Human Reproduction Vol. 15, (Suppl. 5) pp.
26-46, 2000
Aspects of natural cold tolerance in ectothermic animals
Hans Raml0y
Department of Life Sciences and Chemistry, Roskilde University, P.O.Box 260, DK 4000 Roskilde,
Denmark
E-mail: hr@virgil.ruc.dk
Polar, alpine and temperate ectothermic (cold-
blooded) animals encounter temperatures below
the melting point of their body fluids either
diurnally or seasonally. These animals have
developed a number of biochemical and physio-
logical adaptations to survive the low temper-
atures. The problems posed to the animals
during cold periods include changes in mem-
brane and protein structure due to phase
changes in these molecules, changes in electro-
lyte concentrations and other solutes in the body
fluids as well as changes in metabolism. Cold-
tolerant ectothermic animals can be divided into
two groups depending which of two 'strategies'
they employ to survive the low temperatures:
freeze-tolerant animals which survive ice forma-
tion in the tissues and freeze-avoiding animals
which tolerate the low temperatures but not
crystallization of the body fluids. The adapta-
tions are mainly directed towards the control
or avoidance of ice formation and include the
synthesis of low mol. wt cryoprotectants, ice-
nucleating agents and antifreeze proteins. How-
ever, some of the adaptations such as the syn-
thesis of low mol. wt cryoprotectants are also
more specific in their mechanism, e.g. direct
stabilizing interaction with membranes and pro-
teins. The mechanisms employed by such
animals may offer ideas and information on
alternative approaches which might be usefully
employed in the cryopreservation of cells and
tissues frequently required in assisted repro-
ductive technology.
26
Key words: antifreeze protein/cold tolerance/cryo-
protectant/ice-nucleating agent/membrane
Introduction
Animals living in polar, temperate and alpine
environments are either on a daily or yearly basis
subjected to temperatures well below the freezing
point of their body fluids. Liquid water is necessary
for all life processes, therefore these animals must
either avoid freezing of body fluids but survive
the low temperatures or be able to endure ice
formation in body fluids. A number of such animals
are endothermic and survive the low temperatures
by way of insulation such as fur or fat. The
ectothermic (cold-blooded) animals, in which body
temperature follows the surrounding environment,
have a number of options when the temperature
falls: they can hide in microhabitats where they
are not exposed to low temperatures; they can
leave the area and return when conditions become
more hospitable; or they can adapt to the low
temperatures via a number of morphological, ana-
tomical, biochemical and physiological features.
The present review focuses on this last group of
animals and their biochemical and physiological
adaptations. In view of the temperature sensitivity
of mammalian gametes and embryos, important
clues to the potential effects of cold and their
avoidance may be gained from comparative studies.
Damages due to cold per se
Animals living in areas where they are exposed to
low temperatures, or temperatures that are lower
© European Society of Human Reproduction and Embryology

