Differential Gene Expression and Pathway Analysis
There were significant differences in gene expression among the four lakes (Figure 3). The greatest numbers of differentially expressed transcripts were found among the comparisons between L223 and L260 (6,708 transcripts, 1,208 annotated transcripts), L224 and L260 (8,276 transcripts, 1,012 annotated transcripts), L260 and L373 (7,190 transcripts, 879 annotated transcripts), and L223 and L224 (8,123 transcripts, 1,629 annotated transcripts) (Figure 3). The lowest numbers of differentially expressed transcripts were among the comparisons between L223 and L373 (4,659 transcripts; 872 annotated transcripts) and L224 and L373 (4,098 transcripts, 426 annotated transcripts) (Figure 3). Again, the relatively low degree of annotation of transcripts was likely due to the high fragmentation of RNA transcripts in mucus, a result of RNAse present in the mucus (Brinchmann, 2016). A full list of all annotated differentially expressed transcripts for each lake-by-lake comparison may be found in Additional File 1: Table S5. Only transcripts which were annotated were used for further pathway analysis.
Due to the high number of differentially expressed transcripts, pathway analyses were used to understand biological pathways and functions that differ among lake trout populations. A list of all GO molecular functions and biological processes and KEGG pathways for each lake-by-lake comparison can be found in Additional File 1: Table S6. There was a high prevalence of immune-related pathways among the top ten biological process GO terms for most lake-by-lake comparisons (Table 2). Comparing L223 and L224, a notable observation was the presence of pathways related to viral infection within the top ranked GO biological functions, including ‘viral process’ (rank 11), ‘viral gene expression’ (rank 8), and ‘viral transcription’ (rank 9). Furthermore, several KEGG pathways among L223 and L224 were related to pathogens, such as ‘Salmonella infection’ (rank 9), ‘Vibrio choerae infection’ (rank 5), ‘epithelial cell signaling in Helicobacter pylori infection’ (rank 10), bacterial invasion of epithelial cells’ (15), and ‘shigellosis’ (rank 13). Comparing L223 and L260, ‘viral process’ (rank 2) and ‘viral gene expression’ (rank 52) were also identified as altered GO biological functions as well as the KEGG pathways ‘Salmonella infection’ (rank 3), ‘Shigellosis’ (rank 7), ‘hepatitis C’ (rank 14), ‘viral carcinogenesis’ (rank 16), and others (Additional File 1: Table S6). As for the comparison among L233 and L373, several of the top GO biological pathways were related to cell adhesion and cell migration involved in the inflammatory response, such as ‘biological adhesion’ (rank 1), ‘cell adhesion’ (rank 2), ‘cell-cell adhesion’ (rank 4), ‘T cell activation’ (rank 12), and ‘T cell differentiation’ (rank 15) (Table 2; Additional File 1: Table S6). Comparison of L223 with all other lakes identified several immune- and infection-related pathways, suggesting L223 lake trout may be exposed to pathogens. Top GO biological functions among L224 and L373, the most transcriptionally-similar lakes, were mainly related to interleukin-1 beta (Table 2). Finally, comparisons of L260 with L224 and L373 both identified a high prevalence of pathways related to cell death and apoptosis among top GO biological pathways, such as ‘regulation of cell death’ and ‘regulation of apoptotic process’ (Table 2).
As immune- and pathogen-related pathways were identified within the top GO biological processes among L223 and all other lakes, immune-related transcripts differentially regulated in L223 were visualized based on normalized count data (Figure 4). Many immune- and stress-related transcripts were expressed to a greater degree in L223 lake trout compared to all other populations, among them members of the GTPase of immunity-associated protein family, cytokines, interferons, and heat shock proteins (Figure 4). Furthermore, many transcripts involved in viral transcription were found to be consistently upregulated in L223 relative to all other lakes (Figure 5). Aside from immune transcripts, other notable gene expression patterns included that of cytosolic phospholipase A2 gamma (PLA2G4C ), which was the top differentially-expressed transcript and strongly upregulated (fold change > 7) in L260 lake trout in comparison with all other lakes. Up-regulator of cell proliferation (URGCP ) was among the top differentially expressed transcripts in all lake-by-lake comparisons.