Intestinal strangulation in farmed Atlantic cod (Gadus morhua): Pathological changes and possible predisposing anatomical features.

During recent years, there has been a renewed interest in establishing farming of Atlantic cod (Gadus morhua) in Norway. However, a fatal abdominal disorder compromises animal welfare and causes economic losses. A similar problem was present during a previous attempt to establish Atlantic cod farming more than a decade ago. In this paper, we provide the first in-depth description of this intestinal disorder, which is correctly denoted 'strangulating obstruction'. In affected fish, part of the intestine is permanently entrapped (incarcerated) under fibrous strands in the mesentery. The entrapment interferes with blood flow and physically blocks the intestine, causing a strangulating obstruction with severe venous congestion and ischemia of the intestinal wall. Furthermore, comparison of macroscopical and histological anatomy of farmed and wild Atlantic cod is presented and risk factors associated with the anatomical differences are discussed.

A comprehensive transcriptional body map of Atlantic salmon unveils the vital role of the intestine in the immune system and highlights functional specialization within its compartments.

The intestine is a barrier organ that plays an important role in the immune system of Atlantic salmon. The immune functions are distributed among the diffuse gut lymphoid tissue containing diverse immune cells, and other cell types. Comparison of intestinal transcriptomes with those of other organs and tissues offers an opportunity to elucidate the specific roles of the intestine and its relationship with other parts of the body. In this work, a meta-analysis was performed on a large volume of data obtained using a genome-wide DNA oligonucleotide microarray. The intestine ranks third by the expression level of immune genes after the spleen and head kidney. The activity of antigen presentation and innate antiviral immunity is higher in the intestine than in any other tissue. By comparing transcriptome profiles, intestine shows the greatest similarity with the gill, head kidney, spleen, epidermis, and olfactory rosette (descending order), which emphasizes the integrity of the peripheral mucosal system and its strong connections with the major lymphoid organs. T cells-specific genes dominate among the genes co-expressed in these tissues. The transcription signature of CD8+ (86 genes, r > 0.9) includes a master gene of immune tolerance foxp3 and other negative regulators. Different segments of the intestine were compared in a separate experiment, in which expression gradients along the intestine were found across several functional groups of genes. The expression of luminal and intracellular (lysosome) proteases is markedly higher in pyloric caeca and distal intestine respectively. Steroid metabolism and cytochromes P450 are highly expressed in pyloric caeca and mid intestine while the distal intestine harbors genes related to vitamin and iron metabolism. The expression of genes for antigen presenting proteins and immunoglobulins shows a gradual increase towards the distal intestine.

Epigenetic changes in pyloric caeca of Atlantic salmon fed diets containing increasing levels of lipids and choline.

An earlier study of ours investigating the effect of dietary lipid levels on the choline requirement of Atlantic salmon showed increasing severity of intestinal steatosis with increasing lipid levels. As choline is involved in epigenetic regulation by being the key methyl donor, pyloric caeca samples from the study were analysed for epigenetic effects of dietary lipid and choline levels. The diets varied in lipid levels between 16% and 28%, and choline levels between 1.9 and 2.3 g/kg. The diets were fed for 8 weeks to Atlantic salmon of 25 g of initial weight. Using reduced representation bisulfite sequencing (RRBS), this study revealed that increasing dietary lipid levels induced methylation differences in genes involved in membrane transport and signalling pathways, and in microRNAs important for the regulation of lipid homoeostasis. Increasing choline levels also affected genes involved in fatty acid biosynthesis and transport, lipolysis, and lipogenesis, as well as important immune genes. Our observations confirmed that choline is involved in epigenetic regulation in Atlantic salmon, as has been reported for higher vertebrates. This study showed the need for the inclusion of biomarkers of epigenetic processes in studies that must be conducted to define optimal choline levels in diets for Atlantic salmon.

Exploring gut microbiota in adult Atlantic salmon (Salmo salar L.): Associations with gut health and dietary prebiotics.

