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picture of Kenneth Storey picture of Janet Storey Frozen Alive

The wood frog has amazing survival skills: it freezes in winter and thaws when spring arrives. In this story Kenneth and Janet Storey reveal how the frog freezes and how it can survive this process. Following the premiere of this Scientific American Frontiers Special Going to Extremes, the Storeys answered viewers' questions in an Ask the Scientists forum. Here are viewers' questions and their answers:

A message to Frontiers viewers from Kenneth Storey:

Before any of you readers viewers rush out to try freezing animals, you must remember several things.

1. Animals that do not live in places where temperatures normally drop below 0 degrees C for long periods of time DO NOT survive freezing even if the same species survives freezing at a more northern location. Hence, Maine mussels will survive freezing, Florida mussels will not!

2. Animals only survive freezing if they actually hibernate in a habitat where they will encounter freezing temperatures. So, animals that hibernate underwater, like leopard frogs, do not survive freezing nor do animals that burrow underground to hibernate like toads and salamanders.

3. Most animals only endure freezing at the temperatures that they encounter naturally. The lowest temperature that wood frogs can survive, for example, is about -6 degrees C because this is all they ever see in their habitat which is under forest floor leaves and a blanket of snow. A frog will not survive if frozen in a home freezer which is generally about -15 degrees C.

4. Anyone who thinks about doing any experiments where they are exposing animals to conditions that may be lethal to the animal must think long and hard about what they are doing and why they are doing it and carefully design their procedures so as to always optimize the chances for survival. It is unacceptable to waste the life of any animal on frivolous or uninformed experiments.

 

QWhat prevents the outer skin and extremities from becoming frostbitten during the freeze?

Frostbite is the injury done to a tissue of a non-freeze tolerant animal due to the formation of ice crystals in the tissue. A freeze tolerant animal has evolved specific adaptations that keep its tissues from being injured when ice forms in them and therefore it does not get frostbite.

Here is why you get frostbite. Ice forming in your tissues has several bad effects. The crystals themselves can cause physical damage. Crystals growing around and between cells can cause damage due to shear forces, that is squeezing and deforming cells until they are physically damaged. Ice can also puncture cells so that their contents leak out. Because water expands as it forms ice, ice that forms within confined places, like inside capillaries, will expand and break through the walls around it. Then when the tissue thaws it has holes in its blood vessels, as well as lots of broken or damaged cells.

Ice can hurt cells in another way too. Ice usually forms first in the fluid around the outside of cells. Ice is a crystal of pure water and as it forms it leaves behind all the dissolved solutes (like salts, sugars, proteins) that were in the extracellular fluid. These become more and more concentrated in the remaining extracellular fluid. This is why when you make homemade popsicles you find that part of the popsicle is just plain ice but another part of it is thick and syrupy and has all the flavour and color in it. When this happens to the fluid that is outside of a cell, it puts an osmotic stress on the cell causing water to flow out of the cell and solutes to move into the cell until the liquid inside and out reaches the same concentration. What this means is that cells lose a lot of water that leaves to join the growing ice crystals and cells shrink way down in volume. However, if cells lose too much water, the membrane around the cell becomes too compressed and eventually buckles and breaks. When the animal thaws, these damaged membranes cannot reform properly and so again broken cells spill their contents.

To prevent this type of damage, freeze tolerant animals add high concentrations of cryoprotectants to their cells. Usually these are sugars like glucose or glycerol. The presence of high levels of sugars inside cells means that, at any given temperature, cells with high cryoprotectant lose less of their water and the amount of ice that can form is lower, than cells without cryoprotectant. Less ice means less physical damage and a smaller cell volume reduction means that cells are not strained to the point of breaking. The presence of cryoprotectants also tends to blunt the shape of growing ice crystals so again there is less damage. Freeze tolerant animals also seem to have tricks that allow them to move a lot of water out of their tissues so it freezes in places where it does little harm. So in frozen frogs, for example, we find large flat crystals running between the layers of skin and muscle and the abdominal cavity is full of ice. At milder freezing temperatures it even appears that the organs are not frozen at all - organs such as heart and liver appear to be just encased in ice and are very shrunken and have very high concentrations of cryoprotectant in them, but there may not actually be ice within the organ itself. As temperature falls further, however, ice penetrates into all the organs through the blood vessels.



