A genetic method to infer ploidy and aberrant inheritance in triploid organisms.

Polyploidy occurs naturally across eukaryotic lineages and has been harnessed in the domestication of many crops and vertebrates. In aquaculture, triploidy can be induced as a biocontainment strategy, as it creates a reproductive barrier preventing farm-to-wild introgression, which is currently a major conservation issue for the industry. However, recent work suggests that triploidisation protocols may, on occasion, produce 'failed triploids' displaying diploidy, aneuploidy and aberrant inheritance. The potentially negative consequences for conservation and animal welfare motivate the need for methods to evaluate the success of ploidy-manipulation protocols early in the production process. We developed a semi-automated version of the MAC-PR (microsatellite DNA allele counting - peak ratios) method to resolve the allelic configuration of large numbers of individuals across a panel of microsatellite markers that can be used to infer ploidy, pedigree and inheritance aberrations. We demonstrate an application of the approach using material from a series of Atlantic salmon (Salmo salar) breeding experiments where ploidy was manipulated using a hydrostatic pressure treatment. We validated the approach to infer ploidy against blood smears, finding a > 99% agreement between these methods, and demonstrate its potential utility to infer ploidy as early as the embryonic stage. Furthermore, we present tools to assign diploid and triploid progeny to families and to detect aberrant inheritance, which may be useful for breeding programmes that utilise ploidy manipulation techniques. The approach adds to the ploidy verification toolbox. The increased precision in detecting ploidy and inheritance aberrations will facilitate the ability of triploidisation programmes to prevent farm-to-wild introgression.

Effects of dietary fish to rapeseed oil ratio on steatosis symptoms in Atlantic salmon (Salmo salar L) of different sizes.

Choline is recognized as an essential nutrient for Atlantic salmon at all developmental stages. However, its dietary requirement is not well defined. Choline plays a critical role in lipid transport, and the clearest deficiency sign is intestinal steatosis. The present work, aiming to find whether lipid source and fish size may affect steatosis symptoms, was one of a series of studies conducted to identify which production-related conditions may influence choline requirement. Six choline-deficient diets were formulated varying in ratios of rapeseed oil to fish oil and fed to Atlantic salmon of 1.5 and 4.5 kg. After eight weeks, somatic characteristics were observed, and the severity of intestinal steatosis was assessed by histological, biochemical, and molecular analyses. Fatty acid composition in pyloric intestine, mesenteric tissue, and liver samples was also quantified. The increasing rapeseed oil level increased lipid digestibility markedly, enhancing lipid supply to the fish. Moreover, small fish consumed more feed, and consequently had a higher lipid intake. In conclusion, the results showed that choline requirement depends on dietary lipid load, which depends on the fatty acid profile as well as the fish size.

Development and validation of reverse-transcription cross-priming amplification-based lateral flow assay for the detection of infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) severely and lethally infects salmonid fish, including Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) worldwide. Rapid and accurate viral detection is crucial for preventing pathogen spread and minimizing damage. Although several IHNV detection assays have been developed, their analytical and diagnostic performances have not been evaluated and field usability assessments have not been completely validated. Here, we developed a reverse-transcription cross-priming amplification-based lateral flow assay (RT-CPA-LFA) and validated its diagnostic performance. To detect the IHNV, primers were designed based on the consensus sequence of the nucleocapsid (N) gene. Notably, when combined with a lateral flow dipstick, it could visualize the IHNV amplification products within 5 min and the detection limit of the developed RT-CPA-LFA was 3.28×105 copies/µL. The diagnostic sensitivity and specificity in fish samples (n=140) were 98.88 % and 96.08 %, respectively. Moreover, the IHNV detection rate by RT-CPA-LFA in dead rainbow trout artificially injected with the virus was 100 %, consistent with to the results obtained from second conventional and real-time PCR, indicating its applicability for rapid IHNV detection and presumptive IHN diagnosis during the endemic period.

Major vault protein is part of an extracellular cement material in the Atlantic salmon louse (Lepeophtheirus salmonis).