BACKGROUND: The importance of the gut microbiota for physiological processes in mammals is well established, but the knowledge of their functional roles in fish is still limited. The aims of this study were to investigate associations between variation in taxonomical composition of the gut microbiota and gut health status in Atlantic salmon and to explore possible modulatory effects of dietary prebiotics in one net-pen farm in open water. The fish with initial mean body weight of around 240 g were fed diets based on the same basal composition, either without (Ref diet) or with (Test diet) yeast cell wall based-prebiotics, during the marine production phase from December to September the following year. Sampling was conducted at three sampling time points: January, April, and September, with average water temperature of 3.9 ℃, 3.4 ℃ and 9.6 ℃, respectively. RESULTS: As the fish progressed towards September, growth, brush border membrane enzyme activities, and the expression in the gut of most of the observed genes involved in immune (e.g., il8, cd4a, myd88, il1b, gilt, tgfb, cd8b and cd3), barrier (e.g., zo1, occludin, ecad, claudin25b and claudin15), and metabolism increased significantly. Lipid accumulation in pyloric enterocytes decreased remarkably, suggesting improvement of gut health condition. The growth of the fish did not differ between dietary treatments. Further, dietary prebiotics affected the gut health only marginally regardless of duration of administration. Regarding gut microbiota composition, a decrease in alpha diversity (Observed species, Pielou and Shannon) over time was observed, which was significantly associated with an increase in the relative abundance of genus Mycoplasma and decrease in 32 different taxa in genus level including lactic acid bacteria (LAB), such as Lactobacillus, Leuconostoc, and Lactococcus. This indicates that developmental stage of Atlantic salmon is a determinant for microbial composition. Multivariate association analysis revealed that the relative abundance of Mycoplasma was positively correlated with gut barrier gene expression, negatively correlated with plasma glucose levels, and that its relative abundance slightly increased by exposure to prebiotics. Furthermore, certain LAB (e.g., Leuconostoc), belonging to the core microbiota, showed a negative development with time, and significant associations with plasma nutrients levels (e.g., triglyceride and cholesterol) and gene expression related to gut immune and barrier function. CONCLUSIONS: As Atlantic salmon grew older under large-scale, commercial farm settings, the Mycoplasma became more prominent with a concomitant decline in LAB. Mycoplasma abundance correlated positively with time and gut barrier genes, while LAB abundance negatively correlated to time. Dietary prebiotics affected gut health status only marginally.

Development of a Rainbow Trout (Oncorhynchus mykiss) Intestinal In Vitro Platform for Profiling Amino Acid Digestion and Absorption of a Complete Diet.

The ever-increasing number and variation of raw materials utilized to provide alternative feed formulations continues to allow for a more sustainable and flexible approach. Testing all these options in vivo is still the most robust and reliable manner to pick the best raw material candidates, but it requires the use of large numbers of animals and is time-consuming and expensive. Therefore, we are developing an in vitro platform that can provide a reliable evaluation of new ingredients. The main aim of this work was to combine an in vitro digestion protocol of extruded, commercially relevant aquafeeds with the exposure of intestinal epithelial cells to the extracted bio-available fraction (BAF). The results show that 250,000 cells/cm2 represents the optimal seeding density and that up to 50% BAF concentration for up to 24 h had no negative effects on the epithelial barrier morphology and function. It is possible to determine amino acid digestibility and bioavailability in all the experimental conditions (with and without BSA, at 25% and 50% dilution) and at all time points (0, 6, and 24 h). However, BAF concentration, the medium used for its dilution, and the length of exposure to the different epithelial cell lines can all influence the results and, therefore, must be selected according to the final aim of the experiment.

Establishment of an In Vitro Model to Study Viral Infections of the Fish Intestinal Epithelium.

