Marine, freshwater and aerially acclimated mangrove rivulus (Kryptolebias marmoratus) use different strategies for cutaneous ammonia excretion

Abstract

Rhesus (Rh) glycoproteins are ammonia gas (NH(3)) channels known to be involved in ammonia transport in animals. Because of the different osmoregulatory and ionoregulatory challenges faced by teleost fishes in marine and freshwater (FW) environments, we hypothesized that ammonia excretion strategies would differ between environments. Also, we hypothesized that cutaneous NH(3) volatilization in air-acclimated fish is facilitated by base secretion. To test these hypotheses, we used the skin of the euryhaline amphibious mangrove rivulus (Kryptolebias marmoratus). The skin excretes ammonia and expresses Rh glycoproteins. Serosal-to-mucosal cutaneous ammonia flux was saturable (0-16 mmol/l ammonia, K(m) of 6.42 mmol/l). In FW, ammonia excretion increased in response to low mucosal pH but decreased with pharmacological inhibition of Na(+)/H(+) exchangers (NHE) and H(+) ATPase. Conversely, in brackish water (BW), lowering the mucosal pH significantly decreased ammonia excretion. Inhibitors of NHE also decreased ammonia excretion in BW fish. Immunofluorescence microscopy demonstrated that both the Rh isoform, Rhcg1, and NHE3 proteins colocalized in Na(+)/K(+) ATPase expressing mitochondrion-rich cells in the gills, kidney, and skin. We propose that the mechanisms of cutaneous ammonia excretion in FW K. marmoratus are consistent with the model for branchial ammonia excretion in FW teleost fish. NH(4)(+) excretion appeared to play a stronger role in BW. NH(4)(+) excretion in BW may be facilitated by apical NHE and/or diffuse through paracellular pathways. In aerially acclimated fish, inhibition of NHE and H(+) ATPase, but not the Cl(-)/HCO(3)(-) exchanger, significantly affected cutaneous surface pH, suggesting that direct base excretion is not critical for NH(3) volatilization. Overall, K. marmoratus use different strategies for excreting ammonia in three different environments, FW, BW, and air, and Rh glycoproteins and NHE are integral to all.