Smoltification of Atlantic Salmon (Salmo salar L.) Is Associated with Enhanced Traffic and Renewal of B Cell Repertoire.

The smoltification of farmed Atlantic salmon is commonly associated with mild immunosuppression. However, B cells may deviate from this trend, showing increased proliferation and migration during this period. This study assessed the effects of smoltification and adaptation to seawater in a controlled experiment. Analyses were conducted on the head kidney, spleen, gill, and both visceral and subcutaneous fat (VAT, SAT) across four time points: parr, early and complete smoltification, and twelve weeks post-seawater transfer. Gene expression analysis was performed to track the distribution and developmental changes in their B cells. Expression profiles of three types of immunoglobulins (ig), including membrane-bound and secreted forms of igm, as well as B cell-specific markers pax1 and cd79, showed strong correlations and contrasted with profiles of other immune cell markers. The highest levels of expression were observed in the lymphatic tissue, followed by the VAT. Enhanced expression in the gill and adipose tissues of smolts suggested an increase in B cell populations. Parallel sequencing of the variable region of the IgM heavy chain was used to track B cell traffic, assessed by the co-occurrence of the most abundant sequences (clonotypes) across different tissues. Smoltification markedly enhanced traffic between all tissues, which returned to initial levels after twelve weeks in the sea. The preferred migration between the head kidney, spleen, and VAT supports the role of abdominal fat as a reservoir of lymphocytes. These findings are discussed in the context of recent studies that suggested the functional significance of B cell traffic in Atlantic salmon. Specifically, the migration of B cells expressing secreted immunoglobulins to virus-infected hearts has been identified as a key factor in the disease recovery and survival of fish challenged with salmon alphavirus (SAV); this process is accelerated by vaccination. Additionally, the study of melanized foci in the skeletal muscles revealed an association between antigen-dependent differentiation and the migration of B cells, indicating a transfer from local to systemic immune responses. Updating the antibody repertoire in the lymphatic and peripheral tissues of smolts may assist in their adaptation to the marine environment and in encountering new pathogens. Emerging evidence highlights B cell migration as an important and previously unrecognized immune mechanism in salmonids.

Expression and regulation of the CXCL9-11 chemokines and CXCR3 receptor in Atlantic salmon (Salmo salar).

Chemokines are cytokines that mediate leukocyte traffic between the lymphoid organs, the bloodstream, and the site of tissue damage, which is essential for an efficient immune response. In particular, the gamma interferon (IFN- γ) inducible chemokines CXCL9, CXCL10, and CXCL11, and their receptor CXCR3, are involved in T cell and macrophage recruitment to the site of infection. The nature and function of these chemokines and their receptor are well-known in mammals, but further research is needed to achieve a similar level of understanding in fish immunity. Thus, in this study, we seek to identify the genes encoding the components of the Atlantic salmon (Salmo salar) CXCL9, CXCL10, CXCL11/CXCR3 axis (CXCL9-11/CXCR3), predict the protein structure from the amino acid sequence, and explore the regulation of gene expression as well as the response of these chemokines and their receptor to viral infections. The cxcl9, cxcl10, cxcl11, and cxcr3 gene sequences were retrieved from the databases, and the phylogenetic analysis was conducted to determine the evolutionary relationships. The study revealed an interesting pattern of clustering and conservation among fish and mammalian species. The salmon chemokine sequences clustered with orthologs from other fish species, while the mammalian sequences formed separate clades. This indicates a divergent evolution of chemokines between mammals and fish, possibly due to different evolutionary pressures. While the structural analysis of the chemokines and the CXCR3 receptor showed the conservation of critical motifs and domains, suggesting preserved functions and stability throughout evolution. Regarding the regulation of gene expression, some components of the CXCL9-11/CXCR3 axis are induced by recombinant gamma interferon (rIFN-γ) and by Infectious pancreatic necrosis virus (IPNV) infection in Atlantic salmon cells. Further studies are needed to explore the role of Atlantic salmon CXCL9-11 chemokines in regulating immune cell migration and endothelial activation, as seen in mammals. To the best of our knowledge, there have been no functional studies of chemokines to understand these effects in Atlantic salmon.

Characterization of the impact of dietary immunostimulant CpG on the expression of mRNA biomarkers involved in the immune responses in Atlantic salmon (Salmo salar).