Viral infections are still a major concern for the aquaculture industry. For salmonid fish, even though breeding strategies and vaccine development have reduced disease outbreaks, viral diseases remain among the main challenges having a negative impact on the welfare of fish and causing massive economic losses for the industry. The main entry port for viruses into the fish is through mucosal surfaces including that of the gastrointestinal tract. The contradictory functions of this surface, both creating a barrier towards the external environment and at the same time being responsible for the uptake of nutrients and ion/water regulation make it particularly vulnerable. The connection between dietary components and viral infections in fish has been poorly investigated and until now, a fish intestinal in vitro model to investigate virus-host interactions has been lacking. Here, we established the permissiveness of the rainbow trout intestinal cell line RTgutGC towards the important salmonid viruses-infectious pancreatic necrosis virus (IPNV), salmonid alphavirus (subtype 3, SAV3) and infectious salmon anemia virus (ISAV)-and explored the infection mechanisms of the three different viruses in these cells at different virus to cell ratios. Cytopathic effect (CPE), virus replication in the RTgutGC cells, antiviral cell responses and viral effects on the barrier permeability of polarized cells were investigated. We found that all virus species infected and replicated in RTgutGC cells, although with different replication kinetics and ability to induce CPE and host responses. The onset and progression of CPE was more rapid at high multiplicity of infection (MOI) for IPNV and SAV3 while the opposite was true of ISAV. A positive correlation between the MOI used and the induction of antiviral responses was observed for IPNV while a negative correlation was detected for SAV3. Viral infections compromised barrier integrity at early time points prior to observations of CPE microscopically. Further, the replication of IPNV and ISAV had a more pronounced effect on barrier function than SAV3. The in vitro infection model established herein can thus provide a novel tool to generate knowledge about the infection pathways and mechanisms used to surpass the intestinal epithelium in salmonid fish, and to study how a virus can potentially compromise gut epithelial barrier functions.

Effects of dietary lipid level and environmental temperature on lipid metabolism in the intestine and liver, and choline requirement in Atlantic salmon (Salmo salar L) parr.

Choline was recently established as an essential nutrient for Atlantic salmon at all life stages. Choline deficiency is manifested as an excessive accumulation of dietary fat within the intestinal enterocytes, a condition known as steatosis. Most of today's plant-based salmon feeds will be choline-deficient unless choline is supplemented. Choline's role in lipid transport suggests that choline requirement may depend on factors such as dietary lipid level and environmental temperature. The present study was therefore conducted to investigate whether lipid level and water temperature can affect steatosis symptoms, and thereby choline requirement in Atlantic salmon. Four choline-deficient plant-based diets were formulated differing in lipid level of 16, 20, 25 and 28 % and fed to salmon of 25 g initial weight in duplicate tanks per diet at two different environmental temperatures: 8 and 15 °C. After 8 weeks of feeding, samples of blood, tissue and gut content from six fish per tank were collected, for analyses of histomorphological, biochemical and molecular biomarkers of steatosis and choline requirement. Increasing lipid level did not affect growth rate but increased relative weight and lipid content of the pyloric caeca and histological symptoms of intestinal steatosis and decreased fish yield. Elevation of the water temperature from 8 to 15 °C, increased growth rate, relative weight of the pyloric caeca, and the histological symptoms of steatosis seemed to become more severe. We conclude that dietary lipid level, as well as environmental temperature, affect choline requirement to a magnitude of importance for fish biology and health, and for fish yield.

Dietary Fish Meal Level and a Package of Choline, ß-Glucan, and Nucleotides Modulate Gut Function, Microbiota, and Health in Atlantic Salmon (Salmo salar, L.).

Steatosis and inflammation have been common gut symptoms in Atlantic salmon fed plant rich diets. Choline has recently been identified as essential for salmon in seawater, and ß-glucan and nucleotides are frequently used to prevent inflammation. The study is aimed at documenting whether increased fishmeal (FM) levels (8 levels from 0 to 40%) and supplementation (Suppl) with a mixture of choline (3.0 g/kg), ß-glucan (0.5 g/kg), and nucleotides (0.5 g/kg) might reduce the symptoms. Salmon (186 g) were fed for 62 days in 16 saltwater tanks before samples were taken from 12 fish per tank for observation of biochemical, molecular, metabolome, and microbiome indicators of function and health. Steatosis but no inflammation was observed. Lipid digestibility increased and steatosis decreased with increasing FM levels and supplementation, seemingly related to choline level. Blood metabolites confirmed this picture. Genes in intestinal tissue affected by FM levels are mainly involved in metabolic and structural functions. Only a few are immune genes. The supplement reduced these FM effects. In gut digesta, increasing FM levels increased microbial richness and diversity, and changed the composition, but only for unsupplemented diets. An average choline requirement of 3.5 g/kg was indicated for Atlantic salmon at the present life stage and under the present condition.