than the temperature at which the animals are
normally active, may suffer damage due to the
cold per se. The temperature does not need to fall
below the melting point of the body fluids to cause
damage. Damage caused by the cold per se is
due to either changes in metabolism or to phase
transitions in membranes and proteins as a result
of the low temperatures.
Phase changes in membranes
Biological membranes are bilayers of 20-80%
lipids, mostly phosphoglycerides, with one primary
hydroxyl group esterified to phosphoric acid and
the other hydroxyl groups esterified to fatty acids.
The phosphoglycerides also contain a polar head
group often in the form of an amino alcohol, which
is esterified to the phosphoric acid via its hydroxyl
group. This arrangement gives rise to amphipathic
compounds or 'polar lipids', because of their polar
headgroups and their non-polar hydrocarbon tails
consisting of 16, 18, 20 or 22 carbon atoms
(Lehninger, 1975; Grout and Morris, 1987). The
bilayers are formed as a consequence of the amphi-
pathic nature of the phospholipids. In the presence
of water the hydrophilic headgroups are exposed
to the water and the hydrophobic hydrocarbon
chains form the core of the membrane. The integrity
of the biological membrane is determined by
several factors such as Van der Waals forces
(electrostatic interactions), salt bridges, hydrogen
bonds and, perhaps more importantly, thermodyn-
amic relations such as hydrophobic interaction and
entropy. The hydrogen bonding of numerous water
molecules to each other is one of the strongest
forces driving the membrane into its lamellar
configuration (van Oss and Good, 1996). Mem-
brane structure is dependent on temperature, pH,
ionic strength of the surrounding medium and the
state of hydration (Williams, 1990).
During cooling, the initial effect is an increase
in membrane viscosity (Grout and Morris, 1987).
Upon further cooling, phase separations are likely
to occur. A characteristic of bilayers of a pure lipid
is the phase transition temperature (Tc) above
which the lipid bilayers are found in a disordered
phase called the liquid crystalline state and below
which the bilayer is found in the more ordered
gel state (Morris and Clarke, 1987). Efficient
Cold tolerance in ectotherms
membrane function requires the liquid crystalline
phase, in which the membrane is strain-free, so
that hydrophobic regions of proteins and the lipid
bilayer can be matched (Bloom, 1998). It should
be noted that if the hydration of the membrane
changes, the lipids may go through the liquid
crystalline to gel transition and even reach a phase
called the hexagonal II (Hn) phase, where the
lipids organize into a non-lamellar three-dimen-
sional matrix with the hydrophilic headgroups
pointing inwards towards 'channels' of water while
the hydrophobic hydrocarbon chains are pointing
towards each other (Quinn, 1985).
Biological membranes are not composed of only
one pure lipid but rather of many—up to 200
different lipids are found in the membranes of
some biological systems (Morris and Clarke, 1987).
Differential calorimetric studies have shown that
a mixture of a saturated and a non-saturated lipid
gives rise to two distinct endotherms if the mixture
is heated from below the liquid-crystalline phase
temperature (Tc) for both lipids to a temperature
above the Tc for both lipids. These endotherms
arise from the phase transitions of laterally phase-
separated domains of the unsaturated and saturated
lipids respectively. The two lipids were separated
into domains that consisted of the pure lipid of
one or the other. The saturated lipids stayed in
the gel phase at a higher temperature than the
unsaturated lipids but on further heating these also
underwent a phase transition and entered the liquid
crystalline phase (Quinn, 1985; Gennis, 1989).
Chapman et al. (1977) have proposed a model for
the occurrences in the biological membrane during
cooling. They suggest that cooling a biological
membrane below the Tc of the lipids leads to lateral
phase separations and that it also has profound
effects on the distribution of the proteins bound
to or integrated into the membrane. When the
membrane is cooled, some lipids function as 'nuc-
leation sites' and undergo a phase transition, crys-
tallizing into 'islands' of the gel phase in which
proteins become trapped. Along the edges of these
'islands' packing faults are likely and as the
proximity of the proteins increases these may begin
to aggregate (Quinn, 1985).
The consequences of the thermotropic behaviour
of membrane lipids are diverse. A number of the
27
H.Raml0v
membrane components are free to diffuse within
the membrane. Phase separations and the increase
in membrane viscosity will have effects on the
kinetics of diffusion-controlled processes (Grout
and Morris, 1987). Enzymes associated with the
membranes may become clustered into small
domains of liquid lipid and this may have various
effects on membrane function; there may be an
increased probability of the enzymes getting into
contact with their substrates which may lead to
increased enzyme activity (Grout and Morris,
1987). In contrast, such an aggregation of the
enzymes could also impede the transport of various
molecules across the membrane, e.g. where
aggregations of enzymes may deplete an area of
substrate in the immediate vicinity of the enzymes.
Here, transportation rates would decrease and
depend upon the diffusion of new substrate to the
enzyme aggregation in question.
The boundaries between the gel phase and the
liquid-crystalline phase are known to be especially
leaky (Williams, 1990). Leaky areas will tend to
dispel the electrochemical gradients across the
membranes as well as possibly causing the leakage
of potentially damaging substances into the cells
(e.g. increasing intracellular calcium). Around
integral proteins there is another possibility of
leakage because cooling may impair the ability
of non-bilayer forming lipids, also found in the
membranes, to seal the boundaries between these
proteins and the membrane (Quinn and Williams,
1985). Most of the evidence for the above-men-
tioned effects of cooling on membranes was gained
via studies of relatively simple model systems
possibly only consisting of a single or a mixture of
two lipids and perhaps a single protein. Biological
membranes are of course infinitely more complex
with their large array of different lipids and both
bound and integral proteins. However, there are
several examples of effects of cooling predicted
by the above-mentioned models (for a review see
Grout and Morris, 1987).
Structural transitions in proteins
Proteins are of the utmost importance to all biolo-
gical processes and to the structure of cells and
living systems in general. Therefore the mainten-
ance of structure and function of proteins during
28
exposure to low temperature is imperative if the
biological system is to survive.
According to Franks and Hadley (1992), the
cold denaturation temperatures of most proteins
when examined in the pH range of maximal
stability lie below the equilibrium freezing point
of water. For the majority of the examined proteins
this means below -15°C. The stability of proteins
in solution is very limited (Franks, 1985) and
presumably based on a number of contributing
factors, which can be divided into stabilizing and
destabilizing factors. The former are hydrophobic
interactions, intrapeptide attractive effects (hydro-
gen bonding, salt bridges, van der Waals inter-
actions) whilst the latter are core repulsion,
configurational entropy and solvation effects
(Franks and Hadley, 1992). The increasing magni-
tude of the latter, particularly solvation of non-polar
(hydrophobic) moieties upon cooling, is thought to
be the molecular origin of cold denaturation (Hvidt
and Westh, 1998). Denaturation of proteins at low
temperatures is often reversible (Franks, 1985),
e.g. tubulin depolymerizes at low temperatures
but polymerizes when the temperature returns to
normal (Timasheff, 1978). However, some proteins
denature non-reversibly during cooling, due mainly
to the formation of aggregates, e.g. urease (Hofstee,
1949). Another example is phosphofructokinase, a
key enzyme in glycolysis (Carpenter et ai, 1986)
which is dissociated from a tetramer into two
dimers, leading to the synthesis of sucrose from
fructose instead of the phosphorylation of fructose
(Franks, 1985; Storey and Storey, 1992).
Activity and metabolism in animals at low
temperatures above 0°C
When an ectothermic animal is cooled below its
usual temperature of activity, it passes through a
number of states before it eventually dies. The first
state, which is observed below the temperature of
optimal activity, is called cold stupor (Klok and
Chown, 1997). In this state the animal becomes
more and more sluggish and eventually does not
move at all when reaching what is called chill
coma (Vannier, 1987; Block et al., 1992). It should
be noted that some animals do not survive these
stages for more than a few minutes (Lee, 1991)
whereas other animals, namely the cold-adapted

