissue formation, melanocyte migration and up-regulation of genes involved in extracellular matrix formation. The presented description of the wound healing processes in Atlantic salmon gives insight into comparative ulcerative biology in mammals and fish and provides both novel and updated knowledge that can be applied for improved best operational practices for fish welfare in aquaculture.

High fish density delays wound healing in Atlantic salmon (Salmo salar).

In this study, we look closer at how high fish densities influence wound repair mechanisms in post-smolt Atlantic salmon. The fish were wounded with a 5 mm skin punch biopsy needle and stocked at two different densities, a high fish density (100 kg/m3) treatment and a low fish density treatment (20 kg/m3) serving as the control. The healing wounds were followed for 57 days with samples taken 1, 3, 7, 14, 36, 43 and 57 days post wounding. The transcriptomic analysis suggests that high fish density enhance inflammation and represses cell proliferation, tissue secretion and collagen synthesis in the healing wounds. The histological analysis further showed delayed epidermal and dermal repair in the high fish density treatment compared to control. The overall wound contraction was also altered by the treatment. In conclusion, high fish density enhances immune responses and delay tissue repair, which ultimately results in delayed wound healing.

Atlantic salmon skin barrier functions gradually enhance after seawater transfer.

Atlantic salmon farming operates with high production intensities where skin integrity is recognized as a central factor and indicator for animal health and welfare. In the described trial, the skin development and its immune status in healthy Atlantic salmon reared in two different systems, a traditional open net-pen system and a semi-closed containment system, were investigated. Freshwater smolts were compared to post-smolts after 1 and 4 months in seawater. Growth performance, when adjusted for temperature, was equal between the systems. Skin analyses, including epidermis and dermis, showed that thickness and mucus cell numbers increased in pace with the growth and time post seawater transfer (PST). Gene expression changes suggested similar processes with development of connective tissue, formation of extracellular matrix and augmented cutaneous secretion, changes in mucus protein composition and overall increased immune activity related to gradually enforced protection against pathogens. Results suggest a gradual morphological development in skin with a delayed recovery of immune functions PST. It is possible that Atlantic salmon could experience increased susceptibility to infectious agents and risk of diseases during the first post-smolt period.

Antiviral Responses and Biological Concequences of Piscine orthoreovirus Infection in Salmonid Erythrocytes.

Salmonid red blood cells are the main target cells for Piscine orthoreovirus (PRV). Three genotypes of PRV (PRV-1,2,3) infect Atlantic salmon (Salmo salar), Chinook salmon (Onchorhynchus tshawytscha), Coho salmon (Oncorhynchus kisutch), rainbow trout (Onchorhynchus mykiss) and brown trout (Salmo trutta), and can cause diseases like heart and skeletal muscle inflammation (HSMI), jaundice syndrome, erythrocyte inclusion body syndrome (EIBS) and proliferative darkening syndrome (PDS). Purified PRV administrated to fish has proven the causality for HSMI and EIBS. During the early peak phase of infection, salmonid erythrocytes are the main virus-replicating cells. In this initial phase, cytoplasmic inclusions called "virus factories" can be observed in the erythrocytes, and are the primary sites for the formation of new virus particles. The PRV-infected erythrocytes in Atlantic salmon mount a strong long-lasting innate antiviral response lasting for many weeks after the onset of infection. The antiviral response of Atlantic salmon erythrocytes involves upregulation of potential inhibitors of translation. In accordance with this, PRV-1 protein production in erythrocytes halts while virus RNA can persist for months. Furthermore, PRV infection in Coho salmon and rainbow trout are associated with anemia, and in Atlantic salmon lower hemoglobin levels are observed. Here we summarize and discuss the recently published findings on PRV infection, replication and effects on salmonid erythrocytes, and discuss how PRV can be a useful tool for the study of innate immune responses in erythrocytes, and help reveal novel immune functions of the red blood cells in fish.

Training the salmon's genes: influence of aerobic exercise, swimming performance and selection on gene expression in Atlantic salmon.

