Atlantic Salmon Head Kidney Research Articles

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.

Modulation of infectious Salmon Anaemia virus infection by clathrin-mediated endocytosis and macropinocytosis inhibitors

Infectious salmon anaemia virus (ISAV) is a pathogen that causes disease and large mortality in farm-raised Salmo salar L., being considered as a major problem in the salmon industry. However, despite its relevance, there are still numerous knowledge gaps on virus entry and early stages of infection. Previous studies suggested that virus entry into cells occurs via endocytosis, with no description of specific mechanisms. However, it remains unknown if the endocytosis induced by ISAV is a clathrin-dependent or clathrin-independent process. This study aimed to identify cellular mechanisms allowing ISAV entry into Atlantic Salmon head kidney (ASK) cells. Our results showed that ISAV can be found in coated pits and membrane ruffles, the latter being induced by a rearrangement of actin filaments promoted by ISAV infection. Additionally, it was determined that ISAV stimulate the uptake of extracellular fluid in a multiplicity of infection (MOI)-dependent manner. When the clathrin-mediated endocytic pathway was pharmacologically inhibited, ISAV infection was significantly reduced but not entirely inhibited. Similarly, when the Na+/H+ exchanger (NHE), a key component of macropinocytosis, was inhibited, ISAV infection was negatively affected. Our results suggest that ISAV enters cells via both clathrin-mediated endocytosis and most likely macropinocytosis.

Transcriptomics of early responses to purified Piscine orthoreovirus-1 in Atlantic salmon (Salmo salar L.) red blood cells compared to non-susceptible cell lines.

Piscine red blood cells (RBC) are nucleated and have been characterized as mediators of immune responses in addition to their role in gas exchange. Salmonid RBC are major target cells of Piscine orthoreovirus-1 (PRV-1), the etiological agent of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). PRV-1 replicates in RBC ex vivo, but no viral amplification has been possible in available A. salmon cell lines. To compare RBC basal transcripts and transcriptional responses to PRV-1 in the early phase of infection with non-susceptible cells, we exposed A. salmon RBC, Atlantic salmon kidney cells (ASK) and Salmon head kidney cells (SHK-1) to PRV-1 for 24 h. The RNA-seq analysis of RBC supported their previous characterization as pluripotent cells, as they expressed a wide repertoire of genes encoding pattern recognition receptors (PRRs), cytokine receptors, and genes implicated in antiviral activities. The comparison of RBC to ASK and SHK-1 revealed immune cell features exclusively expressed in RBC, such as genes involved in chemotactic activity in response to inflammation. Differential expression analysis of RBC exposed to PRV-1 showed 46 significantly induced genes (≥ 2-fold upregulation) linked to the antiviral response pathway, including RNA-specific PRRs and interferon (IFN) response factors. In SHK-1, PRV induced a more potent or faster antiviral response (213 genes induced). ASK cells showed a differential response pattern (12 genes induced, 18 suppressed) less characterized by the dsRNA-induced antiviral pathway. Despite these differences, the RIG-I-like receptor 3 (RLR3) in the family of cytosolic dsRNA receptors was significantly induced in all PRV-1 exposed cells. IFN regulatory factor 1 (IRF1) was significantly induced in RBC only, in contrast to IRF3/IRF7 induced in SHK-1. Differences in IRF expression and activity may potentially affect viral propagation.

Mapping the cellular landscape of Atlantic salmon head kidney by single cell and single nucleus transcriptomics