Infectious diseases have significantly impacted Atlantic salmon aquaculture worldwide. Modulating fish immunity with immunostimulant-containing functional feeds could be an effective strategy in mitigating disease problems. Previously, we characterized the impact of polyriboinosinic polyribocytidylic acid (pIC) and formalin-killed typical Aeromonas salmonicida bacterin on miRNA expression in Atlantic salmon fed a commercial diet with and without immunostimulant CpG. A set of miRNA biomarkers of Atlantic salmon head kidney responding to pIC and/or bacterin immune stimulations was identified (Xue et al., 2019) [1]. Herein, we report a complementary qPCR study that investigated the impact of the pIC, bacterin and dietary CpG on the expression of immune-relevant mRNAs (n = 31) using the same samples as in the previous study (Xue et al., 2019) [1]. Twenty-six of these genes were predicted target transcripts of the pIC- and/or bacterin-responsive miRNAs identified in the earlier study. The current data showed that pIC and/or bacterin stimulations significantly modulated the majority of the qPCR-analyzed genes involved in various immune pathways. Some genes responded to both stimulations (e.g. tnfa, il10rb, ifng, irf9, cxcr3, campb) while others appeared to be stimulation specific [e.g. irf3, irf7a, il1r1, mxa, mapk3 (pIC only); clra (bacterin only)]. A. salmonicida bacterin stimulation produced a strong inflammatory response (e.g. higher expression of il1b, il8a and tnfa), while salmon stimulated with pIC showed robust interferon responses (both type I and II). Furthermore, the current data indicated significant down-regulation of immune-relevant transcripts (e.g. tlr9, irf5, il1r1, hsp90ab1, itgb2) by dietary immunostimulant CpG, especially among pre-injection and PBS-injected fish. Together with our prior miRNA study, the present research provided complementary information on Atlantic salmon anti-viral and anti-bacterial immune responses and on how dietary CpG may modulate these responses.

Daily crowding stress has limited, yet detectable effects on skin and head kidney gene expression in surgically tagged atlantic salmon (Salmo salar).

To ensure welfare-friendly and effective internal tagging, the tagging process should not cause a long-term burden on individuals given that tagged fish serve as representatives for the entire population in telemetry applications. To some extent, stress is inevitable within regular aquaculture practices, and thus, the consequences of long-term stress should be described in terms of their effects on internal tagging. In fish, stressors activate the Hypothalamus-Pituitary-Interrenal (HPI) and Brain-Sympathetic-Chromaffin Cell (BSC) axes, leading to neuroimmunoendocrine communication and paracrine interactions among stress hormones. The interrelation between wound healing and stress is complex, owing to their shared components, pathways, and energy demands. This study assessed 14 genes (mmp9, mmp13, il-2, il-4, il-8a, il-10, il-12, il-17d, il-1b, tnfa, ifng, leg-3, igm, and crh) in the skin (1.5 cm from the wound) and head kidney over eight weeks. These genes, associated with cell signaling in immunity, wound healing, and stress, have previously been identified as influenced and regulated by these processes. Half of a group of Atlantic salmon (n = 90) with surgically implanted dummy smart-tags were exposed to daily crowding stress. The goal was to investigate how this gene panel responds to a wound alone and then to the combined effects of wounding and daily crowding stress. Our observations indicate that chronic stress impacts inflammation and impedes wound healing, as seen through the expression of matrix metalloproteinases genes in the skin but not in the head kidney. This difference is likely due to the ongoing internal wound repair, in contrast to the externally healed wound incision. Cytokine expression, when significant in the skin, was mainly downregulated in both treatments compared to control values, particularly in the study's first half. Conversely, the head kidney showed initial cytokine downregulation followed by upregulation. Across all weeks observed and combining both tissues, the significantly expressed gene differences were 12 % between the Wound and Stress+ groups, 28 % between Wound and Control, and 25 % between Stress+ and Control. Despite significant fluctuations in cytokines, sustained variations across multiple weeks are only evident in a few select genes. Furthermore, Stress+ individuals demonstrated the most cytokine correlations within the head kidney, which may suggest that chronic stress affects cytokine expression. This investigation unveils that the presence of stress and prolonged activation of the HPI axis in an eight weeklong study has limited yet detectable effects on the selected gene expression within immunity, wound healing, and stress, with notable tissue-specific differences.

Modulation of metabolic and immunoregulatory pathways in the gut transcriptome of Atlantic salmon (Salmo salar L.) after early nutritional programming during first feeding with plant-based diet.

