Martin et al. BMC Genomics 2010, 11:418 http://www.biomedcentral.com/1471-2164/11/418 fish following bacterial [4-7], viral [8-10] and parasitic infections [11]. Mounting an immune response requires energy and an increase in metabolic activity, and the effectiveness of the response may be related to body energy reserves [12]. In mammals dietary restriction can be advanta- geous in relation to autoimmune diseases [13] but is deleterious with respect to defence to infections [14]. In many temperate animals allocation of energy reserves change on a seasonal basis resulting in altered immune status, as seen in deer mice where B cell production is decreased especially during energy demanding periods [15]. Repression of the immune response can be linked to survival in animals that have a high energy reserve demand. For example eider ducks showing a low humoral immune reactivity have significantly greater return rates to breeding areas than those with high immune activity [16]. Although nutritional status of individuals has been shown to have direct effects on their immune response these studies have focused primarily on human health [17] and endothermic animals[18]. Dramatic effects on the transcriptional response in mammals result from either complete food restriction [19] or from calorie restriction [20] with multiple tissues responding. Mice starved for 24 h show activation of genes related to muscle wastage [21] releasing free amino acids for essential metabolic functions. These same pathways are activated during chronic infection [22], demonstrating a link between nutritional status and immune function. Fish are ectothermic and as such control their meta- bolic rate very differently to mammals. They show major changes following starvation [23-25] such as a generalized reduction in protein synthesis and protein turnover [26], however almost nothing is known about the effect of dietary restriction on the immune response in fish. Additionally carnivorous fish such as salmonids rely on protein as the major energy source oxidising proteins for gluconeogenesis [27]. As the liver is a cen- tral organ controlling many physiological functions and synthesizing plasma proteins, under acute infection con- ditions it is required to alter much of its transcriptional and translational machinery to synthesise high levels of acute phase reactants [28,29]. The production of these acute phase proteins requires increased access to free amino acids. Additionally protein synthesis is an energy demanding process [30] thus mounting of a successful innate response may be related to the availability of suf- ficient energy reserves and free amino acids for de novo synthesis. In this paper we have examined how the liver tran- scriptional response to a bacterial pathogen of Atlantic salmon (Salmo salar) is modulated depending on the previous feeding regime. We have used the Atlantic Page 2 of 20 salmon TRAITS-SGP 17 K cDNA microarray [31,32] platform that has been enriched for genes related to lipid and protein metabolism and the immune response [5]. Our results show that key components of the immune system are depressed during starvation but that following infection the starved fish attempt to compen- sate for this by increasing expression of several key immune related genes to a much greater extent than that seen in fish fed prior to infection. Results All fish survived the 4 week trial. The mean (± sem) size of fish starting the trial was 54.2g ± 1.3 and the growth rate of the fed fish was 0.87% ± 0.04 body weight (bw) day-1. Fish that were starved lost weight at -0.57% ± 0.05 bw day-1. The condition factor of the starved fish was also significantly decreased compared to fed fish (0.96 ± 0.02 and 1.27 ± 0. 17, P < 0.01) at the end of the experiment, indicating a decreased level of energy reserves in the starved fish. The condition factor for the fed fish increased significantly during the experiment, increasing from 1.16 ± 0.01 to 1.27 ±0.17 (P < 0.01), showing that the fish were depositing additional energy reserves during growth. Overview of transcriptome analysis The four groups of fish allowed examination of the changes in the liver transcriptome occurring as a result of starvation and acute bacterial infection. Additionally we wished to determine the effect of infection on the liver transcriptome depending on the feeding regime prior to infection. For each sampling, 6 replicates of fish were used, each replicate consisting of four individual fish (24 fish per group). RNA extracted from 4 different fish was pooled equally and then considered as a single sample. This pooling of individuals removed any large individual variation as our intention was not to study individual responses. The experimental design (Fig. 1) was a common reference design for which the reference sample was an equi molar mixture of all the experimen- tal samples and cDNA from the experimental groups was hybridized against this common control RNA sam- ple. Each of the 6 replicates was hybridized using a dye swap protocol resulting in 12 slides per group and 48 slides in total. All 48 microarray slides hybridized were used in the analysis. Following scanning of the slides and quality control 9855 (58%) cDNA features reached a threshold and were maintained for analysis as described in the materials and methods. The challenges resulted in large global alterations in transcriptional activity, with 2363 genes being signifi- cantly altered following infection compared to 959 genes that were altered by the starvation, indicating that 23.9% and 9.7% of those genes that passed filtering were