Samuel A.M. Martin

Protein growth performance, amino acid utilisation and somatotropic axis responsiveness to fish meal replacement by plant protein sources in gilthead sea bream (Sparus aurata)

Partial or total replacement of fish meal by a mixture of plant protein sources (corn gluten meal, wheat gluten, extruded peas, rapeseed meal) balanced with indispensable amino acids (IAA) was examined in juvenile gilthead sea bream (Sparus aurata) over the course of a 12-week growth trial. A diet with fish meal (FM) as the sole protein source was compared to diets with 50%, 75% and 100% of replacement (PP50, PP75, PP100). Protein retention was improved with more plant protein supply, and just a slight decrease in the final weight gain was found in fish fed PP50 and PP75 diets. However, in the PP100 group, weight gain was depressed up to 30% mainly as the result of a marked reduction of feed intake. These fish also showed a lower fat gain along with a marked hypocholesterolemic effect. Dietary treatment did not alter the hepatic activity of amino acid catabolising enzymes (alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), glutamate dehydrogenase (GDH)), although the size of the total muscle free amino acid (FAA) pool was increased by more plant protein supply. The activity of the somatotropic axis also varied among experimental groups, and the up-regulation of circulating growth hormone (GH) levels with a high plant protein supply followed the decrease in growth rates, plasma levels of insulin-like growth factor-I (IGF-I), and liver mRNA transcripts of IGF-I and GH receptors. This catabolic feature evidenced a liver desensitisation to the anabolic action of GH in the PP100 group, and to a lesser extent in the PP75 group. Taken together all these findings, up to 50-75% of fish meal replacement seems to be feasible with IAA supplementation, but further research is needed to fully identify the responsible factors for the depressed feed intake in order to achieve a full replacement in a fish species having high dietary protein requirements.

Proteomic sensitivity to dietary manipulations in rainbow trout

Changes in dietary protein sources due to substitution of fish meal by other protein sources can have metabolic consequences in farmed fish. A proteomics approach was used to study the protein profiles of livers of rainbow trout that have been fed two diets containing different proportions of plant ingredients. Both diets control (C) and soy (S) contained fish meal and plant ingredients and synthetic amino acids, but diet S had a greater proportion of soybean meal. A feeding trial was performed for 12 weeks at the end of which, growth and protein metabolism parameters were measured. Protein growth rates were not different in fish fed different diets; however, protein consumption and protein synthesis rates were higher in the fish fed the diet S. Fish fed diet S had lower efficiency of retention of synthesised protein. Ammonia excretion was increased as well as the activities of hepatic glutamate dehydrogenase and aspartate amino transferase (ASAT). No differences were found in free amino acid pools in either liver or muscle between diets. Protein extraction followed by high-resolution two-dimensional electrophoresis, coupled with gel image analysis, allowed identification and expression of hundreds of protein. Individual proteins of interest were then subjected to further analysis leading to protein identification by trypsin digest fingerprinting. During this study, approximately 800 liver proteins were analysed for expression pattern, of which 33 were found to be differentially expressed between diets C and S. Seventeen proteins were positively identified after database searching. Proteins were identified from diverse metabolic pathways, demonstrating the complex nature of gene expression responses to dietary manipulation revealed by proteomic characterisation.

Effects of dietary amino acid profile on growth performance, key metabolic enzymes and somatotropic axis responsiveness of gilthead sea bream (Sparus aurata)

