ANIMAL COLD
HARDINESS
Introduction
Our world is a cold place. About 90% of
the water in the oceans is colder than 5℃.
Much of the land is even colder. Winter air
temperatures in countries such as Canada
or Russia often fall to −30℃ and, in the
Arctic and Antarctic, −70℃ is not
uncommon. How do animals survive the
cold? Many different strategies are used
ranging from “running away” by migrating
Kenneth B. Storey
Professor of Biochemistry, Carleton University
最 終 学 歴：Ph. D., University of British Columbia
現在の専門：Biochemistry
生物学における分野：Biochemistry
to a warmer climate, entering a dormant
state to “sleep” through the winter, or
enduring the cold by using specialized
biochemical adaptations. However, before
we talk about how animals survive when
environmental temperatures fall below 0℃,
Janet M. Storey
Research associate, Institute of Biochemistry,
Carleton University
最 終 学 歴：M. Sc. University of British Columbia
現在の専門：Biochemistry
生物学における分野：Biochemistry
we need to know why cold is harmful or
even lethal.
Chilling and freezing injury
Temperatures below 0℃ can affect
animals in several ways. Firstly, the cold
temperatures of winter often lead to a
Fig. 1
Cell responses to extracellular freezing. Ice formation in the extracellular space excludes solutes from
the crystal and the solute concentration of the remaining extracellular fluid rises quickly. Water pours out
of cells to try to rebalance solute concentrations inside and outside the cell. Unprotected cells quickly
shrink to such a small volume that permanent damage occurs to cell membranes and when these cells
thaw their integrity is lost. Freeze tolerant animals use protective strategies. Freezing is seeded by
ice-nucleating proteins (INP) so that ice growth is slow and controlled. Antifreeze proteins (AFP)
regulate the shape of crystal growth and prevent the recrystallization of small crystals into larger ones.
Carbohydrate cryoprotectants such as glycerol (g) are distributed to all cells and minimize cell shrinkage,
promote supercooling, and protect protein structures. Trehalose and proline act as membrane protectants
(MP) to stabilize the cell membrane bilayer structure.
scarcity of food or even starvation because
most plant life dies off or is buried under
snow or ice. Warm-blooded animals
(mammals, birds) can actually need more
food in the winter to generate extra body
heat to stay warm. Many species die off
over the winter, leaving only eggs behind
to hatch in the spring. Others store extra
food or synthesize huge amounts of body
fat during the autumn that is used as a
winter fuel. Many animals also enter
dormant states in winter so that their
energy needs are lowered to the minimum
needed to stay alive.
Secondly, cold directly disrupts the
metabolism of animals. For most species,
very little growth, development, or
reproduction can be done near or below 0℃.
Cold stiffens the membranes that surround
40
ANIMAL COLD
HARDINESS
cells and this interferes the import or
grow first on the skin or in the gut and ice
export of nutrients, with ions and other
crystals extend through the large
molecules. Temperature also changes the
extracellular spaces such as between skin
rates of all metabolic reactions. On average,
and muscle layers, in the lumen of the gut,
a decrease of 10℃ lowers the rate of a
or inside the abdominal cavity. Sometimes
reaction by about 50% but selected
this is tolerable for short periods of time if
reactions can be affected much more or
the amount of ice formed is small. For
much less. This can create imbalances in
example, pond frogs often survive short
the rates of different metabolic processes,
freezing when only 10−15% of body water
sometimes with disastrous consequences.
turns to ice but when larger amounts of ice
The human heart, for example, cannot
accumulate, major problems occur due to
maintain its beat below about 25℃ because
cell dehydration. This is because the body
cold temperature disrupts the coordination
fluids of animals are solutions that contain
of the hundreds of metabolic reactions that
many kinds of dissolved molecules including
take place during each heart beat. This and
salts, sugars, and proteins. By contrast, ice
other organ failures are the cause of death
is a crystal of pure water. When ice crystals
due to hypothermia when people fall into
form in a solution, the water molecules lock
cold water or are lost outdoors in the
into position with each other and push all
winter. Although some animal species are
solute molecules out of the crystal. As more
adapted to live in constant cold (e.g. fish in
and more water joins the crystal, the
polar seas), many cold-blooded animals fall
remaining liquid has a higher and higher
victim to injuries or death caused by
concentration of dissolved solutes. When
chilling to low temperatures that they do
this happens in the extracellular fluids that
not normally encounter. Many kinds of
surround tissues, the solute concentration
insects are chill sensitive and sustain lethal
in the fluid outside of cells rises to much
injuries at temperatures well above their
higher values than the fluid inside cells. A
１）
freezing point.
steep gradient is formed that causes a
strong outflow of water from cells, leading
Thirdly, the major problem for animal
to cell dehydration and shrinkage (Fig. 1).
survival below 0℃ is freezing. Think of the
A key consequence of this is compression
devastation of a flower garden by the first
stress on the cell membrane as it shrinks
autumn frost. Freezing causes similar tissue
damage to most animals.２），３） Freezing of
カーレトン大学生化学部
the blood stops the delivery of oxygen and
nutrients to organs. Freezing disrupts or
halts every body function: muscle
movements, heart beat, breathing, and brain
activity among others. Ice crystals can
cause extensive damage such as by
squeezing cells between sheets of ice,
puncturing cells or bursting blood vessels.
