Reproductive colonization of land by frogs: Embryos and larvae excrete urea to avoid ammonia toxicity

Abstract

Vertebrate colonization of land has occurred multiple times, including over 50 origins of terrestrial eggs in frogs. Some environmental factors and phenotypic responses that facilitated these transitions are known, but responses to water constraints and risk of ammonia toxicity during early development are poorly understood. We tested if ammonia accumulation and dehydration risk induce a shift from ammonia to urea excretion during early stages of four anurans, from three origins of terrestrial development. We quantified ammonia and urea concentrations during early development on land, under well‐hydrated and dry conditions. Where we found urea excretion, we tested for a plastic increase under dry conditions and with ammonia accumulation in developmental environments. We assessed the potential adaptive role of urea excretion by comparing ammonia tolerance measured in 96h‐LC50 tests with ammonia levels in developmental environments. Ammonia accumulated in foam nests and perivitelline fluid, increasing over development and reaching higher concentrations under dry conditions. All four species showed high ammonia tolerance, compared to fishes and aquatic‐breeding frogs. Both nest‐dwelling larvae of Leptodactylus fragilis and late embryos of Hyalinobatrachium fleischmanni excreted urea, showing a plastic increase under dry conditions. These two species can develop the longest on land and urea excretion appears adaptive, preventing their exposure to potentially lethal levels of ammonia. Neither late embryos of Agalychnis callidryas nor nest‐dwelling larvae of Engystomops pustulosus experienced toxic ammonia levels under dry conditions, and neither excreted urea. Our results suggest that an early onset of urea excretion, its increase under dry conditions, and elevated ammonia tolerance can all help prevent ammonia toxicity during terrestrial development. High ammonia represents a general risk for development which may be exacerbated as climate change increases dehydration risk for terrestrial‐breeding frogs. It may also be a cue that elicits adaptive physiological responses during early development.

Heat-Induced Hatching of Red-Eyed Treefrog Embryos: Hydration and Clutch Structure Increase Behavioral Thermal Tolerance

“…2017 ; Tippett and Warkentin 2017 ) and also evident in our baseline hatching rates (see Table 1 ). The mechanism underlying this response is unknown but might involve increased osmolality of perivitelline and body fluids or the increased concentration of specific molecules, such as ammonia ( Méndez-Narvaez and Warkentin 2022 ). In addition, these embryos are known to combine information across cue properties and across sensory modalities for their hatching decisions ( Warkentin and Caldwell 2009 ; Güell and Warkentin 2018 ; Jung et al.…”

Elevated ammonia cues hatching in red‐eyed treefrogs: A mechanism for escape from drying eggs

“…Anurans have evolved terrestrial eggs at least 56 times, mainly in humid tropical environments (Gomez‐Mestre et al, 2012). These evolutionary transitions from aquatic to terrestrial egg‐laying created new physiological and ecological challenges for water‐dependent embryos (Martin, 1999; Méndez‐Narváez & Warkentin, 2022; Touchon & Warkentin, 2008). Some terrestrial‐breeding anurans provide prolonged parental care to embryos to supply them with water and, sometimes, protect them from predators (Delia et al, 2020).…”

“…Ammonia is a metabolic waste product that is generated as embryos metabolize yolk proteins (Jorgensen et al, 2009). It has been detected in perivitelline fluid as early as age 2.5 days and accumulates over development (Méndez‐Narváez & Warkentin, 2022). It also becomes more concentrated as eggs dry, with levels increasing almost four‐fold in dry versus well‐hydrated A. callidryas eggs (Méndez‐Narváez & Warkentin, 2022).…”

“…It has been detected in perivitelline fluid as early as age 2.5 days and accumulates over development (Méndez‐Narváez & Warkentin, 2022). It also becomes more concentrated as eggs dry, with levels increasing almost four‐fold in dry versus well‐hydrated A. callidryas eggs (Méndez‐Narváez & Warkentin, 2022). Under high‐ammonia conditions, some terrestrial frog embryos—including the foam‐nesting Leptodactylus fragilis and the glassfrog Hyalinobatrachium fleischmanni —convert ammonia into less toxic urea; however, this has not been documented for A. callidryas (Méndez‐Narváez & Warkentin, 2022).…”

“…It also becomes more concentrated as eggs dry, with levels increasing almost four‐fold in dry versus well‐hydrated A. callidryas eggs (Méndez‐Narváez & Warkentin, 2022). Under high‐ammonia conditions, some terrestrial frog embryos—including the foam‐nesting Leptodactylus fragilis and the glassfrog Hyalinobatrachium fleischmanni —convert ammonia into less toxic urea; however, this has not been documented for A. callidryas (Méndez‐Narváez & Warkentin, 2022). Thus, increasing ammonia concentration could pose a direct threat to embryos and may also serve as a reliable cue indicating the impending risk of mortality by dehydration.…”