Abstract Juvenile gilthead sea bream were fed to visual satiety with isonitrogenous diets based on fish meal and different plant ingredients (33–35% replacement) supplemented with free amino acids to meet the desired indispensable amino acid (IAA) profile and dispensable amino acid (DAA) content. In diets M and WB, IAA profile and DAA content resemble that of the muscle or whole body, respectively. In diets MGlu and WBGlu, DAA content was increased by adding l -glutamic acid (Glu) and thus the IAA/DAA ratio varied from 1.13 (diet M) to 0.80 (diet WBGlu). Growth rates were not significantly different among experimental groups, but feed conversion ratio and nitrogen retention were impaired by the decrease of dietary IAA/DAA ratio. Postprandial ammonia excretion increased with the increase of dietary DAA content irrespective of IAA profile. Conversely, hepatic activity of glutamate dehydrogenase (GDH) was lower in fish fed diet WBGlu than in fish fed diet M. Hepatic growth hormone (GH) binding was not significantly affected by the dietary treatment, but circulating levels of insulin-like growth factor-I (IGF-I) and GH were, respectively, down- and up-regulated in fish fed diet WBGlu, which suggests some defect in the transmission of GH receptor signal. Fat retention and hepatic activities of lipogenic enzymes (glucose-6-phosphate dehydrogenase, G6PD; malic enzyme, ME) were decreased in fish fed diet MGlu. Key metabolic enzymes of hepatic glycolysis (glucokinase, GK) and gluconeogenesis (phosphoenolpyruvate carboxykinase, PEPCK) were also altered in this group of fish. Since soybean meal concentration was highest in diet MGlu, results on lipid and carbohydrate metabolism can be primarily attributed to this component of the diet. In contrast, data on growth performance, ammonia excretion and GH axis mainly reflect changes in the dietary amino acid profile, which reveals that a muscle IAA profile and a high IAA/DAA ratio are important in feeds for gilthead sea bream.

Dietary plant-protein substitution affects hepatic metabolism in rainbow trout (Oncorhynchus mykiss)

The high dietary protein requirements of salmonid fish are met with fishmeal-based feed in commercial aquaculture. The sustainability of this practice is questionable and, therefore, the feasibility of substituting fishmeal with plant-based products needs to be investigated. We investigated growth and metabolism in rainbow trout (Oncorhynchus mykiss) fed a diet composed of a mixture of plant proteins compared with those fed a fishmeal-based diet. Using two-dimensional gel electrophoresis of liver protein extracts, we showed that the liver protein profile changed in response to the alteration in the diet. A number of metabolic pathways were identified as sensitive to the protein source substitution. These included pathways involved in primary energy generation, maintenance of reducing potential, bile acid synthesis, and transport and cellular protein degradation. Interestingly, the pathways shown to be affected in the present study were somewhat different from those identified in our previous work with soyabean-based-protein replacement of fishmeal, with the effects on the abundance of several stress response proteins notably absent. We conclude, therefore, that the metabolic effects of plant protein replacement in aquaculture feed varies with plant-protein source.

Functional Characterization of a Nonmammalian IL-21: Rainbow Trout Oncorhynchus mykiss IL-21 Upregulates the Expression of the Th Cell Signature Cytokines IFN-γ, IL-10, and IL-22

In mammals, IL-21 is a common γ chain cytokine produced by activated CD4+ T cells and NKT cells that acts on multiple lineages of cells. Although IL-21 has also been discovered in birds, amphibians, and fish, to date, no functional studies have been reported for any nonmammalian IL-21 molecule. We have sequenced an IL-21 gene (tIL-21) in rainbow trout, which has a six-exon/five-intron structure, is expressed in immune tissues, and is induced by bacterial and viral infection and the T cell stimulant PHA. In contrast to mammals, calcium ionophore and PMA act synergistically to induce tIL-21. Recombinant tIL-21 (rtIL-21) induced a rapid and long-lasting (4–72 h) induction of expression of IFN-γ, IL-10, and IL-22, signature cytokines for Th1-, Th2-, and Th17-type responses, respectively, in head kidney leukocytes. However, rtIL-21 had little effects on the expression of other cytokines studied. rtIL-21 maintained the expression of CD8α, CD8β, and IgM at a late stage of stimulation when their expression was significantly decreased in controls and increased the expression of the Th cell markers CD4, T-bet, and GATA3. Intraperitoneal injection of rtIL-21 confirmed the in vitro bioactivity and increased the expression of IFN-γ, IL-10, IL-21, IL-22, CD8, and IgM. Inhibition experiments revealed that the activation of JAK/STAT3, Akt1/2, and PI3K pathways were responsible for rtIL-21 action. This study helps to clarify the role of IL-21 in lower vertebrates for the first time, to our knowledge, and suggests IL-21 is a likely key regulator of T and B cell function in fish.