QBeing the wood frog can freeze and thaw, isn't there a clock (internal) that tells the frog the season? Are there any drawbacks if the frog is frozen and thawed out of the cycled seasons? Very, very interesting story and keep up the good work.

Yes, freeze tolerance is a seasonal phenomenon, not just for wood frogs but for almost all freeze tolerant animals. The exceptions to this are high Arctic and Antarctic insects that really could experience freezing temperatures in any month of the year. All animals, even ourselves, respond to internal clocks that are cued to the progression of the seasons and that prepare the animal for the activities or the environmental stresses that are specific to each season. Summer wood frogs are just as susceptible to freezing damage or death as are frogs like the leopard frog that spend the winter unfrozen at the bottom of ponds. In fact, wood frogs lose their tolerance of freezing quite rapidly over just a few weeks after they emerge from hibernation in the spring. One of the reasons for this is that they can no longer make the huge amounts of glucose cryoprotectant that they need to protect their cells during freezing.

All winter long they saved a huge stockpile of carbohydrate in their liver (in the form of glycogen) and used this whenever they had to quickly elevate their blood sugar levels for cryoprotection. In the spring, however, they use up the glycogen and glucose for another purpose and this is as a fuel to give them the energy they need for breeding. Wood frogs breed just after the snow melts with 2-3 nights of very noisy and energetic activity that uses up a lot of their body fuel reserves and these reserves cannot be replaced for quite a while because the weather is still too cold in the early spring for there to be many insects around for the frogs to eat. Various other protective strategies that aid freeze tolerance are also turned off in the summer such as the ability to make the special nucleating proteins that winter wood frogs have in their blood. These proteins aid freezing survival by helping to stimulate and direct ice formation within the blood vessels, presumably molding crystals to grow in the least harmful way.



QAre there other animals, besides the wood frog, that can be frozen for a period of time and then be brought back to life? if so, what are they? If not, why?

There are several other species of North American frogs that hibernate on land that also survive freezing by mechanisms just the same as the wood frog. These are the spring peeper (Pseudacris crucifer), the chorus frog (Pseudacris triseriata), and two forms of the gray tree frog (Hyla versicolor and Hyla chrysoscelis). We have done some work on all of these but the wood frog has become our major model animal mostly because it is the largest frog and the easiest to catch. Several types of northern reptiles also have some freeze tolerance although cannot survive frozen for as long as the frogs. Garter snakes and some lizards can survive for a few hours which may help them endure an overnight frost but for long term hibernation these animals seek frost-free refuges. The best of the reptile freezers are newly hatched juvenile painted turtles (Chysemys picta). In our area of central Canada, these hatch in September but instead of leaving the nest and heading for the nearest water, the turtles stay put in their nests over the winter, where they are safe from predators. Since the nests are in exposed places like on river banks or lake shores and are only about 10 cm underground, the turtles are frequently exposed to subzero temperatures over the winter months and we have found that they can survive several days frozen. However, the ability to survive freezing by painted turtles is less uniform than it is for wood frogs. We find that northern turtles from both eastern and western Canada can survive freezing but other researchers in Nebraska found that they do not survive freezing there. So the development of freeze tolerance probably depends on just how severe the winters are.

Even though frogs and turtles are really neat and interesting to us and to TV viewers, they are by no means the champions of freeze tolerance. HUNDREDS of species of insects spend the winter frozen. One of the species that we work on happily survived 12 weeks in our freezer at -20 degrees C and various Arctic insects endure temperatures of -40 to -50 degrees C. The wooly bear caterpillar from Ellesmere Island in the high Arctic (not the same species that most of us would call a wooly bear), spends many months of every year frozen, surviving temperatures as low as -70 degrees C. Since it only actively eats for one month of the year (July) it spends 14 years as a caterpillar before transforming into a moth that lives for only a few weeks.

Other types of animals also survive freezing including various animals that are found in the intertidal zone on northern seacoasts. This is because these animals can be caught out of water at low tide and in the winter may have to endure air temperatures below 0 degrees C for several hours until the tide returns. Periwinkles, mussels and barnacles are all freeze tolerant.



