Trond M. Kortner

Early response of gene expression in the distal intestine of Atlantic salmon (Salmo salar L.) during the development of soybean meal induced enteritis.

Plant products in general and soybeans in particular can challenge the function and health of the intestinal tract. Salmonids develop an intestinal inflammation when fed diets containing soybean meal (SBM) and certain other legume ingredients. In the present study a 44K oligonucleotide salmonid microarray, qPCR and histology were used to investigate early response mechanisms in the distal intestine of Atlantic salmon (Salmo salar L.) during the first week of oral exposure to a diet containing 20% extracted SBM. The distal intestine transcriptome was profiled on days 1, 2, 3, 5 and 7 and compared to a control group fed fishmeal as the sole protein source. Histological evaluation of the distal intestine revealed the first signs of inflammation on day 5. The most prominent gene expression changes were seen on days 3 and 5. Up-regulation in immune-related genes was observed during the first 5 days, including GTPase IMAP family members, NF-kB-related genes and regulators of T cell and B cell function. Many functional genes involved in lipid metabolism, proteolysis, transport, metabolism and detoxification were initially up-regulated on days 1-3, possibly as an attempt by the tissue to compensate for the initiating immune response. Cell repair and extracellular matrix remodeling genes were up-regulated (heparanase, collagenase) on days 3 and 5. Down regulation of genes related to endocytosis, exocytosis, detoxification, transporters and metabolic processes from day 5 indicated initiation of dysfunction of digestive and metabolic functions that may occur as a result of inflammation or as a response to the introduction of soybean meal in the diet. This is the first study conducting transcriptomic profiling to characterize early responses during the development of SBMIE. Switching Atlantic salmon from a fishmeal to a 20% SBM diet resulted in rapid changes to the intestinal transcriptome, indicating an immune reaction with subsequent impaired epithelial barrier function and other vital intestinal functions.

A high-resolution map of the gut microbiota in Atlantic salmon (Salmo salar): A basis for comparative gut microbial research

Gut health challenges, possibly related to alterations in gut microbiota, caused by plant ingredients in the diets, cause losses in Atlantic salmon production. To investigate the role of the microbiota for gut function and health, detailed characterization of the gut microbiota is needed. We present the first in-depth characterization of salmon gut microbiota based on high-throughput sequencing of the 16S rRNA gene’s V1-V2 region. Samples were taken from five intestinal compartments: digesta from proximal, mid and distal intestine and of mucosa from mid and distal intestine of 67.3 g salmon kept in seawater (12–14 °C) and fed a commercial diet for 4 weeks. Microbial richness and diversity differed significantly and were higher in the digesta than the mucosa. In mucosa, Proteobacteria dominated the microbiota (90%), whereas in digesta both Proteobacteria (47%) and Firmicutes (38%) showed high abundance. Future studies of diet and environmental impacts on gut microbiota should therefore differentiate between effects on mucosa and digesta in the proximal, mid and the distal intestine. A core microbiota, represented by 22 OTUs, was found in 80% of the samples. The gut microbiota of Atlantic salmon showed similarities with that of mammals.

Transcriptional regulation of cholesterol and bile acid metabolism after dietary soyabean meal treatment in Atlantic salmon (Salmo salar L.).