Ice growing inside cells also destroys their
delicate internal architecture. When an
animal begins to freeze, ice usually starts to
41
9
down and below a critical minimum cell
winter survival and pay special attention to
volume, the membrane structure collapses
the strategies used by insects.
and breaks. This damage cannot be
reversed after thawing.
Migration
Migration is best known in birds that
However, despite these problems and
may travel thousands of kilometers to
others that are caused by cold
warmer climates to escape winter. Some
temperatures and freezing, there are still
large mammals also migrate. Caribou spend
many species of animals that live in
the summer on the Arctic tundra but
seasonally cold environments. How do they
migrate south for the winter to take
survive?
advantage of the shelter and the food
supply in the boreal forest. Some cold-
Strategies for winter survival
blooded animals also migrate over shorter
The simplest option for winter survival
distances. In parts of Canada, garter snakes
is to minimize or completely avoid exposure
travel several kilometers in the autumn to
to temperatures below 0℃. Not all animals
gather by the hundreds in deep under-
can do this, but most gain at least some
ground dens where temperature rarely falls
protection by seeking sheltered sites in
below 0℃. Frogs often move from shallow
which to spend the winter. Other species
marshlands where they spent the summer
take more drastic measures. They may
into deeper lakes and rivers where they are
alter their life cycle so that they spend the
protected from severe cold by spending the
winter in the life stage that is easiest to
winter underwater.
protect. They may change their social
Monarch butterflies are a unique example
behavior to huddle together for warmth
of long distance migration by an insect
and insulation. If these strategies are not
(Fig. 2).４） These butterflies leave Canada
enough, then biochemical solutions are used
and the Northern USA in late summer and
to change to the animal’s metabolism. This
fly to central Mexico where they roost by
can include entering dormancy to save
the thousands in cool mountain forests. In
energy and producing specialized molecules
the spring, they journey back north but the
that provide cryoprotection. In the next
butterflies that wintered in Mexico fly only
sections we will discuss these strategies of
as far as the southern USA before they
オタワ上空写真（オタワ空港から離陸した飛行機が水平飛行に移った時に撮影）
stop, lay eggs and then die. Their progeny
then grow, turn into butterflies and fly
farther north where they repeat the cycle.
The butterflies that return to Canada are
two or three generations removed from the
butterflies that flew south the previous
autumn. No one knows yet how the
different generations know whether to
migrate north or south or how they know
when they have reached their destination.
Shelter and insulation of many kinds
Many animals use some form of shelter
42
ANIMAL COLD
HARDINESS
to provide partial or complete protection
from freezing temperatures or from
desiccating in the very dry winter air. Some
frogs, turtles and insects move from the
land to spend the winter underwater in
Fig. 2
Winter life of insects. Several options are available. Monarch butterflies migrate south for thousands of
kilometers to the cool mountains of central Mexico where they gather by the thousands to roost in trees.
The aquatic larvae of many kinds of insects, including damselflies, spend the winter underwater in rivers
and lakes and emerge as adults in the spring. The communal efforts of honeybees allow them to keep
the hive warm all winter by generating heat from muscle shivering. Many species cannot escape winter
and have developed biochemical adaptations that let them endure temperatures of -40℃ or lower. The
spruce budworm keeps its tissues from freezing by using powerful antifreeze proteins.
lakes and rivers. Other animals seek shelter
on land. Many creatures spend the winter
on the forest floor where they are covered
by fallen leaves and a deep layer of snow.
Snow is an excellent insulator and
temperature at the soil surface rarely falls
below −5℃ even when air temperature
above the snow is −30℃.５）Other animals
gain even greater shelter by going
underground, either digging by themselves
(e.g. toads, worms, bumblebees) or going
down burrows made by other animals.