Effects of dietary supplementation with prebiotics and Pediococcus acidilactici on gut health, transcriptome, microbiota, and metabolome in Atlantic salmon (Salmo salar L.) after seawater transfer.

BACKGROUND: Given the importance of gut microbiota for health, growth and performance of the host, the aquaculture industry has taken measures to develop functional fish feeds aiming at modulating gut microbiota and inducing the anticipated beneficial effects. However, present understanding of the impact of such functional feeds on the fish is limited. The study reported herein was conducted to gain knowledge on performance and gut health characteristics in post-smolt Atlantic salmon fed diets varying in content of functional ingredients. Three experimental diets, a diet containing fructo-oligosaccharides (FOS), a diet with a combination of FOS and Pediococcus acidilactici (BC) and a diet containing galacto-oligosaccharides (GOS) and BC, were used in a 10-weeks feeding trial. A commercial diet without functional ingredients was also included as a control/reference. Samples of blood plasma, mucosa and digesta were subjected to microbiota, transcriptome and metabolome profiling for evaluation of the diet effects. RESULTS: No significant growth differences were observed between fish fed the supplemented diets, but FOS-BC fed fish showed significantly faster growth than the control fed fish. The microbiota results showed that the BC was present in both the digesta, and the mucosa samples of fish fed the FOS-BC and GOS-BC diets. Digesta-associated microbiota was altered, while mucosa-associated microbiota was relatively unaffected by diet. Replacing FOS with GOS increased the level of metabolites linked to phospholipid, fatty acid, carnitine and sphingolipid metabolism. Variation in metabolite levels between the treatments closely correlated with genera mainly belonging to Firmicutes and Actinobacteria phyla. The transcriptome analyses indicated diet effects of exchanging FOS with GOS on immune functions, oxidative defense and stress responses. No significant diet effect was observed on intestinal inflammation in the pyloric caeca or in the distal intestine, or on lipid accumulation in the pyloric caeca. CONCLUSIONS: Dietary supplementation with BC induced moderate effects on the microbiota of the digesta, while the effects of replacing FOS with GOS were more marked and was observed also for nutrient metabolism. Our data indicates therefore that the quality of a prebiotic may be of great importance for the effects of a probiotic on gut microbiota, function, and health.

Effects of functional ingredients on gut inflammation in Atlantic salmon (Salmo salar L).

Functional feed ingredients are frequently used in feeds for Atlantic salmon, often claimed to improve immune functions in the intestine and reduce severity of gut inflammation. However, documentation of such effects is, in most cases, only indicative. In the present study effects of two packages of functional feed ingredients commonly used in salmon production, were evaluated employing two inflammation models. One model employed soybean meal (SBM) as inducer of a severe inflammation, the other a mixture of corn gluten and pea meal (CoPea) inducing mild inflammation. The first model was used to evaluate effects of two packages of functional ingredients: P1 containing butyrate and arginine, and P2 containing ß-glucan, butyrate, and nucleotides. In the second model only the P2 package was tested. A high marine diet was included in the study as a control (Contr). The six diets were fed to salmon (average weight of 177g) in saltwater tanks (57 fish per tank), in triplicate, for 69 days (754 ddg). Feed intake was recorded. The growth rate of the fish was high, highest for the Contr (TGC: 3.9), lowest for SBM fed fish (TGC: 3.4). Fish fed the SBM diet showed severe symptoms of inflammation in the distal intestine as indicated by histological, biochemical, molecular, and physiological biomarkers. The number of differently expressed genes (DEG) between the SBM and Contr fed fish was 849 and comprised genes indicating alteration in immune functions, cellular and oxidative stress, and nutrient digestion, and transport functions. Neither P1 nor P2 altered the histological and functional symptoms of inflammation in the SBM fed fish importantly. Inclusion of P1 altered expression of 81 genes, inclusion of P2 altered 121 genes. Fish fed the CoPea diet showed minor signs of inflammation. Supplementation with P2 did not change these signs. Regarding composition of the microbiota in digesta from the distal intestine, clear differences regarding beta-diversity and taxonomy between Contr, SBM, and CoPea fed fish were observed. In the mucosa the microbiota differences were less clear. The two packages of functional ingredients altered microbiota composition of fish fed the SBM and the CoPea diet towards that of fish fed the Contr diet.