can survive for several months or perhaps even
longer (Block, 1990).
The reduction in activity is accompanied by
a temperature-dependent reduction in respiration
(Block, 1990; Block et al., 1998; Raml0v, unpub-
lished observations). This can be assessed by
measurements of Q10, defined as the ratio between
metabolic rates recorded at two temperatures and
extrapolated to a 10°C difference:
Q10 = R,/R 2 *[10/(T 1 -T 2 )].
Block (1977) reported that the Q10 for Antarctic
land invertebrates is 3.04. Similar relationships are
seen in other ectothermic animals although the
value may vary depending on species and the
temperature range over which the Q10 is measured
(Davenport, 1992).
The relationships between decreasing temper-
ature, locomotion and respiration are not surprising
considering changes in membrane and protein
structure mentioned above. Additionally, most
enzymes have a temperature range in which they
function optimally, and when, as the temperature
falls outside this range, kinetics change and activity
also decreases (for discussions see Hochachka and
Somero, 1984; Franks, 1985). Locomotion may
be impaired due to depolymerization of actin
filaments, and cytoplasmic streaming may slow
down or cease (Grout and Morris, 1987). Cyto-
plasmic streaming is highly dependent on Ca 2+
concentrations and ATP production and both may
be changed during cooling, e.g. intracellular Ca 2+
concentration may change because of leakage of
Ca2+ into the cells from the extracellular fluid
because of lateral phase separations in the mem-
brane (see 'Phase changes in membranes' above).
Chilling injury can also be caused by the elastic
stress that occurs when the membrane condenses
to a greater extent than the contents of the cell
(McGrath, 1987).
Protein synthesis may also be impaired or slowed
by a decrease in temperature (Grout and Morris,
1987). 'De novo' synthesis of proteins will not
take place and this may have a bearing on how long
animals are able to tolerate the low temperature. If
leakage occurs, ion gradients and the membrane
potential break down too. As the membrane poten-
tial is important for all transport processes in
Cold tolerance in ectotherms
and out of cells, its breakdown interferes with
metabolite transfer across cell membranes. This in
turn could lead to depletion of necessary substances
inside cells or to the accumulation of potentially
dangerous metabolic waste products. Thus, various
animals are able to endure deceasing temperatures
to various extents and times depending upon their
ability to withstand, counteract or repair the cellular
changes mentioned above.
The freezing process
When the temperature falls below the melting point
of the organism's body fluids there is a potential
for ice formation in the organism. Nucleation of
ice crystals may occur in water solutions at any
temperature below the melting point of the solution.
If ice is not formed in the solution at its melting
point the solution is supercooled or undercooled.
The supercooled solution is metastable as it is
energetically more favourable for the solution to
be in the frozen or partly frozen state than in the
liquid state at this temperature. Crystallization may
therefore occur spontaneously at any time by the
water molecules aggregating into an ice nucleus
(homogeneous nucleation), or by the water molec-
ules aggregating around some substance or irregu-
larity on a surface that lowers the activation energy
of the crystallization (heterogeneous nucleation)
(Franks, 1985; Williams and Carnahan, 1990).
Nucleation is a time-dependent stochastic process
(Vali, 1995) which is dependent on the probability
of a sufficient number of water molecules to form
a structure (cluster) that gives rise to an 'embryo
ice crystal' (Rasmussen and MacKenzie, 1973;
Vali, 1995). These structures arise spontaneously
as a function of random density fluctuations and
their lifetime depends on their self-diffusion rate,
which is temperature dependent (Franks, 1985).
When the ice embryo reaches a critical size it
becomes a nucleus and this event is called nucle-
ation (Vali, 1995).
There are therefore four factors to be considered
when dealing with nucleation events, (i) The tem-
perature itself: the lower the temperature, the
smaller the number of water molecules required
to make an ice embryo, thus the probability of
nucleation increases (Vali, 1995). (ii) The larger
the volume of the sample the higher the probability
29
H.Raml0v
Figure 1. The freezing process. Changes in cell size and solute concentration during freezing: (a) a single cell in solution, (b)
extracellular nucleation occurs (*), (c) freeze concentration of the extracellular fluid with following cell shrinkage due to
osmotic outflow of water from the cell, (d) totally solidified system; cell highly shrunken with intra- and extracellular eutectic
precipitation.
of a sufficient number of water molecules forming
an ice embryo (Vali, 1995). Thus larger volumes
usually freeze closer to the melting point than
small volumes within any given time. In pure
water, the homogeneous nucleation point is about
-39°C, a temperature which can only be reached
in very small volumes (Angell, 1982). (iii) A
certain volume in the metastable supercooled state
will freeze at some time that depends on the
temperature (Vali, 1995). (iv) Presence of nucleat-
ing agents in the system causing heterogeneous
nucleation.
Once freezing is initiated, an amount of ice
forms from water molecules which concentrates
the remaining solution to the extent where its water
vapour pressure equals that of the vapour pressure
of water over the ice at that specific temperature.
This is called freezing concentration and occurs
because ice consists of pure water and only very
few substances can be incorporated into the ice.
A biological entity (Figure 1) contains a large
30
number of various substances, many of which may
give rise to heterogeneous nucleation. Ice formation
is usually initiated in the extracellular fluid (Figure
lb). When ice begins to form, the extracellular
fluid becomes increasingly concentrated (Figure
lc). The vapour pressure of the remaining fluid is
in equilibrium with the vapour pressure of the water
vapour over the ice at the specific temperature. The
increase in solute concentration of the extracellular
fluid results in an osmotic outflow of water from
the cells so that the osmolality of the intracellular
fluid and thus the water vapour pressure is in
equilibrium with the extracellular fluid which again
is in vapour pressure equilibrium with the ice
(Mazur, 1984). If the temperature continues to
decrease further, more ice is formed and eventually
the eutectic point of various substances is reached.
At this point, ice and the solutes precipitate simul-
taneously in a eutectic composition (Moore, 1981)
(Figure Id) and the whole system is solidified.

