Ammonia independent sodium uptake mediated by Na⁺ channels and NHEs in the freshwater ribbon leech Nephelopsis obscura

Freshwater organisms actively take up ions from their environment to counter diffusive ion losses due to inhabiting hypo-osmotic environments. The mechanisms behind active Na⁺ uptake are quite well understood in freshwater teleosts, however, the mechanisms employed by invertebrates are not. Pharmacological and molecular approaches were used to investigate Na⁺ uptake mechanisms and their link to ammonia excretion in the ribbon leech Nephelopsis obscura. At the molecular level, we identified a Na⁺ channel and a Na⁺/H⁺-exchanger (NHE) in the skin of N. obscura, where the NHE was upregulated when acclimated to extremely low [Na⁺] (0.05 mmol l⁻¹, pH 5) conditions. Additionally, we found that leeches in dilute freshwater environments use both, a vacuolar-type H⁺-ATPase (VHA) assisted uptake via a Na⁺ channel and a NHE based mechanisms for Na⁺ uptake. Immunolocalization of VHA and Na⁺/K⁺ ATPase indicated at least two cell types present within leech skin, VHA⁺ and VHA⁻ cells, where the VHA⁺ cells are likely involved in Na⁺ uptake. NKA was present throughout the epithelium. We also found that increasing ammonia excretion by decreasing water pH, ammonia loading leeches or exposing leeches to high environmental ammonia does not affect Na⁺ uptake providing indications that an NHE-Rh metabolon is not present and that ammonia excretion and Na⁺ uptake are not coupled in N. obscura. To our knowledge, this is the first study showing the mechanisms of Na⁺ uptake and their links to ammonia excretion in a freshwater invertebrate, where results suggest an ammonia-independent Na⁺ uptake mechanism relying on both Na⁺ channels and NHEs.