Major vault protein (MVP) is the main component of the vault complex, which is a highly conserved ribonucleoprotein complex found in most eukaryotic organisms. MVP or vaults have previously been found to be overexpressed in multidrug-resistant cancer cells and implicated in various cellular processes such as cell signaling and innate immunity. The precise function of MVP is, however, poorly understood and its expression and probable function in lower eukaryotes are not well characterized. In this study, we report that the Atlantic salmon louse expresses three full-length MVP paralogues (LsMVP1-3). Furthermore, we extended our search and identified MVP orthologues in several other ecdysozoan species. LsMVPs were shown to be expressed in various tissues at both transcript and protein levels. In addition, evidence for LsMVP to assemble into vaults was demonstrated by performing differential centrifugation. LsMVP was found to be highly expressed in cement, an extracellular material produced by a pair of cement glands in the adult female salmon louse. Cement is important for the formation of egg strings that serve as protective coats for developing embryos. Our results imply a possible novel function of LsMVP as a secretory cement protein. LsMVP may play a role in structural or reproductive functions, although this has to be further investigated.

The quality of frozen-thawed Atlantic salmon (Salmo salar L.) fillets as affected by sub-chilling before freezing.

BACKGROUND: To maintain the quality of frozen Atlantic salmon after thawing and highlight the potential for moving from air fright to boat for long-distance export, a study was designed to investigate the effects of sub-chilling before rapid freezing on the quality of thawed fillets. Atlantic salmon chilled on wet ice before filleting and freezing was used as a control for the experimental factor chilling, whereas fresh fillets were used for the frozen-thawed samples. RESULTS: The pre-freezing chilling method interacted with the storage protocol and significantly affected the product. For fresh stored fillets, sub-chilling improved the microbiological and textural stability and degradation of proteins. After 1 month of frozen storage, sub-chilled fillets gave better color and textural properties, less adenosine triphosphate degradation and protein denaturation. In addition, sub-chilled 4-month-frozen fillets also showed improved microbial stability compared to those initially chilled with ice before frozen storage. Quality was lost as a function of storage. Fresh fillets generally had higher bacterial counts, surface breaking force, firmness, hue and contents of inosine monophosphate, and lower drip loss and inosine (HxR) levels than those stored frozen-thawed. Moreover, 4-month-frozen fillets had higher HxR levels and lower psychrotrophic viable count growth than those that were frozen for 1 month. The time fillets were stored frozen did not profoundly affect their quality. CONCLUSION: It is concluded that a frozen product might be competitive with a fresh product when sub-chilling is performed before freezing, especially when including the environmental benefits of frozen export by boat rather than air freight. © 2024 The Author(s). Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Quality Assessment of Fish Oil Obtained after Enzymatic Hydrolysis of a Mixture of Rainbow Trout (Oncorhynchus mykiss) and Atlantic Salmon (Salmo salar) Rest Raw Material Pretreated by High Pressure.

Utilization of fish rest raw material for fish oil extraction has received interest with the increasing demand for sustainable food sources. Enzymatic hydrolysis is an efficient method for the extraction of value-added compounds, but its effectiveness may be enhanced by high-pressure processing (HPP). However, HPP can induce lipid oxidation, affecting the quality of the oil. This study aimed to evaluate the quality of fish oil obtained after enzymatic hydrolysis of a mixture of rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) rest raw material pretreated by HPP. Six pretreatments were tested prior to enzymatic hydrolysis; 200 MPa × 4 min, 200 MPa × 8 min, 400 MPa × 4 min, 400 MPa × 8 min, 600 MPa × 4 min, and 600 MPa × 8 min. The oil samples were analyzed for lipid oxidation parameters, free fatty acid content, fatty acid composition, and color changes over 8 weeks. The results confirmed that HPP may induce lipid oxidation and revealed significant influence of HPP parameters on lipid oxidation, with higher pressures leading to increased oxidation. Fatty acid composition varied among samples, but it was not substantially affected by HPP.

Inclusion of oil from transgenic Camelina sativa in feed effectively supplies EPA and DHA to Atlantic salmon (Salmo salar) grown to market size in seawater pens.

Atlantic salmon were fed either a diet reflecting current commercial feeds with added oil supplied by a blend of fish oil and rapeseed oil (COM), or a diet formulated with oil from transgenic Camelina sativa containing 20% EPA + DHA (TCO). Salmon were grown from smolt to market size (>3 kg) in sea pens under semi-commercial conditions. There were no differences in growth, feed efficiency or survival between fish fed the TCO or COM diets at the end of the trial. Levels of EPA + DHA in flesh of salmon fed TCO were significantly higher than in fish fed COM. A 140 g fillet from TCO-fed salmon delivered 2.3 g of EPA + DHA, 67% of the weekly requirement level recommended by many health agencies, and 1.5-fold more than the 1.5 g of EPA + DHA for COM-fed fish. Oil from transgenic Camelina supported growth and improved the nutritional quality of farmed salmon in terms of increased "omega-3" supply for human consumers.

