About the project

• Background

  Norway is the world’s largest producer of Atlantic salmon and a driver of global aquaculture technology. Today, salmon production is Norway’s number two export in value and the country wants to increase its salmon production from approximately 2 million tons per year to 5 million tons by 2050.

  To ensure sustainable growth in the sector, several challenges need to be solved. One of these is that salmon is faced with multi-stressor conditions during their production cycle, such as high-water temperatures, low oxygen levels, unbalanced nutrition and dietary related health disorders, pathogens outbreaks, and handling during sea-lice treatment and seawater transfer.

  All this can result in poor growth performance, large losses by fish mortality and poor profitability. To help overcome these issues, the Resilient salmon project is using an immuno-nutritional approach where salmon are fed novel functional health-beneficial feeds according to a specific nutritional program with the aim to achieve targeted immune responses such as trained immunity.

• Goal

  The RESILIENT SALMON project is about developing a more robust salmon that can better handle multi-stressor conditions by use of functional feeds combined with nutritional programming to induce trained immunity

• The research

  Resilient salmon project is a spin-off project to Foods of Norway, a Centre for research-based Innovation, where we develop bioactive components from microbial ingredients (e.g. yeast or filamentous fungi) or brown seaweed extracts from brown seaweed such as fucoidan^[1-2] for use in functional feeds.

  These are evaluated in the project with the aim to improve health and robustness of salmon during the stress-full period of seawater transfer

  

  Figure 1. Workflow for RESILIENT SALMON.

  
  In vitro screening (WP1) and targeted delivery (WP2) of bioactive components selected in WP1. In vivo studies (WP3) of A) interactions between diets and fish family groups differing in robustness in fresh water and sea water (WP3.1), B) nutritional programming for improved immunity (WP3.2). Evaluation of the most promising treatment and nutritional program in an infection pathogen challenge trial (WP4).

  Samples collected from WP3 trials will be subjected to a wide range of host phenotyping (histopathology, immunophenotyping, transcriptomics, proteomics) and (WP5) metabolomics as well as metagenomics and metaproteomics of gut microbiota will be performed  to reveal factors determining nutritional effects on salmon developmental biology, environmental requirements and coping strategies in the various life stages.

  In WP6  results will be disseminated and communicated to academia, industry, and society.

• Results

  In vitro screening

  To select most promising bioactive components for the salmon trials, we first screen different microbial ingredients, additives and seaweed extracts (developed in Foods of Norway or by Lallemand AS) in cell lines or primary cells from salmonids to determine potential immunostimulant properties. Based on in vitro data, we selected two promising yeast products from Debaryomyces hansenii yeast and fucoidan extract from sugar kelp for a series of trials with Atlantic salmon^[1].

  The novel D. hansenii yeasts products were either derived from marine (LAN 4) or dairy origin (LAN 6) and they differ in their content and structure of their cell wall constituents (e.g., α-glucans, β-glucans, and mannans)^[3]. The most promising fucoidan product was selected based on its ability to modulate immune-related biomarkers^[2] in Atlantic salmon heady kidney cells^[2]. Fucoidans are complex fucose-rich sulphated water-soluble polysaccharides with many bioactive properties such as immunomodulating properties and anti-viral, anti-bacterial and antioxidant properties, all highly relevant for aquaculture.

  Trials with Atlantic salmon

  To evaluate the functional feeds on stress responses in salmon, we developed a short-term stress model by hypoxia, where we identified several biomarkers that we used as targets in the following studies^[3-4]. The method involved exposing the fish to low oxygen for 60 seconds, followed by plasma and tissue sampling over a 24-hour period to evaluate physiological and immunological stress responses. We measure the stress hormone, cortisol level in plasma, combined with molecular methods to detect biomarkers both at the gene expression and protein production level. Results suggested that the acute-stress model was suitable when evaluating the impact of functional feeds on short-term stress response in salmon such as during vaccination or sea lice treatment.

  Trial 1: A trial was done to evaluate the effect of functional feeds containing the two D. hansenii product on fish health and robustness during seawater transfer^[3-4]. Fish were fed either a control commercial like diet or test diets containing a low level of LAN 4 or 6 for seven weeks in fresh water, followed by seawater transfer, and six weeks in seawater. All fish were vaccinated by a standard vaccine before the trial started, which included antigens against Moritella viscosa, a bacterial pathogen leading to winter ulcers.

  At the end of the freshwater phase, selected fish were exposed to short-term hypoxia stress. Results showed that LAN 4 prevented secretion of the stress hormone cortisol after hypoxia stress in plasma.  From gene expression, we observed that LAN 4 prevented immunosuppression by down-regulating the secretion of the anti-inflammatory marker cytokine IL 10 in plasma and that LAN 4 regulating pathways related to stress tolerance and oxidative regulation in gills. By use of immunohistochemistry, we also found that this diet led to increased goblet cell size with a higher level of mucin in the distal intestine, indicating mucin storage, which can prevent local stress responses^[3].

  During the seawater phase we detected a natural outbreak of M. viscosa. Interestingly, the results showed that vaccinated fish fed D. hansenii yeast LAN 6 had increased level of specific IgM against this pathogen, indicating that LAN 6 may improve the efficacy of the vaccine. Based on transcriptomics, data also suggest that LAN 6 could lead to increased humoral innate immunity by activation of the complement system in the liver^[3].