Inclusion of plant protein sources such as soyabean meal (SBM) in aquafeeds is associated with decreased lipid digestibility, reduced bile acid levels and hypocholesterolaemia. The mechanism for these metabolic abnormalities is unknown. The present study aimed at gaining further insight into how cholesterol and bile acid metabolism is modulated by SBM feeding by quantifying a number of mRNA species corresponding to key proteins involved in cholesterol and bile acid metabolism using quantitative real-time PCR. A 21 d feeding trial with sequential sampling at ten time points following initiation of 20% SBM exposure was conducted on Atlantic salmon. A histological evaluation confirmed distal intestinal enteritis after 5 d of dietary exposure to the SBM, whereas diminished glycogen/lipid deposition was the only relevant finding observed in the liver. SBM inclusion resulted in reduced body pools of cholesterol and bile acids. Hepatic gene expression profiles revealed up-regulation of genes encoding rate-limiting enzymes in cholesterol (3-hydroxy-3-methyl-glutaryl-CoA reductase; HMGCR) and bile acid (cytochrome P4507A1 (CYP7A1)) biosynthesis, as well as up-regulation of their associated transcription factors (sterol regulatory element binding proteins 1 and 2, liver X receptor, farnesoid X receptor and PPAR isoforms). Hepatic gene expressions of cholesterol (ATP binding cassette G5 (ABCG5)) and bile acid (ATP binding cassette B11 (ABCB11)) transporters were, by and large, not influenced by the SBM, but distal intestinal expression patterns of ABCG5 and apical Na-dependent bile acid transporter indicated impaired cholesterol and bile acid reabsorption. In conclusion, hepatic gene expression profiles indicated that the capacity for cholesterol and bile acid synthesis was up-regulated, whereas the indicated impaired cholesterol and bile acid reabsorption probably occurred as a direct result of distal intestinal inflammation.

Dietary soyasaponin supplementation to pea protein concentrate reveals nutrigenomic interactions underlying enteropathy in Atlantic salmon (Salmo salar)

BackgroundUse of plant ingredients in aquaculture feeds is impeded by high contents of antinutritional factors such as saponins, which may cause various pharmacological and biological effects. In this study, transcriptome changes were analyzed using a 21 k oligonucleotide microarray and qPCR in the distal intestine of Atlantic salmon fed diets based on five plant protein sources combined with soybean saponins.ResultsDiets with corn gluten, sunflower, rapeseed or horsebean produced minor effects while the combination of saponins with pea protein concentrate caused enteritis and major transcriptome changes. Acute inflammation was characterised by up-regulation of cytokines, NFkB and TNFalpha related genes and regulators of T-cell function, while the IFN-axis was suppressed. Induction of lectins, complement, metalloproteinases and the respiratory burst complex parallelled a down-regulation of genes for free radical scavengers and iron binding proteins. Marked down-regulation of xenobiotic metabolism was also observed, possibly increasing vulnerability of the intestinal tissue. A hallmark of metabolic changes was dramatic down-regulation of lipid, bile and steroid metabolism. Impairment of digestion was further suggested by expression changes of nutrient transporters and regulators of water balance (e.g. aquaporin, guanylin). On the other hand, microarray profiling revealed activation of multiple mucosal defence processes. Annexin-1, with important anti-inflammatory and gastroprotective properties, was markedly up-regulated. Furthermore, augmented synthesis of polyamines needed for cellular proliferation (up-regulation of arginase and ornithine decarboxylase) and increased mucus production (down-regulation of glycan turnover and goblet cell hyperplasia) could participate in mucosal healing and restoration of normal tissue function.ConclusionThe current study promoted understanding of salmon intestinal pathology and establishment of a model for feed induced enteritis. Multiple gene expression profiling further characterised the inflammation and described the intestinal pathology at the molecular level.Ethical approvalThe present experiment was approved by the Norwegian Animal Research Authority and conducted according to prevailing animal welfare regulations: FOR-1996-01-15-23 (Norway), European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (Strasbourg, 18.III.1986) and COUNCIL DIRECTIVE of 24 November 1986 on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes (86/609/EEC).

Soya Saponins Induce Enteritis in Atlantic Salmon (Salmo salar L.).

Soybean meal-induced enteritis (SBMIE) is a well-described condition in the distal intestine of salmonids, and saponins have been implicated as the causal agent. However, the question remains whether saponins alone cause SBMIE. Moreover, the dose-response relationship has not been described. In a 10 week feeding trial with Atlantic salmon, a highly purified (95%) soya saponin preparation was supplemented (0, 2, 4, 6, or 10 g/kg) to two basal diets, one containing fishmeal as the major protein source (FM) and the other 25% lupin meal (LP). Saponins caused dose-dependent increases in the severity of inflammation independent of the basal diet, with concomitant alterations in digestive functions and immunological marker expression. Thus, saponins induced inflammation whether the diet contained other legume components or not. However, responses were often the same or stronger in fish fed the corresponding saponin-supplemented LP diets despite lower saponin exposure, suggesting potentiation by other legume component(s).