Soya saponins and prebiotics alter intestinal functions in Ballan wrasse (Labrus bergylta).

A 5-week feeding trial was conducted in the cleaner fish Ballan wrasse (Labrus bergylta) for a better understanding of the basic biology of the intestinal functions and health in this stomach less species. During the trial, Ballan wrasse was fed either a reference diet, the reference diet supplemented with (i) a commercial prebiotic (Aquate™ SG, 0·4 %) expected to have beneficial effects, (ii) soya saponins (0·7 %) expected to induce inflammation or (iii) a combination of the prebiotics and the soya saponins to find a remedy for gut inflammation. Blood, intestinal tissue and gut content from four consecutive intestinal segments (IN1 - IN4) were collected. No significant differences in fish growth were observed between the four dietary groups. Saponin supplementation, both alone and in combination with prebiotics, increased weight index of IN2 and IN3 and decreased blood plasma glucose, cholesterol and total protein. Dry matter of intestinal content and activity of digestive enzymes were not affected by diet. Histomorphological analyses revealed a progressing inflammation with increased infiltration by immune cells particularly into the distal parts of the intestine in fish fed diets with saponins, both alone and in combination with prebiotics. Gene expression profiles obtained by RNA sequencing and quantitative PCR mirrored the histological and biochemical changes induced by the saponin load. The study demonstrated that Ballan wrasse gut health and digestive function may be markedly affected by feed ingredients containing antinutrients.

Bile components affect the functions and transcriptome of the rainbow trout intestinal epithelial cell line RTgutGC.

The concomitant increase in cultivation of fish and decrease in supply of marine ingredients, have greatly increased the demand for new nutrient sources. This also regards so-called functional ingredients which may benefit health and welfare of the fish. In vitro cell line-based intestinal epithelial barrier models may serve as tools for narrowing down the broad range of ingredient options, to identify the most promising candidates before in vivo feeding trials are run. In vivo, differentiation of the various epithelial cells in the fish intestine, from the multipotent stem cells, takes place in the presence of a variety of substances from dietary and endogenous origin. Among these, bile salts have recently received attention as regulators of epithelial function in health and disease but have not, until now, been included in the medium when culturing fish gut epithelial cells in vitro. As bile salts are present at high levels in the chyme of the fish intestine, in particular in salmon and rainbow trout, mostly as taurocholate (>90%), their role for effects of diet ingredients on the in vitro gut cell model should be understood. With this study, we wanted to investigate whether inclusion of bile from rainbow trout or pure taurocholate in the culture media would modulate functions of the RTgutGC epithelial cells. Here, we demonstrated that the rainbow trout intestinal epithelial cell line RTgutGC responded significantly to the presence of bile components. Treatment with rainbow trout bile taken from the gall bladder (RTbile) or pure taurocholate (TC) at taurocholate concentrations of ≤0.5 mg/mL retained normal cell morphology, cell viability as in cell oxidation-reduction metabolic activity and membrane integrity, and barrier features, while high concentrations of bile salts (≥1 mg/mL) were cytotoxic to the cells. After long-term (4 days) bile treatment, transcriptome responses showed how bile salts play important roles in intestinal epithelial cell metabolism. qPCR data demonstrated that barrier function genes, brush border enzyme genes and immune genes were significantly affected. Although similar trends were seen, treatment with bile salt as a component of rainbow trout bile or pure taurocholate, induced somewhat different effects. In conclusion, this study clearly indicates that bile salts should be included in the cell medium when running in vitro studies of gut cell functions, not at least immune functions, preferably at the level of ∼0.5 mg/mL supplemented as pure taurocholate to ensure reproducibility.