Problems related to freezing of the body fluids
Freezing in an animal is usually initiated extra-
cellularly either in the gut, due to the presence of
exogenous nucleating agents (Salt, 1953;
Zachariassen, 1985; Duman et al., 1995; Worland
et al., 1997), in the tissues as a consequence of
accidental ice-nucleating substances or specific
ice-nucleating proteins which may be membrane
bound (Baust and Zachariassen, 1983; Duman
et al, 1991a; Tsumuki and Konno, 1991) or in
solution in the blood or haemolymph (Duman
et al., 1995). Freezing may also be initiated via
inoculation, i.e. the nucleation of the body fluids
by contact with external ice (Salt, 1963; Shimada
and Riihimaa, 1988; Layne et al, 1990). Intracellu-
lar freezing, except in a few cases (Salt, 1959a,
1962; Wharton and Ferns, 1995), is lethal
(Mazur, 1984).
When extracellular fluid freezes there are several
possibilities of damage to the system. Freezing of
a part of the body fluid can be looked upon as
drying of the system (Lee, 1991; Ring and Danks,
1998). As crystallization proceeds, more water is
removed from its role as a solvent for the dissolved
solutes. The resulting increase in solute concentra-
tion may lead to changes in enzyme activity and
precipitation or denaturation of proteins (salting
out) (Hochacka and Somero, 1984). These effects
can to a certain extent be predicted by the
Hofmeister series of neutral salts. Changes in
pH are also likely to occur during freezing and
dehydration (Franks et al., 1990), which may
affect enzyme activity and possibly lead to protein
denaturation (Taylor, 1987), changes in membrane
potential and changes in membrane transport
(Franks, 1985).
Osmotic outflow of water from the cells will
cause the cells to shrink. This may decrease the
cell volume below the so-called minimum volume
(Lee, 1991), which is typically reached at a body
ice content of -65% of the total body water (Storey
and Storey, 1993). When the cell volume decreases
to this value the membrane begins to rest on the
intracellular structures, causing hydrostatic stress
on the membrane as the cell cannot shrink further.
The hydrostatic forces eventually rupture the
membrane.
The dehydration of the system by crystallization
Cold tolerance in ectotherms
also causes phase changes in the membranes due
to the removal of the forces keeping the membrane
in its bilayer conformation (see 'Phase changes in
membranes'). Finally, the growth of ice crystals in
the tissues may lead to rupture of these and
sharp ice crystals may penetrate cells (Grout and
Morris, 1987).
Depending on the cooling rate, freezing of a
biological system may affect the cells of the various
tissues differently, and one cooling rate may kill
some cells whereas others survive (Grout and
Morris, 1987). Indeed it seems as if there is an
optimum cooling rate for most cell types (Mazur
et al., 1972). Survival of different cells at various
cooling rates is described by a bell-shaped curve
(Mazur et al, 1972). The shape of these curves is
described as the two-factor hypothesis of freezing
injury (Mazur et al, 1972). The shape of the curves
is explained by the prolonged exposure to high
solute concentrations at low cooling rates (Mazur
et al, 1912) or to the decrease in size of the
unfrozen spaces in which the cells lie (Mazur,
1984) and to intracellular freezing at high cooling
rates. The optimal cooling rate for various cells
relies on a number of factors: the water permeabil-
ity of the cell membrane, the cell surface to volume
ratio and the hydraulic conductivity (Grout and
Morris, 1987). Hence cells with a high surface to
volume ratio and high membrane water permeabil-
ity may tolerate high cooling rates, as such cells
lose water very fast and therefore are not in danger
of intracellular freezing due to supercooling of the
intracellular fluid. The consequence of this is that
the survival of various tissues can vary consider-
ably within an organism or between cells in solution
which are not either adapted to or artificially
protected from freezing at a specific cooling rate.
Adaptations to temperatures below the
melting point of the body fluids in ectothermic
animals
Cold-adapted ectothermic animals employ one of
two 'strategies' when exposed to cold; freeze
avoidance and freeze tolerance (Table I).
