Ontogeny of the Heat-Induced Hatching Response in the Red-Eyed Treefrog, Agalychnis callidryas

In ectothermic vertebrates, temperature impacts the rate and success of embryonic development, and in some species, embryos show adaptive behavioral responses to thermal conditions. The arboreal embryos of red-eyed treefrogs exhibit heat-induced hatching, escaping to cooler water below and demonstrating a limit to their behavioral thermal tolerance, or Voluntary Thermal Maximum (VT_Max). Their escape response to warming has been studied only at developmental stages 31-34 (age 5 days), although these embryos hatch in response to other threats as early as stage 24 (age 3 days). Hence, it is unclear when heat-induced hatching begins or how this behavior may change with further development. We conducted an ontogenetic series of warming trials to determine the onset of heat-induced hatching, assess developmental changes in the proportion of embryos that respond, and test whether expression of VT_Max changes with development. No embryo tested at stage 26 hatched; heat-induced hatching began in some clutches at stage 27. The proportion of embryos hatched increased from 3% at stage 27 (age 4.3 days) to 86% at stage 30 (5.4 days) when all clutches were responsive. The greatest variation in response among clutches occurred at stage 29. Hatching temperature was similar across stages 27-30 (38.2 ± 1.3°C) and consistent with the VT_Max reported for stage 31-34 embryos (38.1 ± 0.8°C) under similar testing conditions. These findings provide new insights into the ontogeny of heat-induced hatching behavior and reveal that embryos can escape from heat more than a day earlier than previously shown. The ontogenetic stability of VT_Max suggests a consistent thermal threshold guiding this response, providing reliable protection against lethal temperatures. This plasticity may help embryos withstand increasing thermal stress under climate change.