Single-cell transcriptomics is the current gold standard for global gene expression profiling, not only in mammals and model species, but also in non-model fish species. This is a rapidly expanding field, creating a deeper understanding of tissue heterogeneity and the distinct functions of individual cells, making it possible to explore the complexities of immunology and gene expression on a highly resolved level. In this study, we compared two single cell transcriptomic approaches to investigate cellular heterogeneity within the head kidney of healthy farmed Atlantic salmon (Salmo salar). We compared 14,149 cell transcriptomes assayed by single cell RNA-seq (scRNA-seq) with 18,067 nuclei transcriptomes captured by single nucleus RNA-Seq (snRNA-seq). Both approaches detected eight major cell populations in common: granulocytes, heamatopoietic stem cells, erythrocytes, mononuclear phagocytes, thrombocytes, B cells, NK-like cells, and T cells. Four additional cell types, endothelial, epithelial, interrenal, and mesenchymal cells, were detected in the snRNA-seq dataset, but appeared to be lost during preparation of the single cell suspension submitted for scRNA-seq library generation. We identified additional heterogeneity and subpopulations within the B cells, T cells, and endothelial cells, and revealed developmental trajectories of heamatopoietic stem cells into differentiated granulocyte and mononuclear phagocyte populations. Gene expression profiles of B cell subtypes revealed distinct IgM and IgT-skewed resting B cell lineages and provided insights into the regulation of B cell lymphopoiesis. The analysis revealed eleven T cell sub-populations, displaying a level of T cell heterogeneity in salmon head kidney comparable to that observed in mammals, including distinct subsets of cd4/cd8-negative T cells, such as tcrγ positive, progenitor-like, and cytotoxic cells. Although snRNA-seq and scRNA-seq were both useful to resolve cell type-specific expression in the Atlantic salmon head kidney, the snRNA-seq pipeline was overall more robust in identifying several cell types and subpopulations. While scRNA-seq displayed higher levels of ribosomal and mitochondrial genes, snRNA-seq captured more transcription factor genes. However, only scRNA-seq-generated data was useful for cell trajectory inference within the myeloid lineage. In conclusion, this study systematically outlines the relative merits of scRNA-seq and snRNA-seq in Atlantic salmon, enhances understanding of teleost immune cell lineages, and provides a comprehensive list of markers for identifying major cell populations in the head kidney with significant immune relevance.

Transcriptome analysis revealed immune responses in the kidney of Atlantic salmon (Salmo salar) co-infected with sea lice (Lepeophtheirus salmonis) and infectious salmon anemia virus

Sea lice (Lepeophtheirus salmonis) and infectious salmon anemia virus (ISAv) are two of the most important pathogens in Atlantic salmon (Salmo salar) farming and typically cause substantial economic losses to the industry. However, the immune interactions between hosts and these pathogens are still unclear, especially in the scenario of co-infection. In this study, we artificially infected Atlantic salmon with sea lice and ISAv, and investigated the gene expression patterns of Atlantic salmon head kidneys in response to both lice only and co-infection with lice and ISAv by transcriptomic analysis. The challenge experiment indicated that co-infection resulted in a cumulative mortality rate of 47.8 %, while no mortality was observed in the lice alone infection. We identified 240 differentially expressed genes (DEGs) under the lice alone infection, of which 185 were down-regulated and 55 were up-regulated, while a total of 994 DEGs were identified in the co-infection, of which 206 were down-regulated and 788 were significantly up-regulated. The pathway enrichment analysis revealed that single-infection significantly suppressed the innate immune system (e.g., the complement system), whereas co-infection induced a strong immune response, leading to the activation of immune-related signaling pathways such as Toll-like receptors and NOD-like receptors pathways, as well as significant upregulation of genes related to the activation of interferon and MH class I protein complex. Our results provide the first global transcriptomic study of gene expression in the Atlantic salmon head kidney in response to co-infection with sea lice and ISAv, and provided the baseline knowledge for understanding the immune responses during co-infection.