Introduction: Plant-based nutritional programming is the concept of exposing fish at very early life stages to a plant-based diet for a short duration to improve physiological responses when exposed to a similar plant-rich diet at a later developmental stage. The mechanisms of action underlying nutritional programming have not been fully deciphered, and the responses may be controlled at multiple levels. Methods: This 22-week study examines gut transcriptional changes after nutritional programming. Triplicate groups of Atlantic salmon were fed with a plant (V) vs. a marine-rich (M, control) diet for 2 weeks (stimulus phase) at the first exogenous feeding. Both stimulus fish groups (M and V fish) were then fed the M diet for 12 weeks (intermediate phase) and lastly fed the V diet (challenge phase) for 6 weeks, generating two dietary regimes (MMV and VMV) across phases. This study used a whole-transcriptome approach to analyse the effects of the V diet at the end of stimulus (short-term effects) and 22 weeks post-first feeding (long-term effects). After the stimulus, due to its developmental stage, the whole intestine was used, whereas, after the challenge, pyloric caeca and middle and distal intestines were examined. Results and discussion: At the stimulus end, genes with increased expression in V fish enriched pathways including regulatory epigenetic responses and lipid metabolism, and genes involved in innate immune response were downregulated. In the middle intestine at the end of the challenge, expression levels of genes of lipid, carbohydrate, and energy metabolism were increased in V fish, while M fish revealed increased expression of genes associated with autoimmune and acute adaptive immune response. The distal intestine of V fish showed increased expression of genes associated with immune response and potential immune tolerance. Conversely, the distal intestine of M fish at challenge revealed upregulation of lipid and carbohydrate metabolic pathways, tissue degeneration, and apoptotic responses. The present study demonstrated nutritional programming-associated changes in the intestinal transcriptome, with altered expression of genes involved in both immune responses and different metabolic processes. While there were limited changes in growth between the groups, the results show that there were transcriptional differences, suggesting a programming response, although the mechanism of this response still requires to be fully elucidated.

Leukolectins support lifelong innate immunity in lower vertebrates, and reveal dichotomies of several leukocytic lineages.

Innate immunity is vital for animal homeostasis and survival. First-line immuno-defense for fish larvae involves mucus enriched with leukolectin (LL) secreted by dermal lectocytes. Later during the critical transition from yolk-nutrition to feeding, additional larval immuno-protection in zebrafish (zF) is provided by macrophages containing LL (lectophages). This work investigated new LL-expression in embryos and in blood, structures of fish leukocytic LL and LL-genes, and LL-presence in chicken leukocytes. In zF-embryos, lectophages appear ∼10 hpf, while later, cells co-expressing myeloperoxidase- and LL-mRNA were detected (∼19 hpf). Furthermore, protein-extracts of Atlantic salmon (Ssal) leukocytes contained LL-proteins, compartmentalized in the cytosol. Cloning and sequencing revealed 94 % nt-sequence identity between variants of Ssal-leukolectins. Highly conserved LLs allowed production of epitope-specific anti-LL IgGs. Immuno-fluorescence-analysis demonstrated that most Ssal-bloodcells were LL-negative, but both some large cells with protrusions and some small, rounded cells did express LL. Immunoperoxidase-staining method confirmed LL-expression in some Ssal-leukocytes, identified as macrophages, PMN-leukocytes, thrombocytes and dendritic cells. However, closer examination revealed a dichotomy of these cell-categories into either LL-positive, or LL-negative variants. In situ hybridization demonstrated profuse LL-expression in Ssal head kidney interstitial tissue, while LL-transcripts were absent in large kidney tubules. Both hematopoietic (non-pigmented) marrow cells and melano-macrophages expressed LL-mRNA, implying that leukolectins provide lifelong innate immuno-protection. PCR-amplification using Ssal-leukocytic DNA as template, and direct sequencing yielded a leukocytic ll-gene. Some cells in salmon, cod, halibut, oikopleura and zebrafish embryos express LL-proteins and/or LL-mRNA, and LL-mRNA is detected in salmon, cod and chicken leukocytes. However, current genomes for these species lack recognizable LL-loci except the Ssal_v3.1 Genome-assembly. The data demonstrate an unexpected dichotomy of some leukocyte lineages into LL-positive or LL-negative cell-variants. Such dichotomies suggest exploring differential impacts from the duplicated leukocyte-lineages in health and disease.

Atlantic salmon type I interferon genes revisited.

