Plastic gene expression profiles within ecotypes
A fish adapted to a particular salinity need to have complex physiological regulatory mechanisms, at both the organismal and cellular level, in order to maintain water homeostasis. Changes in salinity will induce alterations in the nature and direction of ion transport, and genes linked to the maintenance of water homeostasis, cell signalling and structural permeability of cell membranes and stress responses, are likely targets of short-term salinity responses. To start a physiological response, the fish must be able to recognize osmolarity change, where input from the osmolarity-sensors also need to encode the magnitude, direction and ionic basis of the perceived change. Of the few genes with significant regulatory differences in both comparison 1 and 2, arrestin and SLC-genes are linked to early osmosensory signal transduction. The solute carriers are membrane transport proteins mostly located in the cell membrane where they facilitate movement of small solutes across cell membranes in response to chemiosmotic gradients. A total of 27 SLC-related transcripts were linked to salinity changes within the freshwater ecotype (contrast 2), including the Na+/H+-exchanger (SLC9a2 ) with a higher expression in freshwater that also have been found in long term studies of freshwater acclimation in stickleback (although different isoforms; Gibbons et al. 2017). As expected, the Na+/K+/2CL-cotransporter (NKCC1 / SLC12a2 ) had higher expression in saltwater, which also is consistent with findings in long term salinity exposures in stickleback (Gibbons et al. 2017) and other species of saltwater fish (Shaughnessy and McCormick 2020). That NKCC1 has a central role in salinity acclimation is further supported with no expression in gills of the salmonid grayling (Thymallus thymallus ), which is a strict freshwater fish (Varadharajan et al. 2018). Three members of the monocarboxylate transporter family (MCT/SLC16 ) were upregulated in saltwater (Figure 6a), whereSLC16a9a was one of the few genes differentially regulated in contrast 1. MCT’s are involved in H+-linked transport of monocarboxylic anions (Verri et al. 2012), that again are linked to the level of carnitide and energy metabolism by the transport of long fatty acid chains, like lactate, into mitochondria for energy production and between cell-types. Fish gills are highly oxidative tissues, and oxygen requirements increase with increasing salinities (Vijayan et al. 1996), which again increase the natural concentrations of both plasma and gill-cellular lactate (Mommsen 1984; Sangiao-Alvarellos et al. 2005; Sangiao-Alvarellos et al. 2003). Lactate might hence be the primary candidate for rapid carbohydrate fuel in the gill tissue, especially for the saltwater fish in the early responses to reduced salinity.
Arrestin (arrdc2, arrdc3a and arrdc3b ) was downregulated in saltwater and arrdc3a were also included amongst the ten transcripts differentially regulated with salinity in saltwater fish (Figure 6a). Arrestins have been found to be involved in the modulation of diverse cellular processes through their adaptor functions, facilitating the localization and function of other proteins. Arrdc3a is linked to the GPCR-regulation of the adrenergic signalling pathway, which again is linked to cellular Na+-regulation (Kumai et al. 2012), to increased insulin and glucose metabolism in mice livers (Batista et al. 2020), to growth in plants under salinity stress (Colaneri et al. 2014) and to stress and phosphorylation of the actin-cytoskeleton in a soil amoeba, Dictyostelium (Habourdin et al. 2013). Arrestins have also been linked to positive activation of MAP kinases (Lefkowitz and Shenoy 2005), a family of enzymes involved in osmosensory signal transduction (Fiol and Kültz 2007), several which are differentially regulated between comparison 2 and 3 in this study. Arrestins have also previously been linked to other short time osmoregulatory experiments, being downregulated in turbot (Scophthalmus maximus ) livers, when turbot acclimated to 30‰ salinity was exposed to 5‰ for 24 hours (Cui et al. 2020), and four homologs of Arrestin was upregulated in the shrimp (Halocaridina rubra ) (Havird et al. 2019) and crab (Portunus trituberculatus ) (Lv et al. 2016) when they were transferred from 32‰ to 15‰ salinity, also for 24 hours (similar results as in this study). That Arrestins could have an important role in being “osmosensory genes” are also supported by DNA sequences for the arrestin-gene arrb2b , as the sequences for stickleback and another euryhaline fish, the tiger pufferfish (Takifugu rubripes ), were found to be more diverse from their alpha counterpart, arrb2a , than for several other fish species, which likely is a result of directional selection (Indrischek et al. 2017).
