Individuals Maintain Similar Rates of Protein Synthesis over Time on the Same Plane of Nutrition under Controlled Environmental Conditions.

Consistent individual differences in animal performance drive individual fitness under variable environmental conditions and provide the framework through which natural selection can operate. Underlying this concept is the assumption that individuals will display consistent levels of performance in fitness-related traits and interest has focused on individual variation and broad sense repeatability in a range of behavioural and physiological traits. Despite playing a central role in maintenance and growth, and with considerable inter-individual variation documented, broad sense repeatability in rates of protein synthesis has not been assessed. In this study we show for the first time that juvenile flounder Platichthys flesus reared under controlled environmental conditions on the same plane of nutrition for 46 days maintain consistent whole-animal absolute rates of protein synthesis (As). By feeding meals containing 15N-labelled protein and using a stochastic end-point model, two non-terminal measures of protein synthesis were made 32 days apart (d14 and d46). As values (mass-corrected to a standard mass of 12 g) showed 2- to 3-fold variation between individuals on d14 and d46 but individuals showed similar As values on both days with a broad sense repeatability estimate of 0.684 indicating significant consistency in physiological performance under controlled experimental conditions. The use of non-terminal methodologies in studies of animal ecophysiology to make repeat measures of physiological performance enables known individuals to be tracked across changing conditions. Adopting this approach, repeat measures of protein synthesis under controlled conditions will allow individual ontogenetic changes in protein metabolism to be assessed to better understand the ageing process and to determine individual physiological adaptive capacity, and associated energetic costs of adaptation, to global environmental change.

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

BACKGROUND: The 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. RESULTS: Total 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. CONCLUSIONS: We 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.

BACKGROUND: The early stages of the immune response are regulated by key cytokines including both interleukin 1beta (IL-1beta) and interferon-gamma (IFN-gamma) 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-1beta and IFN-gamma in the trout macrophage cell line RTS-11. RESULTS: RNA 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-1beta elicited a response where genes involved in the acute phase response were up-regulated, whilst the recombinant IFN-gamma 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. CONCLUSION: We demonstrated that in fish both rIL-1beta and rIFN-gamma stimulated discrete panels of mRNA transcripts which indicted the cells were being directed towards different cellular functions, with IL-beta inducing genes involved in the inflammatory response, whereas IFN-gamma induced genes associated with antigen presentation.

Proteome analysis of the Atlantic salmon (Salmo salar) cell line SHK-1 following recombinant IFN-gamma stimulation.

Type II IFN exists as a single molecule (IFN-gamma) in contrast to type I IFN, of which there are a number of different forms. IFN-gamma is involved both directly and indirectly in antiviral activity, stimulation of bactericidal activity, antigen presentation and activation of macrophages. Recently IFN-gamma was cloned from a salmonid fish, the rainbow trout and a functional recombinant protein produced exhibited IFN-gamma activity. This recombinant IFN-gamma was used to stimulate an Atlantic salmon cell line, SHK-1, to monitor the changes in protein expression by proteomic analysis 24 h after stimulation compared to unstimulated control cells. An SHK-1 cell proteome map was developed and proteins altered in abundance by the IFN-gamma stimulation were identified. Under the analytical conditions used, 22 proteins were found to be altered in abundance, 15 increased and 7 decreased. Several proteins were excised from the gel and identified, following trypsin digestion and MALDI-MS/MS/LC-MS and database interrogation. Transcriptional analysis of five mRNAs encoding proteins increased in abundance by IFN-gamma in the proteome analysis was determined by real-time PCR. We assessed the correlation between gene expression and change in abundance of proteins for these genes.

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.

Influence of oxygen partial pressures on protein synthesis in feeding crabs.

Many water-breathing animals have a strategy that consists of maintaining low blood PO2 values in a large range of water oxygenation level (4-40 kPa). This study examines the postprandial changes in O2 consumption, arterial blood PO2, and tissue protein synthesis in the shore crab Carcinus maenas in normoxic, O2-depleted, and O2-enriched waters to study the effects of this strategy on the O2 consumption and peptide bond formation after feeding. In normoxic water (21 kPa), the arterial PO2 was 1.1 kPa before feeding and 1.2 kPa 24 h later. In water with a PO2 of 3 kPa (arterial PO2 0.6 kPa), postprandial stimulation of protein synthesis and O2 consumption were blocked. The blockade was partial at a water PO2 of 4 kPa (arterial PO2 0.8 kPa). An increase in environmental PO2 (60 kPa, arterial PO2 10 kPa) resulted in an increase in protein synthesis compared with normoxic rates. It is concluded that the arterial PO2 spontaneously set in normoxic Carcinus limits the rates of protein synthesis. The rationale for such a strategy is discussed.

Cypermethrin induces glutathione S-transferase activity in the shore crab, Carcinus maenas.

Cypermethrin is a synthetic pyrethroid that is particularly toxic to crustacea. It is therefore applied as a chemotherapeutant in the salmonid aquaculture industry for the treatment of sea lice infestations. After use, cypermethrin is released directly into the marine environment, to be diluted by fresh seawater. The shore crab, Carcinus maenas is found in the vicinity of fish farms, and may come into contact with released cypermethrin. The detoxification enzyme, glutathione S-transferase (GST) has been implicated in cypermethrin metabolism in terrestrial arthropods, but this has not yet been demonstrated in crustacea. In this paper we investigate the response of GST activity in Carcinus to cypermethrin exposure, and also the time course of the induction process. GST activity is significantly increased in Carcinus exposed to nominal concentrations of 50 and 500 ng/l of water-borne cypermethrin. Carcinus demonstrate a significant elevation in GST activity following intra-cephalothoracic injection with 10 ng of cypermethrin. GST activity returns to basal levels after 36 h. The potential application of GST activity in Carcinus as a biomarker of cypermethrin exposure is discussed.