QHow long are the frogs typically in the frozen state? Is there a "time limit" to the length of time they can remain frozen, and then thaw? Also, approximately how long does it take for them to become thoroughly frozen?

There is no field data on how long frogs stay frozen in nature. This is because there is no good way of finding frogs outdoors in natural woodlands in the winter. From early autumn onwards they are hidden away in hibernation sites under a good pile of leaf litter or a log, etc. and once you get a deep blanket of snow over top, there is no way to find them. The longest that we have frozen wood frogs for in the lab is 2 weeks and this is the longest time that has been reported in the scientific literature. The frogs were just fine after thawing so we suspect that they could remain frozen for considerably longer. With a good covering of snow and leaf litter, the forest floor and animals hibernating there can remain unfrozen well into the winter and there can be a huge gradient (as much as 20 or 30 degrees C) between the temperature under the snow and that above. So, realistically, for the northern U.S.A. or southern Canada I would suggest that 2-3 months maximum would be an absolute upper limit on the time that freezing temperatures could occur under the snow and it would also be unlikely to be continuous freezing.

When a frog freezes the process is quite slow and this is undoubtedly advantageous for it allows lots of time to initiate adaptations that protect the frog in the frozen state. We often freeze frogs at -2.5 degrees C and at this temperature it takes about an hour after freezing begins before the skin even feels crunchy. Animals can still slowly extend their partially frozen legs and arms as much as 4-6 hours later and maximal ice content is not reached until about 24 hours. At lower the temperatures the whole process is faster but below about -6 or -8 degrees C, both the rate of freezing and the amount of ice that accumulates is too great for the frogs to survive. At -2.5 degrees C frogs accumulate ice at a rate of about 3 % of total body water freezing per hour and end up with a maximum ice content of about 65 % of their total body water.



QCould extra glucose injected into humans give them similar resistance to freezing temperatures? If not, could it be used to help minimize the effects of frostbite?

Probably not. For freezing protection you need to be able to pack high concentrations of cryoprotectants into cells. This is most easily done using compounds that move easily across cell membranes by passive diffusion. Two of the cryoprotectants that are commonly used for freezing cells and tissues in cryomedicine are glycerol and dimethylsulfoxide, chosen for just this ability. Indeed, glycerol is also the most common natural cryoprotectant among the hundreds of freeze tolerant insect species. However, very little glucose enters mammalian cells by passive diffusion. Most of the glucose is carried across the membrane by transporter proteins and the activity of these proteins is regulated by hormones like insulin. Hence, it is very hard to overload a mammalian cell with glucose because as soon as too much sugar goes in, the cells turn off their transporters. Any extra glucose left in the blood is then taken to the liver (one organ whose transporters are not inhibited by high glucose) and in the liver the excess glucose is converted to the polysaccharide glycogen and stored away. Frogs have made specific adaptations to their plasma membrane transporters so that they can effectively overload their cells with glucose and they have also made other adaptations to their metabolism that allow them to keep sugar levels high instead of just using up the glucose as a fuel or converting it back to glycogen. Given these problems and also the many metabolic problems that arise in diabetics due to high glucose (and diabetics have problems when glucose reaches 15-40 mM whereas frogs accumulate 200-300 mM glucose), it does not seem too hopeful that glucose would be a good cryoprotectant in cryomedicine. However, its clearly the compound of choice for the best of the freeze-tolerant vertebrate animals so perhaps we have yet to figure out how to use glucose effectively in cryomedicine.



QMight the use of glucose as an "anti-freeze" serve a second benefit to the frog in that it could also be metabolized anaerobically, providing an energy source to thawing extremities prior to the circulation of blood?

Yes, it is very possible that some of the glucose is used as a fuel to generate energy in cells while they are surrounded by ice and cut off from fuels or oxygen that would otherwise be delivered by the blood. Organs can also use their own supply of glycogen (the polysaccharide carbohydrate reserve) for this purpose. When glucose or glycogen are fermented without oxygen, lactic acid accumulates as the end product in tissues and lactate does build up slowly in all frog organs while the animal is frozen. So clearly carbohydrate fermentation fuels the low metabolic rate in frozen animals. The pattern of lactate accumulation over time generally matches better with the pattern of glycogen loss in most organs whereas glucose tends to stay high and stable over the duration of the freeze. But, nonetheless, all organs are capable of fermenting both glucose and glycogen and hence both are potential fuels to keep metabolism going while the animal is frozen.



