Glassfrogs hide red blood cells in their liver to become transparent
Researchers finally decipher how a species of frog becomes a master of camouflage with the help of modern biomedical imaging techniques
Peer-Reviewed PublicationVIDEO: A GLASSFROG DURING DIFFERENT LEVELS OF ACTIVITY, SHOWING THE CHANGE IN RBC PERFUSION WITHIN ITS CIRCULATORY SYSTEM. THE SAME ANIMAL WAS FILMED ACROSS ALL THREE CONDITIONS USING TRANSMITTED LIGHT. view more
CREDIT: JESSE DELIA
DURHAM, N.C. -- Glassfrogs make themselves transparent while they rest by taking red blood cells from circulation and concealing them in their livers. A multi-disciplinary team of biologists and biomedical engineers has shown how these frogs make themselves see-through in research that appears December 23 in the journal Science.
It’s easy to miss a glassfrog in its natural environment. The northern glassfrog, Hyalinobatrachium fleischmanni, measures no more than a few centimeters, and they are most active at night, when their green skin helps them blend in with the surrounding leaves and foliage.
But these amphibians become true masters of camouflage during the day when they’re asleep.
“When glassfrogs are resting, their muscles and skin become transparent, and their bones, eyes and internal organs are all that’s visible,” said Carlos Taboada, a post-doctoral fellow at Duke and a co-first author of the paper. “These frogs sleep on the bottoms of large leaves, and when they’re transparent, they can perfectly match the colors of the vegetation.”
Many animals in the sea can change the color of their skin or become completely transparent, but it’s a far less common skillset on land. One reason transparency is so difficult to achieve is because of red blood cells in the circulatory system. Red blood cells are adept at absorbing green light, which is the color of light usually reflected by plants and other vegetation. In return, these oxygen-rich cells reflect red light, making 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. Taboada first began studying glass frogs as a post-doctoral fellow in the lab of Sönke Johnsen, a professor of biology at Duke who specializes in studying transparency. Working with Jesse Delia, who traveled around the world collecting different glassfrogs for the study, they observed that red blood cells seemed to be disappearing from the circulating blood whenever the frogs became transparent.
They conducted additional imaging tests on the animals, proving via 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 inner organs which are packaged in a reflective membrane.
For a see-through animal, its biology was shockingly challenging to decipher. The research drew on the expertise of biologists and biomedical engineers not only at Duke but 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, now a post-doctoral fellow at the American Museum of Natural History. “The only way to study transparency is if these animals are happily asleep, which is difficult to achieve in a research lab. We were really banging our heads against the wall for a solution.”
But Taboada had learned about an imaging technology called photoacoustic microscopy, or PAM, when he was studying biliverdin, the compound that gives certain species of frogs their signature green color. PAM involves shooting a safe laser beam of light into tissue, which is then absorbed by molecules and converted into ultrasonic waves. These sound waves are then used to make detailed biomedical images of the molecules. The imaging tool is non-invasive, quiet, sensitive and, in 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, an assistant professor of Biomedical Engineering at Duke who specializes in PAM technologies. “The red blood cells themselves provide the contrast, because different types of cells absorb and reflect different wavelengths of light. We could optimize our imaging systems to specifically look for red blood cells and track how much oxygen was circulating in the frog’s bodies.”
In their imaging set-up, the frogs slept upside down in a petri dish, similar to how they would sleep on a leaf, and the team shined a green laser at the animal. The red blood cells in the frog’s body absorbed the green light and emitted ultrasonic waves, which were then picked up by an acoustic sensor to trace their whereabouts, with high spatial resolution and high sensitivity.
The results were startlingly clear: When the frogs were asleep, they removed nearly 90 percent of their circulating red blood cells and stored them in their liver.
In further tests, the team also saw that red blood cells flowed out of the liver and circulated when the frogs were active, and then re-aggregated in the liver while the frogs were recovering.
“The primary result is that whenever glassfrogs want to be transparent, which is typically when they're at rest and vulnerable to predation, they filter nearly all the red blood cells out of their blood and hide them in a mirror-coated liver -- somehow avoiding creating a huge blood clot in the process,” said Johnsen. “Whenever the frogs need to become active again, they bring the cells back into the blood stream, which gives them the metabolic capacity to move around.”
According to Delia and Taboada, this process raises questions about how the frogs can safely store almost all their red blood cells in their liver without clotting or damaging their peripheral tissues. One potential next step, they said, could be to study this mechanism and how it could one day apply to vascular issues in humans.
This work also introduces glassfrogs as a useful model for research, especially when paired with the state-of-the-art photoacoustic imaging. As long-time glassfrog researchers, they are excited about the new avenues of study now available to them and interested collaborators.
“We can learn more about the glassfrog’s physiology and behavior, or we can use these models to optimize imaging tools for biomedical engineering,” Delia said. “This started because Carlos and I thought this frog was doing something weird with its blood, and it led to productive collaborations both at Duke and across the globe.”