They adjust their position to keep
themselves below the frost line, digging
down farther as the frost penetrates deeper
and then rising again as the soil above them
melts in the spring. Warm-blooded
mammals and birds also need extra
protection to minimize passive heat loss
winter. For these animals, winter conditions
from their bodies. They use strategies such
are very difficult. The depth of cold
as huddling together in communal dens and
exposure can be extreme (−30℃ or below)
adding extra body insulation in the form of
and temperatures can also change
a thick layer of fat under the skin or a
dramatically over the day from deep cold
thickened undercoat of fur or down
overnight to melting on a sunny day.
feathers. Vegetation may also be used to
Another major problem is desiccation
add extra insulation to winter nests,
because cold air is very dry and animals
burrows, or dens.
may have to spend weeks or months
without access to liquid water. These
Relatively few types of animals live in
highly exposed sites all winter and most of
those that do are insects. Some insects
spend the winter under the bark of trees or
high in the branches. For example, larvae of
insects have to use some extraordinary
adaptations of their biochemistry to survive.
Life cycles
Many kinds of animals make
the spruce budworm spend the winter in
adjustments to their life cycles so that a
the forest canopy attached to the needles of
particular life stage overwinters and is
spruce trees (Fig. 2). Other insects live
relatively easy to protect. For example,
inside galls (swollen nodules) on the stems
insect species that have both aquatic and
of woody plants like goldenrod, willow and
terrestrial life stages frequently spend the
rose and are held up above the snow all
winter in their aquatic larval form and then
６）
43
9
metamorphose into flying adults in the
can feed and, even then, they can feed for
spring. This is the strategy of dragonflies
only about 20% of the day and need to
and mayflies, among others (Fig. 2). Many
spend the rest of the day basking in the
kinds of terrestrial invertebrates including
sun to again enough heat to move and to
diverse insects, slugs, snails and spiders
digest food.
spend the winter months as eggs. Eggs can
usually be well hidden, are often highly
Staying warm
resistant to dehydration and to freezing,
Winter cold can be defeated by keeping
can be kept in a dormant state, and are
the body warm. This is a major strategy of
easy to protect with simple biochemical
birds and mammals, including humans, but
adaptations. Eggs of the silkmoth are an
it is a very energy expensive strategy.
excellent example and are discussed in
However, given plentiful food or body fat
other chapters of this book. A mechanism
supplies, mammals and birds can exploit
called diapause is often use to interrupt the
many kinds of cold environments that are
life cycle of the last generation of insects
not open to reptiles and amphibians. For
produced in the late summer or autumn
example, there are virtually none of these
and induce a dormancy that will last until
cold-blooded creatures above the Arctic
７）
the springtime.
So instead of progressing
circle.
to the next life stage (e.g. pupa → adult) in
a matter of days, diapause halts develop-
Honeybees have a novel strategy for
ment for many months until the weather is
winter survival. Unlike all other insects,
again favorable. Each species typically has
they keep warm all winter by heating the
one life stage that can enter diapause (e.g.
colony.９） This strategy only works because
in silkmoths it is the egg; in spruce
of the communal effort of thousands of bees
budworms it is the young larva) and the
(Fig. 2). The bees shiver to produce heat by
critical trigger for initiating diapause is
muscle contraction and by increasing or
usually day length. Animals enter diapause
decreasing the number of bees shivering or
when the number of hours of daylight
how closely the bees huddle together, they
drops below a critical minimum.
can keep the brood area of the hive at a
constant 34−35℃. This protects the queen,
Another strategy for life in cold climates
is to greatly extend the life cycle. For
depends on another communal activity−
example, bullfrog tadpoles in the southern
the collection and storage of honey over
USA easily grow to maximum size and
the summer months.
metamorphose into adults within one
summer but in Canada the tadpoles need
two years to grow to full size. Wooly bear
caterpillars on Ellsemere Island in the high
８）
44
eggs and larvae. The fuel for this effort
Dormancy and Hibernation
The normal life of an animal can be
challenged by many kinds of environmental
Arctic have taken this to the extreme.
stresses such as extreme temperatures,
These caterpillars need at least 7 years to
oxygen deprivation, desiccation, and lack of
grow before they can pupate and turn into
food. When these stresses are prolonged,
the adult moth which lives only a few days.
the best hope for survival is for the animal
This is because there are only about 4
to cut down the amount of energy that it
weeks each summer when the caterpillars
uses in order to ration its remaining food
ANIMAL COLD
HARDINESS
stores or body fuel reserves (glycogen,
hibernation by eating large amounts of food
lipids, proteins) to stay alive as long as
during the late summer and converting this
possible. Animals in these situations take
to body fat, often increasing their body
two main actions: (a) a general suppression
mass by about 50%. They then retreat to
of energy use by all metabolic activities,
sheltered sites (burrows, caves) and soon
and (b) a reorganization of the priorities for
enter torpor. They turn down the biological
energy use to preserve core metabolic
thermostat in their brain so that body
functions and virtually halt energy
temperature falls from 37℃ down to the
expenditure on optional activities such as
10）
temperature of their environment and they
growth, development and reproduction.