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 gill epithelial cell line ASG-10, an in vitro model for studying effects of microplastics in gills.

Microplastics are ubiquitous environmental pollutants frequently detected in aquatic environments. Here we used the Atlantic salmon epithelial gill cell line (ASG-10) to investigate the uptake and effects of polystyrene (PS) microplastic. The ASG-10 cell line has phagocytotic/endocytic capacities and can take up clear PS particles at 0.2 and 1.0 µm, while PS at 10 µm was not taken up. As a response to the uptake, the ASG-10 cells increased their lysosomal activity. Furthermore, no effects on the mitochondria were found, neither on the mitochondrial membrane potential nor the mitochondria morphology (branch length and diameter). Interestingly, even a very high concentration of PS (200 µg/ml) with all tested particle sizes had no effects on cell viability or cell cycle. The environmental toxin Benzo(a)pyrene (B(a)P), a known inducer of CYP1A, is highly hydrophobic and thus sticks to the PS particles. However, co-exposure of B(a)P and PS the particles did not increase the induction of CYP1A activity compared to B(a)P alone. Our study contributes to the understanding of the cellular effects of PS particles using a highly relevant Atlantic salmon gill epithelium in vitro model.

Effects of aquaculture effluents on the slender sea pen Virgularia mirabilis.

This study aims to assess in situ the impact of effluents originating from an Atlantic salmon (Salmo salar) farm on a nearby slender sea pen (Virgularia mirabilis) field. We evidenced (1) the presence and persistence of emamectin residues (i.e. a common chemotherapeutants used for treating ectoparasites in salmons) in V. mirabilis tissue 56 days after treatment and (2) lethal and sublethal responses of V. mirabilis to effluents discharged by the salmon farm. Particularly, sea pens near the fish farm exhibited significant overproduction of mucus, contraction of polyps' tentacles, and disappearance of associated fauna. Furthermore, sea pens located directly underneath the farm showed substantial tissue necrosis and, in the most severe case, complete tissue loss and mortality. Our results suggest that lethal damages on sea pens occur directly below the farm, and that sublethal effects are visible up to 500 m from the farm. However, the presence of V. mirabilis below the studied farm, which has been active for more than twenty years, suggests that V. mirabilis population possesses the capacity to recover from the impacts of the farm, thereby preventing the complete disappearance from the area. In this context, it would be particularly interesting to run a temporal survey following the health state of V. mirabilis during an entire production cycle to have a more precise overview of fish farm impacts on this species, including during and after the post-production fallowing period.

Glucoregulatory Properties of a Protein Hydrolysate from Atlantic Salmon (Salmo salar): Preliminary Characterization and Evaluation of DPP-IV Inhibition and Direct Glucose Uptake In Vitro.

Metabolic disorders are increasingly prevalent conditions that manifest pathophysiologically along a continuum. Among reported metabolic risk factors, elevated fasting serum glucose (FSG) levels have shown the most substantial increase in risk exposure. Ultimately leading to insulin resistance (IR), this condition is associated with notable deteriorations in the prognostic outlook for major diseases, including neurodegenerative diseases, cancer risk, and mortality related to cardiovascular disease. Tackling metabolic dysfunction, with a focus on prevention, is a critically important aspect for human health. In this study, an investigation into the potential antidiabetic properties of a salmon protein hydrolysate (SPH) was conducted, focusing on its potential dipeptidyl peptidase-IV (DPP-IV) inhibition and direct glucose uptake in vitro. Characterization of the SPH utilized a bioassay-guided fractionation approach to identify potent glucoregulatory peptide fractions. Low-molecular-weight (MW) fractions prepared by membrane filtration (MWCO = 3 kDa) showed significant DPP-IV inhibition (IC50 = 1.01 ± 0.12 mg/mL) and glucose uptake in vitro (p ≤ 0.0001 at 1 mg/mL). Further fractionation of the lowest MW fractions (<3 kDa) derived from the permeate resulted in three peptide subfractions. The subfraction with the lowest molecular weight demonstrated the most significant glucose uptake activity (p ≤ 0.0001), maintaining its potency even at a dilution of 1:500 (p ≤ 0.01).