BACKGROUND: Farmed and wild Atlantic salmon are exposed to many infectious and non-infectious challenges that can cause mortality when they enter the sea. Exercise before transfer promotes growth, health and survival in the sea. Swimming performance in juveniles at the freshwater parr stage is positively associated with resistance to some diseases. Genetic variation is likely to affect response to exercise. In this study we map genetic differences associated with aerobic exercise, swimming performance and genetic origin. Eggs from the selectively bred Bolaks salmon and wild Lærdal River salmon strains were reared until parr in a common environment. Swimming performance was assessed by subjecting the fish to either continuous hard exercise or control conditions for 18 days. Heart was sampled for examination of gene expression using RNA-seq (~60 fish/treatment). RESULTS: Lower expression of genes affecting immune function was found in domesticated than wild parr. Among wild parr under control exercise the expression of a large number of genes involved in general metabolism, stress and immune response was lower in superior swimmers suggesting that minimisation of energy expenditure during periods of low activity makes parr better able to sustain bursts of swimming for predator avoidance. A similar set of genes were down-regulated with training among wild parr with inferior swimming performance. These parr react to training in a way that their cardiac expression patterns become like the superior performing wild parr under control exercise conditions. Diversifying selection caused by breeding of domesticated stock, and adaptive pressures in wild stock, has affected the expression and frequency of single nucleotide polymorphisms (SNPs) for multiple functional groups of genes affecting diverse processes. SNPs associated with swimming performance in wild parr map to genes involved in energetic processes, coding for contractile filaments in the muscle and controlling cell proliferation. CONCLUSIONS: Domesticated parr have less phenotypic plasticity in response to training and lower expression of genes with functions affecting immune response. The genetic response to training is complex and depends on the background of parr and their swimming ability. Exercise should be tailored to the genetics and swimming performance of fish.

Resolving the complexity of vitellogenins and their receptors in the tetraploid Atlantic salmon (Salmo salar): Ancient origin of the phosvitin-less VtgC in chondrichthyean fishes.

Egg yolk proteins are mainly derived from vitellogenin (Vtg), and serve as essential nutrients during early development in oviparous organisms. Vertebrate Vtgs are predominantly synthesized in the liver of maturing females, and are internalized by the oocyte after binding to specific surface receptors (VtgR). Here, we clarify the evolutionary history of vertebrate Vtgs, including the teleost VtgC, which lacks phosvitin, and investigate the repertoire of Vtgs and VtgRs in the tetraploid Atlantic salmon (Salmo salar). Conserved synteny of the vtg genes in elephant fish (Callorhinchus milii) strongly indicates that the vtg gene cluster was present in the ancestor of tetrapods and ray-finned fish. The shortened phosvitin in the VtgC ortholog of this chondrichthyean fish may have resulted from early truncation events that eventually allowed the total disappearance of phosvitin in teleost VtgC. In contrast, the tandem-duplicated VtgCs identified in the spotted gar (Lepisosteus oculatus) both contain the phosvitin domain. The Atlantic salmon genome harbors four vtg genes encoding the complete VtgAsa1, phosvitin-less VtgC, and truncated VtgAsb proteins; vtgAsa2 is a pseudogene. The three vtg genes were mainly expressed in the liver of maturing females, and the vtgAsa1 transcript predominated prior to spawning. The splice variant lacking the O-linked sugar domain dominated ovarian expression of vtgr1 and vtgr2. Strongly increased vtgAsa1 expression during vitellogenesis contrasted with the peaks of vtgr1 and vtgr2 in the previtellogenic oocytes, which gradually decreased over the same period. Recycling of the oocyte VtgRs is probably not sufficient to maintain receptor number during vitellogenesis.

Hypoxia tolerance and responses to hypoxic stress during heart and skeletal muscle inflammation in Atlantic salmon (Salmo salar).

