DOI: 10.5061/dryad.bzkh189bk
title: Early onset of urea synthesis and ammonia detoxification pathways in three terrestrially developing frogs

description: 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.
description: We conducted experiments during the rainy seasons of 2018 and
 2019 in Panama with early ontogenetic stages of three terrestrial-breeding
 frogs: <em>Leptodactylus fragilis </em>(which lay eggs in
 subterranean foam-nests)<em>, Agalychnis
 callidryas </em>(which lay gelatinous clutches on leaves over ponds)
 and <em>Hyalinobatrachium fleischmanni </em>(which lay
 gelatinous clutches on leaves over streams, then provide paternal
 egg-care).<em> </em>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 <em>L. fragilis</em>
 and <em>H. fleischmanni</em>, 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
 NH<sub>4</sub>Cl 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 <em>L.
 fragilis </em>and <em>H. fleischmanni</em> and the two
 terrestrial contexts in <em>A. callidryas.</em> 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 Scientific<sup>TM</sup> Coonassie Protein
 Assay kit. We used these values to correct enzyme activities (μmol or nmol
 min<sup>-1</sup> mg<sup>-1</sup> of protein) and
 the concentration of urea and non-essential amino acids (μmol or nmol
 mg<sup>-1</sup> of protein).
 <strong>References</strong> Méndez‐Narváez, J. and
 Warkentin, K.M., 2022. Reproductive colonization of land by frogs: Embryos
 and larvae excrete urea to avoid ammonia toxicity. <em>Ecology and
 Evolution</em>, <em>12</em>(2), p.e8570.