A new role for carbonic anhydrase 2 in the response of fish to copper and osmotic stress: implications for multi-stressor studies

Abstract

The majority of ecotoxicological studies are performed under stable and optimal conditions, whereas in reality the complexity of the natural environment faces organisms with multiple stressors of different type and origin, which can activate pathways of response often difficult to interpret. In particular, aquatic organisms living in estuarine zones already impacted by metal contamination can be exposed to more severe salinity variations under a forecasted scenario of global change. In this context, the present study aimed to investigate the effect of copper exposure on the response of fish to osmotic stress by mimicking in laboratory conditions the salinity changes occurring in natural estuaries. We hypothesized that copper-exposed individuals are more sensitive to osmotic stresses, as copper affects their osmoregulatory system by acting on a number of osmotic effector proteins, among which the isoform two of the enzyme carbonic anhydrase (CA2) was identified as a novel factor linking the physiological responses to both copper and osmotic stress. To test this hypothesis, two in vivo studies were performed using the euryhaline fish sheepshead minnow (Cyprinodon variegatus) as test species and applying different rates of salinity transition as a controlled way of dosing osmotic stress. Measured endpoints included plasma ions concentrations and gene expression of CA2 and the α1a-subunit of the enzyme Na+/K+ ATPase. Results showed that plasma ions concentrations changed after the salinity transition, but notably the magnitude of change was greater in the copper-exposed groups, suggesting a sensitizing effect of copper on the responses to osmotic stress. Gene expression results demonstrated that CA2 is affected by copper at the transcriptional level and that this enzyme might play a role in the observed combined effects of copper and osmotic stress on ion homeostasis.

Figure 1. Copper in liver and plasma – Exp.1.

(A) 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 (n = 10/12). The asterisk denotes statistically significant difference from the control value (P<0.05, Tukey t-test) (B1–2) 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 (n = 4/7). The asterisk denotes statistically significant difference from the control value post switch (P<0.05, Tukey t-test). Solid horizontal lines represent median values and dashed lines represent mean values.

Figure 2. Plasma sodium and chloride – Exp.1.

Plasma sodium (A) and chloride (B) levels (µg/ml) measured in fish exposed to 0 (Ctrls), 10 and 100 µg/L copper before (light grey bars) and after (dark grey bars) the salinity transition. Values are means ± SE (n = 6/7).

Figure 3. Pre-post change in NKA and CA2 expression – Exp.1.

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 (n = 6/7). In each graph, bars sharing the same symbol (asterisk, cross or dot) are significantly different one from the other (P<0.05, Tukey t-test).

Figure 4. Plasma copper – Exp.2.

Plasma copper levels (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 (n = 18/20, except for FW Cu320, where n = 3). The asterisk denotes statistically significant difference from the control value (P<0.05, Tukey t-test). Solid horizontal lines represent median values and dashed lines represent mean values.

Figure 5. Plasma sodium – Exp.2.

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 (n = 10, except for FW Cu320, where n = 3). The asterisk denotes statistically significant difference from the control value (P<0.05, Tukey t-test). Solid horizontal lines represent median values and dashed lines represent mean values.

Figure 6. Degree of change in sodium levels.

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.

Figure 7. NKA expression – Exp.2.

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 (n = 10/12, except for the FW Cu320 group, where n = 6). The asterisk denotes statistically significant difference from the control value (P<0.05, Tukey t-test). Solid horizontal lines represent median values and dashed lines represent mean values.

Figure 8. Pre-post change in NKA expression – Exp.2.

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. Within each copper treatment, the pair of bars represents NKA 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 (n = 10/12). No values are reported for the high copper dose as no fish survived after the salinity change.

Figure 9. CA2 expression – Exp.2.

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 (n = 10/12, except for the FW Cu320 group, where n = 6). The asterisk denotes statistically significant difference from the control value (P<0.05, Tukey t-test). Solid horizontal lines represent median values and dashed lines represent mean values.

Figure 10. Pre-post change in CA2 expression – Exp.2.

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 (n = 10/12). No values are reported for the high copper dose as no fish survived after the salinity change.