About 400 million years ago, when fish first crawled out of the primordial ooze and onto land, they faced a slew of new challenges. One pressing issue was getting rid of ammonia, a toxic waste product of protein metabolism, without water flowing across their gills to flush it out. Although terrestrial lineages have long since converted to a land-friendly form of waste management, some 40 families of fish continue to relive the glory days of tetrapod evolution by taking short sojourns onto land. Amphibious fishes rely on many strategies to dodge ammonia toxicity, including converting ammonia into less-toxic urea, temporarily suppressing protein breakdown and even releasing ammonia as a gas. Intrigued by the diversity of solutions to this recurring physiological problem, a group of Canadian researchers from the University of Guelph and Wilfrid Laurier University looked for rhyme and reason in the ammonia disposal strategies of six aplocheiloid killifishes selected from across a 1000-plus-member family tree.
First, the team had to verify that their killifish showed amphibious behaviour. They ‘encouraged’ the animals to be adventurous and emerge from the water by cranking up the thermostat or dropping the oxygen level in the water, mimicking the environmental challenges common in their natural habitat of swampy, stagnant pools. Five of the six species emerged when the water got too hot and all of them vacated the water when exposed to hypoxia, suggesting that the ability to pack up and leave inhospitable conditions may be a driver in the evolution of an amphibious lifestyle.
As expected, air exposure strongly handicapped ammonia excretion across the gills in all six species. The researchers then investigated each of the alternative ammonia handling strategies that the killifish could use while out of water: retention of ammonia in the tissues for ‘washout’ upon return to the water, conversion to urea, temporary suppression of protein metabolism, and the release of ammonia as a gas. Although one species showed evidence for ammonia retention and another for urea excretion, ‘gassing off’ was the primary strategy of all six killifishes, accounting for 57% to 89% of total ammonia excretion during emersion.
We know of very few fish that release ammonia gas, but the process seems to depend on ammonia-transporting Rhesus glycoproteins embedded in the skin or gut lining. Using dye that is specially targeted to Rhesus glycoproteins, the team found that the skin of all six killifish species stained positive for two of the proteins, Rhcg1 and Rhcg2, which likely act as release valves for the ammonia gas. However, the positioning of Rhcg1 and Rhcg2 varied between species: some fish glowed with an abundance of Rhesus proteins all over the skin, while others showed only pinpricks of stain, with their proteins being closely associated with ion-transporting cells. These different patterns may reflect some specialization in excreting ammonia in habitats with different salinity, or in how long a species typically spends out of the water.
Fish are remarkable colonists and have evolved many clever solutions for dealing with the new habitats. Despite the many possible options for handling ammonia on land, the aplocheiloid killifish adopted a common strategy for nitrogenous waste management when exposed to air. Whether this shared mechanism comes from evolutionary ‘family resemblance’, convergent evolution or both, there is no denying that the ability to survive on land is a powerful tool for an otherwise aquatic organism. Perhaps the secret to being a successful fish out of water is to just let the nasty stuff roll off your back.