Consistent changes in the intestinal microbiota of Atlantic salmon fed insect meal diets.

BACKGROUND: Being part of fish's natural diets, insects have become a practical alternative feed ingredient for aquaculture. While nutritional values of insects have been extensively studied in various fish species, their impact on the fish microbiota remains to be fully explored. In an 8-week freshwater feeding trial, Atlantic salmon (Salmo salar) were fed either a commercially relevant reference diet or an insect meal diet wherein black soldier fly (Hermetia illucens) larvae meal comprised 60% of total ingredients. Microbiota of digesta and mucosa origin from the proximal and distal intestine were collected and profiled along with feed and water samples. RESULTS: The insect meal diet markedly modulated the salmon intestinal microbiota. Salmon fed the insect meal diet showed similar or lower alpha-diversity indices in the digesta but higher alpha-diversity indices in the mucosa. A group of bacterial genera, dominated by members of the Bacillaceae family, was enriched in salmon fed the insect meal diet, which confirms our previous findings in a seawater feeding trial. We also found that microbiota in the intestine closely resembled that of the feeds but was distinct from the water microbiota. Notably, bacterial genera associated with the diet effects were also present in the feeds. CONCLUSIONS: We conclude that salmon fed the insect meal diets show consistent changes in the intestinal microbiota. The next challenge is to evaluate the extent to which these alterations are attributable to feed microbiota and dietary nutrients, and what these changes mean for fish physiology and health.

Digestive and immune functions in the intestine of wild Ballan wrasse (Labrus bergylta).

This study was carried out to profile key characteristics of intestinal functions and health in wild-caught Ballan wrasse. To describe functional variation along the intestine, samples were collected from four intestinal segments, named from the proximal to the distal segment: IN1, IN2, IN3 and IN4. The sections showed quite similar structure, i.e. regarding mucosal fold height and branching, lamina propria and submucosal width and cellular composition and thickness of the muscle layers. Leucine aminopeptidase and maltase capacity decreased from IN1 to IN4, suggesting a predominant role of IN1 in digestion. Gene expression levels of vitamin C transporter (slc23a1) and fatty acid transporters (cd36 and fabp2) were higher in IN1 than in IN4, indicating a more important role of the proximal intestine regarding transport of vitamins and fatty acids. Higher expression of the gene coding for IgM heavy chain constant region (ighm) was found in IN4 than in IN1, suggesting an important immune function of the distal intestine. Other immune related genes il1b, il6, cd40, showed similar expression in the proximal and the distal part of the intestine. Parasite infection, especially the myxozoan parasite Enteromyxum leei, coincided with infiltration of lymphocytic and eosinophilic granular cells in the submucosa and lamina propria. The present study established reference information necessary for interpretation of results of studies of intestinal functions and health in cultured Ballan wrasse.

The plasma metabolome of Atlantic salmon as studied by 1H NMR spectroscopy using standard operating procedures: effect of aquaculture location and growth stage.

INTRODUCTION: Metabolomics applications to the aquaculture research are increasing steadily. The use of standardized proton nuclear magnetic resonance (1H NMR) spectroscopy can provide the aquaculture industry with an unbiased, reproducible, and high-throughput screening tool, which can help to diagnose nutritional and disease-related metabolic disorders in farmed fish. OBJECTIVE: Standard operating procedures developed for analysing (human) plasma by 1H NMR were applied to fingerprint the metabolome in plasma samples collected from Atlantic salmon. The aim was to explore the metabolome of salmon plasma in relation to growth stage and sampling site. METHODS: A total of 72 salmon were collected from three aquaculture sites in Norway (Lat. 65, 67, and 70 °N) and over two sampling events (December 2017 and November 2018). Plasma drawn from each salmon was measured by 1H NMR and metabolites were quantified using the SigMa software. The NMR data was analysed by principal component analysis (PCA) and ANOVA-simultaneous component analysis (ASCA). RESULTS: Important metabolic differences were evidenced, with adult salmon having a much higher content of very low-density lipoproteins and cholesterol in their plasma, while smolts displayed significantly higher levels of propylene glycol. Overall, 24% of the metabolite variation was due to the growth stage, whereas 12% of the metabolite variation was related to the aquaculture site and practice (p < 0.001). CONCLUSION: This study provides a baseline investigation of the plasma metabolome of the Atlantic salmon and demonstrates how 1H NMR metabolomics can be used in future investigations for comparing aquaculture practices and their influence on the fish metabolome.

