Ontogeny of the Heat‐Induced Hatching Response in the Red‐Eyed Treefrog, <i>Agalychnis callidryas</i>

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.