Heart and skeletal muscle inflammation (HSMI) is associated with Piscine orthoreovirus (PRV) infection and is an important disease in Atlantic salmon (Salmo salar) aquaculture. Since PRV infects erythrocytes and farmed salmon frequently experience environmental hypoxia, the current study examined mutual effects of PRV infection and hypoxia on pathogenesis and fish performance. Furthermore, effects of HSMI on hypoxia tolerance, cardiorespiratory performance and blood oxygen transport were studied. A cohabitation trial including PRV-infected post-smolts exposed to periodic hypoxic stress (4 h of 40% O2; PRV-H) at 4, 7 and 10 weeks post-infection (WPI) and infected fish reared under normoxic conditions (PRV) was conducted. Periodic hypoxic stress did not influence infection levels or histopathological changes in the heart. Individual incipient lethal oxygen saturation (ILOS) was examined using a standardized hypoxia challenge test (HCT). At 7 WPI, i.e. peak level of infection, both PRV and PRV-H groups exhibited reduced hypoxia tolerance compared to non-infected fish. Three weeks later (10 WPI), during peak levels of pathological changes, reduced hypoxia tolerance was still observed for the PRV group while PRV-H performed equal to non-infected fish, implying a positive effect of the repeated exposure to hypoxic stress. This was in line with maximum heart rate (fHmax) measurements, showing equal performance of PRV-H and non-infected groups, but lower fHmax above 19°C as well as lower temperature optimum (Topt) for aerobic scope for PRV, suggesting reduced cardiac performance and thermal tolerance. In contrast, the PRV-H group had reduced hemoglobin-oxygen affinity compared to non-infected fish. In conclusion, Atlantic salmon suffering from HSMI have reduced hypoxia tolerance and cardiac performance, which can be improved by preconditioning fish to transient hypoxic stress episodes.

Short-term starvation at low temperature prior to harvest does not impact the health and acute stress response of adult Atlantic salmon.

A period of starvation is regarded as a sound practice in aquaculture prior to handling, transportation and harvest, to minimise impacts on welfare and ensure proper hygiene after harvest. However, documentation of welfare issues such as stress following starvation and handling in adult Atlantic salmon are lacking. This study aimed to examine gut emptying and potential stress during a two week starvation period, and whether this starvation period changed the tolerance for physical stress. The study confirmed slower emptying of the gut segments at low temperature. Plasma and bile cortisol, and selected clinical analyses were used to characterize potential stress, as well as the response to acute physical crowding stress during the starvation period. Neither the general stress level nor the ability to cope with handling stress was affected by a 14 day starvation period. Down-regulation of selected nutritional related gene markers in liver indicated classical starvation responses, with reduced metabolism and oxidative pressure, and sparing of nutrients. The response to acute handling stress was not affected by two weeks of starvation. There were minor effects of starvation on stress and health markers, as evaluated by plasma lysozyme activity and gene expression of selected inflammation marker proteins in heart and skin tissues.

Experimental Piscine orthoreovirus infection mediates protection against pancreas disease in Atlantic salmon (Salmo salar).

Viral diseases are among the main challenges in farming of Atlantic salmon (Salmo salar). The most prevalent viral diseases in Norwegian salmon aquaculture are heart and skeletal muscle inflammation (HSMI) caused by Piscine orthoreovirus (PRV), and pancreas disease (PD) caused by Salmonid alphavirus (SAV). Both PRV and SAV target heart and skeletal muscles, but SAV additionally targets exocrine pancreas. PRV and SAV are often present in the same locations and co-infections occur, but the effect of this crosstalk on disease development has not been investigated. In the present experiment, the effect of a primary PRV infection on subsequent SAV infection was studied. Atlantic salmon were infected with PRV by cohabitation, followed by addition of SAV shedder fish 4 or 10 weeks after the initial PRV infection. Histopathological evaluation, monitoring of viral RNA levels and host gene expression analysis were used to assess disease development. Significant reduction of SAV RNA levels and of PD specific histopathological changes were observed in the co-infected groups compared to fish infected by SAV only. A strong correlation was found between histopathological development and expression of disease related genes in heart. In conclusion, experimentally PRV infected salmon are less susceptible to secondary SAV infection and development of PD.