Differential response of digesta- and mucosa-associated intestinal microbiota to dietary insect meal during the seawater phase of Atlantic salmon.

BACKGROUND: Intestinal digesta is commonly used for studying responses of microbiota to dietary shifts, yet evidence is accumulating that it represents an incomplete view of the intestinal microbiota. The present work aims to investigate the differences between digesta- and mucosa-associated intestinal microbiota in Atlantic salmon (Salmo salar) and how they may respond differently to dietary perturbations. In a 16-week seawater feeding trial, Atlantic salmon were fed either a commercially-relevant reference diet or an insect meal diet containing ~ 15% black soldier fly (Hermetia illucens) larvae meal. The digesta- and mucosa-associated distal intestinal microbiota were profiled by 16S rRNA gene sequencing. RESULTS: Regardless of diet, we observed substantial differences between digesta- and mucosa-associated intestinal microbiota. Microbial richness and diversity were much higher in the digesta than the mucosa. The insect meal diet altered the distal intestinal microbiota resulting in higher microbial richness and diversity. The diet effect, however, depended on the sample origin. Digesta-associated intestinal microbiota showed more pronounced changes than the mucosa-associated microbiota. Multivariate association analyses identified two mucosa-enriched taxa, Brevinema andersonii and Spirochaetaceae, associated with the expression of genes related to immune responses and barrier function in the distal intestine, respectively. CONCLUSIONS: Our data show that salmon intestinal digesta and mucosa harbor microbial communities with clear differences. While feeding insects increased microbial richness and diversity in both digesta- and mucosa-associated intestinal microbiota, mucosa-associated intestinal microbiota seems more resilient to variations in the diet composition. To fully unveil the response of intestinal microbiota to dietary changes, concurrent profiling of digesta- and mucosa-associated intestinal microbiota is recommended whenever feasible. Specific taxa enriched in the intestinal mucosa are associated to gene expression related to immune responses and barrier function. Detailed studies are needed on the ecological and functional significance of taxa associated to intestinal microbiota dwelling on the mucosa.

Microbiota in intestinal digesta of Atlantic salmon (Salmo salar), observed from late freshwater stage until one year in seawater, and effects of functional ingredients: a case study from a commercial sized research site in the Arctic region.

BACKGROUND: The importance of the gut microbiota for health and wellbeing is well established for humans and some land animals. The gut microbiota is supposedly as important for fish, but existing knowledge has many gaps, in particular for fish in the Arctic areas. This study addressed the dynamics of Atlantic salmon digesta-associated gut microbiota assemblage and its associations with host responses from freshwater to seawater life stages under large-scale, commercial conditions in the Arctic region of Norway, and explored the effects of functional ingredients. The microbiota was characterized by 16S rRNA gene sequencing in distal intestinal digesta at four time points: 2 weeks before seawater transfer (in May, FW); 4 weeks after seawater transfer (in June, SW1); in November (SW2), and in April (SW3) the following year. Two series of diets were fed, varying throughout the observation time in nutrient composition according to the requirements of fish, one without (Ref diet), and the other with functional ingredients (Test diet). The functional ingredients, i.e. nucleotides, yeast cell walls, one prebiotic and essential fatty acids, were supplemented as single or mixtures based on the strategies from the feed company. RESULTS: Overall, the fish showed higher microbial richness and lactic acid bacteria (LAB) abundance after seawater transfer, while Simpson's diversity decreased throughout the observation period. At SW1, the gut microbiota was slightly different from those at FW, and was dominated by the genera Lactobacillus and Photobacterium. As the fish progressed towards SW2 and SW3, the genera Lactobacillus and Mycoplasma became more prominent, with a corresponding decline in genus Photobacterium. The overall bacterial profiles at these time points showed a clear distinction from those at FW. A significant effect of functional ingredients (a mixture of nucleotides, yeast cell walls and essential fatty acids) was observed at SW2, where Test-fed fish showed lower microbial richness, Shannon's diversity, and LAB abundance. The multivariate association analysis identified differentially abundant taxa, especially Megasphaera, to be significantly associated with gut immune and barrier gene expressions, and plasma nutrients. CONCLUSIONS: The gut microbiota profile varied during the observation period, and the Mycoplasma became the dominating bacteria with time. Megasphaera abundance was associated with gut health and plasma nutrient biomarkers. Functional ingredients modulated the gut microbiota profile during an important ongrowing stage.

