Martin et al. BMC Genomics 2010, 11:418 http://www.biomedcentral.com/1471-2164/11/418 Page 13 of 20 Table 5 GO Biological processes significantly altered in infected fish depending on prior feeding regime (Continued) GO:0030335 GO:0040012 GO:0040017 GO:0051270 positive regulation of cell migration regulation of locomotion positive regulation of locomotion regulation of cellular component movement 3 3 3 3 9 17 9 20 0.01 0.04 0.01 0.07 Gene ontology (GO) identifiers over represented in genes found differentially expressed following infection depending on prior feeding regime. For this analysis both increased and decreased genes were included. 1Gene ontology identifier, 2Gene Ontology term, 3number of times the GO term is present in the gene list, 4number of times the GO term is present on the filtered array list, 5statistical enrichment for GO term. Only GO terms that appear 3 or more times are shown. Table 6 Real time PCR analysis of genes selected for confirmation of microarray data Group: AFL vs PFL ASL vs PSL PSL vs PFL SAA 31.7 ± 4.4* 128.2 ± 43.8 -6.6 ± 3.7 TRL 5 17.2 ± 4.6* 60.5 ± 26.8*0 -1.0 ± 0.3 Hepcidin 100.0 ± 20.7* 424.5 ± 66.2* -1.5 ± 0.4 JunB 18.1 ± 3.3* 8.7 ± 1.4* -0.6 ± 0.3 CAAT/EBP 2.5 ± 0.4 5.6 ± 3.0 -1.0 ± 0.6 COUP 3.0 ± 1.5 0.9 ± 0.5* -13.4 ± 7.7 C type lectin Precerebellin 3.6 ± 0.6* 4.3 ± 2.0* -1.1 ± 0.2 15.9 ± 7.5* 27.1 ± 13.6* -0.5 ± 0.2* Candidate gene expression in liver RNA isolated from Atlantic salmon fed or starved prior to bacterial infection. Values are fold increase (± sem). The groups are: AFL fish that were fed on normal diet prior to bacterial infection compared to fed uninfected fish, PFL. ASL: fish that were starved for 28 days prior to infection compared to uninfected starved fish PSL. PSL uninfected starved fish compared to uninfected fed fish, PFL. Gene expression was determined by real time PCR and normalized to the elongation factor 1a gene. Statistical analysis of expression data was by t-test, * indicates P < 0.05. translation occurring in the liver. During starvation there was a marked decrease in the transcription of these proteins. Both a and b globins were down regu- lated in the starved animals, reflecting the presumed decrease in whole animal metabolism and decreased oxygen demand. They were also further decreased when the starved animals were immune challenged (ASL group), but the globins were not decreased in the fish which were fed before infection (AFL group). Serum albumin was depressed in starved fish (1.7 fold, data not shown) as also observed in mammals fed low protein diets [53]. The modest decrease in serum albumin in starved fish showed a further decrease in the ASL group which could indicate decreases in total circulating pro- tein. The decrease in circulating globins and serum albumin was also observed in brook trout following bac- terial infection [54] and ascribed to a reduction in tran- scription and potentially additional catabolism induced by the inflammation. In mammals reduced circulating serum albumin levels correlates with increased suscept- ibility to infection [55] which may also be the case in fish. In parallel to serum albumin, apoliproteins, the major lipid and cholesterol transporters were reduced following infection. It is likely this was brought about by either reduction in lipid metabolism and transport or to a down regulation of these genes to make way for the synthesis of APR proteins. The greatest increase in transcription in both AFL and ASL groups was for genes related to the APR as has been widely reported in a number of previous studies in liver [4,5,28,29,56]. There was a trend for there to be a greater increase in the ASL as seen for SAA, serotrans- ferrin, b 2 microglobulin and a major antimicrobial pep- tide (hepcidin) which is often increased in parallel with the APR following bacterial challenge [57]. Interestingly, SAA, serotransferrin and hepcidin were all decreased in the starved fish suggesting the starved fish were down regulating their constitutively expressed immune mole- cules, most likely as an energy conserving mechanism. Upon infection the fish needed to dramatically increase production of the APR proteins and appear to sacrifice the transcription of normal plasma proteins as seen for serum albumin, globins and apoliproteins. These dra- matic changes were likely to have more major conse- quences for the physiology of the fish in the ASL group than the AFL group. One further APR protein of note is precerebellin [58]. The mRNA for this gene was increased following starvation in uninfected fish, sug- gesting that there is a requirement for this protein to be maintained at a higher constitutive level, although to date the exact function of the protein remains unknown. The APR is predominantly stimulated by cytokines, in particular IL-6 which acts via its receptor gp130/IL-6R to transduce the signal and stimulate STAT transcrip- tion factors to target responsive genes. A number of transcription factors were increased in expression fol- lowing the infection, including CAAT/enhancer binding protein (also termed C/EBP or nuclear factor IL-6) which binds to promoters of APR proteins and IL-6 tar- get genes [59]. Although c/EBP increases APR transcrip- tion it also binds to the promoter of serum albumin decreasing its expression [60]. Toll like receptors (Toll like 5 and a Toll like leucine rich repeat protein) which are related to IL-1 family receptors, signal via NF B and are key in eliciting an immune response [61], were increased in ASL and AFL. As with several APR pro- teins, the increase in these cell surface receptors was greater in ASL for both genes which could help explain the magnitude of expression of the APR genes. A further inflammatory regulatory gene, suppressor of