“Our successful collaboration has been a great example of how multiple disciplines can jointly advance science in the most synergistic way,” said Yao. “We are extremely excited about the future interactions between the biology and engineering teams. With all the strength on board, the sky is the limit.”
This work was supported the National Geographic Society grant (NGS-65348R-19), the Human Frontier Science Program postdoctoral fellowship (LT 000660/2018-L), the Gerstner Scholars Fellowship provided by the Gerstner Family Foundation and the Richard Gilder Graduate School at the American Museum of Natural History, start-up funds from Stanford University and start-up funds from Duke University, the National Institutes of Health grants (R01 EB028143 R01 NS111039 and RF1 NS115581 BRAIN Initiative), the National Science Foundation CAREER Award (2144788), the Duke Institute of Brain Science Incubator award, the American Heart Association Collaborative Sciences award (18CSA34080277), and the Chan Zuckerberg Initiative (2020-226178).
CITATION: “Glassfrogs Conceal Blood in Their Liver to Maintain Transparency,” Carlos Taboada, Jesse Delia, Maomao Chen, Chenshuo Ma, Xiaorui Peng, Xiaoyi Zhu, Liaming Jiang, Tri Vu, Qifa Zhou, Junjie Yao, Laureo O’Connell, Sonke Johnsen. Science, December 22, 2022. DOI: 10.1126/science.abl6620
JOURNAL
Science
METHOD OF RESEARCH
Experimental study
SUBJECT OF RESEARCH
Animals
ARTICLE TITLE
Glassfrogs conceal blood in their liver to maintain transparency
RCBs in vessels.mp4 [VIDEO]
sleeping glassfrog scanning on [VIDEO] |
a male glassfrog photographed in ventral view using a flash, showing its transparency. The team measured muscle and ventral skin and found that these tissues transmit over 90% of visible light and are still perfectly functional. These tissues are so transparent that their organs and bones can be viewed through them.
Photoacoustic Microscopy images showing circulating red blood cells within a glassfrog while asleep and under anesthesia (which breaks blood storage). This system uses light to induce absorption in a hemoglobin and an ultrasound transducer to map the soundwaves back to the point of optical absorption. The color scheme shows the percent of hemoglobin bound with oxygen. These images were acquired on an OR-PAM system developed by Dr. Junjie Yao’s lab.
CREDIT
Junjie Yao, Duke University
Glassfrogs “hide” blood in their liver while sleeping to stay transparent
Tiny, translucent glassfrogs increase their transparency two- to threefold while sleeping by temporarily storing red blood cells in their liver, according to a new study. The findings – which provide insight into this unique adaptation in vertebrates – could be used in understanding blood flow more broadly and in the development of new anticoagulants or other cardiovascular drugs. The glassfrog (Hyalinobatrachium fleischmanni) is a tropical, leaf-dwelling frog species with a transparent body and translucent skin – adaptations these frogs use as camouflage against would-be predators as they sleep on green leaves during the day. For many vertebrates, particularly terrestrial species, achieving a glassfrog’s level of transparency is challenging because of the numerous red blood cells (RBCs) that circulate continuously throughout the body, rendering even highly transparent tissues opaque. In this study, Carlos Taboada and colleagues investigated how glassfrogs seemingly overcome this physiological barrier. Using calibrated color photography to measure animal transparency and photoacoustic imaging to trace the movement of RBCs in living frogs, Taboada et al. found that glassfrogs become 34 to 61% more transparent, on average, while sleeping, suggesting that the animals actively maintain their dynamic transparency. According to the authors, they do this by removing roughly 89% of their RBCs from circulation and “hiding” them in the liver during sleep, without any detrimental vascular or metabolic effects. As the frogs wake up and become more active, the number of RBCs in circulation increases greatly, as does their opacity. Since such high local concentrations of RBCs in most vertebrates often results in vaso-occlusion, or clotting, these findings may offer insights into the mechanisms involved in preventing these and other vascular pathologies. “The mechanism that drives RBC re-distribution in glassfrogs is not understood. It is unclear if the glassfrog can actively manipulate the changes in RBC circulation – for example, in the presence of a predator,” write Nelly Cruz and Richard White in a related Perspective. “Another intriguing question is how sequestration of RBCs in the liver affects cellular respiration and whether the glassfrog has a special metabolism that adapts to the drastic changes in RBC circulation.
JOURNAL
Science
ARTICLE TITLE
Glassfrogs conceal blood in their liver to maintain transparency
ARTICLE PUBLICATION DATE
23-Dec-2022
Glassfrogs achieve transparency by packing red blood cells into mirror-coated liver
New study documents glassfrogs storing red blood cells in liver while they sleep; opens avenues of research for preventing blood clots
Peer-Reviewed PublicationIMAGE: A GROUP OF GLASSFROGS SLEEPING TOGETHER UPSIDE DOWN ON A LEAF. view more
CREDIT: © JESSE DELIA
New research shows that glassfrogs—known for their highly transparent undersides and muscles—perform their “disappearing acts” by stowing away nearly all of their red blood cells into their uniquely reflective livers. The study, led by scientists at the American Museum of Natural History and Duke University, is being published Friday in the journal Science. The work could lead to new avenues of research tied to blood clots, which the frogs somehow avoid while packing and unpacking about 90 percent of their red blood cells into their livers on a daily basis.
