mRNA expression and activity of ion-transporting proteins in gills of the blue crab callinectes sapidus: effects of waterborne copper

Abstract

Waterborne Cu effects on the transcription of genes encoding ion-transporting proteins and the activities of these proteins were evaluated in gills of the blue crab Callinectes sapidus acclimated to diluted (2%) and full (30%) seawater. Crabs were exposed (96 h) to an environmentally relevant concentration of dissolved Cu (0.78 mM) and had their posterior (osmoregulating) gills dissected for enzymatic and molecular analysis. Endpoints analyzed were the activity of key enzymes involved in crab osmoregulation (sodiumpotassium adenosine triphosphatase [Naþ/Kþ-ATPase], hydrogen adenosine triphosphatase [Hþ-ATPase], and carbonic anhydrase [CA]) and the mRNA expression of genes encoding these enzymes and the sodium-potassium-chloride (Naþ/Kþ/2Cl_) cotransporter. Copper effects were observed only in crabs acclimated to diluted seawater (hyperosmoregulating crabs) and were associated with an inhibition of the expression of mRNA of genes encoding the Naþ/Kþ-ATPase and the Naþ/Kþ/2Cl_ cotransporter. However, Cu did not affect Naþ/Kþ-ATPase activity, indicating that the gene transcription is downregulated before a significant inhibition of the enzyme activity can be observed. This also suggests the existence of a compensatory response of this enzyme to prevent osmoregulatory disturbances after short-term exposure to environmentally relevant Cu concentrations. These findings suggest that Cu is a potential ionoregulatory toxicant in blue crabs C. sapidus acclimated to low salinity. The lack of Cu effect on blue crabs acclimated to full seawater would be due to the reduced ion uptake needed for the regulation of the hemolymph osmotic concentration in full seawater (30%). Also, this could be explained considering the lower bioavailability of toxic Cu (free ion) associated with the higher ionic content and dissolved organic matter concentration in high salinity (30%) than in diluted seawater (2%).