Freeze-avoiding animals do not tolerate crystal-
lization of their body fluids and thus their super-
cooling point (SCP) is equal to their lower lethal
temperature (LLT). Often such animals have a
31
H.Raml0v
Table I. Adaptations to temperatures below the melting point of the body fluids in ectothermic animals
Freezing avoidance
Supercooling point = lower lethal temperature
Ice formation is lethal
High supercooling capacity
Polyols function as antifreeze (may also stabilize
membranes and proteins)
- Ice-nucleating agents
Antifreeze proteins often present
RI = recrystalisation inhibition.
large supercooling capacity (SCC), which means
that there is a large temperature difference between
the melting point of the body fluids and the
SCR Freeze-avoiding animals have to survive cold
periods in the metastable supercooled state. They
are therefore in constant danger of ice formation
in their tissues. However, there are a number of
examples of freeze-avoiding animals surviving
temperatures as low as approximately -25 °C for
prolonged periods or even as low as -50 to -60°C
(Miller, 1982; Ring, 1982) or periods of up to
possibly 30-40 years supercooled by about 1 °C in
the Antarctic ocean (e.g. Dissostichus mawsoni
which reaches weights of up to around 80 kg in
Antarctic waters).
To survive low temperatures in the supercooled
state, nucleating agents either have to be absent
from the tissues or masked during the cold period
(Lee, 1991). It has been observed that some freeze-
avoiding species cease to eat and empty their gut
during autumn before the temperatures fall below
the melting point of the body fluids, thereby
enhancing these organisms SCC (Cannon and
Block, 1988).
Freeze-tolerant animals, however, survive the
formation of ice within their tissues, consequently
the SCP of these animals is different from their
LLT. Due to the presence of ice-nucleating agents
(INA) (Zachariassen and Hammel, 1976) or inocu-
lation of the body fluid, the SCC of these animals
is small and the body fluids typically crystallize at
a relatively high temperature below the melting
point (Zachariassen, 1980), usually -5 and -10°C
(Zachariassen and Hammel, 1976; Zachariassen,
1980) [e.g. the New Zealand alpine weta Hemide-
ina maori (Raml0v etal., 1992)] and the crysomelid
32
Freeze tolerance
Supercooling point =£ lower lethal temperature
Survival of extracellular ice
Poor supercooling capacity
Polyols function as colligative and non-colligative
cryoprotectants
+ Ice-nucleating agents
Antifreeze proteins intracellular and inhibits RI
beetle Phyllodecta laticollis (Laak, 1982) (see also
Table II). Some freeze-tolerant animals, however,
have very low supercooling points due to the
removal of virtually all ice nucleators from the
system (Miller, 1982; Ring, 1982). A number of
ectothermic animals are freeze-tolerant even during
the summer period (Laak, 1982; Raml0v et al.,
1992), presumably because they are exposed to low
temperatures even during summer (Raml0v, 1999).
The reason why the two strategies mentioned
above have evolved in parallel is not known.
Freeze-avoiding animals are more likely to experi-
ence freezing events the further the temperature
decreases; it could therefore be speculated that
these animals are found in areas where temper-
atures do not fall to very low extremes, whereas
freeze-tolerant animals whose body fluids are in
thermal equilibrium with their surroundings may
be found in areas with extremely low winter
temperatures (Zachariassen, 1980; Ring, 1982).
However, recently a number of species inhabiting
areas in which the temperature only rarely falls
below -10°C have been shown to be freeze tolerant
(Sinclair, 1997; Raml0v, 1999). Lundheim and
Zachariassen (1993) proposed that freeze tolerance
is an adaptation to the desiccating conditions
encountered during winter. At low temperatures
the air is usually very dry and unless an animal
has an impermeable integument it risks losing
water to the frozen surroundings if its body fluids
are supercooled [due to the higher chemical poten-
tial (vapour pressure) of supercooled water than
that of ice]. Indeed Lundheim and Zachariassen
(1993) have shown that some freeze-avoiding
beetles have low cuticular water permeability.
Another explanation for this observation may be

