Widespread use of emersion and cutaneous ammonia excretion in Aplocheiloid killifishes

The invasion of land required amphibious fishes to evolve new strategies to avoid toxic ammonia accumulation in the absence of water flow over the gills. We investigated amphibious behaviour and nitrogen excretion strategies in six phylogenetically diverse Aplocheiloid killifishes (Anablepsoides hartii, Cynodonichthys hildebrandi, Rivulus cylindraceus, Kryptolebias marmoratus, Fundulopanchax gardneri, and Aplocheilus lineatus) in order to determine if a common strategy evolved. All species voluntarily emersed (left water) over several days, and also in response to environmental stressors (low O₂, high temperature). All species were ammoniotelic in water and released gaseous ammonia (NH₃ volatilization) during air exposure as the primary route for nitrogen excretion. Metabolic depression, urea synthesis, and/or ammonia accumulation during air exposure were not common strategies used by these species. Immunostaining revealed the presence of ammonia-transporting Rhesus proteins (Rhcg1 and Rhcg2) in the skin of all six species, indicating a shared mechanism for ammonia volatilization. We also found Rhcg in the skin of several other fully aquatic fishes, implying that cutaneous ammonia excretion is not exclusive to amphibious fishes. Overall, our results demonstrate that similar nitrogen excretion strategies while out of water were used by all killifish species tested; possibly the result of shared ancestral amphibious traits, phenotypic convergence, or a combination of both.