Type I interferons (IFN-I) play a pivotal role in vertebrate innate immunity against viruses. This study is an analysis of IFN-I genes in an updated version of the Atlantic salmon genome published in 2021 (version Ssal_v3.1), revealing 47 IFN-I genes in the Atlantic salmon genome. The GH1 locus of chromosome (Chr) 3 harbors 9 IFNa genes, 5 IFNb genes, 6 IFNc genes, 11 IFNe genes and 1 IFNf gene. The GH2 locus on Chr6 contains 1 IFNa gene, 12 IFNc genes and 1 IFNf gene while Chr19 carries a single IFNd gene. Intraperitoneal injection of Atlantic salmon presmolts with poly I:C, a mimic of virus double-stranded RNA, significantly up-regulated IFNc genes from both Chr3 and Chr6 in heart, with lower expression in head kidney. IFNe expression increased in the heart, but not in the head kidney while IFNf was strongly up-regulated in both tissues. Antiviral activity of selected IFNs was assessed by transfection of salmon cells with IFN-expressing plasmids followed by infectious pancreatic necrosis virus infection, and by injection of fish with IFN-plasmids followed by measuring expression of the antiviral Mx1 gene. The results demonstrated that IFNc from both Chr3 and Chr6 provided full protection of cells against virus infection, whereas IFNe and IFNf showed lesser protection. IFNc from Chr3 and Chr6 along with IFNe and IFNf, up-regulated the Mx1 gene in the muscle, while only the IFNcs caused induction of Mx1 in liver. Overall, this study reveals that Atlantic salmon possesses an even more potent innate immune defense against viruses than previously understood.

Environmental influence on the Atlantic salmon transcriptome and methylome during sea lice infestations.

The fish's immune response is affected by different factors, including a wide range of environmental conditions that can also disrupt or promote changes in the host-pathogen interactions. How environmental conditions modulate the salmon genome during parasitism is poorly understood here. This study aimed to explore the environmental influence on the Salmo salar transcriptome and methylome infected with the sea louse Caligus rogercresseyi. Atlantic salmon were experimentally infected with lice at two temperatures (8 and 16 °C) and salinity conditions (32 and 26PSU). Fish tissues were collected from the infected Atlantic salmon for reduced representation bisulfite sequencing (RRBS) and whole transcriptome sequencing (RNA-seq) analysis. The parasitic load was highly divergent in the evaluated environmental conditions, where the lowest lice abundance was observed in fish infected at 8 °C/26PSU. Notably, transcriptome profile differences were statistically associated with the number of alternative splicing events in fish exposed to low temperature/salinity conditions. Furthermore, the temperature significantly affected the methylation level, where high values of differential methylation regions were observed at 16 °C. Also, the association between expression levels of spliced transcripts and their methylation levels was determined, revealing significant correlations with Ferroptosis and TLR KEEG pathways. This study supports the relevance of the environmental conditions during host-parasite interactions in marine ecosystems. The discovery of alternative splicing transcripts associated with DMRs is also discussed as a novel player in fish biology.

Discovery and Characterization of the ddx41 Gene in Atlantic Salmon: Evolutionary Implications, Structural Functions, and Innate Immune Responses to Piscirickettsia salmonis and Renibacterium salmoninarum Infections.

The innate immune response in Salmo salar, mediated by pattern recognition receptors (PRRs), is crucial for defending against pathogens. This study examined DDX41 protein functions as a cytosolic/nuclear sensor for cyclic dinucleotides, RNA, and DNA from invasive intracellular bacteria. The investigation determined the existence, conservation, and functional expression of the ddx41 gene in S. salar. In silico predictions and experimental validations identified a single ddx41 gene on chromosome 5 in S. salar, showing 83.92% homology with its human counterpart. Transcriptomic analysis in salmon head kidney confirmed gene transcriptional integrity. Proteomic identification through mass spectrometry characterized three unique peptides with 99.99% statistical confidence. Phylogenetic analysis demonstrated significant evolutionary conservation across species. Functional gene expression analysis in SHK-1 cells infected by Piscirickettsia salmonis and Renibacterium salmoninarum indicated significant upregulation of DDX41, correlated with increased proinflammatory cytokine levels and activation of irf3 and interferon signaling pathways. In vivo studies corroborated DDX41 activation in immune responses, particularly when S. salar was challenged with P. salmonis, underscoring its potential in enhancing disease resistance. This is the first study to identify the DDX41 pathway as a key component in S. salar innate immune response to invading pathogens, establishing a basis for future research in salmonid disease resistance.

Organisation of the Atlantic salmon (Salmo salar) thymus and its content of Ig-expressing cells.

