Differentially regulated genes and pathways among lakes
Differential gene expression analysis and pathway analysis was used to
understand the underlying biological meaning of transcriptional
differences among lake trout populations. Among each lake-by-lake
comparison, there was a high prevalence of transcripts and gene ontology
biological processes related to the immune system, suggesting an active
response to stress in lake trout populations (Addition File 1: Table S5;
Table S6) (Tort, 2011). Transcripts that were commonly differentially
regulated among most lake-by-lake comparisons included members of the
GTPase of immunity-associated protein family (GIMAP2 ,GIMAP4 , GIMAP7 , GIMAP8 , etc.), chemokine
transcripts (CCL2 , CCL3 , CCL12 , CCL20 ,
etc.), interferon-related transcripts (IRF1 , IRF3 ,IFI44 , etc.), and others (Figure 4). Expression of immune-related
genes is not surprising as fish epidermal mucus is full of various
immune relevant molecules (Brinchmann, 2016) and differential regulation
of immune-related genes in mucus has been identified in previous studies
(Greer et al., 2019; Ren et al., 2015). Aside from immune response
transcripts, expression of viral RNA was also observed among lake trout
populations.
RNA sequencing of epidermal mucus suggested the presence of retroviral
infection in the sampled lake trout populations. Presence of
retroviruses among lakes was evidenced by the expression of transcripts
involved in transcription and replication of viral RNA, including
transcripts coding for pol polyprotein, gag-pol polyprotein, replicase
polyprotein, RNA-directed RNA polymerase, and RNA-dependent RNA
polymerase (Additional File 1: Table S5) (Ahlquist, 2002; Fodor, 2013;
Lepa & Siwicki, 2011). Interestingly, the transcript gag-polencodes the gag-pol polyprotein of the Walleye dermal sarcoma virus
(WDSV) (UniProt ID: POL_WDSV), an exogenous retrovirus inducing dermal
lesions and sarcomas in walleye (Sander vitreus ) (Holzschu et
al., 1995; K. Xu et al., 2013). Gag, a major structural protein, and
pol, the reverse transcriptase, are essential proteins encoded within
retroviral genomes and within the WDSV genome specifically (Holzschu et
al., 1995; Katz, 1994; Rovnak & Quackenbush, 2010). Elevated expression
of viral RNA has been identified in dermal sarcomas of WDSV-infected
fish (Poulet, Vogt, Bowser, & Casey, 1995). While WDSV is specific to
walleye, only six tumorigenic piscine retroviruses have been fully or
partially sequenced and, thus, it is likely that the gag-poltranscript detected in the present study is endogenous to a closely
related retrovirus that has not yet been sequenced (Quackenbush et al.,
2001).
Following infection, cyclins encoded by WDSV induce cells to proliferate
abnormally, resulting in lesions and tumors (LaPierre, Casey, &
Holzschu, 1998). Interestingly, URGCP was consistently among the
top differentially regulated transcripts among all lake-by-lake
comparisons. URGCP stimulates cyclin to accelerate cell growth
and promote tumor formation, and overexpression of URGCP has been
implicated in virus-induced carcinomas in humans (Dodurga et al., 2014;
Lale Satiroglu Tufan et al., 2002). As lymphocytes have been shown to
aggregate around WSDV dermal lesions, the differential regulation of
transcripts involved in lymphocyte development, trafficking, and
function (e.g. GIMAP2 , GIMAP4 , GIMAP7 ,GIMAP8, CCL2 , CCL3 , CCL12 , CCL20 ,IL1B , etc.) may also indicate antiviral response to epidermal
lesions (Ciucci & Bosselut, 2014; Filén & Lahesmaa, 2010; Reyes-Cerpa
et al., 2012). Differential regulation of interferon-related transcripts
(IRF1 , IRF3 , IFI44 , etc.) further suggests
antiviral response as interferon regulatory factors play a critical role
in the antiviral immune response in fish and transcription of interferon
regulatory factors, interferons, and interferon-stimulated genes has
been shown to be upregulated following viral infection in fish (Collet
& Secombes, 2002; Huang et al., 2015; Robertsen, 2018; Yao, Huang, Fan,
Kong, & Wang, 2012; Zou & Secombes, 2011). Overall, expression of
WDSV-specific gag-pol , transcripts involved in cell
proliferation, and immune transcripts associated with antiviral response
suggest that viral carcinogenesis may be present in the studied lake
trout populations.
