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.