  Overall (Fig. 2), the results suggest that functional feed containing both yeast strains led to a more robust salmon, but their immune response differed: LAN 4 led was more effective in regulating short-term stress responses, while LAN6 was more active in coordinating the immune response against a pathogen after seawater transfer. The differences in the mode of action could be associated with the differences in the content and structure of the two D. hansenii yeasts^{[1, 3]}.

  

  Trial 2. A trial was performed to evaluate the effect of functional feed containing the brown seaweed extract fucoidan in Atlantic salmon in freshwater^[5]. Fish were fed a control diet and functional diets containing either increasing levels of fucoidan or a commercial yeast-based β-glucan. After the end of the trial, Salmon head kidney cells were isolated from fish fed either a control or functional feeds with fucoidan and stimulated by an inactivated pathogen, Aeromonas salmonicida. Results showed that higher inclusion level resulted in immune suppression, while a moderate level of fucoidan led to an increase in pro-inflammatory cytokines involved an early immune activation and increased the protection against stress as indicated by increased production of the chaperon.

  At the in vivo level, we found that fucoidan (moderate level) activated responses related to adaptative immunity in the distal intestine, including development and modulation of T-cells and activation of professional antigen presenting cells, which suggest T-cell polarization since we also detected an up-regulation of gata3, a global transcriptional factor related to T helper 2 cells (Th2). By 16 s rRNA sequencing, we also found that fucoidan a lead to changes in gut microbiota, with a dose-dependent increase in Bacillus spp, which are known to have positive health effects^[5].

  These results (Fig. 3) suggest that fucoidan is a promising candidate for functional feeds for salmon that performed even better than the commercial available β-glucan product. However, the efficiency of fucoidan dependent on the processing method used during the seaweed extraction and the dosage used in the feed.

  

  Trial 3. In a follow-up trial, we evaluate if functional feed containing fucoidan and use of nutritional programming could lead to trained immunity and boost the immune system of salmon when exposed to an inactivated pathogen challenge with Tenacibaculum maritimum during seawater.

  The preliminary data showed that one of the regimens used was capable of modulate this type of response in the fish (related to cytokines) and that the inclusion of fucoidan was able of increasing the humoral response by increasing antimicrobial peptides in the head kidney and intestine. Also, results showed that fish fed diets containing fucoidan had higher feed intake, grew faster and were more efficient than those fed the control diet^[6].

  Trial 4. To evaluate further evaluate the health effect of fucoidan in functional diets, we concluded a live pathogen challenge trial in Chile using the same diet and nutritional programming as in trial 3. In this trial we looked and survival rate and immune modulating responses of Atlantic salmon challenged fish with the pathogen, Tenacibaculum dicentrarchi. Preliminary results showed that the mortality rates of salmon fed the functional diet containing fucoidan was lower. We are currently evaluating at the cellular and molecular level the possible immunological mechanisms involved in this response. Overall, results suggest that fucoidan hold promise in functional feeds aiming at improving health and robustness of Atlantic salmon.

• References

  1. Morales-Lange B, et al. 2024. Immunomodulatory effects of hydrolyzed Debaryomyces hansenii in Atlantic salmon (Salmo salar L): From the in vitro model to a natural pathogen challenge after seawater transfer. Aquaculture, 578: 740035.
  2. Michalak L, et al. 2023. Impact of biorefinery processing conditions on the bioactive properties of fucoidan extracts from Saccharina latissima on SHK-1 cells. Algal Research, 75: 103221
  3. Morales-Lange B, et al. 2022. Dietary Inclusion of Hydrolyzed Debaryomyces hansenii Yeasts Modulates Physiological Responses in Plasma and Immune Organs of Atlantic Salmon (Salmo salar) Parr Exposed to Acute Hypoxia Stress. Frontiers in Physiology, 13:83681.
  4. Djordjevic B, et al. 2021. Comparison of Circulating Markers and Mucosal Immune Parameters from Skin and Distal Intestine of Atlantic salmon in Two Models of Acute Stress. Int. J. Mol. Sci., 22: 1028.
  5. Morales-Lange B, et al. 2022. Dietary inclusion of fucoidan from Sugar kelp (Saccharina latissima) modulates immune responses in distal intestine of Atlantic salmon (Salmo salar) pre-smolt. 2nd International Symposium Mucosal Health in Aquaculture. October 3-6, 2022. Madrid, Spain.
  6. Morales-Lange B, et al. 2023. Nutritional programming using Fucoidan from Sugar kelp as a feed additive to improve Trained Immunity in Atlantic salmon against Tenacibaculum maritimum. Aquaculture Europe. September 18 - 21, 2023. Vienna, Austria.
  7. Øverland M., et al, 2022. Role of functional aquafeeds on mucosal immunity: from bioactive compounds to a resilient salmon. 2nd International Symposium Mucosal Health in Aquaculture 2022, Madrid, October 3-6, 2022

Participants

Partners

Academic partners: Prof. Charles Press, Faculty of Veterinary Sciences, Dr. Jake Olsen, University of Wisconsin, Prof. Mónica Imarai, University of Santiago, Chile, Prof. Luis Marcado, Pontifical Catholic University of Valparaíso, Chile.

Industrial partners: Lallemand, AquaGene, Biomar, Borregaard, Seaweed solution.