Analysis of gene expression by microarray

Liver gene expression in two families of Atlantic salmon, LEAN and FAT, selected on the basis of flesh adiposity was investigated by transcriptomics using the SGP/TRAITS 17k cDNA microarray. To determine the effects of diet and family on gene expression, a two way ANOVA without multiple testing correction of the cDNA array data was performed. The analyses identified 712 gene features that were differentially expressed between the two families, and 539 gene features that were significantly different (P < 0.05) due to the effects of diet. In addition, a significant interaction between the effects of family and diet was observed for 787 of these gene features. All the Atlantic salmon gene clones were identified by BLASTN and BLASTX homolog searching against Tentative Consensus (TC) contigs from the Atlantic salmon gene index (ASGI) (http://compbio.dfci.harvard.edu/tgi/tgipage.html) as described in Chapter 2. For each factor (i.e. family, diet, and their interaction), the top 100 clones (i.e. based on statistical significance, the most highly significant) that were identified as being significantly different were categorised according to function and the results presented as pie charts in Figure 5.1. Note that the pie charts do not include gene features in the top 100 that were of unknown (i.e. unannoted clones).

The functional categories most affected by family and diet were those associated with metabolism. Over a third (35 %) of the gene features affected by diet were associated with metabolic processes, with the majority of these (23 %) linked to lipid metabolism, with 5 % involved in carbohydrate metabolism, 5 % associated with protein and AA metabolism and 3 % related to vitamin metabolism. Family had less of an effect on metabolism than diet, with just over a quarter (27 %) of gene features affected being related to metabolic processes of which 9 % were linked to lipid metabolism, 6 % to metabolic energy pathways and 12 % to protein and AA metabolism. Most of the observed interactions between diet and family were in genes associated with metabolic functions, with almost a third (32 %) of the top 100 genes affected being associated with metabolism. Of these, 18 % of the genes were linked with lipid metabolism, 4 % with carbohydrate metabolism and 10 % with protein and AA metabolism. In addition to metabolic genes, groups of genes related to immune / stress responses, and thus fish health and welfare, were also affected. Therefore, approximately 18 % of the top 100 responding genes affected by strain/family 14 % of the genes affected by diet and 8% of genes affected by their interaction were associated with immune/stress responses (Figure 5.1).

Given that the two-way ANOVA found significant effects associated with both family and diet but that there was significant interaction between them, a series of two-by-two comparisons were performed fixing one of the variables and using t-test to analyse the statistical significance of the differences. Therefore, to examine the specific effects of diet, that is, feeding either a FO or VO, on liver gene expression was separately investigated in each individual family, FAT and LEAN. Furthermore, the effect of family on liver gene expression was investigated when fed either a FO or a VO diet. Therefore, four comparisons were performed; 1) comparison of dietary FO vs VO

Signal

Figure 5.1. The functional categories of the top 100 significant cloned genes that were differentially expressed by the FO and VO diets, the strain and the interaction between diet and strain. Non-annotated and unknown cloned genes are not included.

in the LEAN family, 2) comparison of dietary FO vs VO in the FAT family, 3) comparison of LEAN and FAT families fed a FO diet and, 4) comparison of LEAN and FAT families fed a VO diet. Diet FO and FAT fish were used as control. The results from the t-tests, without multiple testing correction, found that there were 272 gene clones whose expression was significantly different (P < 0.05) in LEAN fish fed either a FO or a VO diet or, in other words, diet significantly affected the expression of 272 gene features in LEAN fish. Similarly, diet significantly affected the expression of 441 gene features in FAT fish. Family significantly affected the expression of 563 gene features in fish fed the VO diet, and 246 genes in fish fed the FO diet. As with the two-way ANOVA, the top 100 significantly different gene clones (based on statistical significance) in each of the four comparisons were classified according to biological function and are presented in Tables 5.2-5.5. Beside the top 100 responding genes, other lipid metabolism/transport-related and cholesterol/steroid biosynthesis-related genes that were found to be significantly different following the t-test analysis of the four comparisons were also categorised and reported in Table 5.6.

The results of the two-by-two comparisons indicated that the levels of expression of metabolism-related genes in the liver differed between the two families of salmon when fed either a FO or a VO diet. Effects of diet on the expression of the HUFA biosynthesis genes, 6 and 5 fatty acyl desaturases, were observed in the LEAN family (Table 5.2) whereas effects on 5 desaturase were not observed in the FAT family (Table 5.3). Thus, the expression of 6 desaturase was approximately 1.5 to 2.8 fold higher and that of 5 desaturase was about 2.0 fold higher in the LEAN family fed the VO diet than fish fed the FO diet (Table 5.2). In the FAT family, the expression of the 6 desaturase was around 1.9 fold higher in fish fed the VO diet compared to fish fed the FO diet (Table 5.3). Differences between families in the expression of FA and

HUFA biosynthesis genes were observed in fish fed the FO diet (Table 5.4) whereas there were no significant differences in those genes in fish fed the VO diet (Table 5.5).

Thus, the liver transcript levels of the 5 desaturase and FA synthase genes were approximately -1.7 and -1.2 fold lower, respectively, in the LEAN family than in the FAT family fed the FO diet (Table 5.4). Therefore, the difference in expression of 5 desaturase between the two families observed in the diet comparisons in Tables 5.2 and 5.3 was reflected in the family comparisons of fish fed FO (Table 5.4) and not of fish fed VO (Tabel 5.5) indicating that it was the different expression level in fish fed FO that led to the differential expression between the families.

Other differences in liver gene expression were observed between the families.

The expression levels of acyl-CoA binding protein, isopentenyl-diphosphate, angiopoitin-like 3 (ANGPTL3) and squalene monooxygenase in the LEAN family were up-regulated in fish fed the VO diet compared to fish fed the FO diet (Tables 5.2 and 5.6). The expression levels of ANGPTL3 and lipoprotein lipase (LPL) were also up-regulated in the FAT family in fish fed VO compared to fish fed FO, whereas no significant effects of diet on the expression of acyl-CoA binding protein, isopentenyl-diphosphate and squalene monooxygenase were observed in the FAT family (Tables 5.3 and 5.6). Furthermore, in fish fed the FO diet, the expression of LPL was higher in the LEAN family than in the FAT family, whereas the expression levels of isopentenyl-diphosphate, ANGPTL4 and squalene monooxygenase were lower in the LEAN family compared to the levels in the FAT family (Tables 5.4 and 5.6). In fish fed the VO diet, the levels of expression of LPL, ANGPTL3 and ANGPTL4 were higher in the LEAN family compared to the levels found in the FAT family (Tables 5.5 and 5.6).

Other dietary differences in liver gene expression also showed differential

LEAN family showed lower levels of expression in liver of fish fed the VO diet than in liver of fish fed the FO diet (Table 5.2). The only exception was the heat shock protein (hsp90), whose expression was 1.3-fold greater in the LEAN family in fish fed VO compared to fish fed FO. In contrast, the levels of expression of the immune response-related genes in the FAT family were higher in fish fed the VO diet than in fish fed the FO diet (Table 5.3). It should be noted that the dietary effects on the expression of hsp90 were not found in the FAT family. Consistent with this difference, the liver expression of hsp 90 in the LEAN family was 1.5 fold higher than that of the FAT family in fish fed the FO diet (Table 5.4) while the expression of hsp70 was 1.2 fold greater in the LEAN family than that in the FAT family in fish fed the VO diet (Table 5.5).