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

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.