Cryobiology: Frozen Wood Frogs and Adaptations for Survival
An Amazing Animal
Cryobiology is the study of biological material that is at below normal temperatures. One example of this material is the body of the wood frog in winter. This amazing animal survives months of hibernation with much of its body frozen and without a beating heart. In most other animals, when the heart stops beating the animal is dead. This is not true for wood frogs, however. Despite the almost complete shut-down of their bodies, the frogs aren't harmed by freezing and become active again when the warmer temperatures of spring arrive.
The wood frog is a fascinating organism to study in its own right. In addition, the adaptations that enable it to survive freezing may be helpful in understanding or even in dealing with human medical problems. These problems include the safe freezing and thawing of organs for cryopreservation and transplants, a high glucose level in the body, and the safe resumption of blood flow after a heart attack or stroke.
The Wood Frog
There are two scientific names for the wood frog—Lithobates sylvaticus and Rana sylvatica. It's a small animal that is around 1.4 to 3.25 inches in length. The frog is brown, orange-red, or tan in colour. There is a dark line in front of each eye and a dark blotch behind it. There may be dark, horizontal bars across the hind legs, a dark patch on the upper inside corner of each leg, and dark patches or speckles on other parts of the body.
The animal's range covers the majority of Canada and extends into Alaska and down into the northeastern United States. There is also a small area in the central United States where wood frogs can be found. The animals live mainly in woods, as their name suggests, but they also inhabit grasslands and the tundra. The adults eat insects and other small invertebrates. The tadpoles eat only plants. The male's call is notable because it resembles a duck's quack.
Wood Frog Call
Cryobiology is the study of proteins, cells, tissues, organs, and organisms that are at an unusually low temperature.
In the northern part of its range, the wood frog experiences very low winter temperatures. Most frogs in this situation bury themselves in the mud at the bottom of a lake, pond, or other body of water. This stops the animals from freezing during hibernation. As winter approaches, the wood frog buries itself in a shallow burrow on land, however.
The leaf litter that covers the frog and the snow that falls on top provide a little insulation from the cold winter temperatures, but not much. In fact, there is so little insulation that the animal soon freezes. The heart stops beating, the lungs and other organs stop working, and a large proportion of the water in the body freezes. The frozen liquid includes the blood.
Wood frogs have evolved ways to freeze solid for up to eight months each year. They’ve accomplished what would seem to be a biological miracle.— National Park Service
Dangers of Freezing Living Tissue
Freezing of living tissue is normally a dangerous process due to the ice crystals that form as the water in the cells freezes. The crystals can rupture materials and cause rearrangement of cell structures, which can lead to permanent damage. They can also cause water loss and dehydration of cells. If blood vessels are ruptured, cells in the body will no longer receive oxygen and nutrients. The wood frog has overcome these problems, however.
Researchers have found that wood frogs can survive when sixty-five to seventy percent of their body is frozen. They can also safely undergo multiple freeze-thaw cycles in a winter.
Preventing Cells From Freezing in the Winter
The frog's liver produces a large amount of glucose as winter approaches. This is transported by the blood and enters the animal's cells, where it acts as an antifreeze. When substances dissolve in water, they lower its freezing temperature. The high glucose concentration in cells prevents their interior from freezing as the temperature drops. The glucose concentration in the cells of the hibernating frog may be a hundred times higher than normal during the winter.
An increased concentration of a waste substance called urea also helps to prevent freezing in the cells. Urea is normally excreted in urine. The high glucose and urea levels don't appear to hurt the frog.
Even though the animal's cells are not frozen, they are either inactive or have extremely low activity. Active cells need oxygen and other nutrients from the blood and must send their waste substances into the blood. The blood doesn't flow when a wood frog is frozen, however.
Freeze tolerant animals typically confine ice growth to extracellular spaces of their bodies while using protective mechanisms to keep the water inside their cells from freezing (extracellular freeze tolerance plus intracellular freeze avoidance).— Janet M. Storey (Carleton University) and Nature North
Frozen But Still Alive
Although the water in the frog's cells doesn't freeze, at least some of the water outside the cells does. This includes water on the skin, between the skin and muscle, surrounding the organs in the abdominal cavity, and in the lens of the eye. As a result, a hibernating frog looks as though it's frozen and feels like a solid block. Researchers have discovered that much of the extracellular water is moved to places where its freezing is least likely to damage cells.
Scientists have found that the frog seems to encourage the start of ice formation outside its cells. Its skin is highly permeable to water and its body contains ice-nucleating agents. These act as a seed for ice growth in the water that has collected in the extracellular spaces. The nucleating agents include certain minerals and bacteria that the frog has ingested as well as specific proteins in its body.