Characterization and transcript expression analyses of four Atlantic salmon (Salmo salar) serpinh1 paralogues provide evidence of evolutionary divergence

Atlantic salmon (Salmo salar) are not only the world’s most economically important farmed fish in terms of total value, but also a salmonid, which means that they are invaluable for studies of the evolutionary fate of genes following multiple whole-genome duplication (WGD) events. In this study, four paralogues of the molecular chaperone serpinh1 were characterized in Atlantic salmon, as while this gene is considered to be a sensitive biomarker of heat stress in salmonids, mammalian studies have also identified it as being essential for collagen structural assembly and integrity. The four salmon paralogues were cloned and sequenced so that in silico analyses at the nucleotide and deduced amino acid levels could be performed. In addition, qPCR was used to measure: paralogue- and sex-specific constitutive serpinh1 expression across 17 adult tissues; and their expression in the liver and head kidney of male Atlantic salmon as affected by stress phenotype (high vs. low responder), increased temperature, and injection with a multi-valent vaccine. Compared to the other three paralogues, serpinh1a-2 had a unique constitutive expression profile across the 17 tissues. Although stress phenotype had minimal impact on the transcript expression of the four paralogues, injection with a commercial vaccine containing several formalin inactivated bacterins increased the expression of most paralogues (by 1.1 to 4.5-fold) across both tissues. At 20 °C, the expression levels of serpinh1a-1 and serpinh1a-2 were generally lower (by −1.1- to −1.6-fold), and serpinh1b-1 and serpinh1b-2 were 10.2- to 19.0-fold greater, in comparison to salmon held at 12 °C. With recent studies suggesting a putative link between serpinh1 and upper thermal tolerance in salmonids, the current research is a valuable first step in elucidating the potential mechanisms involved. This research: supports the use of serpinh1b-1 and serpinh1b-2 as a biomarkers of heat stress in salmon; and provides evidence of neo- and/or subfunctionalization between the paralogues, and important insights into how multiple genome duplication events can potentially lead to evolutionary divergence.

Alternative splicing in Atlantic salmon head kidney and SHK-1 cell line during the Piscirickettsia salmonis infection: A comparative transcriptome survey

Piscirickettsia salmonis, an intracellular bacterium in salmon aquaculture, is a big challenge because it is responsible for 54.2% of Atlantic salmon mortalities. In recent years, the high relevance of Alternative Splicing (AS) as a molecular mechanism associated with infectious conditions and host-pathogen interaction processes, especially in host immune activation, has been observed. Several studies have highlighted the role of AS in the host's immune response during viral, bacterial, and endoparasite infection. In the present study, we evaluated AS transcriptome profiles during P. salmonis infection in the two most used study models, SHK-1 cell line and salmon head kidney tissue. First, the SHK-1 cell line was exposed to P. salmonis infection at 0-, 7-, and 14-days post-infection (dpi). Following, total RNA was extracted for Illumina sequencing.On the other hand, RNA-Seq datasets of Atlantic salmon head kidney infected with the same P. salmonis strayingwase used. For both study models, the highest number of differentially alternative splicing (DAS) events was observed at 7 dpi, 16,830 DAS events derived from 9213 DAS genes in SHK-1 cells, and 13,820 DAS events from 7684 DAS genes in salmon HK. Alternative first exon (AF) was the most abundant AS type in the three infection times analyzed, representing 31% in SHK-1 cells and 228.6 in salmon HK; meanwhile, mutually exclusive exon (MX) was the least abundant. Notably, functional annotation of DAS genes in SHK-1 cells infected with P. salmonis showed a high presence of genes related to nucleotide metabolism. In contrast, the salmon head kidney exhibited many GO terms associated with immune response. Our findings reported the role of AS during P. salmonis infection in Atlantic salmon. These studies would contribute to a better understanding of the molecular bases that support the pathogen-host interaction, evidencing the contribution of AS regulating the transcriptional host response.

