Table 1.
Details of feeding period and tank replicates for the feeding trials.
The base diet was manufactured by Biomar and described in three terms: the product range name (CPK), the pellet diameter and the oil to protein ratio. The control and experimental diets were designed to be iso-nitrogenous and iso-lipidic. YCW = yeast cell wall extracts. FOS = fructooligosaccharides.
Table 2.
Primer sequences, together with their optimum annealing temperature and product size, used for the gene expression studies in Atlantic salmon skin cDNA.
Fig 1.
Representative gel of a two-dimensional SDS-PAGE of the epidermal mucus of Atlantic salmon.
The first dimension was run on a pH4-7 IPG strip and the second dimension was run on an Anykd Criterion gel. Molecular weights in kDa are denoted on the right side of the image as indicated by Precision Plus Dual Color protein standards (Bio-Rad). The circled spots represent those that show significant differential expression between dietary treatments as given by SameSpots analysis (n = 6, p<0.05). The spots that are circled and numbered refer to those that were subsequently identified by LC-MS/MS. The boxed area refers to Fig 2 where this part of the image has been magnified.
Table 3.
Differentially expressed proteins in the skin of Atlantic salmon fed prebiotic dietary supplements, as identified by two-dimensional electrophoresis.
The relevant spots were cut out from the gel and sequenced by LC-MS/MS, after which the peptides were identified by a MASCOT search. The spot numbers refer to Fig 1, MW stands for molecular weight and fold change represents the expression level in the experimental fish group as compared to the control.
Fig 2.
Magnification of the gel image in the area where the calreticulin spot (no.757) was located in Fig 1.
The table indicates the identities the other protein spots migrating in this region of the profile along with their fold-change and p-value from the SameSpots analysis of mucus samples from Trial#1.
Fig 3.
Phylogenetic trees showing the evolutionary relationship of the calreticulin family of proteins in vertebrates, rooted by the mammalian calreticulin-3.
The tree was used to name the calreticulin isoforms of Salmo salar. The tree was constructed using multiple alignments of the vertebrate calreticulin. The neighbour-joining method in MEGA 5 [37] was selected. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (10,000 replicates) is shown next to the branches [40]. The salmon sequences are boxed. Each entry is described by the species common name and protein name, followed by the Genbank or Ensembl accession number.
Fig 4.
Effect of dietary YCW extracts on calrl expression in the skin of Atlantic salmon during two independent feeding trials.
Fish were fed either a control diet (Control) or the same base diet supplemented with 0.4% YCW for 4–7 weeks. Expression is given as a ratio of the arbitrary unit for calrl to the arbitrary units of two reference genes, elongation factor 1-alpha and beta-actin. Data are present as Means ± SEM (n = 9). Statistical significance was calculated by independent sample t-test, after checking for normality and outliers. One star (*) denotes p<0.10 and two stars (**) denotes p<0.05.
Fig 5.
Effect of dietary YCW extracts on calrl expression in the gut of Atlantic salmon during Trial#1.
Fish were fed either a control diet (Control) or the same base diet supplemented with 0.4% YCW. Expression is given as a ratio of the arbitrary unit for CALRL to the arbitrary units of two reference genes, elongation factor 1-alpha and beta-actin. Data are present as Means ± SEM (n = 6). The expression of CALRL is not significantly different between diets (p>0.05), as established by independent sample t-test after checking for normality and outliers.
Table 4.
Validation of proteomic results by analysing the gene expression of the skin mRNA corresponding to the respective proteins.
Fold changes at the proteomic level was calculated on SameSpots using normalised spot volume data of two-dimensional gels (2DGE). Gene expression fold changes were calculated from Ct values of qPCR assays by deriving arbitrary units using the standards graph equation and normalising using two housekeeping genes, EF-1α and β-actin. (n = 9).