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.

Differential expression of immune-related biomarkers in primary cultures from Atlantic salmon (Salmo salar) exposed to processed Paecilomyces variotii with or without inactivated Moritella viscosa.

Paecilomyces variotii (a filamentous fungus), is a promising novel protein source in fish feeds due to its high nutritional value. Also, P. variotii has Microbial-Associated Molecular Patterns (MAMPs) such as glucans and nucleic acids that could modulate the host's immune response. To understand the potential bioactive properties of this fungus in Atlantic salmon (Salmo salar), our study was conducted to evaluate the gene expression of immune-related biomarkers (e.g., cytokines, effector molecules and receptors) on primary cultures from salmon head kidney (HKLs) and spleen leukocytes (SLs) exposed to either UV inactivated or fractions from P. variotii with or without inactivated Moritella viscosa (a skin pathogen in salmonids). Moreover, the effect of the fermentation conditions and down-stream processing on the physical ultrastructure and cell wall glucan content of P. variotii was characterized. The results showed that drying had a significant effect on the cell wall ultrastructure of the fungi and the choice of fermentation has a significant effect on the quantity of ß-glucans in P. variotii. Furthermore, stimulating Atlantic salmon HKLs and SLs with P. variotii and its fractions induced gene expression related to pro-inflammatory (tnfα, il1ß) and antimicrobial response (cath2) in HKLs, while response in SLs was related to both pro-inflammatory and regulatory response (tnfα, il6 and il10). Similarly, the stimulation with inactivated M. viscosa alone led to an up-regulation of genes related to pro-inflammatory (tnfα, il1ß, il6) antimicrobial response (cath2), intra-cellular signalling and recognition of M. viscosa (sclra, sclrb) and a suppression of regulatory response (il10) in both HKLs and SLs. Interestingly, the co-stimulation of cells with P. variotii and M. viscosa induced immune homeostasis (il6, tgfß) and antimicrobial response (cath2) in SLs at 48h. Thus, P. variotii induces immune activation and cellular communication in Atlantic salmon HKLs and SLs and modulates M. viscosa induced pro-inflammatory responses in SLs. Taken together, the results from physical and chemical characterization of the fungi, along with the differential gene expression of key immune biomarkers, provides a theoretical basis for designing feeding trials and optimize diets with P. variotii as a functional novel feed ingredient for Atlantic salmon.

The melano-macrophage: The black leukocyte of fish immunity.

Melanin and the process of melanin synthesis or melanogenesis have central roles in the immune system of insects, and production of melanin-synthesizing enzymes from their haemocytes may be induced following activation through danger signals. Melanin-containing macrophage-like cells have been extensively studied in amphibians and they are also present in reptiles. In fish, melano-macrophages are especially recognized with respect to melano-macrophage centres (MMCs), hypothesized to be analogues of germinal centres in secondary lymphoid organs of mammals and some birds. Melano-macrophages are in addition present in several inflammatory conditions, in particular melanised focal changes, or black spots, in the musculature of farmed Atlantic salmon, Salmo salar. Melanins are complex compounds that may be divided into different forms which all have the ability to absorb and scatter light. Other functions include the quenching of free radicals and a direct effect on the immune system. According to the common view held in the pigment cell community, vertebrate melanin synthesis with melanosome formation may only occur in cells of ectodermal origin. However, abundant information suggests that also myeloid cells of ectothermic vertebrates may be classified as melanocytes. Here, we discuss these opposing views and review relevant literature. Finally, we review the current status on the research concerning melanised focal muscle changes that represent the most severe quality problem in Norwegian salmon production, but also other diseases where melano-macrophages play important roles.

No additional stress of sublethal gas supersaturation in a landlocked population of Atlantic salmon (Salmo salar) exposed to environmental acidification.