that it reduces the risk of inoculation from external
ice crystals (Lundheim and Zachariassen, 1993).
An explanation for the observation that some
animals are freeze-tolerant even though ambient
temperatures do not fall to low extremes may
therefore be that these animals overwinter in hab-
itats which are very moist and thus the chance of
inoculation is high (Klok and Chown, 1997;
Raml0v, 1999). This seems to be the case both for
H.maori (Raml0v, 1999) and the New Zealand
alpine cockroach Celattoblatta quinquemaculata
(Sinclair, 1997).
Freeze tolerance may also have evolved as an
adaptation to areas where ambient temperatures
show considerable variation. In such areas, animals
may experience diurnal freeze/thaw cycles or per-
haps prolonged periods of temperatures above the
melting point of the body fluids. Such conditions
may call for the possibility of these animals to
forage and thus they are exposed to ingestion of
INA during periods when freezing temperatures
may occur. Again, this is the case for H.maori
which may encounter sub-freezing temperatures at
any time of the year and where the animals are
regularly found with the cuticle covered in ice
crystals during winter (Raml0v, 1999).
The control of ice formation
One of the most important features of adaptations
to cold in ectothermic animals is the control of ice
formation. This is achieved either by complete
avoidance of ice formation and inoculation or,
alternatively, control of the site of ice formation
(extracellular/intracellular), the crystallization tem-
perature (and hence the ice growth rate and osmotic
equilibration intra- and extracellularly), the amount
of ice formed (and hence the extent of dehydration
and freezing concentration of the body fluids) and
finally the control of recrystallization. Recrystal-
lization is the growth of large ice crystals at the
expense of smaller ones. Cold-adapted ectothermic
animals have evolved a number of physiological
and biochemical adaptations to achieve this control
(Figure 2).
Cryoprotective low mol. wt substances
Most cold-tolerant ectothermic animals synthesize
high or low mol. wt substances (Zachariassen,
1985) that protect the organisms either against ice
Cold tolerance in ectotherms
formation or damage due to ice occurring in
their tissues.
Protective low mol. wt substances in cold-
adapted animals can be divided into two classes
based upon their actions: (i) colligative cryoprotec-
tants* (which affect vapour pressure or freezing
point, depending upon the number of molecules
involved) and (ii) cryoprotectants which stabilize
membranes and proteins (Storey and Storey, 1992).
A number of requirements have to be fulfilled if
these substances are to control ice formation: (i)
they have to be highly soluble in aqueous solution;
(ii) they are relatively non-toxic and non-reactive
towards cells and macromolecules, even in high
concentrations; (iii) they have to be compatible
solutes (for definition see Hochachka and Somero,
1984) so that they do not perturb protein structure
and function; (iv) they have to counteract the
denaturing effects on proteins of cold, dehydration
and high ionic concentrations (Storey and Storey,
1992).
A number of species employ dual or multiple
cryoprotectant systems [multifactorial systems
(S0mme, 1982)]. This may have the advantage
that the concentration of none of the substances
reaches poisonous levels in the organism's body
fluids (Ring, 1980). It has also been proposed
that a combination of certain commonly found
cryoprotective substances increases the possibility
of vitrification of the body fluid (Wasylyk et al.,
1988). However, this theory is based upon data
with artificial 'haemolymph' where the solute
species and concentrations are chosen to emulate
those in the freeze-tolerant larva of the gall fly
Eurosta solidaginis. Vitrification has not been
observed under natural conditions in animals.
According to Storey and Storey (1992) the employ-
ment of multiple cryoprotectant systems may also
have metabolic advantages. For example, sorbitol
and glycerol pools have different fates in spring;
sorbitol being converted into glycogen, whereas
*Colligative comes from the word 'colligare' meaning
'to glue together'. In the chemical context this means
the properties of a solution that depend on the number
of particles (molecules) involved and not on the
quality of the particles. The properties 'glued together'
are, for example, vapour pressure, freezing (melting)
point depression and boiling point elevation.
33
H.Raml0v