QI am a high school senior and I am doing a science project on the effects of cryogenics on the normal behavior of tardigrades and I was wondering if you could help my find any information on cryogenics or tardigrades that I can use in my review of literature?

Tardigrades and other microfauna are not animals that we work on or follow the literature on so I can not offer any direct information from our experience. However, I searched our library database and came up with the following 4 citations as probably the most relevant recent papers/books over the last 2 years that deal with tardigrade cold hardiness. If you have access to a university science library, then you should be able to get at least some of these recent articles.

1. The following seems to be a special issue of the Zoological Journal of the Linnean Society devoted to Tardigrade biology as there were many citations to this particular issue when I searched for tardigrades. However, I do not know whether some or any articles deal with cold hardiness or anhydrobiosis but very likely there will be something useful.

TARDIGRADE BIOLOGY - FOREWORD written by MCINNES SJ IN: ZOOLOGICAL JOURNAL OF THE LINNEAN SOCIETY 1996 JAN-FEB Vol.116 No.1-2

2. Dr. Somme is one of the foremost authorities on the cold hardiness of invertebrates so I am sure that this will be an excellent summary of the current state of knowledge about the cryobiology of tardigrades and should contain an extensive reference list to take you back to the older literature.

ANHYDROBIOSIS AND COLD TOLERANCE IN TARDIGRADES author: L. SOMME IN: EUROPEAN JOURNAL OF ENTOMOLOGY 1996 Vol.93 No.3 Pages 349 - 357

ABSTRACT: A review of the literature regarding anhydrobiosis and cold tolerance in tardigrades is presented. During increasing desiccation, invertebrates like tardigrades, rotifers, nematodes and some collembolans are able to shut down metabolism to undetectable levels. When tardigrades are entering anhydrobiosis, a tun-like structure is formed, facilitated by structural adaptations of the cuticle. Slow dehydration is essential for tun formation, and the accumulation of trehalose during this process may help to stabilize phospholipids and proteins. Wax extrusion on the cuticle surface reduces transpiration. A fraction of 5-15% of the initial body water is retained during anhydrobiosis. Tardigrades are principally aquatic organisms, but anhydrobiosis makes it possible for some species to live in habitats with changing moisture conditions. Tardigrades in anhydrobiosis may tolerate exposure to freezing temperatures of liquid gases, and some species also survive such temperatures in their hydrated state. Few investigations are available on the relation of tardigrades to temperatures more representative to their natural environments. Experimental studies, however, from Greenland and the Antarctic Continent suggest that some species overwinter both in a hydrated frozen state and in anhydrobiosis. During the summer, a number of tardigrade species have been recorded from cryoconite holes, formed on the surface of glaciers. These species are freeze tolerant since their habitats are permanently frozen during the winter.

3. This citation seems to be to a book review published in the journal Science (which any university library will have). You can use this citation to get more information on the book (like publisher, year, etc.) if you need it or you can just search your library for the title of this book by the stated authors.

THE BIOLOGY OF TARDIGRADES, Authors: KINCHIN,IM LIVINGSTON K IN: SCIENCE 1995 MAY 5 Vol.268 No.5211 Pages 745 - 745

4. This is a specific research paper dealing with Antarctic tardigrades. There were several other papers about Arctic and Antarctic tardigrades in the search I did but most just seemed to be identification / taxonomy / ecology type papers. This was the only one that seemed to address the cold hardiness of the animals.

COLD TOLERANCE IN TARDIGRADA FROM DRONNING-MAUD-LAND, ANTARCTICA, Author: SOMME L, Meier T. IN: POLAR BIOLOGY 1995 FEB Vol.15 No.3 Pages 221 - 224

ABSTRACT: Survival at low temperatures was studied in three species of Tardigrada from Muhlig-Hofmannfjella, Dronning Maud Land, Antarctica. Both hydrated and dehydrated specimens of Echiniscus jenningsi, Macrobiotus furciger and Diphascon chilenense had high survival rates following exposure to -22 degrees C for ca. 600 days, and dehydrated specimens following 3040 days at this temperature. In hydrated E. jenningsi, mortality increased with the duration of exposure from 7 to 150 days at -80 degrees C, while mortalities of the two other species did not change. Hydrated specimens of all species were rapidly killed at -180 degrees C, but all species exhibited good survivorship in the dehydrated state after 14 days at -180 degrees C. In conclusion, hydrated tardigrades are able to survive extended periods at low temperatures, and dehydrated specimens are even better adapted to survive overwintering on Antarctic nunataks.