Gut immune functions and health in Atlantic salmon (Salmo salar) from late freshwater stage until one year in seawater and effects of functional ingredients: A case study from a commercial sized research site in the Arctic region.

The present study was conducted to strengthen the knowledge on gut immune functions and health in Atlantic salmon under large scale, commercial conditions in the Arctic region of Norway. Two groups of fish were monitored, one fed a series of diets without functional ingredients (Ref) and the other diets with functional ingredients (Test). The nutritional composition of the two diet series varied in parallel according to the nutrient requirements of the fish during the observation time. The content of functional ingredients in the Test diets, i.e. nucleotides, yeast cell walls, a prebiotic and essential fatty acids, varied in accordance with a strategy developed by the feed company. The fish were observed at four sampling time points, the first (FW) in May 2016 two weeks before seawater transfer, the other three throughout the following seawater period until the fish reached a size of about 2 kg, i.e. in June, four weeks after seawater transfer (SW1); in November (SW2), and in April the following year (SW3). Gut health was assessed based on histopathological indicators of lipid malabsorption and gut inflammation, expression of gut immune, barrier and other health related genes, plasma biomarkers, somatic indices of intestinal sections, as well as biomarkers of digestive functions. Seawater transfer of the fish (SW1 compared to FW) caused a marked lowering of expression of genes related to immune and barrier functions in the distal intestine, i.e. cytokines (il1ß, il10, tgfß, ifnγ), T-cell markers (cd3γδ), myd88 and tight junction proteins (zo-1, claudin-15, claudin-25b), indicating suppressed immune and barrier functions. At SW2 and SW3, most of the immune biomarkers showed values similar to those observed at FW. The development of plasma cholesterol and triglyceride levels showed similar picture, with markedly lower levels after seawater transfer. Lipid malabsorption was observed in particular in fish from SW1 and SW2, as indicated by hyper-vacuolation of the pyloric caeca enterocytes with concurrently increased expression levels of plin2. Regarding effects of functional ingredients, significantly lower condition factor and plasma triglyceride level were observed for Test-fed fish at SW2, indicating a metabolic cost of use of a mixture of nucleotides, yeast cell walls and essential fatty acids. No clear effects of functional ingredients on expression of gut immune genes and other health indexes were observed through the observation period. The great, temporary lowering of expression of gut immune and barrier genes at SW1 is suggested to be an important factor underlying the increased vulnerability of the fish at this time point. Our findings regarding supplementation with functional ingredients raise questions whether some of these ingredients overall are beneficial or might come with a metabolic cost. Our results highlight the need for a better understanding of the cause and consequences of the suppression of gut immune functions of farmed Atlantic salmon just after seawater transfer, and the use of functional ingredients under commercial conditions.

Anatomy, immunology, digestive physiology and microbiota of the salmonid intestine: Knowns and unknowns under the impact of an expanding industrialized production.

Increased industrialized production of salmonids challenges aspects concerning available feed resources and animal welfare. The immune system plays a key component in this respect. Novel feed ingredients may trigger unwarranted immune responses again affecting the well-being of the fish. Here we review our current knowledge concerning salmon intestinal anatomy, immunity, digestive physiology and microbiota in the context of industrialized feeding regimes. We point out knowledge gaps and indicate promising novel technologies to improve salmonid intestinal health.