Branchial ammonia excretion in the Asian weatherloach Misgurnus anguillicaudatus

Abstract

The weatherloach, Misgurnus anguillicaudatus, is a freshwater, facultative air-breathing fish that lives in streams and rice paddy fields, where it may experience drought and/or high environmental ammonia (HEA) conditions. The aim of this study was to determine what roles branchial Na +/K +-ATPase, H +-ATPase, and Rhcg have in ammonia tolerance and how the weatherloach copes with ammonia loading conditions. The loach's high ammonia tolerance was confirmed as was evident from its high 96 h LC 50 value and high tissue tolerance to ammonia. The weatherloach does not appear to make use of Na +/NH 4 +-ATPase facilitated transport to excrete ammonia when exposed to HEA or to high environmental pH since no changes in activity were observed. Using immunofluorescence microscopy, distinct populations of vacuolar (V)-type H +-ATPase and Na +/K +-ATPase immunoreactive cells were identified in branchial epithelia, with apical and basolateral staining patterns, respectively. Rhesus C glycoprotein (Rhcg1), an ammonia transport protein, immunoreactivity was also found in a similar pattern as H +-ATPase. Rhcg1 (Slc42a3) mRNA expression also increased significantly during aerial exposure, although not significantly under ammonia loading conditions. The colocalization of H +-ATPase and Rhcg1 to the similar non-Na +/K +-ATPase immunoreactive cell type would support a role for H +-ATPase in ammonia excretion via Rhcg by NH 4 + trapping. The importance of gill boundary layer acidification in net ammonia excretion was confirmed in this fish; however, it was not associated with an increase in H +-ATPase expression, since tissue activity and protein levels did not increase with high environmental pH and/or HEA. However the V-ATPase inhibitor, bafilomycin, did decrease net ammonia flux whereas other ion transport inhibitors (amiloride, SITS) had no effect. H +-ATPase inhibition also resulted in a consequent elevation in plasma ammonia levels and a decrease in the net acid flux. In gill, aerial exposure was also associated with a significant increase in membrane fluidity (or increase in permeability) which would presumably enhance NH 3 permeation through the plasma membrane. Taken together, these results indicate the gill of the weatherloach is responsive to aerial conditions that would aid ammonia excretion.