Study reveals how glass frogs are transparent; see research here

A multi-disciplinary team of biologists and biomedical engineers has shown how these frogs make themselves visible.

It’s easy to miss a glass frog in its natural environment. The northern glass frog, Hyalinobatrachium fleischmannii, measures no more than a few centimetres, and they are most active at night when its green skin helps them blend in with the surrounding leaves and foliage, the study said.

But these amphibians become true masters of camouflage during the day when they are sleeping.

“When glassfrogs are at rest, their muscles and skin become transparent, and their bones, eyes and internal organs are visible,” said Carlos Taboda, a post-doctoral fellow at Duke and co-author of the paper.

“These frogs sleep on the underside of large leaves, and while they are transparent, they can match the colors of the vegetation perfectly,” he said.

Many animals in the sea can change the color of their skin or become completely transparent, but this is a much less common skill on land. One of the reasons transparency is so difficult to achieve is because of the red blood cells in the circulatory system.

Red blood cells specialize in absorbing green light, which is usually the color of light reflected by plants and other vegetation. In turn, these oxygen-rich cells reflect red light, making the blood – and by extension the circulatory system – highly visible, especially against a bright green leaf.

Glassfrogs are some of the only land-based vertebrates that can achieve transparency, which has made them a target for study. Taboda first began studying glass frogs as a post-doctoral fellow in the lab of Sonke Johnson, a biology professor at Duke who specializes in studying transparency. Working with Jesse Delia, who traveled around the world to collect individual glassfrogs for study, he observed that red blood cells began to disappear from the circulating blood whenever the frogs became transparent.

They performed additional imaging tests on animals, proving through optical models that the animals were able to achieve transparency because they were pushing red blood cells out of their vessels. He suspected that the cells were being stored in one of the frog’s internal organs that was packaged in a reflective membrane.

For a see-seeing animal, understanding its biology was surprisingly challenging. The research drew on the expertise of biologists and biomedical engineers not only at Duke but also at the American Museum of Natural History, Stanford University and the University of Southern California.

“If these frogs are awake, stressed or under anesthesia their circulatory system is full of red blood cells and they are opaque,” explained Delia, who is now a post-doctoral fellow at the American Museum of Natural History. “The only way to study transparency is if these animals are happily sleeping, which is difficult to achieve in a research lab. We were really banging our heads against the wall for solutions.”

But Taboda had learned about an imaging technique called photoacoustic microscopy, or PAM, when he was studying biliverdin, the compound that gives some species of frogs their distinctive green color. PAM involves shooting a safe laser beam of light into tissue, which is then absorbed by the molecules and converted into ultrasonic waves. These sound waves are then used to create detailed biomedical images of the molecules. The imaging tool is non-invasive, quiet, sensitive and, with a stroke of luck, available at Duke.

“PAM is the ideal tool for non-invasive imaging of red blood cells because you don’t need to inject contrast agents, which would be very difficult for these frogs,” explained Junjie Yao, assistant professor of biomedical engineering at Duke. who specialize in PAM technologies. “The red blood cells themselves provide contrast because different types of cells absorb and reflect different wavelengths of light. We specifically designed our imaging system to look for red blood cells and to monitor the flow of oxygen throughout the frog’s body.” Can adapt to track.”

In their imaging set-up, the frogs slept upside down in a petri dish, just like they would on a leaf, and the team shone a green laser at the animal. The red blood cells in the frog’s body absorbed green light and emitted ultrasonic waves, which were picked up by an acoustic sensor with high spatial resolution and high sensitivity to locate their whereabouts.

The results were startlingly clear: When the frogs were asleep, they removed about 90 percent of their circulating red blood cells and stored them in their livers.

In further tests, the team also observed that red blood cells move out of the liver and circulate when the frogs are active, and then reassemble into the liver while the frogs recover.

“The primary result is that whenever glassfrogs want to be transparent, which is usually when they are at rest and vulnerable to prey, they filter out almost all red blood cells from their blood and hide in the mirror-coated liver – somehow avoiding creating a huge blood clot in the process,” Johnson said. “Whenever the frogs need to be active again, they bring the cells back into the bloodstream, giving them the metabolic ability to move around.”

According to Delia and Taboda, this process raises the question of how frogs can safely store nearly all their red blood cells in their livers without clotting or damaging their peripheral tissues. A possible next step, he said, could be to study this mechanism and how it might one day apply to vascular issues in humans.

This work also introduces the glassfrog as a useful model for research, especially when combined with state-of-the-art photoacoustic imaging. As longtime glass frog researchers, they are excited about the new avenues of study now available to them and interested collaborators.

“We can learn more about the physiology and behavior of the glass frog, or we can use these models to optimize imaging tools for biomedical engineering,” Delia said. “It started because Carlos and I thought this frog was doing something weird with its blood, and it led to productive collaborations with Duke and around the world.”

“Our successful collaboration has been a great example of how multiple disciplines can jointly advance science in the most synergistic way possible,” Yao said. “We are extremely excited about future interactions between the biology and engineering teams. With full force on board, the sky is the limit.”

(With inputs from ANI)