Although transcripts for viral RNA transcription were differentially
regulated among all lake-by-lake comparisons, these transcripts were
only consistently upregulated within L223 lake trout, providing
compelling evidence that prevalence of viral infection may be greatest
within L223 (Figure 5). Comparison of L233 lake trout to all other lakes
indicated that URGCP was consistently among the top ten
transcripts differentially expressed in L223 and was strongly
upregulated in L223 lake trout (FC > 5). Along with this,
viral-related processes were among the top altered GO biological
processes in L223 comparisons, including viral gene expression, viral
transcription, and viral process among L223 and L224 and among L223 and
L260 (Table 2). While immune response was evident among most lakes,
immune-related transcripts were most consistently upregulated in L223
lake trout and upregulation of certain transcripts was unique to L223
(e.g. interleukin-8 (CXCL8 ), interleukin 1 beta (IL1B ),
etc.) (Figure 4).
Aside from immune-specific transcripts, transcripts coding for various
heat shock proteins were upregulated in lake trout from L223 compared to
all other lakes, including HSP90AA1 , hsp90a.1 ,hsp90ab1 , and HSPA12A (Figure 4; Additional File 1: Table
S5). In fish, heat shock proteins are expressed in response to a range
of biotic and abiotic stressors, such as environmental contaminants,
heat and cold shock, food deprivation, and infectious pathogens (Basu et
al., 2002; Cara, Aluru, Moyano, & Vijayan, 2005; Iwama, Thomas,
Forsyth, & Vijayan, 1998; Roberts, Agius, Saliba, Bossier, & Sung,
2010). Given the evidence for viral transcription in L223 lake trout,
the upregulation of heat shock protein transcripts may be in response to
pathogens. For example, Hsp90 isoforms have been shown to be upregulated
in several fish species following viral infection and bacterial
challenge (Y. M. Chen et al., 2010; Wei, Gao, Wang, & Xu, 2013; Xie,
Song, Weng, Liu, & Liu, 2015). Upregulation of Hsp90 alpha and beta
proteins has also been observed in epidermal mucus of sea-lice infected
Atlantic salmon (Salmo salar L.) (Provan et al., 2013). Along
with this, Hsp90 is one of the most frequently observed host chaperone
for viruses, aiding in synthesis, localization, and folding of viral
proteins (Geller, Taguwa, & Frydman, 2012; Hu & Seeger, 1996;
Kampmueller & Miller, 2005; Nagy, Wang, Pogany, Hafren, & Makinen,
2011). As dependence of viruses on Hsp90 appears to be nearly universal
(Geller et al., 2012), the upregulation of Hsp90 isoforms in L223 may
further suggest viral response. However, these results are not
conclusive as Hsp90 is responsive to multiple different stressors. For
example, the expression of heat shock proteins may also be due to heat
stress, as L223 is relatively shallow and the available optimal habitat
for lake trout is limited during the stratified season; this phenomenon
is reflected in the fact that L223 contained the greatest water
temperatures, though this difference was not significantly different
from L224 and L260 water temperatures (Table 1). Heat stress coupled
with a higher load of pathogens may explain this together, though
further research is needed to elucidate the upregulation of heat shock
proteins in L223 lake trout.
Discussion of PLA2G4C , encoding cytosolic phospholipase A2
gamma (cPLA2γ), is warranted as it was consistently among the top
upregulated gene in L260. While the function of cPLA2γ specifically has
not been characterized in fish, it is known that cPLA2γ a lipolytic
enzyme that catalyzes the release of arachidonic acid from membrane
phospholipids in humans (Murakami, Masuda, & Kudo, 2003). In fish,
arachidonic acid is a precursor for a wide spectrum of eicosanoids
involved in hemodynamic regulation, immune and inflammatory responses,
reproduction, renal and neural functions (Tocher, 2003). It has been
suggested that cPLA2γ specifically is involved in the inflammatory
response, as PLA2G4C was upregulated in rodents following
pathogen infection and also has been shown to contribute to formation,
replication, and assembly of hepatitis C virus (Brown et al., 2008; S.
Xu et al., 2012). Due to the number of biological processes affected by
arachidonic acid release, further work will be required to identify the
downstream pathways associated with PLA2G4C upregulation in lake
trout from L260.