Table II. Examples of cold-tolerant organisms from various taxons for which low (LMW) or high (HMW) mol. wt
cryoprotectants, supercooling point (SCP) or lower lethal temperature (LLT) have been published

Freeze- Freeze- LMW cryo- HMW cryo- SCP LLT References

avoiding tolerant protectant protectant (°C) (°C)

Nematoda
Wetanema sp. -4 -61 Tyrell et al. (1994)
Dochanoides -20 Balasingham (1964)
stenocephala
Tardigrada
Adorybiotus Trehalose INA -6.5 -196 Westh and Raml0v
(1991),
coronifer Raml0v and Westh
(1992),
Westh etal. (1991),
Wright et al. (1992)
Arthropoda
Arachnida
Acari
Nanorchestes + Glycerol -24 Block and S0mme
(1982)
antarcticus Mannitol
Alaskozetes + Glycerol -30.5 Young and Block (1980)
antarcticus
Aranea
Araneus + Glycerol -23.2 Kirchner and Kestler
(1969)
cornutus
Insecta
Collembola
Tetracanthella + Glycerol -31.6 S0mme and Conradi-
Larsen (1977)
wahlgreni
Cryptopygus + Glycerol -26.7 S0mme and Block
(1982)
antarcticus Mannitol
Trehalose
Orthoptera
Hemideina maori Prolin INA -3.9 -12 Raml0v et al. (1992),
(A) Trehalose? Raml0v (1999),
Wilson and Raml0v
(1995),
Neufeld and Leader
(1998)
Meridacris c.-9 S0mme (1986)
subaptera (A)
Lepidoptera
Isia Isabella (L) Glycerol -18.2 Mansingh and Smallman
Sorbitol (1972)
Pieris brassicae Sorbitol -26.4 Hansen and Merivee
(1971)
(P)
Coleoptera
Rhagium Glycerol AFP -27.0 Zachariassen (1973),
inquisitor Sorbitol Gehrken (1992)
(A and L)
Pytho Glycerol -54 -55 + Ring (1982)
deplanatus (A) Trehalose
Ips acuminatus Ethylene -24 Gehrken (1984)
(A) glycol
Carabus -5 -12 Merivee (1978)
granulatus (A) -6.5 -13
 Cold tolerance in ectotherms

Table II. Continued

Freeze- Freeze- LMW cryo- HMW cryo- SCP LLT References

avoiding tolerant protectant protectant (°C) (°C)

Diptera
Rhabdophaga + Glycerol -61.6 Ring (1981)
sp. (L)
Hymenoptera
Diastrophus Glycerol -32.0 Ring (1981)
kincaidii (L)
Ceratina sp. (A) Glucose -20 Tanno (1964)
Fructose
Trehalose
Vertebrata
Osteichthyes
Boreogadus + AFP Denstad et al. (1987)
saida
Gadus morh.ua + AFGP Goddard et al. (1992)
Trematomus + AFGP DeVries (1971)
bernacchi
Pagothenia + AFGP -2.1 DeVries (1971)
borchgrevinci
Caudata
Salamandrella Glucose -35 Berman et al. (1984),
keyserlingi Glycerol -15.3 Jensen (1999)
Salientia
Hyla versicolor + Glycerol -3 Schmid (1982),
Storey and Storey (1985)
Rana sylvatica Glucose -6 Schmid (1982), Storey
(1984),
Layne and Lee (1987)
Pseudacris crucifer + Glucose -2.5 Churchill and Storey
(1996)
Rana arvalis Glucose -A
Testudines
Chrysemys picta -2.5 Storey et al. (1988),
marginata Churchill and Storey
(1992)
Chrysemys picta -2.5 Churchill and Storey
belli (1992)

A = adult; L = larva; P = pupa; INA = ice-nucleating agents; AFP = antifreeze peptide; AFGP = antifreeze glycoprotein.

glycerol is not. Restoration of the glycogen reserve
during spring therefore depends on the relative
amounts of carbon channelled into each pool during
autumn (Storey and Storey, 1992).
The low mol. wt substances most commonly
found in cold-adapted ectothermic animals are
polyols and sugar alcohols. Glycerol is the most
common of these substances (Zachariassen, 1977;
Miller, 1982; Storey and Storey, 1992) but other
polyalcohols have also been found (Gehrken,
1984), as well as sugars such as trehalose and
glucose (Block, 1982; Storey and Storey, 1993)
and free amino acids such as proline (Storey et al.,
1981; Laak, 1982; Storey, 1983; Lefevere et al.,
1989; Raml0v, 1999) (see also Table II).
In cold-adapted ectotherms, low mol. wt sub-
stances can be found in very high concentrations;
for example, glycerol is found in concentrations
of > 2 mol/1 in the freeze-avoiding bark beetle
Rhagium inquisitor (Zachariassen, 1973). Low
mol. wt substances control ice formation via their
colligative properties. They decrease the melting
point by 1.86°C/molal (the molal freezing point
depression [see also Mazur, 1984)] and the SCP by
approximately double the melting point depression
(Salt, 1959b; MacKenzie, 1977; Gehrken, 1984).