Impact of biorefinery processing conditions on the bioactive properties of fucoidan extracts from Saccharina latissima on SHK-1 cells

Fucoidans from brown macroalgae exhibit a wide array of bioactivities, from antioxidant to immunostimulatory effects. Their specific effects and efficacy depend on the molecular weight and structure of the polysaccharide chains, which in turn depends on the seaweed species and the methods used for fucoidan extraction and purification. In this study, a bench scale, three factor, three level full factorial experiment was used to determine the relationship between the extraction parameters (duration, hydrochloric acid concentration in extraction liquor, and temperature), the sulfation level and fucoidan yield. Three of the extractions representing the least severe and most severe extractions as well as a calculated sweet spot were scaled up to a pilot biorefinery (5 kg biomass per extraction). To evaluate the potential of using these complex polysaccharides as functional ingredients in salmon aquaculture, bioactivity of the three different fucoidan preparations was examined by in vitro assays with cultures of Atlantic salmon head kidney cells (SHK-1). All pilot extraction products induced an increase in SHK1 cell viability. In addition, “Too careful” also induced an upregulation of il1-β and sod1, both immune-related biomarkers. Interestingly, the expression of these biomarkers showed a positive correlation in SHK-1 cells stimulated with products from both “Too careful” and “Sweet spot” extractions. The data presented here show the extent of shift in bioactivity resulting from changes in the extraction severity in fucoidan biorefining and present the potential for fucoidan application as an immunostimulant for salmon cells.

Conserved and divergent arms of the antiviral response in the duplicated genomes of salmonid fishes

Antiviral innate immunity is orchestrated by the interferon system, which appeared in ancestors of jawed vertebrates. Interferon upregulation induces hundreds of interferon-stimulated-genes (ISGs) with effector or regulatory functions. Here we investigated the evolutionary diversification of ISG responses through comparison of two salmonid fishes, accounting for the impact of sequential whole genome duplications ancestral to teleosts and salmonids. We analysed the transcriptomic response of the IFN pathway in the head kidney of rainbow trout and Atlantic salmon, which separated 25–30 Mya. We identified a large set of ISGs conserved in both species and cross-referenced them with zebrafish and human ISGs. In contrast, around one-third of salmonid ISG lacked orthologs in human, mouse, chicken or frog, and often between rainbow trout and Atlantic salmon, revealing a fast-evolving, lineage-specific arm of the antiviral response. This study also provides a key resource for in-depth functional analysis of ISGs in salmonids of commercial significance.

Comparative Transcriptomics in Atlantic Salmon Head Kidney and SHK-1 Cell Line Exposed to the Sea Louse Cr-Cathepsin.

The development of vaccines against sea lice in salmon farming is complex, expensive, and takes several years for commercial availability. Recently, transcriptome studies in sea louse have provided valuable information for identifying relevant molecules with potential use for fish vaccines. However, the bottleneck is the in vivo testing of recombinant protein candidates, the dosage, and the polyvalent formulation strategies. This study explored a cell-based approach to prospect antigens as candidate vaccines against sea lice by comparison with immunized fish. Herein, SHK-1 cells and Atlantic salmon head kidney tissue were exposed to the antigen cathepsin identified from the sea louse Caligus rogercresseyi. The cathepsin protein was cloned and recombinantly expressed in Escherichia coli, and then SHK-1 cell lines were stimulated with 100 ng/mL cathepsin recombinant for 24 h. In addition, Atlantic salmons were vaccinated with 30 ug/mL recombinant protein, and head kidney samples were then collected 30 days post-immunization. SHK-1 cells and salmon head kidney exposed to cathepsin were analyzed by Illumina RNA sequencing. The statistical comparisons showed differences in the transcriptomic profiles between SHK-1 cells and the salmon head kidney. However, 24.15% of the differentially expressed genes were shared. Moreover, putative gene regulation through lncRNAs revealed tissue-specific transcription patterns. The top 50 up and downregulated lncRNAs were highly correlated with genes involved in immune response, iron homeostasis, pro-inflammatory cytokines, and apoptosis. Also, highly enriched pathways related to the immune system and signal transduction were shared between both tissues. These findings highlight a novel approach to evaluating candidate antigens for sea lice vaccine development, improving the antigens screening in the SHK-1 cell line model.