only regulate body temperature again if it
Not surprisingly, the cold temperatures and
falls near to 0℃ and puts them at risk of
food restriction of the winter season require
freezing. By midwinter, their metabolic rate
many animals to reduce their energy use
in deep torpor can be as low as 1−5% of
by employing some form of torpor or
normal and the total energy savings gained
dormancy. The difference between winter
by using hibernation can be as much as
survival and death for many small
90% of the energy that would otherwise be
mammals and birds is the energy savings
needed over the winter season.
available from a mild nightly torpor. For
Interestingly, the hibernating season is
example, while sleeping, chickadees reduce
not just one long session of cold torpor but
their metabolic rate by 20−30% and let their
involves multiple cycles of 1−3 weeks of
body temperature drop by a few degrees.
torpor followed by 12−24 hours back at 37℃
The birds quickly rewarm themselves again
(Fig. 3).12） The heat needed to rewarm the
in the morning. As we already discussed
animal at the end of each cycle is huge and
above, many insects enter a state of
diapause over the winter and during this
time their metabolic rate is usually less
than 10% of normal.
Fig. 3
Body temperature as a function of time in a ground squirrel over the year from July to May. The photo
shows a torpid 13-lined ground squirrel, one of the main species used by hibernation researchers. The
hibernating season runs from mid-September to early April. The inset shows a magnified view of three
torpor-arousal cycles highlighting the different stages: the entrance into torpor (EN) which lasts up to 12
hours, early torpor (ET) within 48 hours of entering torpor, late torpor (LT), arousal (AR) with a duration
of 〜2 hours, and finally interbout arousal (IBA) with a duration of 〜20 hours. Taken from ref.12）
The best-known and extreme example of
winter dormancy is mammalian hibernation
which is most famous in bears but actually
occurs widely in many kinds of rodents,
bats and marsupials.11） The huge energy
expenditure of mammalian life is the
production of body heat. This is illustrated
by the fact that the metabolic rate (total
oxygen consumption) of a small mammal is
about 7 times higher than that of a lizard of
similar size. Cold weather demands even
greater energy expenditure on heating the
body because heat loss to the environment
increases hugely. If food is also scarce,
hibernation can be the only way to survive
through the winter. Animals prepare for
45
9
comes mostly from a special tissue called
or remove molecules of phosphate to
brown fat that surrounds the heart and
selected enzymes, generally those enzymes
major thoracic blood vessels so that it can
that are the master regulators of key
transfer heat directly to the blood which
pathways. This temporarily reduces the
then warms other tissues. In normal
activity of the enzymes but can be rapidly
circumstances, the energy released in the
reversed when dormancy is broken.
oxidation of lipid by the mitochondria of
Energy-expensive optional types of cell
cells is captured and used to synthesize
activities are most strongly suppressed
ATP (adenosine triphosphate) which is then
such as new protein synthesis or cell
used as the energy currency to drive many
division so that energy use can be diverted
other cellular reactions. However, in brown
instead to the fundamental activities that
fat, a special protein uncouples the energy
are absolutely required to sustain life. A
transfer to ATP so that all the energy
good example is the maintenance of the
produced from lipid oxidation is released as
electrical potential difference across the cell
heat that is then used to rewarm the
membrane which is critical for many
animal. The reason why hibernators wake
activities such as nerve impulses, muscle
up intermittently is not known for certain
contraction, and the import/export of
but it must be important since it uses so
molecules to/from cells. Another universal
much energy. Some suggestions are to
principle of dormancy is long term
sense whether spring has arrived or to
protection of cells. Under normal circum-
refresh key vital functions (e.g. brain
stances, damaged cell proteins and other
activity, immune system, kidney function).
components are rapidly broken down and
Brain activity might be critical since
recycled or excreted and new components
researchers have found that hibernators
are made. Both synthesis and degradation
experience intense REM sleep during the
are energy-expensive and are largely shut
arousal period.
down in dormant systems. Our work has
shown that what happens instead is that
Our laboratory is very interested in
identifying the principles of dormancy
greatly extend the useful “shelf life” of
including how animals regulate their
cellular components. This includes several
metabolism to enter or leave dormancy and
kinds of chaperone proteins that help to
how they stabilize and preserve their cells
refold other proteins if they are damaged,
10）,13）
during long term dormancy.