The thymus of fishes is located as a dual organ in a rostrodorsal projection within the gill chamber and is covered by the operculum. The histological organization of the teleost fish thymus displays considerable diversity, particularly in salmonids where a clear distinction between the thymus cortex and medulla is yet to be defined. Recent interest has focused on the role of B cells in thymic function, but the presence of these cells within the salmon thymus remains poorly understood. In this morphological study, we applied in situ hybridization to investigate developing Atlantic salmon thymi for the expression of recombination activating (Rag) genes 1 and 2. We identified the location of the cortex, aligning with the previously described inner zone. Expression of IgM and IgD transcripts was predominantly observed in cells within the outer and subcapsular zones, with lesser expression in the cortex and inner zone. IgT expression was confined to a limited number of cells in the inner zone and capsule. The location of the thymus medulla could not be established. Our results are discussed in the context of the recently identified lymphoid organs, namely the intrabranchial lymphoid tissue (ILT) and the salmon bursa.

Impact of freshwater rearing history on Atlantic salmon gill response to viral stimulation post seawater transfer.

Land-based recirculating aquaculture systems (RAS) have risen in prevalence in recent years for Atlantic salmon production, enabling intensive production which allows increased growth and environmental control, but also having the potential for reducing water use and eutrophication. The Atlantic salmon has an anadromous life history with juvenile stages in freshwater (FW) and on-growing in seawater (SW), enabled by a transformational process known as smoltification. The timing of smoltification and transfer of smolts from FW to SW is critical under commercial production with high mortalities during this period. The impact of FW rearing system on immune function following seawater transfer (SWT) is not well understood. In this study parr were raised in either RAS or a traditional open-LOCH system until smolting and then transferred to a common marine environment. Two-weeks post-SWT fish were immune stimulated with a viral mimic (poly I:C) for 24 h to assess the ability to mount an antiviral immune response, assessed by whole transcriptome analysis of gill tissue, an important immune organ in fish. We show that unstimulated smolts reared in the LOCH had higher immune gene expression than those reared in RAS as determined by functional analysis. However, following stimulation, smolts reared in the RAS mounted a greater magnitude of response with a suite of immune genes displaying higher fold induction of transcription compared to LOCH reared smolts. We suggest RAS smolts have a lower steady state immune-associated transcriptome likely due to an unvarying environment, in terms of environmental factors and lack of exposure to pathogens, which shows a compensatory mechanism following stimulation allowing immune 'catch-up' with those reared in the LOCH. Alternatively, the RAS fish are experiencing an excessive response to the immune stimulation.

A comparative analysis of alternative splicing patterns in Atlantic salmon (Salmo salar) in response to Moritella viscosa and sea lice (Lepeophtheirus salmonis) infection.

Moritella viscosa (M. viscosa) and sea lice (Lepeophtheirus salmonis) are severe pathogens that primarily infect the skin of Atlantic salmon (Salmo salar), which cause significant economic losses in the farming industry. However, the pathogenesis and molecular mechanisms underlying the host's immune defence at the post-transcriptional level remain unclear. Alternative splicing (AS) is an evolutionarily conserved post-transcriptional mechanism that can greatly increase the richness of the transcriptome and proteome. In this study, transcriptomic data derived from skin tissues of Atlantic salmon after M. viscosa and sea lice infections were used to examine the AS profiles and their differential expression patterns. In total, we identified 33,044 AS events (involving 13,718 genes) in the control (CON) group, 35,147 AS events (involving 14,340 genes) in the M. viscosa infection (MV) group, and 30,364 AS events (involving 13,142 genes) in the sea lice infection (LC) group, respectively. Among the five types of AS identified in our study (i.e., SE, A5SS, A3SS, MXE, and RI), SE was the most prevalent type in all three groups (i.e., CON, MV, and LC groups). Decreased percent-spliced-in (PSI) levels were observed in SE events under both MV- and LC-infected conditions, suggesting that MV or LC infection elevated exon-skipping isoforms and promoted the selection of shorter transcripts in numerous DAS genes. In addition, most of the differential AS genes were found to be associated with pathways related to mRNA regulation, epithelial or muscle development, and immune response. These findings provide novel insights into the role of AS in host-pathogen interactions and represent the first comparative analysis of AS in response to bacterial and parasitic infections in fish.

Effects of eicosapentaneoic acid on innate immune responses in an Atlantic salmon kidney cell line in vitro.