Transcriptomic response to ISAV infection in the gills, head kidney and spleen of resistant and susceptible Atlantic salmon

BackgroundInfectious Salmon Anaemia virus (ISAV) is an orthomyxovirus responsible for large losses in Atlantic salmon (Salmo salar) aquaculture. Current available treatments and vaccines are not fully effective, and therefore selective breeding to produce ISAV-resistant strains of Atlantic salmon is a high priority for the industry. Genomic selection and potentially genome editing can be applied to enhance the disease resistance of aquaculture stocks, and both approaches can benefit from increased knowledge on the genomic mechanisms of resistance to ISAV. To improve our understanding of the mechanisms underlying resistance to ISAV in Atlantic salmon we performed a transcriptomic study in ISAV-infected salmon with contrasting levels of resistance to this virus.ResultsThree different tissues (gills, head kidney and spleen) were collected on 12 resistant and 12 susceptible fish at three timepoints (pre-challenge, 7 and 14 days post challenge) and RNA sequenced. The transcriptomes of infected and non-infected fish and of resistant and susceptible fish were compared at each timepoint. The results show that the responses to ISAV are organ-specific; an important response to the infection was observed in the head kidney, with up-regulation of immune processes such as interferon and NLR pathways, while in gills and spleen the response was more moderate. In addition to immune related genes, our results suggest that other processes such as ubiquitination and ribosomal processing are important during early infection with ISAV. Moreover, the comparison between resistant and susceptible fish has also highlighted some interesting genes related to ubiquitination, intracellular transport and the inflammasome.ConclusionsAtlantic salmon infection by ISAV revealed an organ-specific response, implying differential function during the infection. An immune response was observed in the head kidney in these early timepoints, while gills and spleen showed modest responses in comparison. Comparison between resistance and susceptible samples have highlighted genes of interest for further studies, for instance those related to ubiquitination or the inflammasome.

The impact of Piscirickettsia salmonis infection on genome-wide DNA methylation profile in Atlantic Salmon

Salmon rickettsial septicaemia (SRS), caused by the bacteria Piscirickettsia salmonis (P. salmonis), is responsible for significant mortality in farmed Atlantic salmon in Chile. Currently there are no effective treatments or preventive measures for this disease, although genetic selection or genome engineering to increase salmon resistance to SRS are promising strategies. The accuracy and efficiency of these strategies are usually influenced by the available biological background knowledge of the disease. The aim of this study was to investigate DNA methylation changes in response to P. salmonis infection in the head kidney and liver tissue of Atlantic salmon, and the interaction between gene expression and DNA methylation in the same tissues. The head kidney and liver methylomes of 66 juvenile salmon were profiled using reduced representation bisulphite sequencing (RRBS), and compared between P. salmonis infected animals (3 and 9 days post infection) and uninfected controls, and between SRS resistant and susceptible fish. Methylation was correlated with matching RNA-Seq data from the same animals, revealing that methylation in the first exon leads to an important repression of gene expression. Head kidney methylation showed a clear response to the infection, associated with immunological processes such as actin cytoskeleton regulation, phagocytosis, endocytosis and pathogen associated pattern receptor signaling. Our results contribute to the growing understanding of the role of methylation in regulation of gene expression and response to infectious diseases and could inform the incorporation of epigenetic markers into genomic selection for disease resistant and the design of diagnostic epigenetic markers to better manage fish health in salmon aquaculture.

Transcriptomic characterization of Atlantic salmon (Salmo salar) head kidney following administration of Aeromonas salmonicida subsp. masoucida vaccine