We have
antioxidants that fight chemical damage to
found that the biochemistry of dormancy is
proteins and membranes, and protein
very similar in all animal groups even
stabilizers such as glycerol (more about
though the stress that can trigger
glycerol later).
dormancy can be very different for different
species (e.g. low temperature, low oxygen,
low food, etc.). In general, the basic
46
extra stabilizing molecules are made that
Winter life in freshwater
Water has its greatest density at 4℃ and
biochemistry of a cell is all left in place
so cold water sinks to the bottom of a pond
when an animal enters dormancy but the
or lake. A gradient then forms with water
rate at which cell reactions run is strongly
temperature rising to about 0℃ just under
reduced. To do this, cells use a chemical
the surface ice. In severe winters, ice may
modification called phosphorylation to add
penetrate all the way to the bottom of a
ANIMAL COLD
HARDINESS
pond and then the animals freeze and die.
the fish masters of this strategy which is
But mostly this does not happen, and
why they are often the only kinds of fish
animals spend the winter in a protected
that survive the winter in small ponds.
environment at a stable 0−4℃. However,
They have modified their energy-producing
winter living under water has another
fermentation reactions to make a mix of
challenge and that is low oxygen. Animals
ethanol (alcohol) and carbon dioxide and
that normally breathe with lungs (frogs,
they excrete both of these out into the
turtles) cannot use their lungs under water.
water across their gills. The most successful
Even species that breathe with gills can be
example of insects that can live without
in trouble. When a pond is covered by ice
oxygen are the larvae of chironomid midges
and snow, the water can no longer be
which use this ability to live in the sediment
oxygenated by the air. The animals, plants
on the bottom of ponds and lakes all over
and microorganisms in the water use up
the world.14） They also produce ethanol as
the oxygen through respiration but the
the product of their energy metabolism.
snow/ice cover makes it too dark for algae
14）
This ability also helps chironomids to
to replace the oxygen by photosynthesis.
survive freezing and has made them the
There are two solutions to this – either get
most widespread and successful insect
oxygen in a different way or live without
group in the Arctic because they can
oxygen. As long as there is still oxygen in
survive all winter frozen into the sediment
the water, frogs switch to breathing across
of small ponds.
15）
their skin.
The amount of oxygen
dissolved in water increases as water gets
Winter life on land
colder whereas the amount of oxygen
Although the strategies that we have
needed by animals decreases because
already discussed provide many animals
metabolic rate falls in the cold. So, in cold
with partial or complete protection when
water, frogs can absorb all the oxygen that
environmental temperatures drop below
they need across their skin. Some turtles
0℃, many cold-blooded terrestrial animals
have a similar solution and absorb oxygen
must be able to endure extreme cold that is
across the lining of their mouth and throat.
far below the normal freezing point of their
body fluids. For this, they use adaptations
However, once the oxygen in the water
of their biochemistry to protect them from
is all used up, many species are in trouble
injury or death due to freezing. In general
but others can survive. Some kinds of
there are two ways of doing this. One is to
turtles can live for about 3 months without
16）
use a variety of antifreeze strategies to
oxygen when submerged in cold water.
prevent body fluids from freezing – this is
As soon as they submerge they greatly
called freeze avoidance. The other is called
decrease their metabolic rate to only about
freeze tolerance. This may seem like
10% of their previous energy needs and
science fiction to us but there are lots of
they switch to producing energy (ATP)
animals that can survive for weeks or
from carbohydrate fuel (called glycogen).
months with 65−70% of their total body
This can be done by fermentation reactions
water frozen as ice. For the remainder of
that do not need oxygen. They store the
this article we will investigate how these
waste product (lactic acid) from this process
strategies work.
in their huge shell. Carp and goldfish are
47
9
Freeze avoidance
We all think of water as freezing at 0℃.
helps to keep their soft tissues from coming
into contact with environmental ice which
is an extremely potent nucleator. Extra
However, this actually occurs because the
protection against contact with ice can also
formation of ice crystals is seeded by the
come from the addition of a water-repelling
presence of a surface (e.g. dust, dirt, rock,
cocoon or a wax coating on the exoskeleton.
bacteria) that can align water molecules
Secondly, animals get rid of nucleators from
into the crystal shape for long enough to
their bodies. Uncontrollable nucleation
trigger an explosion of ice growth. In actual
usually begins in the gut because food
fact, if a very small volume of ultrapure
particles, feces and bacteria can trigger
water is cooled very precisely in the
freezing. To fix this, animals usually stop
absence of any nucleating surfaces, it can
eating in early autumn and completely clear
remain liquid down to −40℃. So, if animals
out their gut. Finally, the most powerful
have a way of eliminating or masking
anti-nucleation device is the production of
nucleators, they can also chill their body
antifreeze proteins.６） These specialized
fluids by several degrees below the natural
proteins work by binding directly to
freezing point (which is about −0.5 to −1℃
microscopic ice crystals and preventing
for most land animals) without freezing.
them from growing any larger. The bonds
This phenomenon is called supercooling.
are formed between ice and the polar side
Most small insects actually do this easily
chains of some amino acids in the protein.