Studies of the interplay between metabolism and immunity, known as immunometabolism, is steadily transforming immunological research into new understandings of how environmental cues like diet are affecting innate and adaptive immune responses. The aim of this study was to explore antiviral transcriptomic responses under various levels of polyunsaturated fatty acid. Atlantic salmon kidney cells (ASK cell line) were incubated for one week in different levels of the unsaturated n-3 eicosapentaneoic acid (EPA) resulting in cellular levels ranging from 2-20% of total fatty acid. These cells were then stimulated with the viral mimic and interferon inducer poly I:C (30 ug/ml) for 24 hours before total RNA was isolated and sequenced for transcriptomic analyses. Up to 200 uM EPA had no detrimental effects on cell viability and induced very few transcriptional changes in these cells. However, in combination with poly I:C, our results shows that the level of EPA in the cellular membranes exert profound dose dependent effects of the transcriptional profiles induced by this treatment. Metabolic pathways like autophagy, apelin and VEGF signaling were attenuated by EPA whereas transcripts related to fatty acid metabolism, ferroptosis and the PPAR signaling pathways were upregulated. These results suggests that innate antiviral responses are heavily influenced by the fatty acid profile of salmonid cells and constitute another example of the strong linkage between general metabolic pathways and inflammatory responses.

Virus-specific antibody secreting cells reside in the peritoneal cavity and systemic immune sites of Atlantic salmon (Salmo salar) challenged intraperitoneally with salmonid alphavirus.

The development and persistence of antibody secreting cells (ASC) after antigenic challenge remain inadequately understood in teleosts. In this study, intraperitoneal (ip) injection of Atlantic salmon (Salmo salar) with salmonid alphavirus (WtSAV3) increased the total ASC response, peaking 3-6 weeks post injection (wpi) locally in the peritoneal cavity (PerC) and in systemic lymphoid tissues, while at 13 wpi the response was only elevated in PerC. At the same time point a specific ASC response was induced by WtSAV3 in PerC and systemic tissues, with the highest frequency in PerC, suggesting a local role. Inactivated SAV (InSAV1) induced comparatively lower ASC responses in all sites, and specific serum antibodies were only induced by WtSAV3 and not by InSAV1. An InSAV1 boost did not increase these responses. Expression of immune marker genes implies a role for PerC adipose tissue in the PerC immune response. Overall, the study suggests the Atlantic salmon PerC as a secondary immune site and an ASC survival niche.

Vegetable omega-3 and omega-6 fatty acids differentially modulate the antiviral and antibacterial immune responses of Atlantic salmon.

The immunomodulatory effects of omega-3 and omega-6 fatty acids are a crucial subject of investigation for sustainable fish aquaculture, as fish oil is increasingly replaced by terrestrial vegetable oils in aquafeeds. Unlike previous research focusing on fish oil replacement with vegetable alternatives, our study explored how the omega-6 to omega-3 polyunsaturated fatty acid (PUFA) ratio in low-fish oil aquafeeds influences Atlantic salmon's antiviral and antibacterial immune responses. Atlantic salmon were fed aquafeeds rich in soy oil (high in omega-6) or linseed oil (high in omega-3) for 12 weeks and then challenged with bacterial (formalin-killed Aeromonas salmonicida) or viral-like (polyriboinosinic polyribocytidylic acid) antigens. The head kidneys of salmon fed high dietary omega-3 levels exhibited a more anti-inflammatory fatty acid profile and a restrained induction of pro-inflammatory and neutrophil-related genes during the immune challenges. The high-omega-3 diet also promoted a higher expression of genes associated with the interferon-mediated signaling pathway, potentially enhancing antiviral immunity. This research highlights the capacity of vegetable oils with different omega-6 to omega-3 PUFA ratios to modulate specific components of fish immune responses, offering insights for future research on the intricate lipid nutrition-immunity interplay and the development of novel sustainable low-fish oil clinical aquaculture feeds.

New insight into the biological activity of Salmo salar NK-lysin antimicrobial peptides.