Atlantic salmon is one of the most famous and economically important fish species globally. However, bacterial diseases constantly constrain salmon aquaculture. Thereinto, Aeromonas salmonicida subsp. masoucida (ASM), classified as atypical A. salmonicida, caused huge losses to salmonid industry in China. In this regard, we conducted transcriptome analysis in Atlantic salmon head kidney following the administration of ASM vaccination to reveal genes, their expression patterns, and pathways involved in immune responses. A total of 448.71 million clean reads were obtained, and 397.69 million reads were mapped onto the Atlantic salmon reference genome. In addition, 117, 1891, 741, 207, and 377 genes were significantly up-regulated, and 183, 1920, 695, 83, and 539 genes were significantly down-regulated post ASM vaccination at 12 h, 24 h, 1 m, 2 m, and 3 m, respectively. Furthermore, KEGG pathway analysis revealed that many differentially expressed genes (DEGs) following ASM vaccination were involved in cell adhesion molecules (H2-Aa-l and CD28-l),cytokine-cytokine receptor interaction (IL10, CXCL9, CXCL11, CXCR3, and CCL19), herpes simplex infection (IL1B, SOCS3-l, and C3-l), HTLV-I infection (Il1r2 and BCL2L1), influenza A (CXCL8 and Il12b), and PI3K-Akt signaling pathway (PIK3R3-l and Ddit4-l). Finally, the results of qRT-PCR showed a significant correlation with RNA-Seq results, suggesting the reliability of RNA-Seq for gene expression analysis. This study sets the foundation for further study on the vaccine protective mechanism in Atlantic salmon as well as other teleost species.

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

β‐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.

A Bioactive Extract Rich in Triterpenic Acid and Polyphenols from Olea europaea Promotes Systemic Immunity and Protects Atlantic Salmon Smolts Against Furunculosis.

In the present study, the modulation of the transcriptional immune response (microarray analysis) in the head kidney (HK) of the anadromous fish Atlantic salmon (Salmo salar) fed a diet supplemented with an olive fruit extract (AQUOLIVE®) was evaluated. At the end of the trial (133 days), in order to investigate the immunomodulatory properties of the phytogenic tested against a bacterial infection, an in vivo challenge with Aeromonas salmonicida was performed. A total number of 1,027 differentially expressed genes (DEGs) (805 up- and 222 downregulated) were found when comparing the transcriptomic profiling of the HK from fish fed the control and AQUOLIVE® diets. The HK transcripteractome revealed an expression profile that mainly favored biological processes related to immunity. Particularly, the signaling of i-kappa B kinase/NF-kappa and the activation of leukocytes, such as granulocytes and neutrophils degranulation, were suggested to be the primary actors of the innate immune response promoted by the tested functional feed additive in the HK. Moreover, the bacterial challenge with A. salmonicida that lasted 12 days showed that the cumulative survival was higher in fish fed the AQUOLIVE® diet (96.9 ± 6.4%) than the control group (60.7 ± 13.5%). These results indicate that the dietary supplementation of AQUOLIVE® at the level of 0.15% enhanced the systemic immune response and reduced the A. salmonicida cumulative mortality in Atlantic salmon smolts.

Chitosan nanoparticle formulation attenuates poly (I:C) induced innate immune responses against inactivated virus vaccine in Atlantic salmon (Salmo salar)

Many vaccine formulations, in particular vaccines based on inactivated virus, needs adjuvants to boost immunogenicity. In aquaculture, mineral and plant oil are used as adjuvant in commercial vaccines, and the advent of oil-adjuvanted vaccines was crucial to aquaculture development. Nevertheless, some of these approved vaccines display suboptimal performance in the field compared to experimental conditions. Therefore, there is a need to improve adjuvants and delivery methods for fish vaccines against viruses. We used RNA sequencing of Atlantic salmon head kidney to analyse the difference in gene expression 24 h after injection of different experimental vaccine formulations. We compared five different formulations in addition to a PBS control: inactivated virus alone (group V), soluble poly (I:C) (group P), nanoparticles containing poly (I:C) (group N), soluble poly (I:C) + inactivated virus (group PV) and finally nanoparticles containing poly (I:C) + inactivated virus (group NV). Our results showed poly (I:C)’s ability as adjuvant and its capacity influence innate immune genes expression in Atlantic salmon. Soluble poly (I:C) upregulated multiple immune related genes and was more effective compared to poly (I:C) formulated into chitosan nanoparticles (more than 10 fold increase in differentially expressed genes, DEGs). However, inclusion of inactivated ISA virus in the nanoparticle vaccine, increased the number of DEGs fivefold suggesting a synergistic effect of adjuvant and antigen. Our results indicate that the way poly (I:C) is formulated and the presence of antigen is important for the magnitude of the innate immune response in Atlantic salmon.

