As the frog embryos grew, tiny parts came into view. Biology graduate student Estefany Caroline Guevara-Molina watched them develop at the Smithsonian Tropical Research Institute (STRI) in Panama. On day two, small hearts began blinking like signals beneath translucent chests. On day three, eyes blackened and swaying sets of external gills lengthened in branches. On day five, sensory systems like the vestibular system and its inner ear organs were in place to detect the strike of a snake, the bite of a wasp, or the creep of fungus over the eggs. That’s when Guevara-Molina exposed the eggs to heat. In droves, the embryos hatched before they were due.
This behavior makes red-eyed treefrogs the first species whose embryos are shown to hatch to escape overheating, and it adds rising temperature to the frogs’ known triggers of early hatching, including vibrations made by predation and oxygen loss in flooding.
Guevara-Molina reported the finding alongside Boston University biologist Karen Warkentin, who conducts research at STRI, and physiologist Fernando Ribeiro Gomes of the University of São Paulo in a study published in Integrative Organismal Biology this week. “The more questions we ask of them, the more we find out about what [the frogs] can do,” says Warkentin, who led the research team. “The fact that the embryos have a behavioral response to thermal stress, which gets them out of the egg and into the presumably cooler pond, is really interesting because it opens a bunch of other questions.”
Warkentin has studied environmentally cued hatching in red-eyed treefrogs since the 1990s, when they learned that their embryos could hatch to evade snake attacks. The discovery showed an exception to the once-prevailing notion in evolutionary biology that embryos do little more than play out a predetermined program for development. Instead, Warkentin found that while red-eyed treefrog embryos were still growing those tiny parts from inside the clear jelly of their eggs, they reached a point where they could receive and act on cues from the world outside.
Every question Warkentin has since asked of the embryos has come from observations in nature. What threats approached the eggs, and from which of them could the still-growing frogs defend themselves? In Costa Rica’s Corcovado National Park, cat-eyed snakes sucked down whole chunks of clutches. In Gamboa, wasps landed on leaves and broke into egg after egg, taking embryo after embryo. Fungal infections spread over eggs, and rainstorms rose ponds that flooded clutches on low-hanging leaves. The researchers recreated each of these threats in the lab and found that embryos accelerated their hatching in response. Hatching seemed to be the frogs’ broad-spectrum defense mechanism.
Whereas snakes and wasps fill their bellies after attacking a few clutches, the conditions of drought and high temperatures can descend on the entire rainforest. And those conditions are on the rise during the frog’s typically cool, wet breeding season as a result of climate change. “That is a threat to all the eggs that are there, unlike any of the predators or pathogens,” Warkentin says. “Even flooding—it’s not globally applied to the whole population.”
In 2017, Warkentin’s team added dehydration to the red-eyed treefrog’s environmental hatching cues. When the scientists withheld water from clutches they had collected in nature, embryos began hatching from dry eggs around ten hours earlier than embryos from hydrated eggs. But Guevara-Molina wanted to know more. She believed that warming could also accelerate hatching, and that its natural interplay with drying could affect how early embryos hatched.
Guevara-Molina had previously been part of a team that studied the heat responses of juvenile bullfrogs. They had found the maximum temperatures the bullfrogs tolerated before moving to a cooler place. She believed that red-eyed treefrog embryos could use hatching to similarly move somewhere cooler, making the behavior an indicator of the maximum temperatures they tolerated. She teamed up with Warkentin to test heat-induced hatching and see if dehydration led to even earlier heat-induced hatching.
In 2018, Guevara-Molina collected clutches of eggs laid overnight by red-eyed treefrogs at a pond in the wet tropical forest surrounding STRI. Some eggs she kept plump and wet in the lab’s automated egg humidor system, and others she treated with controlled drying. She kept some eggs in clutches, while isolating others to more evenly control their dehydration.
Guevara-Molina then warmed the eggs over several hours once they were five days old. When Warkentin’s team had pitted the frogs against previous threats, they found the five-day mark to be a reliable starting point for cued hatching experiments. The embryos would normally undergo a weeklong development cycle, but at five days they were developed enough to hatch in response to most cues and survive once out of the egg—though not as well as seven-day-olds. “They're little and they're just not as skilled and robust as if they had had a few more days to develop in the egg,” Warkentin explains. “There’s a price to pay there. But if you know you’re going to die, you take your chances with what might be next.”
As Guevara-Molina brought temperatures up from the frog’s wet-season comfort level of around 78 degrees Fahrenheit, she saw the embryos become restless, moving around until the heat eventually pushed them to rupture their egg capsules and wriggle their way out. Embryos hatched at around 100 degrees Fahrenheit in wet clutches, and at around 93 degrees in wet, isolated eggs. The frogs hatched from dry clutches at around 97 degrees, and from dry, isolated eggs at around 88 degrees. The dips in numbers between wet and dry eggs confirmed to Guevara-Molina that embryos developing under dehydration hatch at lower temperatures to protect themselves.
Though she had anticipated the response, Guevara-Molina was still stunned. “I know that in adult lizards and snakes and frogs, they can respond to temperature and avoid overheating,” Guevara-Molina says. “But for embryos, it’s an amazing response.”
Brooke Bodensteiner, a thermal physiology graduate student at Yale University who was not involved in the study, says its findings bring a “solid piece of the puzzle” to the study of thermal tolerances in animals. Bodensteiner studies how animals like anoles lizards in the Greater Antilles respond to temperature in a rapidly-changing world. When biologists pinpoint an animal’s heat limits, she explains, scientists can then use those behavioral details to inform models that map out life in future climate change scenarios. “Understanding what those temperature limits are can be important to understanding where an organism can occur, where it can’t, and if it can move through certain environments,” Bodensteiner says.
But questions remain about how red-eyed treefrog embryos can act on their temperature limits. The researchers want to find the mechanisms by which the embryos can sense heat, dehydration or both. And they don’t yet know exactly how early the embryos can hatch in response to heat.
Guevara-Molina focused on testing five-day-olds, and while the embryos may respond to heat earlier, the course of their development gives them a limited window of opportunity. As embryos grow from balls of yolk in their first couple of days, they’re too young to act. That waiting period could be enough to make them vulnerable to a hot, dry spell. “From an environmental threat perspective, it seems to me that a lot of eggs could die when they’re younger, or be so dried out that they die before they get to a point where they could deploy this defense,” Warkentin says.
Warkentin's team will continue to work with frogs and heat in Gamboa to learn more. By studying embryos, they’re extending our understanding of animals’ responses to heat to earlier life stages, says Bodensteiner. We need to study those stages for a full picture, she explains. “Some of these other life stages like eggs and tadpoles may have very different responses to temperature than another life stage, or they may be more vulnerable to climate change,” Bodensteiner says. “We as a field are getting better at filling in those gaps. But I think there's still a long way to go.”