“There are more than 150 species of known glassfrogs in the world, and yet we’re really just starting to learn about some of the really incredible ways they interact with their environment,” said co-lead author Jesse Delia, a Gerstner postdoctoral fellow in the Museum’s Department of Herpetology.
Glassfrogs, which live in the American tropics, are nocturnal amphibians that spend their days sleeping upside down on translucent leaves that match the color of their backs—a common camouflage tactic. Their tummies, however, show something surprising: translucent skin and muscle that allows their bones and organs to be visible, giving the glassfrog its common name. Recent research has proposed that this adaptation masks the frogs’ outlines on their leafy perches, making them harder for predators to spot.
Transparency is a common form of camouflage among animals that live in water, but it’s rare on land. In vertebrates, attaining transparency is difficult because their circulatory system is full of red blood cells that interact with light. Studies have shown that ice fish and larval eels achieve transparency by not producing hemoglobin and red blood cells. But glassfrogs use an alternative strategy, according to the findings of the new study.
“Glassfrogs overcome this challenge by essentially hiding red blood cells from view,” said Carlos Taboada, the study’s co-lead author from Duke University. “They almost pause their respiratory system during the day, even at high temperatures.”
At Duke, the researchers used a technique called photoacoustic imaging, which uses light to induce sound-wave propagation from red blood cells. This allows researchers to map the location of the cells within sleeping frogs without restraint, contrast agents, sacrifice, or surgical manipulation—particularly important to this study because glassfrog transparency is disrupted by activity, stress, anesthesia, and death.
The researchers focused on one particular species of glassfrog, Hyalinobatrachium fleischmanni. They found that resting glassfrogs increase transparency two- to threefold by removing nearly 90 percent of their red blood cells from circulation and packing them within their liver, which contains reflective guanine crystals. Whenever the frogs need to become active again, they bring the red blood cells back into the blood, which gives the frogs the ability to move around—at which point, light absorption from these cells breaks transparency.
In most vertebrates, aggregating red blood cells can lead to potentially dangerous blood clots in veins and arteries. But glassfrogs don’t experience clotting, which raises a set of significant questions for biological and medical researchers.
“This is the first of a series of studies documenting the physiology of vertebrate transparency, and it will hopefully stimulate biomedical work to translate these frogs’ extreme physiology into novel targets for human health and medicine,” Delia said.
Other authors on the study include Maomao Chen, Chenshuo Ma, Xiaorui Peng, Xiaoyi Zhu, Tri Vu, Junjie Yao, and Sönke Johnsen from Duke University; Laiming Jiang and Qifa Zhou, from the University of Southern California, Los Angeles; and Lauren O’Connell, from Stanford University.
This study was supported in part by the National Geographic Society, grant # NGS-65348R-19; the Human Frontier Science Program postdoctoral fellowship # LT 000660/2018-L; the Gerstner Scholars Fellowship provided by the Gerstner Family Foundation and the Richard Gilder Graduate School at the American Museum of Natural History; start-up funds from Stanford University; start-up funds from Duke University; the National Institutes of Health, grant #s R01 EB028143, R01 NS111039, RF1 NS115581 BRAIN Initiative; a Duke Institute of Brain Science Incubator award; the American Heart Association Collaborative Sciences award 18CSA34080277; and a Chan Zuckerberg Initiative grant 2020- 226178.
Study DOI: www.science.org/doi/10.1126/science.abl6620
A pair of mating glassfrogs sleep together on a leaf.
CREDIT
© Jesse Delia
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The American Museum of Natural History, founded in 1869, is one of the world’s preeminent scientific, educational, and cultural institutions. The Museum encompasses more than 40 permanent exhibition halls and galleries for temporary exhibitions, the Rose Center for Earth and Space and the Hayden Planetarium, and the Richard Gilder Center for Science, Education, and Innovation, which opens February 2023. The Museum’s scientists draw on a world-class permanent collection of more than 34 million specimens and artifacts, some of which are billions of years old, and on one of the largest natural history libraries in the world. Through its Richard Gilder Graduate School, the Museum grants the Ph.D. degree in Comparative Biology and the Master of Arts in Teaching (MAT) degree, the only such freestanding, degree-granting programs at any museum in the United States. The Museum’s website, digital videos, and apps for mobile devices bring its collections, exhibitions, and educational programs to millions around the world. Visit amnh.org for more information.
JOURNAL
Science
METHOD OF RESEARCH
Experimental study
SUBJECT OF RESEARCH
Animals
ARTICLE TITLE
Glassfrogs conceal blood in their liver to maintain transparency
ARTICLE PUBLICATION DATE
23-Dec-2022
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