Copper uptake in blue crabs is independent of sodium transport under hyposaline conditions

In vivo and in vitro experiments were conducted to test whether copper (Cu) uptake occurs via sodium (Na⁺) transporters in the gills of Callinectes sapidus acclimated to dilute seawater (2 ppt), a condition in which the species hyper-osmoregulates. Specific inhibitors targeting Na⁺/H⁺ exchangers (amiloride, 100 μM) and Na⁺, K⁺, 2 Cl^- cotransporters (NKCC) (furosemide, 120 μM) were used. In vivo, adult crabs were exposed for 6 h to 1 μM radiolabeled Cu (⁶⁴Cu) in artificial seawater or Na-free media, both at 2 ppt. In vitro, isolated posterior gills were perfused with hemolymph-like saline and exposed to external solutions containing ⁶⁴Cu. Na⁺ uptake was first validated using radiolabeled Na (²⁴Na) and the inhibitors: in vivo Na⁺ uptake was significantly reduced by amiloride (68 %) and furosemide (23 %) and in vitro amiloride reduced Na⁺ uptake by 40 %. Cu uptake, however, remained unaffected by the Na⁺ presence/absence or by the inhibitors in both experimental approaches. The ⁶⁴Cu accumulated mainly in the carapace (49 %) and posterior gills (22 %), regardless of Na⁺ availability. The findings clearly demonstrate that Cu uptake, irrespective of the uptake pathway, proceeds independently of Na.