Genes differentially expressed in response to salinity (comparison 1 and 2)
When contrasting SwC and SwFw (comparison 1), a total of 10 transcripts were significantly differentially regulated, six which were annotated. Of the 10 transcripts, 3 transcripts were significantly different only for comparison 1 (Figure 4 ). Two of the transcripts were annotated; a gene predicted to be involved in the elongation factor process (si:ch211-13k12.2 ) and the enzyme Galactosylceramide sulfotransferase (GAL3ST1 ). Five transcripts were shared with comparison 2: an arrestin domain (arrdc3a ), a solute carrier (SLC16a9a) (Figure 6a ), a cytochrome P450, family 1 (CYP1a ) (Figure 6b ), a docking protein (dok4 ) and a novel gene, ENSGACG00000019379, which shares sequence homology with TBC1 family member 24 (BlastP GenBank) that has a potential function in intracellular trafficking. Two additional transcripts, which were also not annotated, were shared with comparison 3 (Figure 4 ).
Of the 1535 genes reported in comparison 2, between freshwater ecotypes, a total of 691 transcripts (569 annotated) were upregulated in FwSw when compared to FwC (Table 1 ). The upregulated gene-list included several ion transporting ATPases; ATPase Na+/K+ transporting subunit beta 1a (ATP1b1a ), ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 1 (ATP2a1 , Figure 6b ) and ATPase phospholipid transporting 11C (ATP11c ); t wo ABC-transporter genes were upregulated in FwSw (ABCf2a andABCb6a ) which belong to a family of proteins that utilize the energy of ATP binding and hydrolysis to transport various substrates across cellular membranes. A total of 20 different solute carriers were also upregulated in FwSw, and two genes involved in vesicular trafficking; transmembrane emp24 trafficking protein 2 (tmed2 ,Figure 6b ) and 10 (tmed10 ). Genes linked to stress included five Heat Shock Protein (HSP)-genes (HSPa4b, HSPa5, HSPa8 (Figure 6b ), HSPa8b, HSPd1 ) and seven ubiquitin specific peptidase (USP)-genes. The HSPa8 and HSPa8bencodes members of HSP70, and four DnaJ homologs which are co-chaperones of the HSP70-family were also upregulated in FwSw (dnaja1, dnajb1a, dnaja2a, dnajb9b ). Gene ontology enrichment of the upregulated genes involved processes such as peptide biosynthetic process, cellular response to xenobiotic stimulus and glutathione metabolic process (Table 2 ).
A total of 671 annotated genes were downregulated in FwSw when compared to FwC (Table 1 ). Several known genes had reduced expression for freshwater fish when in saltwater; six genes related to ion transporting ATPases, including five related to phospholipid transporting (ATP8a1, ATP8b2, ATP9a, ATP9b and ATP10d ), and one linked to metallopeptidase and ATP-synthase assembly factor homologs (ATP23 ). Further, seven different solute carriers were downregulated, as were genes linked to the potassium voltage-gated channel (KCNd3 ), the potassium inwardly rectifying channel (KCNj2a ), a potassium channel tetramerisation domain (KCTd12b ) and the chloride channel, voltage-sensitive 6 (CKCn6 ). Two genes linked to calcium regulation; an EF-hand calcium binding domain (EFcab7 ) and a transient receptor potential cation channel gene (TRMP7 ), and two genes linked to thyroid hormone signalling, thyroid hormone receptor interactor 10a (trip10a ) and thyroid hormone receptor interactor 4 (trip4 ). Related to osmosensing, and involved in the activation and differentiation of immune cells, five interlukins all had similarly expression across groups, except for FwSw where they were downregulated (ILf3a, ILf3b, IL113ra2, IL15, and IL19 ). No HSP-genes were downregulated, but one ATP-binding cassette (ABCh1 ), three DnaJ homologs (co-chaperones of the HSP70- family: dnajc7, dnajc9 anddnajc11b ), together with three USP-genes (USP31, USP40 and USP45 ) all had lower expression in FwSw. When testing all downregulated genes in this comparison, transcripts for GO-enrichments processes such as DNA repair, nucleosome assembly, cilium assembly were included on top of the list (Table 2 ).