Salinity-genes with contrasting ecotype expression profiles
Genetic assimilation occurs when a plastic ancestral trait becomes environmentally stable, resulting in a loss of plasticity (Lande 1976). Many of the environmentally expressed genes were linked to known osmoregulatory- and immune functions. Intracellular levels of calcium play an important role in responses to osmotic stress and functions in volume-regulation of the cells (Erickson et al. 2001), and the ability to take up calcium at low Ca2+-concentration have likely been selected for in freshwater. Genes that were differentially expressed in salt- and freshwater linked to the uptake of Ca2+, and to a certain degree also for Na+ (Horng et al. 2007), included different ATP6Vs, subunits of a V-type proton located at the basolateral membrane of mitochondria rich cells (Figure 7). Consisting of two main parts, the ATP6V1 comprise at least eight- and the ATP6V0 include at least five different subunits (Sun-Wada et al. 2004), where five V1 and two V0 subunits had a higher expression in freshwater fish in this study (contrast 3 and 4), clearly indicating that this gene has been important for both freshwater and saltwater acclimation, as was also found for killifish (Fundulus heteroclitus ) (Whitehead et al. 2012) . Other genes related to calcium was ano1 and cacbn1 (Figure 6d), both with an increased expression in saltwater fish.
Pathogen diversity in freshwater is often found to be higher than in salt water (Wang et al. 2012), and in teleosts the skin, gills and gut are continuously exposed to the external aquatic environment and are therefore the main mucosal surfaces that represent potential entry ports for pathogens (Gomez et al. 2013). Many of the mechanisms for antigen sampling in the mucosal epithelium of teleost fish are mostly unknown, as they lack many of the mammalian molecules for transporting pathogens across the epithelia. Recent evidence suggests that two specific antigen-sampling cell types exist in the gill, where one is expressing protein tyrosine phosphatase receptor type C (PTPrc ) and IL-1β (Kato et al. 2018). The PTPs catalyzes the dephosphorylation of protein tyrosine kinases (PTKs ) directly or through their downstream targets, and play key regulatory roles in multiple signal transduction pathways, where most are expressed in immune cells (Mustelin et al. 2005). In this study, increasing expression of PTP’s were linked to high salinity, as two members of the PTP-family were found to be differentially upregulated in the saltwater ecotype (PTPN14 andPTPrfa ), and three were upregulated in comparison 2 (FwSw;PTPN2a, PTPN21, PTPrna ). In contrast, IL-1β had overall higher expression in the freshwater ecotype. The second significant type of teleost antigen-sampling cell types that were recently identified in gills was a microfold cell (M-cell), expressing Anexin5 (ANXA5 , Figure 6d) (Kato et al. 2018), a gene that has been linked to apoptosis by its ability to be recruited to the cell surface and co-localize with phosphatidylserine; the “eat-me”-signal for macrophages (Lizarbe et al. 2013). In the present study, ANXA5b was significantly upregulated in the freshwater ecotype, which could indicate that the two different antigen sampling cell types might have been under directional selection in the opposite environments. ANXA5 has also been linked to changes in calcium concentration, as they can bind around twelve Ca2+ ions and exhibit calcium channel activity in plasma membranes and in matrix vesicles (Lizarbe et al. 2013), so it is unclear if ANXA5 has a immunological or osmoregulatory function in freshwater fish (or both).