QOut in the woods, do the frogs make preparations for winter and bury themselves before they freeze?

Frogs do not bury themselves as in digging underground. They hibernate at the soil surface but they choose places in the woods where the soil is covered by a good coat of leaf litter, possibly several inches thick. They may also get under rotting logs or into crevices around tree roots, etc. Some amphibians get well underground for the winter: toads can actively dig down as much as 3 feet underground whereas salamanders retreat down rodent tunnels. These strategies generally put toads and salamanders well below the frost line. Frogs, however, have only partial protection from winter cold although the leaf litter and the snow that piles on top of it are very good insulators. For much of the winter the subnivean (under snow) environment can remain unfrozen allowing many animals to stay active under the snow. There may be a difference of 20 degrees Celsius or more between air temperature above the snow and ground temperature under the snow. For frogs this seems to make the difference between life and death. The lowest temperatures that are generally encountered under the snow are about -6 to -8 degrees Celsius and freezing at this temperature is survivable by frogs whereas they can not survive freezing at the much colder temperatures found above the snowpack.



QCan a frog be frozen underwater or in a block of solid ice? Or would it destroy the frog from being crushed or suffocated?

Theoretically, we could probably freeze a frog into a block of ice although this is not how they would freeze naturally. I do not think the frog would be either crushed or suffocated. When a frog is frozen it is not breathing and has no blood circulation so the animal has to be able to survive for a long time without oxygen and this is an adaptation that wood frogs have. However, often when people ask us about freezing a frog in a block of ice (and we get asked quite often!!), they are (a) thinking about freezing the animal in their home freezer and (b) thinking about freezing a common pond frog like a leopard frog or bullfrog. Neither of these will work. Firstly, your home freezer is usually about -15 degrees Celsius which is far too cold for a frog to survive; you need to freeze the animal at about -2 to -3 degrees Celsius to guarantee its survival. Secondly, only a very few species of frogs survive freezing in nature and they are generally quite hard to find and never the common frogs that you find around ponds and rivers. So, the message is - do not try this at home!



QHow did you first find out about the Wood Frog and how he freezes? Why did you decide to study this species?

We first found out about the frog from an article in the journal Science by an ecologist from North Dakota. He had heard of anecdotal evidence from various people about frogs freezing and then had made the mistake of leaving a collecting bag full of frogs in the trunk of his car overnight. After an unexpected frost, he found all his animals frozen the next morning but was amazed when they revived on the lab bench. He then did a variety of tests to sketch out the freezing capabilities of several species.

We were absolutely fascinated when we read his work. We had been working with freeze tolerant insects for several years but these are very small. The frogs offered us the opportunity to work with a much larger animal (about 100-times greater mass) and gave us a vertebrate model animal that has all the same major organs as humans do. Hence, we believed that the lessons we could learn from finding out how a frog liver or heart freezes could be applied in trying to find ways to freeze mammal or human organs.

We have worked with 4 species of freeze tolerant frogs at different times but mainly use the wood frog for 2 reasons. First, its the largest animal - adult wood frogs in Canada range from 5-7 grams for males to 8-12 grams for females whereas spring peepers and chorus frogs are only about 1-2 grams. Secondly, its the easiest frog to catch. Wood frogs live on the forest floor whereas the other 3 types of frogs are tree frogs. Tree frogs are very noisy and you hear them a lot but they are often way above your head in the trees and hence virtually impossible to collect.



QI was wondering if you dropped one of the wood frogs when they were frozen, if it would break like a piece of glass?

No, they are not brittle like something that has been in liquid nitrogen. They are also very compact when they freeze, with their legs tucked in under their body so there are not body parts sticking out that could break off.




 

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