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.