Reproduction After Thawing
Thawing Safely in the Spring
Although researchers partially understand the processes that occur in a wood frog's body as it freezes, the signals that stop the heart from beating and the lungs from working are still mysterious. Some aspects of the thawing process are still puzzling as well.
It takes about a day for the wood frog to both thaw and return to normal activity and a bit longer before it's ready to reproduce. The thawing process starts from the inside of the animal's body and moves outwards, causing the frog to gradually come out of suspended animation. The signals that stimulate the heart to start beating again and the lungs to start working is unknown.
The frog appears to be in good condition once it's thawed. There is some evidence that body repair processes become more active than usual during and immediately after thawing, however.
In the northern part of its range, the wood frog has a major advantage over other frogs. In the spring, the land and the frog's body thaw before the icy covering of lakes, ponds, and rivers. Wood frogs are therefore able to breed before most other frog species. They lay their eggs primarily in temporary meltwater ponds, also known as vernal pools. The eggs are also laid in permanent bodies of water, however, especially in the warmer part of the animal's range.
The heart resumes beating even before ice in the body has completely melted, and pulmonary respiration and blood circulation are restored soon thereafter.— Miami University
Wood Frog Development: Egg, Tadpole, and Adult
Similarities and Differences Between Frogs and Humans
A frog is a vertebrate, like humans. Although the animal looks very different from a human externally, there are many similarities in the internal organs of a frog and a human. Both follow the basic vertebrate plan for internal anatomy. Their bodies also have many chemicals and chemical reactions in common.
One difference between the two organisms is that humans are endothermic (warm blooded) and frogs are ectothermic (cold blooded). An endothermic organism maintains the same internal temperature whatever the environmental temperature, except in special circumstances, due to processes that occur in the body. The temperature of ectothermic organisms is generally the same as that of the environment. Some ectotherms modify their temperature by their behaviour, however, such as by sunbathing when they're cold and entering a shelter of some kind when they're hot. The term "cold blooded" is not always accurate for them.
A study of wood frogs may help scientists improve the cryopreservation of tissues and organs, glucose management in diabetes, and the prevention of reperfusion injuries.
Understanding how the frog's body responds to temperatures below and then above freezing may help us improve the cryopreservation (preservation at low temperatures) of human cells, tissues, and organs. These need to be preserved in excellent condition so that they can be transplanted into the patients that need them.
Improving the preservation of organs would be especially helpful. At the moment, these are cooled but not frozen, which limits their availability to patients who need them. The organs eventually die unless they are frozen. Freezing and thawing are much more successful for small items such as eggs, sperm, and embryos than for large items such as organs. Frozen organs are damaged by cracking during the thawing process.
A High Glucose Level in the Blood and in Neurons
Discovering the details of glucose management in the frog may help doctors deal with diabetes. Insulin transports glucose into most of the cells in our body. The glucose molecules are used as an energy source. In diabetes, blood glucose (also known as blood sugar) rises, either because insulin is no longer being made by the pancreas or because insulin is no longer doing its job.
Neurons, or nerve cells, don't use insulin to absorb glucose, but they do have molecules called glucose transporters that serve a somewhat similar function. In diabetes, a high blood sugar level sometimes causes neurons to have a higher than normal glucose concentration. Intracellular glucose in neurons can be toxic at high concentrations and is thought to contribute to diabetic neuropathy. A high glucose level doesn't seem to be toxic in wood frogs, however, at least heading up to and during hibernation. It would be interesting and possibly useful to know why.
There is another way in which the study of the frogs could help humans. Humans may experience a reperfusion injury, or tissue damage, when blood returns to an area after being absent for a while. The absence of a blood supply may be caused by a heart attack or by a stroke.
The lack of blood flow to a part of the body means that the area lacks oxygen and nutrients and that toxins build up. These factors may damage the area. The area is then susceptible to being further damaged by reactive oxygen species when blood returns. The reason for the appearance of these chemicals is still being investigated.
The wood frog doesn't appear to experience any harm when its blood starts to flow again in the spring, or if it does, the damage is quickly repaired. Understanding how damage from the stoppage and restarting of blood flow is prevented or very significantly reduced could be useful.
An Intriguing Amphibian
The wood frog is an intriguing animal that may have much to teach us. Hopefully, understanding its biology will help us deal with medical problems. Even if this doesn't prove to be true, the frog is a fascinating little creature that is worth studying. Its adaptations for survival in winter are very impressive.
- Information about wood frogs in winter from the National Park Service
- Facts about freeze tolerance in wood frogs from Nature North magazine
- Strategies for frog survival during freezing from the Laboratory of Ecophysiological Cryobiology at Miami University
- Reperfusion injury and reactive oxygen species from the National Institutes of Health
© 2017 Linda Crampton