Enpoints results from in vivo 1 and 2

The file contains all the measured endpoints of the two in vivo experiments (Worksheet 1 and 2) described in the manuscript, including: plasma ions concentrations and gene expression levels of NKA and CA2 in samples of gills and intestine. The spreadsheet also reports fish sex, copper exposure concetrations and salinity. The datasets are presented in a format that can be easily used for PCA analysis

Degree of change in sodium levels.

<p>Difference (delta) in plasma sodium levels (µg/ml) before and after the salinity transition in fish exposed to 0 (Ctrls), 32 and 100 µg/L copper. Light grey and dark grey bars represent the delta change in the transition respectively from FW to SW and from SW to FW. No values are reported for the high-copper group as no fish survived after the salinity change. Values are calculated as difference between means and as such have no SE.</p

Primers sequences.

<p>Primers used for qPCR and respective optimal annealing temperatures.</p><p>Primers sequences.</p

CA2 expression – Exp.2.

<p>Relative Mean Normalized Expression (MNE) levels of CA2 measured in the gills (A and B) and in the mid-anterior tract of the intestine (C and D, striped bars) of sheepshead minnows exposed to 0 (Ctrls), 32, 100 and 320 µg/L copper for 19 days. Light grey boxes (graphs A and C) and dark grey boxes (B and D) represent NKA levels respectively in FW and SW groups. Relative MNE levels were determined by qPCR, normalized to control gene (18S) and expressed as fold change relative to FW control value, which was set at 1. Values are means ± SE (<i>n</i> = 10/12, except for the FW Cu320 group, where <i>n</i> = 6). The asterisk denotes statistically significant difference from the control value (<i>P</i><0.05, Tukey <i>t</i>-test). Solid horizontal lines represent median values and dashed lines represent mean values.</p

Copper in liver and plasma – Exp.1.

<p>(<b>A</b>) Copper liver burden (expressed as µg/g wet weight) in fish exposed to 0 (Ctrls), 10 and 100 µg/L copper for 9 days. Values are means ± SE (<i>n</i> = 10/12). The asterisk denotes statistically significant difference from the control value (<i>P</i><0.05, Tukey <i>t</i>-test) (<b>B1–2</b>) Plasma copper levels (µg/ml) in fish exposed to 0, 10 and 100 µg/L copper and sampled either before (B1) or after (B2) the salinity transition. Values are means ± SE (<i>n</i> = 4/7). The asterisk denotes statistically significant difference from the control value post switch (<i>P</i><0.05, Tukey <i>t</i>-test). Solid horizontal lines represent median values and dashed lines represent mean values.</p

Pre-post change in NKA and CA2 expression – Exp.1.

<p>Relative Mean Normalized Expression (MNE) levels of NKA (graphs A and B), and CA2 (graphs C and D) measured in the gills (A and C) and in the mid-anterior tract of the intestine (B and D, striped bars) of sheepshead minnows exposed to 0 (Ctrls), 10 and 100 µg/L copper. Within each copper treatment, the pair of bars represents expression levels before (left side) and after (right side) the salinity transition. Relative MNE levels were determined by qPCR, normalized to control gene (18S) and expressed as fold change relative to pre-switch control value, which was set at 1 (dotted horizontal line). Values are means ± SE (<i>n</i> = 6/7). In each graph, bars sharing the same symbol (asterisk, cross or dot) are significantly different one from the other (<i>P</i><0.05, Tukey <i>t</i>-test).</p

Inorganic copper speciation.

<p>Most relevant copper forms are reported (expressed as % of total dissolved copper concentrations), repectively in FW and SW, as calculated by Visual MINTEQ using measured water chemistry parameteres of Experiment 1.</p><p>Inorganic copper speciation.</p

Pre-post change in CA2 expression – Exp.2.

<p>Relative Mean Normalized Expression (MNE) levels of CA2 measured in the gills (A and B) and in the mid-anterior tract of the intestine (C and D, striped bars) of sheepshead minnows exposed to 0 (Ctrls), 32, 100 and 320 µg/L copper. Within each copper treatment, the pair of bars represents CA2 expression levels before (left side) and after (right side) the salinity transition. The colour of the bars represents the salinity conditions: light-grey for FW and dark-grey for SW. Relative MNE levels were determined by qPCR, normalized to control gene (18S) and expressed as fold change relative to pre-switch FW control value, which was set at 1 (dotted horizontal line). Values are means ± SE (<i>n</i> = 10/12). No values are reported for the high copper dose as no fish survived after the salinity change.</p

Plasma sodium – Exp.2.

<p>Plasma sodium concentrations (µg/ml) measured in fish exposed to 0 (Ctrls), 32, 100 and 320 µg/L copper in either FW (A) or SW (B). Values are means ± SE (<i>n</i> = 10, except for FW Cu320, where <i>n</i> = 3). The asterisk denotes statistically significant difference from the control value (<i>P</i><0.05, Tukey <i>t</i>-test). Solid horizontal lines represent median values and dashed lines represent mean values.</p

NKA expression – Exp.2.

<p>Relative Mean Normalized Expression (MNE) levels of NKA measured in the gills (A and B) and in the mid-anterior tract of the intestine (C and D, striped bars) of sheepshead minnows exposed to 0 (Ctrls), 32, 100 and 320 µg/L copper for 19 days. Light grey boxes (graphs A and C) and dark grey boxes (B and D) represent NKA levels respectively in FW and SW groups. Relative MNE levels were determined by qPCR, normalized to control gene (18S) and expressed as fold change relative to FW control value, which was set at 1. Values are means ± SE (<i>n</i> = 10/12, except for the FW Cu320 group, where <i>n</i> = 6). The asterisk denotes statistically significant difference from the control value (<i>P</i><0.05, Tukey <i>t</i>-test). Solid horizontal lines represent median values and dashed lines represent mean values.</p