and even in summer they may supercool to
Most antifreeze proteins have a lot of
−5℃ or even −10℃. Another property of
repeats of the same amino acids because
water that can be exploited for cold
the side chains of those amino acids line up
hardiness is the fact that the addition of
correctly with the distance between two
solutes to water lowers both its freezing
water molecules in the ice crystal. For
point and its supercooling point. For
example, the antifreeze protein of the
example, the salt in the ocean makes
spruce budworm has about 20% cysteine
seawater freeze at about −2℃ and the
content. The production of antifreeze
huge amount of ethylene glycol that we
proteins in insects is triggered by the
pour into car radiators keeps that water
decrease in daylength in the autumn,
from freezing down to −40℃ or lower.
usually when there is less than 10−11 h of
So, if animals can pack enough solutes into
light. The change in daylength is detected
their body water, they can also chill to
by the brain and stimulates the release of
low temperatures without freezing. Indeed,
juvenile hormone from the insect brain. The
a combined strategy that blocks ice
hormone travels to the fat body (the liver
nucleation and greatly increases the
equivalent of insects) and turns on the
concentrations of solutes in body fluids is
genes that code for antifreeze proteins. The
exactly what many animals do, particularly
proteins are synthesized and released into
terrestrial arthropods (insects, spiders,
the blood and into the lumen of the gut
mites, ticks, etc.) .
where they go to work to prevent ice
growth. Production of the proteins can also
Prevention of nucleation is acheived in
48
be sped up or slowed down by environ-
several ways. Firstly, the water-
mental temperature so if there is a very
impermeable exoskeleton of arthropods
early frost, the insect will quickly make
ANIMAL COLD
HARDINESS
more antifreeze proteins.
Antifreeze proteins are also very
important to marine fish that live in cold
water.17） Fish that swim just under the
polar ice cap or that live in shallow water
near to shore often have to swim among ice
Fig. 4
Larvae of the goldenrod gall moth, Epiblema scudderiana, use the freeze-avoidance strategy of cold
hardiness. (A) The larvae eat and grow within elliptical galls on the woody stems of goldenrod. (B) The
final instar larva overwinters, spinning a thin cocoon that separates it from waste materials and protects
it from nucleation by ice crystals growing in wet plant material. C. Beginning in mid-October, the larvae
start to synthesize glycerol from the stores of glycogen that were built up during summer feeding. By
midwinter, about 20% of the body mass of the animal is glycerol. As spring approaches glycerol levels
fall again and cryoprotectant is catabolized for energy to support renewed development to the pupal and
adult stages. D. The supercooling point of the larvae decreases over the winter down to about -38℃ due
to the accumulation of antifreeze proteins and glycerol. At all times the supercooling point at which the
larvae would spontaneously freeze is well below the mean daily temperature. Data are compiled from
18）.
crystals. Ocean water is still liquid at −2℃
but the freezing point of fish blood is about
−0.5℃. So, the fish are in constant danger
of freezing, and ice touching the skin and
gill surfaces of the fish can easily trigger
freezing, with lethal results. The fish
probably also take in sea ice when they
swallow food and, interestingly, research on
one group of Antarctic fish (the nototheniids) found that their antifreeze protein
had evolved from a normal digestive
protein found in the gut called trypsinogen.
Probably trypsinogen had a small antifreeze
activity by itself and this was helpful in
masking ice crystals that the fish
glycerol, a 3-carbon sugar alcohol, that is
swallowed. Over evolutionary time, the
analogous to ethylene glycol, a 2-carbon
trypsinogen gene was copied, modified and
sugar alcohol. Alcohols with 4, 5 or 6 carbon
amplified until new genes were created that
atoms also occur in some species. Glycerol
coded for a highly efficient antifreeze
is favored because it is non-toxic, easily
protein but no longer had any digestive
passes across cell membranes, and is
function.
energy-efficient to synthesize from the
stored carbohydrate reserves (glycogen)
The other biochemical adaptation that is
that insects build up in their bodies during
critical to the freeze avoidance strategy is
summer feeding. In midwinter, as much as
to pack body water fluids with huge
20−25% of the body mass may be glycerol
quantities of solutes which lower both the
in a freeze-avoiding insect such as the gall
freezing point and the supercooling point.
moth, Epiblema scudderiana (Fig. 4).13），18）
Sugars or sugar alcohols are typically used
Carbohydrate cryoprotectants also have an
as cryoprotectants and these lower the
added action. Not only do they suppress the
freezing point of water in exactly the same
freezing and supercooling points of body
way that ethylene glycol works in a car
water but because they hold onto water,
radiator. They work by colligative action−
they greatly reduce evaporative water loss
the power of huge numbers of solute mole-
from the body. Indeed, the most ancient
cules to interfere with the close interactions
and universal use of these molecules in
between water molecules that are needed
insects is to provide dessication resistance
to start ice crystallization. The most
for species that live in arid environments.
common cryoprotectant in insects in
Hence, insects that spend the winter in
49
9
exposed sites such as in tree tops seem to
maintain a supercooled state. Chiefly, they
employ glycerol for both purposes,
fail because they cannot make their body
cryoprotection and desiccation resistance.