NK-lysin is a potent antimicrobial peptide (AMP) with antimicrobial activity against bacteria, fungi, viruses, and parasites. NK-lysin is a type of granulysin, a member of the saposin-like proteins family first isolated from a pig's small intestine. In previous work, for the first time, we identified four variants of nk-lysin from Atlantic salmon (Salmo salar) using EST sequences. In the present study, we reported and characterized two additional transcripts of NK-lysin from S. salar. Besides, we evaluated the tissue distribution of three NK-lysins from S. salar and assessed the antimicrobial, hemolytic, and immunomodulatory activities and signaling pathways of three NK-lysin-derived peptides. The synthetic peptides displayed antimicrobial activity against Piscirickettsia salmonis (LF-89) and Flavobacterium psychrophilum. These peptides induced the expression of immune genes related to innate and adaptive immune responses in vitro and in vivo. The immunomodulatory activity of the peptides involves the mitogen-activated protein kinases-mediated signaling pathway, including p38, extracellular signal-regulated kinase 1/2, and/or c-Jun N-terminal kinases. Besides, the peptides modulated the immune response induced by pathogen-associated molecular patterns (PAMPs). Our findings show that NK-lysin could be a highly effective immunostimulant or vaccine adjuvant for use in fish aquaculture.

Mucosal barrier status in Atlantic salmon fed rapeseed oil and Schizochytrium oil partly or fully replacing fish oil through winter depression.

The study was designed to investigate the effects of replacing fish oil by algal oil and rapeseed oil on histomorphology indices of the intestine, skin and gill, mucosal barrier status and immune-related genes of mucin and antimicrobial peptide (AMP) genes in Atlantic salmon (Salmo salar). For these purposes, Atlantic salmon smolts were fed three different diets. The first was a control diet containing fish oil but no Schizochytrium oil. In the second diet, almost 50 % of the fish oil was replaced with algal oil, and in the third diet, fish oil was replaced entirely with algal oil. The algal oil contained mostly docosahexaenoic acid (DHA) and some eicosapentaenoic acid (EPA). The study lasted for 49 days in freshwater (FW), after which some fish from each diet group were transferred to seawater (SW) for a 48-h challenge test at 33 ppt to test their ability to tolerate high salinity. Samples of skin, gills, and mid intestine [both distal (DI) and anterior (AI) portions of the mid intestine] were collected after the feeding trial in FW and after the SW-challenge test to assess the effects of the diets on the structure and immune functions of the mucosal surfaces. The results showed that the 50 % VMO (Veramaris® algal oil) dietary group had improved intestinal, skin, and gill structures. Principal component analysis (PCA) of the histomorphological parameters demonstrated a significant effect of the algal oil on the intestine, skin, and gills. In particular, the mucosal barrier function of the intestine, skin, and gills was enhanced in the VMO 50 % dietary group after the SW challenge, as evidenced by increased mucous cell density. Immunolabelling of heat shock protein 70 (HSP70) in the intestine (both DI and AI) revealed downregulation of the protein expression in the 50 % VMO group and a corresponding upregulation in the 100 % VMO group compared to 0 % VMO. The reactivity of HSP70 in the epithelial cells was higher after the SW challenge compared to the FW phase. Immune-related genes related to mucosal defense, such as mucin genes [muc2, muc5ac1 (DI), muc5ac1 (AI), muc5ac2, muc5b (skin), and muc5ac1 (gills)], and antimicrobial peptide genes [def3 (DI), def3 (AI), and cath1 (skin)] were significantly upregulated in the 50 % VMO group. PCA of gene expression demonstrated the positive influences on gene regulation in the 50 % VMO dietary group. In conclusion, this study demonstrated the positive effect of substituting 50 % of fish oil with algal oil in the diets of Atlantic salmon. The findings of histomorphometry, mucosal mapping, immunohistochemistry, and immune-related genes connected to mucosal responses all support this conclusion.

Exogenous sulphide donors modify the gene expression patterns of Atlantic salmon nasal leukocytes.

Hydrogen sulphide (H2S) is a known mediator of immunity, but the regulatory function of its exogenous form is not well understood in fish particularly in the mucosa. Here we report transcriptomic changes in the nasal leukocytes of Atlantic salmon (Salmo salar) following exposure to two forms of H2S donors - the salt sodium hydrosulfide (NaHS) and the organic analogue morpholin-4-ium 4-methoxyphenyl (morpholino) phosphinodithioate (GYY4137). Nasal leukocytes were exposed to three concentrations (1, 10 and 100 µM) of either of the two H2S forms for 24 h before the cells were checked for viability and collected for microarray analysis. Though cellular viability was minimally affected by the exposure to two H2S donors, GYY4137-exposed cells exhibited reduced viability compared with the NaHS group at the highest dose. The H2S-induced transcriptomic changes in the nasal leukocytes were concentration-dependent regardless of the sulphide forms. However, a larger number of differentially expressed genes (DEGs) were identified in the NaHS-exposed versus GYY4137-exposed groups across concentrations. In all comparisons, at least 53% of the DEGs identified were significantly upregulated. Gene ontology (GO) terms enriched in the lists of upregulated DEGs at higher concentrations included ferric iron binding. A comparison of the two H2S forms showed a clear grouping of different GO terms relative to concentrations. Pathway enrichment analysis revealed a significant influence in VEGF ligand-receptor interactions, oxidative stress, innate and adaptive immunity, and interleukin signalling especially at higher concentrations. Congruence analysis demonstrated that there were 16 GO terms overlapping; of these, 12 were upregulated by both sulphide donors including several involving iron binding and transport. The study offers the first molecular insights into how fish nasal leukocytes respond to exogenous H2S, and the results will be vital in resolving the regulatory function of H2S on mucosal immunity in fish.