The landlocked Atlantic salmon population "bleke" faces extinction due to environmental acidification (EA) and hydropower expansion in the Norwegian river Otra. Despite of restoration, unexpected mortality has been reported for this population, possibly due to gas bubble trauma (GBT) from gas supersaturation (GSS) downstream of hydroelectric plants, or EA induced aluminum toxicity. In this study, we applied the allostasis concept to investigate interactions between EA and GBT. This concept comprises additive effects of stressors, which can lead to allostatic overload. Stress coping mechanisms become maladaptive in such situations, which can be indicated by an inability to mount a proper cortisol response in fish. Fish were exposed to sublethal levels of simulated EA (SEA), GSS (a total gas pressure; TGP; of 110%) or a combination of these stressors for six days. Effects on allostatic load were subsequently investigated by assessing the cortisol response to an acute stress test. SEA increased cortisol responsiveness and GSS induced clinical signs of GBT, but no interacting effects between GSS and SEA were observed. This suggests that that 110% TGP did not have an additive effect on the allostatic load imposed by SEA.

Biocompatibility of intraperitoneally implanted TEMPO-oxidized cellulose nanofiber hydrogels for antigen delivery in Atlantic salmon (Salmo salar L.) vaccines.

Disease outbreaks are a major impediment to aquaculture production, and vaccines are integral for disease management. Vaccines can be expensive, vary in effectiveness, and come with adjuvant-induced adverse effects, causing fish welfare issues and negative economic impacts. Three-dimensional biopolymer hydrogels are an appealing new technology for vaccine delivery in aquaculture, with the potential for controlled release of multiple immunomodulators and antigens simultaneously, action as local depots, and tunable surface properties. This research examined the intraperitoneal implantation of a cross-linked TEMPO cellulose nanofiber (TOCNF) hydrogel formulated with a Vibrio anguillarum bacterin in Atlantic salmon with macroscopic and microscopic monitoring to 600-degree days post-implantation. Results demonstrated a modified passive integrated transponder tagging (PITT) device allowed for implantation of the hydrogel. However, the Atlantic salmon implanted with TOCNF hydrogels exhibited a significant foreign body response (FBR) compared to sham-injected negative controls. The FBR was characterized by gross and microscopic external and visceral proliferative lesions, granulomas, adhesions, and fibrosis surrounding the hydrogel using Speilberg scoring of the peritoneum and histopathology of the body wall and coelom. Acutely, gross monitoring displayed rapid coagulation of blood in response to the implantation wound with development of fibrinous adhesions surrounding the hydrogel by 72 h post-implantation consistent with early stage FBR. While these results were undesirable for aquaculture vaccines, this work informs on the innate immune response to an implanted biopolymer hydrogel in Atlantic salmon and directs future research using cellulose nanomaterial formulations in Atlantic salmon for a new generation of aquaculture vaccine technology.

The use of salmonid epithelial cells to characterize the toxicity of Tenacibaculum maritimum soluble extracellular products.

AIMS: This study assessed how the etiological agent of mouth rot in farmed Atlantic salmon, Tenacibaculum maritimum, induces toxicity in host salmonid barrier cells, and determined whether environmental changes are relevant for these effects. METHODS AND RESULTS: Tenacibaculum maritimum soluble extracellular products (ECPs) were collected and used to treat Atlantic salmon and rainbow trout intestinal barrier cell lines as a comparative model of bacterial-salmonid cell interactions. Cellular assays that examine cell membrane integrity, marker expression, and metabolic activity revealed that T. maritimum ECPs induced salmonid epithelial cell death through an apoptosis mechanism. Changes in salinity (25, 29, and 33 ppt) and temperature (12°C, 18°C, and 24°C) within the natural ranges observed in Pacific Northwest aquaculture facilities affected bacterial growth and cytotoxicity of T. maritimum ECPs. CONCLUSIONS: Our results suggest epithelial barriers as targets of T. maritimum-mediated toxicity in farmed mouth rot-infected Atlantic salmon. The induction of apoptosis by T. maritimum soluble ECPs may also help to explain the absence of overt inflammation typically reported for these fish.

Gill epithelial cell line ASG-10 from Atlantic salmon as a new research tool for solving water quality challenges in aquaculture.