surface impermeable to ice. This is
Freeze tolerance
especially true of animals that spend the
winter in moist habitats. For example,
The freeze avoidance strategy works
several kinds of woodland frogs spend the
excellently as long as air temperature does
winter on the forest floor under a layer of
not drop below the supercooling point of an
damp vegetation. Their skin is very
animal and as long as the animal does not
permeable to water and if they did not seek
come in direct contact with a potent ice
shelter in a damp place, they would soon
nucleator such as ice crystals in their
dry out and die due to desiccation.
surrounding environment. If either of these
However, when frost penetrates into
happen, the animal’s body fluids are
their habitat, the permeable skin of these
instantly nucleated and the animal freezes
frogs means that they cannot avoid
in seconds and dies. Hence, there is a risk
nucleation if they touch ice. To survive they
to the freeze avoidance strategy but the
have evolved adaptations that allow them
adaptations used by freeze avoiding species
to endure the formation of ice in
typically provide excellent protection for a
extracellular spaces of their body.19），20）
high percentage of the individuals in a
Often 65−70 % of total body freezes.
population. However, some animals cannot
In frogs, ice crystals grow between the skin
Fig. 5
Some examples of freeze tolerant animals. A number of amphibian and reptile species that live in
seasonally cold climes are freeze tolerant. Shown here are wood frogs (Rana sylvatica) that are the
best-studied model of vertebrate freeze tolerance and newly hatched painted turtles (Chrysemys picta)
that spend their first winter hidden in shallow nests on land. Many insects are freeze tolerant and gall fly
larvae (Eurosta solidaginis) are one of the major model species used by researchers. They spend the
winter inside ball galls on the stems of goldenrod plants. The last instar larva eats out a tunnel to the
surface of the gall in the autumn, then retreats to the center and enters diapause. Pupation occurs in the
spring and the adult fly walks up the tunnel and pushes through the thin skin of the gall. The larvae use
a mixture of glycerol and sorbitol for cryoprotection and actually elevate the supercooling point of their
body fluids (from about −15℃ in summer to −8℃ in winter) so that they so that they gain better control
over ice growth through their tissues. About 65% of total body water turns to ice and in Canada the
larvae survive temperatures of at least −30℃. Larvae of the hermit flower beetle (Osmoderma eremicola)
live in the heart wood of deciduous trees such as maples and also endure extreme cold. A number of
mollusks, barnacles and other creatures that live in the intertidal zone of northern oceans also survive
freezing when they are exposed to air temperatures below 0℃ at low tide. Shown here are periwinkle
snails (Littorina littorea). For more information see ３）, 19）−21）or to go www.carleton.ca/~kbstorey.
and muscle layers of the body, in the
bladder and in the lens of the eye. A huge
mass of ice also fills the abdominal cavity
encasing all of the internal organs which
shrink down to small sizes due to water
loss into the surrounding ice mass.
Freeze tolerance occurs in many
different groups of animals (Fig. 5).19） Six
species of frogs and several kinds of lizards,
snakes and turtles that live in cold climates
are freeze tolerant. Many insects and other
invertebrates also use this strategy. Snails
and barnacles that live in the intertidal
zone of northern oceans also survive
freezing when they are exposed to subzero
air temperatures at low tide. Freeze
tolerance is a more difficult strategy to
achieve than is freeze avoidance because
animals must deal with many extra issues
including (1) regulating ice growth and
limiting physical damage to their tissues by
ice crystals, (2) adjusting to extreme cell
dehydration when water exits to join
extracellular ice masses, (3) surviving for
50
ANIMAL COLD
HARDINESS
long times without breathing or blood
circulation, and (4) reactivating all vital
processes after thawing including breathing,
heart beat, muscle movement and brain
activity. Hence, a broad range of adaptations are needed to regulate the freezing
process, protect tissues while the animal is
frozen, and help the animal to fully recover
after thawing.13），20）
The first issue that needs to be
addressed is ice management. All freeze
tolerant species allow ice to accumulate in
extracellular spaces but freezing inside of
cells is lethal to them, just as it is to all
other animals. So, animals need to manage
where ice grows and this is done by using
ice nucleators to trigger ice growth in
extracellular spaces (Fig. 1).
