Early onset of urea synthesis and ammonia detoxification pathways in three terrestrially developing frogs

Frogs evolved terrestrial development multiple times, necessitating mechanisms to avoid ammonia toxicity at early stages. Urea synthesis from ammonia is a key adaptation that reduces water dependence after metamorphosis. We tested for early expression and plasticity of enzymatic mechanisms of ammonia detoxification in three terrestrial-breeding frogs: foam-nest-dwelling larvae of Leptodactylus fragilis (Lf) and arboreal embryos of Hyalinobatrachium fleischmanni (Hf) and Agalychnis callidryas (Ac). Activity of two ornithine-urea cycle (OUC) enzymes, arginase and CPSase, and levels of their products urea and CP in tissues were high in Lf regardless of nest hydration, but reduced in experimental low- vs. high-ammonia environments. High OUC activity in wet and dry nests, comparable to that under experimental high ammonia, suggests terrestrial Lf larvae maintain high capacity for urea excretion regardless of their immediate risk of ammonia toxicity. This may aid survival through unpredictably long waiting periods before rain enables their transition to water. Moderate levels of urea and CP were present in Hf and Ac tissues and enzymatic activities were lower than in Lf. In both species, embryos in drying clutches can hatch and enter the water early, behaviorally avoiding ammonia toxicity. Moreover, glutamine synthetase was active in early stages of all three species, condensing ammonia and glutamate to glutamine as another mechanism of detoxification. Enzyme activity appeared highest in Lf, although substrate and product levels were higher in Ac and Lf. Our results reveal that multiple biochemical mechanisms of ammonia detoxification occur in early life stages of anuran lineages that evolved terrestrial development.