We conducted experiments during the rainy seasons of 2018 and 2019 in Panama with early ontogenetic stages of three terrestrial-breeding frogs: Leptodactylus fragilis (which lay eggs in subterranean foam-nests), Agalychnis callidryas (which lay gelatinous clutches on leaves over ponds) and Hyalinobatrachium fleischmanni (which lay gelatinous clutches on leaves over streams, then provide paternal egg-care). We collected clutches from the field and moved them to an ambient-conditions laboratory (~26°C, ~85% RH) at the Smithsonian Tropical Research Institute (STRI) in Gamboa. For all three species, we exposed developing animals to species-specific well-hydrated (wet) and dry terrestrial conditions in the laboratory and sampled them at ages after the detection of ammonia or urea excretion in prior work (Méndez-Narváez and Warkentin, 2022). For L. fragilis and H. fleischmanni, we also exposed individuals from well-hydrated (wet) terrestrial conditions to species-specific low and high environmental ammonia (LEA and HEA) in water (Méndez-Narváez and Warkentin, 2022), during their extended terrestrial development period. For controlled exposure to HEA, we used NH4Cl to prepare experimental concentrations of ammonia in aged, dechlorinated tap water, placing test groups of siblings in small plastic cups with 20 ml of LEA or HEA solution for 96 hours. We assessed CPSase 1 and arginase activity and the concentrations of their products, CP and urea, in tissues of all three species, across the two terrestrial and two aquatic environmental contexts in L. fragilis and H. fleischmanni and the two terrestrial contexts in A. callidryas. We also assessed glutamine and glutamate concentrations and GSase activity in control and dry terrestrial environments for all three species. We performed all enzymatic assays at 26 °C, from fresh homogenates stored at –80°C for not more than one month after homogenization. We followed the colorimetric method to measure enzymatic activities, using the biochemical conversion of arginine to urea to measure arginase activity, carbamoyl phosphate to hydroxyurea to measure CPSase I activity, and glutamate and ammonia to glutamine and ADP to measure the activity of GSase. To measure the concentration of urea and CP in larval tissues, we used a colorimetric method for ureido compounds. To assess the concentration of glutamine and glutamate in larval tissues, we used BioVision diagnostic kits. We determined protein concentration in each sample by the dye-binding method with the Thermo ScientificTM Coonassie Protein Assay kit. We used these values to correct enzyme activities (μmol or nmol min-1 mg-1 of protein) and the concentration of urea and non-essential amino acids (μmol or nmol mg-1 of protein). References Méndez‐Narváez, J. and Warkentin, K.M., 2022. Reproductive colonization of land by frogs: Embryos and larvae excrete urea to avoid ammonia toxicity. Ecology and Evolution, 12(2), p.e8570.

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 1, 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 is 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.