６）
カエルと卵の採取
For example,
frogs use ice-nucleating bacteria on their
clotting proteins in their blood to quickly
skin or in their gut to trigger the freezing
patch any internal bleeding that occurs
process whereas most freeze tolerant
when animals thaw. In frogs, the first
insects produce special ice-nucleating
freezing responsive genes to be identified
proteins. By using ice nucleators, animals
were for fibrinogen, a central protein in the
can also initiate ice growth just below 0℃
formation of a blood clot.３），13）
and avoid extensive supercooling and the
risk of instant flash freezing. When freezing
Freeze tolerant animals also use another
starts between 0 and −5℃, the rate of ice
trick that we have discussed before and
growth is low and animals have lots of time
that is the use of carbohydrate
to achieve a controlled water loss from cells
cryoprotectants. However, instead of using
and make other adjustments to their
cryoprotectants to keep the whole body
metabolism before they are fully frozen.
Surprisingly, many freeze tolerant insects
カエルの解剖の様子
also make antifreeze proteins and
researchers were initially confused by the
presence of proteins with opposite actions.
However, it seems that antifreeze proteins
have a different role in freeze tolerant
insects. They help engineer the size and
shape of ice crystals, keeping individual
crystals from growing so big that they
cause physical damage to the tissues. For
further damage control, freeze tolerant
animals also increase the amounts of
51
9
from freezing, they use them to keep the
３），13）
inside of cells from freezing.
Ice forms
special sugar called trehalose and the amino
acid proline are produced and these directly
outside of cells but the high levels of sugars
interact the phospholipid molecules in
or sugar alcohols inside the cells keeps
membranes to stabilize them.３），20）
intracellular water liquid. The
cryoprotectants also help to resist the loss
of too much water from cells so that the
stops, the heart beat stops, and blood no
cells never shrink so much that their
longer circulates. Oxygen and nutrients can
membranes are broken (Fig. 1). Glycerol is
no longer be delivered to tissues and so
again the most common cryoprotectant
each cell must be able to survive on its own
used by freeze tolerant insects but a pair of
for the whole length of the freezing episode
cryoprotectants, glycerol and sorbitol, is
which could be as long as several weeks.
also common, such as in silkmoth eggs or in
The human brain is damaged after only
larvae of the gall fly (Eurosta solidaginis)
about 4 minutes without oxygen and most
(Fig. 5). The important feature of sorbitol
of our other organs cannot survive too
seems to be that it cannot move across cell
much longer. So freeze tolerant animals
membranes easily so sorbitol that is packed
need to be able to live for a long time
into the cells of freeze tolerant animals
without oxygen. As we also discussed for
stays there to defend the cell from
animals that spend the winter under water,
excessive volume loss during extracellular
several factors contribute to survival
ice formation. By contrast, glycerol can pass
without oxygen. Firstly, because metabolic
easily across cell membranes and so its
rate decreases with decreasing tempera-
concentration would equilibrate in extra-
ture, the demand for oxygen is low when
and intracellular fluids. Most freeze tolerant
body temperature is less than 0℃.
frogs accumulate glucose as their cryo-
Secondly, many animals are already in a
３），13），20）
protectant.
Glucose is the normal
dormant state during the winter months so
blood sugar of all vertebrate animals and its
their metabolic needs are already very low.
levels are normally closely regulated
For example, insects in diapause often have
because high blood glucose causes diabetes.
a metabolic rate that is less than 5% of the
Diabetic humans are very sick if their blood
normal value. Thirdly, freeze tolerant
glucose levels are 5−10 times higher than
animals have a well-developed capacity for
normal but frogs are uninjured by levels
producing ATP energy using fermentative
that are 100 times greater than normal.
pathways. Indeed, the same pathway of
Researchers are very interested in how
fermentative metabolism that allows adult
frogs can resist damage by high glucose
turtles to survive underwater all winter
because this could lead to new ways to stop
without breathing oxygen, also allows
glucose damage to the tissues of diabetics.
juvenile turtles to survive when they are
Freeze tolerant animals also have a second
frozen on land in their natal nests.３），21）
type of cryoprotectant that protects
membranes. The structure of cell
membranes needs to be stabilized when
cells shrink during freezing so that the
52
When animals freeze, their breathing
Lessons from studies of
animal cold hardiness
Animal cold hardiness is a fascinating
membrane does not break or collapse under
subject on its own but many of the things
the compression stress that occurs. A
that we are learning about how animals
ANIMAL COLD
HARDINESS
survive in the cold also have applications to
industry, agriculture and medicine.22）
Proteins that interact with ice have a
variety of uses. For example, industrial
snow-making machines use ice-nucleating
bacteria to improve snow production for ski
hills. Ice-nucleating bacteria are also
sprayed on agricultural insect pests to help
freeze and kill insects that would normally
supercool.１） Oppositely, other research has
aimed to create transgenic species that
produce antifreeze proteins that can
improve the cold tolerance of beneficial
冬のカナダ国会議事堂（オタワ）
insects or provide increased protection to
oceanic salmon that are now farmed in pens
close to shore where they encounter sea ice
in winter.22） Studies of the mechanisms of
freezing tolerance in animals have also been
critical for developing cryopreservation and
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