Effects of dietary plant meal and soya-saponin supplementation on intestinal and hepatic lipid droplet accumulation and lipoprotein and sterol metabolism in Atlantic salmon (Salmo salar L.).

Altered lipid metabolism has been shown in fish fed plant protein sources. The present study aimed to gain further insights into how intestinal and hepatic lipid absorption and metabolism are modulated by plant meal (PM) and soya-saponin (SA) inclusion in salmon feed. Post-smolt Atlantic salmon were fed for 10 weeks one of four diets based on fishmeal or PM, with or without 10 g/kg SA. PM inclusion resulted in decreased growth performance, excessive lipid droplet accumulation in the pyloric caeca and liver, and reduced plasma cholesterol levels. Intestinal and hepatic gene expression profiling revealed an up-regulation of the expression of genes involved in lipid absorption and lipoprotein (LP) synthesis (apo, fatty acid transporters, microsomal TAG transfer protein, acyl-CoA cholesterol acyltransferase, choline kinase and choline-phosphate cytidylyltransferase A), cholesterol synthesis (3-hydroxy-3-methylglutaryl-CoA reductase) and associated transcription factors (sterol regulatory element-binding protein 2 and PPARγ). SA inclusion resulted in reduced body pools of cholesterol and bile salts. The hepatic gene expression of the rate-limiting enzyme in bile acid biosynthesis (cytochrome P450 7A1 (cyp7a1)) as well as the transcription factor liver X receptor and the bile acid transporter abcb11 (ATP-binding cassette B11) was down-regulated by SA inclusion. A significant interaction was observed between PM inclusion and SA inclusion for plasma cholesterol levels. In conclusion, gene expression profiling suggested that the capacity for LP assembly and cholesterol synthesis was up-regulated by PM exposure, probably as a compensatory mechanism for excessive lipid droplet accumulation and reduced plasma cholesterol levels. SA inclusion had hypocholesterolaemic effects on Atlantic salmon, accompanied by decreased bile salt metabolism.

Effects of oral Bt-maize (MON810) exposure on growth and health parameters in normal and sensitised Atlantic salmon, Salmo salar L.

Responses to GM maize Bt-maize, MON810) expressing Cry1Ab protein from the soil bacterium Bacillus thuringiensis (Bt) in diets for both normal and immune-sensitised (with soyabean meal (SBM)-induced enteropathy) post-smolt Atlantic salmon were investigated following 33 and 97 d of exposure. Triplicate tanks of salmon were fed one of four diets, all containing 20% whole-kernel meal maize, either Bt-maize or its near-isogenic maternal line, without or with 15% extracted SBM inclusion. The fish fed Bt-maize utilised the feed less efficiently, as revealed by lower protein and mineral digestibilities and lower lipid and energy retention efficiencies. Higher intestinal weight, as well as increased interferon-γ and decreased sodium-glucose co-transporter mRNA expression, and a transient increase in T-helper cell presence, as measured by cluster of differentiation 4 (CD4) protein in the distal intestine (DI), may partly explain the lower nutrient digestibilities and retentions. The Bt-maize seemed to potentiate oxidative cellular stress in the DI of immune-sensitised fish, as indicated by increases in superoxide dismutase and heat shock protein 70 mRNA expression. The data suggest that Cry1Ab protein or other antigens in Bt-maize have local immunogenic effects in salmon DI. No systemic immune responses could be detected, as indicated by haematology, differential leucocyte counts, plasma clinical chemistry, as well as absence of Cry1Ab-specific antibodies and Cry1Ab protein in plasma. The responses to Bt-maize observed in the present study differed from results from earlier studies in salmon and other animals fed the same event Bt-maize. Longer-term experiments and more in-depth studies on intestinal physiology and immune responses are needed to evaluate health implications.

Dietary cholesterol supplementation to a plant-based diet suppresses the complete pathway of cholesterol synthesis and induces bile acid production in Atlantic salmon ( Salmo salar L.)