Tailoring freshwater diets towards boosted immunity and pancreas disease infection robustness in Atlantic salmon post smolts.

The aim of the current study was to investigate how freshwater diets impact on immunity in Atlantic salmon smolts in freshwater, during transfer to seawater and in post smolts during the seawater stage with and without pancreas disease (PD) infection. Three specific freshwater diets were prepared: (i) A diet similar in composition to commercial salmon freshwater diets (Standard diet); (ii) A diet composed of vegetable oils (rapeseed, palm and linseed oils) mimicking the fat composition in aquatic insects - the natural diet of wild salmon in freshwater (Fatty acid diet); (iii) A diet enriched with possible immune modulating amino acids including dl-methionine, l-lysine, l-threonine and taurine (Amino acid diet). After seawater transfer, all fish were fed the same commercial diet. Head kidneys were extracted, and their leukocytes isolated from smolts right before transfer to seawater, from post smolts one and six weeks after transfer to seawater, and from post smolts in seawater after 8 weeks of ongoing PD infection. In addition, to provoke bacterial or virus induced inflammation in vitro, the individual leukocyte suspension from all fish were stimulated by lipopolysaccharide (LPS) or polyinosinic acid: polycytidylic acid (PIC). The transfer of smolts from fresh-to seawater changed the transcription of several types of genes. Particularly in isolates from fish fed the Standard or Fatty acid diet in freshwater, overall gene transcription (IL-1ß, CD83, INF-γ, cox2, cd36, MGAT2, catalase) declined. However, the Amino acid diet stimulated the LPS induced gene transcription of IL-1ß, CD83, Cox2, and INF-γ at this stage. In freshwater smolts, PIC stimulated leukocytes showed higher transcription level of Mx and viperin in the Fatty acid and Amino acid diet groups compared to the Standard diet group. In seawater post smolts, Mx and viperin responded similarly to PIC challenge in all diet groups. Furthermore, leukocytes isolated from PD infected fish, continued responding to PIC, regardless of freshwater diet.

Secretome Profiling of Atlantic Salmon Head Kidney Leukocytes Highlights the Role of Phagocytes in the Immune Response to Soluble ß-Glucan.

ß-Glucans (BG) are glucose polymers which are produced in bacteria and fungi but not in vertebrate organisms. Being recognized by phagocytic leukocytes including macrophages and neutrophils through receptors such as dectin-1 and Complement receptor 3 (CR3), the BG are perceived by the innate immune system of vertebrates as foreign substances known as Pathogen Associated Molecular Patterns (PAMPs). The yeast-derived BG has been recognized for its potent biological activity and it is used as an immunomodulator in human and veterinary medicine. The goal of the current study was to characterize the immunostimulatory activity of soluble yeast BG in primary cultures of Atlantic salmon (Salmo salar) head kidney leukocytes (HKLs) in which phagocytic cell types including neutrophils and mononuclear phagocytes predominate. The effect of BG on the secretome of HKL cultures, including secretion of extracellular vesicles (EVs) and soluble protein55s was characterized through western blotting and mass spectrometry. The results demonstrate that, along with upregulation of proinflammatory genes, BG induces secretion of ubiquitinated proteins (UbP), MHCII-containing EVs from professional antigen presenting cells as well as proteins derived from granules of polymorphonuclear granulocytes (PMN). Among the most abundant proteins identified in BG-induced EVs were beta-2 integrin subunits, including CD18 and CD11 homologs, which highlights the role of salmon granulocytes and mononuclear phagocytes in the response to soluble BG. Overall, the current work advances the knowledge about the immunostimulatory activity of yeast BG on the salmon immune system by shedding light on the effect of this PAMP on the secretome of salmon leukocytes.