Here we evaluated the gill epithelial cell line ASG-10 from Atlantic salmon, as an in vitro model for research on known water quality challenges in aquaculture. Ammonia/ammonium (NH3/NH4+), a recognized challenge in water-intensive recirculating aquaculture systems (RAS), induced lysosomal vacuolization, reduced protein degradation and cell migration of the ASG-10 cells. Aluminium (Aln+), another challenge in freshwater aquaculture facilities had only minor effects. Next, we investigated the tolerance for direct water exposure of ASG-10. The cells tolerated water with osmolarity between 169 and 419 mOsmol/kg for 24 h. However, cells exposed for 3 h to water at 863 mOsmol/kg changed cellular morphology and induced gene expression related to stress (gpx1, casp3, hsp70), and after 24 h exposure cellular viability was severely reduced. Nevertheless, when the cells were grown in transwell inserts, they tolerated 863 mOsmol/kg for 3 h and induction of stress response associated genes was considerably reduced. Lastly, the ASG-10 cells were exposed to water samples, with no known quality issues, from different aquaculture facilities. The cells showed no differences in viability or morphology compared to their representative control. In conclusion, the ASG-10 cell line is a promising in vitro model to study water quality challenges and whole water samples.

Design and functional characterization of Salmo salar TLR5 agonist peptides derived from high mobility group B1 acidic tail.

Toll-like receptor 5 (TLR5) responds to the monomeric form of flagellin and induces the MyD88-depending signaling pathway, activating proinflammatory transcription factors such as NF-κB and the consequent induction of cytokines. On the other hand, HMGB1 is a highly conserved non-histone chromosomal protein shown to interact with and activate TLR5. The present work aimed to design and characterize TLR5 agonist peptides derived from the acidic tail of Salmo salar HMGB1 based on the structural knowledge of the TLR5 surface using global molecular docking platforms. Peptide binding poses complexed on TLR5 ectodomain model from each algorithm were filtrated based on docking scoring functions and predicted theoretical binding affinity of the complex. Circular dichroism spectra were recorded for each peptide selected for synthesis. Only intrinsically disordered peptides (6W, 11W, and SsOri) were selected for experimental functional assay. The functional characterization of the peptides was performed by NF-κB activation assays, RT-qPCR gene expression assays, and Piscirickettsia salmonis challenge in SHK-1 cells. The 6W and 11W peptides increased the nuclear translation of p65 and phosphorylation. In addition, the peptides induced the expression of genes related to the TLR5 pathway activation, pro- and anti-inflammatory response, and differentiation and activation of T lymphocytes towards phenotypes such as TH1, TH17, and TH2. Finally, it was shown that the 11W peptide protects immune cells against infection with P. salmonis bacteria. Overall, the results indicate the usefulness of novel peptides as potential immunostimulants in salmonids.

Cell atlas of the Atlantic salmon spleen reveals immune cell heterogeneity and cell-specific responses to bacterial infection.

The spleen is a conserved secondary lymphoid organ that emerged in parallel to adaptive immunity in early jawed vertebrates. Recent studies have applied single cell transcriptomics to reveal the cellular composition of spleen in several species, cataloguing diverse immune cell types and subpopulations. In this study, 51,119 spleen nuclei transcriptomes were comprehensively investigated in the commercially important teleost Atlantic salmon (Salmo salar L.), contrasting control animals with those challenged with the bacterial pathogen Aeromonas salmonicida. We identified clusters of nuclei representing the expected major cell types, namely T cells, B cells, natural killer-like cells, granulocytes, mononuclear phagocytes, endothelial cells, mesenchymal cells, erythrocytes and thrombocytes. We discovered heterogeneity within several immune lineages, providing evidence for resident macrophages and melanomacrophages, infiltrating monocytes, several candidate dendritic cell subpopulations, and B cells at distinct stages of differentiation, including plasma cells and an igt + subset. We provide evidence for twelve candidate T cell subsets, including cd4+ T helper and regulatory T cells, one cd8+ subset, three γδT subsets, and populations double negative for cd4 and cd8. The number of genes showing differential expression during the early stages of Aeromonas infection was highly variable across immune cell types, with the largest changes observed in macrophages and infiltrating monocytes, followed by resting mature B cells. Our analysis provides evidence for a local inflammatory response to infection alongside B cell maturation in the spleen, and upregulation of ccr9 genes in igt + B cells, T helper and cd8+ cells, and monocytes, consistent with the recruitment of immune cell populations to the gut to deal with Aeromonas infection. Overall, this study provides a new cell-resolved perspective of the immune actions of Atlantic salmon spleen, highlighting extensive heterogeneity hidden to bulk transcriptomics. We further provide a large catalogue of cell-specific marker genes that can be leveraged to further explore the function and structural organization of the salmonid immune system.