For such a tiny animal, Caenorhabditis elegans must be one of the best understood creatures on the planet. Its development has been painstakingly scrutinized cell by cell and its 100 million base pair genome has been deconstructed in fine detail; yet little is known about the physiology of these microscopic soil dwellers that reside in the water film surrounding soil particles. ‘It's not just a worm in a fridge’, chuckles Dirk Weihrauch from the University of Manitoba, Canada, who adds that he is always keen to learn about how animals from exotic environments dispose of toxic nitrogenous waste. Yet, it wasn't even clear whether the nematodes excrete ammonia or urea. So, Weihrauch and his student Aida Adlimoghaddam rolled up their sleeves and decided to get to grips with nitrogen excretion in this super-star model organism (p. 675).
Caenorhabditis elegans usually dine on bacteria in soil, so Adlimoghaddam and Ann-Karen Brassinga made sure that the lab worms were well fed with nutritious E. coli before collecting the nematodes’ waste for 1 day. Analysing the media that was the worms’ home, the duo found that instead of producing costly urea, they were excreting ammonia.
Next, Adlimoghaddam tested whether the worms were expressing the genes encoding proteins that are known to participate in nitrogen excretion in other animals. As Weihrauch explains, the Na+/K+-ATPase enzyme pumps ammonium ions across the cell membranes of various animals, while the V-type H+-ATPase produces a pH gradient across membranes that ammonia can diffuse along, and Rhesus proteins form channels that ammonia gas travels through. When Adlimoghaddam looked for evidence of expression of each of these proteins by measuring mRNA expression of the C. elegans equivalents, she found that the worms expressed them all.
However, Weihrauch wanted to know more about the worms’ excretion mechanism, even though it was impossible to measure directly the transport processes in the minute animals. So he and Adlimoghaddam resorted to measuring the ammonia excreted by large numbers of the animals bathed in drugs that targeted specific proteins in ammonia excretion in other organisms to find out which participated in ammonia excretion. The duo discovered that inhibiting the V-ATPase, carbonic anhydrase (which provides protons to fuel the V-ATPase) and the worm's microtubule network all reduced ammonia excretion. The drug targeting the Na+/K+-ATPase (ouabain) had no effect on ammonia excretion, suggesting that it was unable to access the pump in the cell membrane. However, when Jason Treberg directly tested whether Na+/K+-ATPase could transport ammonium, the protein successfully transported the ions, so it also contributes to excretion.
Next Mélanie Boeckstaens and Anna-Maria Marini tested whether one of the worm Rhesus channel proteins – CeRhr-1 – actually transports ammonia. Inserting the channel into yeast mutants that were unable to import ammonia – which is essential for their survival – the duo found that the mutant yeast carrying the CeRhr-1 protein survived. So CeRhr-1 is an ammonia transporter.
Tying all of their results together, Weihrauch concludes that C. elegans excrete ammonia through their skin using two mechanisms. The first – known as ammonia trapping – occurs when the V-ATPase, fuelled by protons provided by the carbonic anhydrase, creates a pH gradient across the cell membrane allowing the gas to diffuse through Rhesus proteins to cross membranes in the skin. In the second mechanism, ammonium is transported in lipid vesicles through the cell's microtubule network to the external membrane, ready for excretion. And Weihrauch is excited to have discovered the ammonia trapping mechanism in such an ancient animal, saying: ‘Ammonia excretion mechanisms… evolved early on’.