Figures:
Figure 1: Survival and organismal phenotypes during hypersalinity acclimation. a) Blood osmolality values for five 14-day acclimations after 24 hours at the target salinity (n=6 except at 75g/kg with n=4) and following extended exposure for 12 weeks (75g/kg, n=8) and at MP (85/105g/kg, n=6). b) Body condition for the same treatments as c . Letters in C and D were assigned using a post-hoc Tukey test following one-way ANOVA.
Figure 2: Growth and feeding during 12-week exposure to 75g/kg salinity. a) Size comparison of smallest and largest fish in the freshwater and 75g/kg treatment at the end of the experiment. b) Weight gain for FW control and at 75g/kg, with comparison between treatments. Significance was determined using Kruskal-Wallis test by ranks with “ns”- not significant, “*”- p<0.05, “**”- p<0.005, “***”- p<0.0005. c) Percent of fish body weight consumed daily over the course of the experiments, with weekly weighing identified by dashed vertical lines.
Figure 3 : Salinity effects on the gill proteome for five treatments. a) Volcano plots for each treatment with log2(Fold Change) plotted against log10(p-value). n=8 for 85 and 105g/kg 14-day acclimation and 75g/kg extended treatment, and n=6 for extended 85 and 105g/kg treatments. Significantly upregulated proteins are plotted in red with the total number of sig. upregulated proteins labelled, and significantly downregulated proteins are in blue. b) Overlap in all significantly regulated proteins between five treatments with the number of proteins in each segment labelled. c) Top significantly regulated proteins with the FC difference with controls shown. Redder boxes are more highly upregulated while bluer boxes are more highly downregulated.
Figure 4: STRING network showing all proteins which were significantly regulated in all treatments. Nodes represent one STRING protein ID and edges represent a connection based on protein-protein
interactions including known or predicted interactions based on published literature. Solid lines connect all members of one cluster, while dashed lines indicate connections that are not strong enough to be included in the same cluster based on a Markov Cluster inflation factor of 3. Central members of the Electron Transport Chain (red), glycolysis (green), and citric acid cycle (yellow) are indicated, along with specific proteins labelled. Insets used to more clearly display protein names.
Figure 5: STRING networks for pejus and pessimum stress stages and network; a) Networks with more than three nodes for all significantly regulated proteins found in the 12-week exposure to 75g/kg salinity representing pejus level salinity stress. Protein names in red were upregulated, those in blue were downregulated, and those outlined in red were upregulated in this treatment while being significantly downregulated in extended exposures to MP at 85g/kg and/or 105g/kg.b) Networks with more than three nodes for significantly regulated proteins found in all four pessimum level salinity stress treatments (14-day acclimation and extended exposure to 85g/kg and 105g/kg). Legend indicates the evidence used to connect protein nodes in each network.