Several cytochrome P450 genes were upregulated in saltwater, includingCYP1a that were one of the ten transcripts with differential regulation in contrast 1 (Figure 6a). CYP1 is a superfamily of enzymes that catalyses the oxidation of many reactions, and is widely used as an indicator of environmental pollution, also for stickleback (Knag and Taugbøl 2013). The historic focus on CYP1 as “only” a pollutant biomarker might have constricted the assessments of many related results to other potential pathways (Evans et al. 2005), as recent findings indicate a more direct link to general stress- and immune responses (Lenoir et al. 2021). The translation and expression pattern of CYP1a is being regulated by the aryl hydrocarbon receptor, AHR , which after heterodimerizing with ARNT , also is functional in immune cells of Atlantic salmon (Salmo salar ) (Song et al. 2020), and when overexpressed, CYP1a has been found to actively suppress the expression of inferon type 1 (IFNI ) (but not IRF7 ) in grass carp (Ctenopharyngodon idella ), inferons that are secreted by infected cells (Chu et al. 2019). In previous salinity treatment experiments including fish, CYP1a has been found to be both upregulated and downregulated with salinity; Wang et al. (2014) found sticklebacks to have the highest expression in their original water quality (salt- and freshwater) when compared to freshwater fish in both 11‰ and 34‰ after 30 days of exposure (saltwater fish was only exposed to saltwater in this study), whereas CYP1a was found to be upregulated in tiger puffer after 30 days exposure in the low salinity group (Jiang et al. 2020), and opposite, to increase with increasing salinities in coho salmon (Oncorhynchus kisutch ) (Lavado et al. 2014) and rainbow trout (Leguen et al. 2010), as is similar to this study.
Maintaining cell volume is critical during salinity changes. Tight junction proteins such as claudins and occludins were upregulated in freshwater (comparison 4), similar to a long-term salinity study on stickleback (Gibbons et al. 2017). Aquaporin 3a (AQ P3a), a water channel protein linked to cell volume regulation and sensing, also had higher expression in freshwater, which is commonly found in euryhaline fish (Cutler et al. 2007; Velotta et al. 2017). In this study, AQP3a had a slight plastic change within the freshwater ecotype, as FwS had lower expression than FwC (Figure 6c). Aquaporin expression has been found to be involved in the meditation of osmoreception in the tilapia prolactin-secretion and gill chloride cell differentiation (Yan et al. 2013), and the DNA sequence for aquaporin in sticklebacks has previously been associated with positive selection between marine- and freshwater populations (DeFaveri et al. 2013; Shimada et al. 2011), as has the gene expression patterns (Gibbons et al. 2017). Interestingly, in a purebred stickleback cross-fostering experiment in 20‰ and 5‰, the expression pattern for AQP3a was equally expressed in the freshwater ecotype, and the saltwater ecotype had a higher expression with increased salinity (Hasan et al. 2017). This is the opposite pattern of what was found here. Wang et al. (2014) identified AQP4 , another member of the aquaporin family, as a salt-responsive gene in the kidneys of sticklebacks, although significant differences were only observed for freshwater fish in fresh- and saltwater (saltwater fish was only exposed to saltwater in Wang et al. 2014). In the present study, AQP4 was filtered out due to low overall expression, but did have increased expression in the saltwater ecotype (data not shown).
Slow-working hormones are involved in rearrangements during long-term acclimation, by altering the abundance of ion transporters and cell proliferation, and differentiation of ionocytes and other osmoregulatory cells (Takei and McCormick 2012). Prolactin is one of the slow-working hormones, since long known to have a function in salinity acclimation (Pickford and Phillips 1959). The concentration of Prolactin (PRLR) is typically increased following freshwater acclimation (Takei and McCormick 2012) and more directly, prolactin has been linked to the chloride cell regulation; if injected with prolactin, the number of chloride cells in the gills of seawater acclimated tilapia decreases to the levels characterized by freshwater acclimated tilapia (Yan et al. 2013). Prolactin consists of two receptors; PRLRa and PRLRb, for which the expression patterns in gills have been found to be individually linked to salinity in tilapia (Fiol et al. 2009). Similar to this study, Fiol et al. (2009), we found the expression of PRLRa to be overall higher in freshwater (Figure 6c), with FwC-expression being significantly different from both SwC and SwFw. Oppositely, the PRLRb-receptor had an increased expression in saltwater, but the difference was only significant in comparison2 (increased in FwSw). Specific activation of the two PRLR-receptors was also found to activate different downstream signalling pathways, likely activating alternative routes leading to osmoprotection of gill-cells during the period of active restructuring of gill epithelium in response to salinity stress (Fiol et al. 2009).