Nutrient Digestibility, Growth, Mucosal Barrier Status, and Activity of Leucocytes From Head Kidney of Atlantic Salmon Fed Marine- or Plant-Derived Protein and Lipid Sources.

Nutrient digestibility, growth, and mucosal barrier status of fish skin, gills, and distal intestine were studied in Atlantic salmon fed feeds based on marine or plant-derived ingredients. The barrier status was assessed by considering the expression of four mucin genes, five genes that encode antimicrobial proteins, distal intestine micromorphology, and design-based stereology of the midgut epithelium. In addition, the head kidney leukocytes were examined using flow cytometry; to understand the differences in their counts and function. Five experimental feeds containing the main components i) fishmeal and fish oil (BG1), ii) soybean meal (BG2; to induce enteritis), iii) fishmeal as the main protein source and rapeseed oil as the main lipid source (BG3), iv) a mix of plant protein concentrates as the protein sources and fish oil as the lipid source (BG4), and v) plant and marine ingredients in the ratio 70:30 (BG5) were produced for the study. Atlantic salmon with initial weight 72.7 ± 1.2 g was offered the experimental feeds for 65 days. The results revealed that the weights of all fish groups doubled, except for fish fed BG2. Fish fed the BG2 diet had lower blood cholesterol concentration, developed enteritis, had lower expression of muc2 in the distal intestine, and had a compromised barrier status in the intestine. Expression of both the mucin genes and genes that encode antimicrobial peptides were tissue-specific and some were significantly affected by diet. The fish fed BG1 and BG3 had more head kidney lymphocyte-like cells compared to BG5-fed fish, and the phagocytic activity of macrophage-like cells from the head kidney was the highest in fish fed BG1. The intestinal micromorphology and the mucosal mapping suggest two different ways by which plant-based diets can alter the gut barrier status; by either reducing the mucous cell sizes, volumetric densities and barrier status (as noted for BG2) or increasing volumetric density of mucous cells (as observed for BG4 and BG5). The results of the compromised intestinal barrier in fish fed plant ingredients should be further confirmed through transcriptomic and immunohistochemical studies to refine ingredient composition for sustainable and acceptable healthy diets.

Influence of Dietary Long-Chain Polyunsaturated Fatty Acids and ω6 to ω3 Ratios on Head Kidney Lipid Composition and Expression of Fatty Acid and Eicosanoid Metabolism Genes in Atlantic Salmon (Salmo salar).

