Two types of hatching gland cells facilitate escape-hatching at different developmental stages in red-eyed treefrogs, Agalychnis callidryas (Anura: Phyllomedusidae)

The optimal time to hatch can vary, favouring embryos that maintain the ability to hatch or remain in ovo across a broad developmental period. Terrestrial embryos of Agalychnis callidryas hatch spontaneously at 6-7 days, but leave flooded (hypoxic) eggs as young as 3 days and escape egg predators from 4 days. We investigated the hatching mechanisms enabling this plasticity. Although many frog eggs weaken and expand continuously until hatching, egg capsules of A. callidryas remain robust and stable in size throughout their hatching period. We found two morphologically distinct hatching gland cell (HGC) types: early HGCs appear at 3 days on the snout and dorsum, begin regressing at 4 days and are scarce by 5 days, whereas late HGCs appear at 4 days, concentrated on the snout, and regress only after hatching. Both cell types show regulated, acute release of hatching enzyme, which enables rapid hatching. Expression of a single hatching enzyme gene appears on the snout and dorsal midline before HGCs are visible at the surface, subsequently matches the distributions of both HGC types, and diminishes before spontaneous hatching. In morphology and ontogeny, A. callidryas early HGCs resemble the single type of HGC described in other treefrogs; we hypothesize that these are the ancestral form. They enable the earliest hypoxia-cued hatching, but disappear, unused, before most hatching occurs. The late HGCs, which mediate most hatching, appear derived, perhaps after the evolution of terrestrial eggs altered selection on the timing of hatching.