Starvation alters the liver transcriptome of the innate immune response in Atlantic salmon (Salmo salar)

BackgroundThe immune response is an energy demanding process, which has effects in many physiological pathways in the body including protein and lipid metabolism. During an inflammatory response the liver is required to produce high levels of acute phase response proteins that attempt to neutralise an invading pathogen. Although this has been extensively studied in both mammals and fish, little is known about how high and low energy reserves modulate the response to an infection in fish which are ectothermic vertebrates. Food withdrawal in fish causes a decrease in metabolic rate so as to preserve protein and lipid energy reserves, which occurs naturally during the life cycle of many salmonids. Here we investigated how the feeding or fasting of Atlantic salmon affected the transcriptional response in the liver to an acute bacterial infection.ResultsTotal liver RNA was extracted from four different groups of salmon. Two groups were fed or starved for 28 days. One of each of the fed or starved groups was then exposed to an acute bacterial infection. Twenty four hours later (day 29) the livers were isolated from all fish for RNA extraction. The transcriptional changes were examined by micro array analysis using a 17 K Atlantic salmon cDNA microarray. The expression profiling results showed major changes in gene transcription in each of the groups. Enrichment for particular biological pathways was examined by analysis of gene ontology. Those fish that were starved decreased immune gene transcription and reduced production of plasma protein genes, and upon infection there was a further decrease in genes encoding plasma proteins but a large increase in acute phase response proteins. The latter was greater in magnitude than in the fish that had been fed prior to infection. The expression of several genes that were found altered during microarray analysis was confirmed by real time PCR.ConclusionsWe demonstrate that both starvation and infection have profound effects on transcription in the liver of salmon. There was a significant effect on the transcriptional response to infection depending on the prior feeding regime of the fish. It is likely that the energy demands on protein synthesis for acute phase response proteins are relatively high in the starved fish which have reduced energy reserves. This has implications for dietary control of fish if an immune response is anticipated.

Directional responses following recombinant cytokine stimulation of rainbow trout ( Oncorhynchus mykiss ) RTS-11 macrophage cells as revealed by transcriptome profiling

BackgroundThe early stages of the immune response are regulated by key cytokines including both interleukin 1β (IL-1β) and interferon-γ (IFN-γ) which stimulate panels of responsive genes via conserved signal transduction pathways. To further our understanding of the transcriptional response to these cytokines in lower vertebrates we have utilized microarray analysis to characterize the transcriptional response to recombinant rainbow trout IL-1β and IFN-γ in the trout macrophage cell line RTS-11.ResultsRNA was extracted from stimulated or control cells following 6 h incubation and used to hybridize to a salmonid cDNA microarray containing 16,006 different genes. Analysis of the arrays revealed mRNA transcripts that were differentially expressed as a result of exposure to the recombinant proteins, with some responses common for both cytokines. In general the recombinant IL-1β elicited a response where genes involved in the acute phase response were up-regulated, whilst the recombinant IFN-γ induced strong up-regulation of genes involved in the MHC class I antigen presentation pathway. Key genes were chosen that were differentially regulated and analysed by real time PCR at additional time points, up to 48 h following stimulation. This allowed a deeper insight into the kinetics of the response to the cytokines in this cell line.ConclusionWe demonstrated that in fish both rIL-1β and rIFN-γ stimulated discrete panels of mRNA transcripts which indicted the cells were being directed towards different cellular functions, with IL-β inducing genes involved in the inflammatory response, whereas IFN-γ induced genes associated with antigen presentation.

Transcriptomic responses to functional feeds in Atlantic salmon (Salmo salar).