Plants now supply more than 50 % of protein in Norwegian salmon aquafeeds. The inclusion of plant protein in aquafeeds may be associated with decreased lipid digestibility and cholesterol and bile salt levels, indicating that the replacement of fishmeal with plant protein could result in inadequate supplies of cholesterol in fish. A reduction in feed efficiency, fish growth and pathogen resistance is often observed in parallel to alterations in sterol metabolism. Previous studies have indicated that the negative effects induced by plant components can be attenuated when diets are supplemented with cholesterol. The present study evaluated the effects of dietary cholesterol supplementation (1·5 %) in Atlantic salmon fed a plant-based diet for 77 d. The weights of body, intestines and liver were recorded and blood, tissues, faeces, chyme and bile were sampled for the evaluation of effects on growth, nutrient utilisation and metabolism, and transcriptome and metabolite levels, with particular emphasis on sterol metabolism and organ structure and function. Cholesterol supplementation did not affect the growth or organ weights of Atlantic salmon, but seemed to promote the induction of cholesterol and plant sterol efflux in the intestine while suppressing sterol uptake. Cholesterol biosynthesis decreased correspondingly and conversion into bile acids increased. The marked effect of cholesterol supplementation on bile acid synthesis suggests that dietary cholesterol can be used to increase bile acid synthesis in fish. The present study clearly demonstrated how Atlantic salmon adjusted their metabolic functions in response to the dietary load of cholesterol. It has also expanded our understanding of sterol metabolism and turnover, adding to the existing, rather sparse, knowledge of these processes in fish.

Gene expression and activity of digestive enzymes during the larval development of Asian seabass (Lates calcarifer)

Knowledge of digestive enzyme development during larval stages provides a better understanding of the digestive and nutritional physiology of fish larvae. This study characterized the ontogeny of key digestive enzymes in Asian seabass larvae from hatching to juvenile stage (30 days post hatch, dph) using molecular and biochemical approaches. Gene expression and activity of pepsinogen (pg), trypsinogen (try), chymotrypsinogen (ctr), bile salt-activated lipase (bal), α-amylase (amy), leucine aminopeptidase (lap) and alkaline phosphatase (alp) were determined. Gene expression and enzyme activity of all digestive enzymes were detectable from hatching. Pepsinogen mRNA levels were close to detection limit during 0-15 dph, but were highly expressed from 18 dph and onwards. This coincided with a marked increase in specific and individual pepsin activity, indicating complete development of digestive function. Expression levels of try, ctr, amy and bal were high between 3 and 15 dph and thereafter a decreasing trend was observed. Intestinal enzymes, lap and alp, showed highest expression levels during the yolk sac stage, and thereafter levels decreased. Activity of all digestive enzymes increased from around 18 dph and onwards. In conclusion, the development of main digestive enzymes in Asian seabass larvae shows a similar pattern to that of other marine fish species.

Intestinal fatty acid binding protein (fabp2) in Atlantic salmon (Salmo salar): Localization and alteration of expression during development of diet induced enteritis

In the present study full-length cDNAs corresponding to three isoforms of intestinal fatty acid binding protein (fabp2) in Atlantic salmon were cloned and characterized. Gene expression of fabp2 was observed in all tissues investigated, although differences were observed between isoforms. The highest fabp2a1, fabp2a2, and fabp2b expression was in the intestine. A 15kDa protein, corresponding to putative Fabp2 protein, was identified by immunoblotting using anti-human Fabp2 antibody. Immunoblotting and immunohistochemistry confirmed that Fabp2 protein was present in most Atlantic salmon tissues. Similar to gene expression, intestinal tissues had the highest Fabp2 protein levels, decreasing gradually from proximal to distal intestine. During development of distal intestinal inflammation caused by dietary soybean meal from 0 to 21days, Fabp2 decreased significantly on both transcriptional and protein levels. The reduction in Fabp2 was preceded by a down regulation of peroxisome proliferator activated receptor (ppar) alpha and gamma, fabp2s presumed regulatory proteins, and followed by a progressive increase in proliferating cell nuclear antigen (Pcna) staining. Results illustrate that the early decline of distal intestinal fabp2 was likely caused by a down regulation of their regulatory proteins, but at later time points reduced Fabp2 may largely be due to a less mature enterocyte population resulting from rapid cell turnover.