The interaction of dietary eicosapentaenoic acid and docosahexaenoic acid (EPA+DHA) levels with omega-6 to omega-3 ratios (ω6:ω3), and their impact on head kidney lipid metabolism in farmed fish, are not fully elucidated. We investigated the influence of five plant-based diets (12-week exposure) with varying EPA+DHA levels (0.3, 1.0, or 1.4%) and ω6:ω3 (high ω6, high ω3, or balanced) on tissue lipid composition, and transcript expression of genes involved in fatty acid and eicosanoid metabolism in Atlantic salmon head kidney. Tissue fatty acid composition was reflective of the diet with respect to C18 PUFA and MUFA levels (% of total FA), and ω6:ω3 (0.5–1.5). Fish fed 0.3% EPA+DHA with high ω6 (0.3% EPA+DHA↑ω6) had the highest increase in proportions (1.7–2.3-fold) and in concentrations (1.4-1.8-fold) of arachidonic acid (ARA). EPA showed the greatest decrease in proportion and in concentration (by ~½) in the 0.3% EPA+DHA↑ω6 fed fish compared to the other treatments. However, no differences were observed in EPA proportions among salmon fed the high ω3 (0.3 and 1.0% EPA+DHA) and balanced (1.4% EPA+DHA) diets, and DHA proportions were similar among all treatments. Further, the transcript expression of elovl5a was lowest in the 0.3% EPA+DHA↑ω6 fed fish, and correlated positively with 20:3ω3, 20:4ω3 and EPA:ARA in the head kidney. This indicates that high dietary 18:3ω3 promoted the synthesis of ω3 LC-PUFA. Dietary EPA+DHA levels had a positive impact on elovl5a, fadsd5 and srebp1 expression, and these transcripts positively correlated with tissue ΣMUFA. This supported the hypothesis that LC-PUFA synthesis is positively influenced by tissue MUFA levels in Atlantic salmon. The expression of pparaa was higher in the 0.3% EPA+DHA↑ω6 compared to the 0.3% EPA+DHA↑ω3 fed fish. Finally, significant correlations between head kidney fatty acid composition and the expression of eicosanoid synthesis-related transcripts (i.e., 5loxa, 5loxb, cox1, cox2, ptges2, ptges3, and pgds) illustrated the constitutive relationships among fatty acids and eicosanoid metabolism in salmon.

Characterization of miRNAs in Extracellular Vesicles Released From Atlantic Salmon Monocyte-Like and Macrophage-Like Cells.

Cell-derived extracellular vesicles (EVs) participate in cell-cell communication via transfer of molecular cargo including genetic material like miRNAs. In mammals, it has previously been established that EV-mediated transfer of miRNAs can alter the development or function of immune cells, such as macrophages. Our previous research revealed that Atlantic salmon head kidney leukocytes (HKLs) change their morphology, phagocytic ability and miRNA profile from primarily “monocyte-like” at Day 1 to primarily “macrophage-like” at Day 5 of culture. Therefore, we aimed to characterize the miRNA cargo packaged in EVs released from these two cell populations. We successfully isolated EVs from Atlantic salmon HKL culture supernatants using the established Vn96 peptide-based pull-down. Isolation was validated using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. RNA-sequencing identified 19 differentially enriched (DE) miRNAs packaged in Day 1 versus Day 5 EVs. Several of the highly abundant miRNAs, including those that were DE (e.g. ssa-miR-146a, ssa-miR-155 and ssa-miR-731), were previously identified as DE in HKLs and are associated with macrophage differentiation and immune response in other species. Interestingly, the abundance relative of the miRNAs in EVs, including the most abundant miRNA (ssa-miR-125b), was different than the miRNA abundance in HKLs, indicating selective packaging of miRNAs in EVs. Further study of the miRNA cargo in EVs derived from fish immune cells will be an important next step in identifying EV biomarkers useful for evaluating immune cell function, fish health, or response to disease.

Growth hormone (GH) and growth hormone release factor (GRF) modulate the immune response in the SHK-1 cell line and leukocyte cultures of head kidney in Atlantic salmon

To clarify the role of growth hormone (GH) in the immune system of fish, we examine the comparative effect of GH and Growth Hormone Release Factor (GRF) on leukocytes culture of the head kidney of Atlantic salmon and the SHK-1 cell line. There are studies that associate the growth hormone (GH) / insulin-like growth factor (IGF) axis with the immune regulation of fish. However, there is no evidence that GH and GRF stimulate Atlantic salmon leukocyte cell lines, where there areńt reports on expression changes in different immune response markers. Thus, we investigated the effect of GH and GRF in Atlantic salmon leukocytes extracted from head kidney and the SHK-1 cell line on the different immune response markers such as: NLRC5, NLRC3, IL-1β, TNF-α, and IL-8 through qPCR. Our data suggest that GH increases the expression of NLRC5, NLRC3, and IL-1β mainly at 16 h post-stimulation in Atlantic salmon leukocytes. This indicates differential regulation between the two models used, helping us to better understand the independent action of GH on the immune system and the GH / IGF axis for future research.