Functional feeds are diets that have positive effects on both health and growth promoting performance of the animals ingesting them, by supplying additional compounds above and beyond the basic nutritional requirements for animal growth alone. The most common additives used in aquaculture diets are probiotics, prebiotics, immunostimulants, vitamins and nucleotides. Inclusion of these components to fish diets can increase feed conversion efficiency and growth, as well as having positive effects on the fish immune system. This review discusses the results from previous studies on fish nutrition and includes a novel genomic approach, using microarray analysis, to elucidate nutritional responses in Atlantic salmon (Salmo salar) fed a newly developed functional feed health premix diet. The transcriptome analysis demonstrated that compared to the standard diet feeding with the functional feed had significant effects on biological processes in the liver. This resulted in a reduction of the expression of genes related to protein turnover, reduced circulating plasma proteins and a down regulation of genes involved in the immune response. These results suggest that the functional feed may infer a decrease in whole body metabolic demands, suppressing both protein turnover and whole body oxygen demand, as well as down regulating several genes involved in the innate immune system. Together these changes appear to result in less energy wastage in fish and an enhanced growth and performance.

Nasal immunity is an ancient arm of the mucosal immune system of vertebrates

The mucosal surfaces of all vertebrates have been exposed to similar evolutionary pressures for millions of years. In terrestrial vertebrates such as birds and mammals, the nasopharynx-associated lymphoid tissue (NALT) represents a first line of immune defence. Here we propose that NALT is an ancient arm of the mucosal immune system not restricted to terrestrial vertebrates. We find that NALT is present in rainbow trout and that it resembles other teleost mucosa-associated lymphoid tissues. Trout NALT consists of diffuse lymphoid cells and lacks tonsils and adenoids. The predominant B-cell subset found in trout NALT are IgT(+) B cells, similar to skin and gut. The trout olfactory organ is colonized by abundant symbiotic bacteria, which are coated by trout secretory immunoglobulin. Trout NALT is capable of mounting strong anti-viral immune responses following nasal delivery of a live attenuated viral vaccine. Our results open up a new tool for the control of aquatic infectious diseases via nasal vaccination.

Two interleukin-17C-like genes exist in rainbow trout Oncorhynchus mykiss that are differentially expressed and modulated ☆

Interleukin (IL)-17 family members (IL-17A-F) are key players in adaptive immune responses and have a central role in coordinating innate and adaptive immunity. Here, we report on two novel IL-17 homologues in rainbow trout Oncorhynchus mykiss, trout (t) IL-17C1 and tIL-17C2, that share 73.7% amino acid identity. The two tIL-17C-like molecules have relatively higher sequence identities to IL-17Cs from fish and mammals and the fish IL-17C-like molecules phylogenetically form a specific clade that groups with the mammalian IL-17C and IL-17E clades. However, the gene organisation of the fish IL-17C-like molecules is closer to mammalian IL-17Es than to IL-17Cs, and this taken together with other factors suggest the fish IL-17C-like genes may have arisen from an ancestral gene that gave rise to mammalian IL-17C and IL-17E. The expression of tIL-17Cs was detectable in all the eight tissues examined, with the expression of tIL-17 mainly contributed by tIL-17C1 in gills and skin, and by tIL-17C2 in spleen, head kidney and brain. The expression of tIL-17Cs was modulated by inflammatory stimulants, including IL-1beta, interferon-gamma, LPS and PolyIC, in a trout macrophage cell line (RTS-11). IL-1beta was the most potent inducer of tIL-17C2 but only had a minor effect on the expression of tIL-17C1. LPS and PolyIC were also potent inducers of tIL-17C2. The expression of tIL-17Cs was also up-regulated by bacterial infection, with the extent and increase more dramatic for tIL-17C2. The broad distribution of expression and differential modulation of tIL-17Cs by inflammatory stimulants and infection suggest important roles of the two tIL-17Cs in the salmonid immune system.