35
H.Raml0v
CONTROL OF ICE FORMATION
Low molecular weight cryoprotective substances
• Polyols
(e.g. Glycerol, Ethylene glycol, Sorbitol)
• Sugars
(e.g. Glucose, Trehalose)
• Free amino acids
(e.g. Proline)
COLLIGATIVE CONTROL OF:
Melting point
Rate of ice formation
Ice fraction size
Supercooling point
Figure 2. Various adaptations to the control of ice in cold-tolerant ectothermic animals.
These two properties are important in freeze-
avoiding animals which have to maintain liquid
body fluids during cold periods.
In freeze-tolerant animals, low mol. wt sub-
stances decrease the amount of ice formed at any
given temperature (Zachariassen, 1979) and slow
the rate of ice formation, e.g. the ice content of
E.solidaginis reaches -65% in -40 h (Lee and
Lewis, 1985). This animal accumulates sorbitol
and glycerol during autumn amounting to 400 fimol
glycerol/g dry weight and -120 |umol sorbitol/g
dry weight as well as lower concentrations of
trehalose and glucose (Storey and Storey, 1992).
In contrast, H.maori does not accumulate high
concentrations of low mol. wt substances. The
osmolality of its haemolymph increases from about
350 mOsm in summer to -700 mOsm in winter,
mainly due to the accumulation of proline (Raml0v,
1999). In this animal, ice formation after nucleation
is rapid and the ice content reaches -80% of the
body water in -6 h (Raml0v and Westh, 1993).
The reduction of ice formed in the organism
36
Proteinaceous cryoprotective substances
• Ice nucleating proteins
Synthesized or ingested
Proteins/lipoproteins
77 kDa - 800 kDa
CONTROL OF:
Supercooling point
• Antifreeze proteins
Proteins/glycoproteins
3.2 k D a - 3 2 kDa
CONTROL OF:
Ice growth and
recrystallisation
limits the dehydration and is of significance in
maintaining a critical minimum cell volume. In
the case of H.maori it must be assumed that its
cells can survive extensive dehydration and thus
have a very small critical minimum volume
(Raml0v and Westh, 1993). Fat body cells and
Malpighian tubules from H.maori undergo extens-
ive dehydration during freezing, as observed by
cryo-microscopy. Cells frozen to -8°C showed
high survival upon thawing whereas cells cooled
to -15°C and below showed a decline in survival
correlated with temperature (Sinclair and
Wharton, 1997).
Proteinaceous substances as special adaptations
to the control of ice formation
Cold-adapted ectothermic organisms synthesize a
number of proteinaceous substances that in various
ways participate in the control of ice formation. The
substances in question are INA and the antifreeze/
thermal hysteresis proteins (for reviews see
DeVries, 1982, 1986; Zachariassen, 1985; Cheng

and DeVries, 1991; Duman et al, 1991a, 1993,
1995; Hew and Yang, 1992; Costanzo and Lee,
1995).
Ice-nucleating agents
Extracellular INAs are found in a large number of
freeze-tolerant animals (Aunaas, 1982; Duman,
1982; Zachariassen, 1982, 1985; Duman and
Horwath, 1983; Loomis, 1985; Duman et al.,
1991a; Westh et al., 1991), but not in all species
(Miller, 1982; Ring, 1982; Costanzo and Lee,
1996). INAs inhibit extensive supercooling by
initiating ice formation via heterogeneous nucle-
ation at relatively high temperatures below the
melting point of the extracellular fluid
(Zachariassen and Hammel, 1976; Zachariassen,
1982; Duman and Horwath, 1983), preventing
lethal osmotic shock (Lee, 1991; Zachariassen,
1992) and intracellular ice (Zachariassen and Ham-
mel, 1976; Duman, 1982; Zachariassen, 1982).
INAs are thought to provide a template around
which an embryonic ice crystal can form and grow
to become large enough to ensure freezing of a
supercooled liquid; that is, a supercritical radius at
a given supercooling (Burke and Lindow, 1990).
INAs are found in the haemolymph (Duman et al.,
1995), associated with different tissues (Baust and
Zachariassen, 1983; Duman et al., 1991a; Tsumuki
and Konno, 1991) or in the gut contents (Duman
et al., 1995; Worland et al., 1997). There is much
evidence that INAs in the haemolymph of insects
and other animals are either proteins or lipoproteins
(Duman and Horwath, 1983; Hayes and Loomis,
1985; Neven et al., 1989; Duman et al, 1991b;
Wilson and Raml0v, 1995) but can also be found
in some tissues as crystals (Mugnano et al., 1996).
Ice-nucleating proteins are large, ranging in size
from 74 to 800 kDa, the latter having a diameter
of 135A (Duman et al, 1984; Neven et al, 1989)
presumably with rough surfaces and with many
hydrophilic residues extending into the solvent
(Burke and Lindow, 1990; Wilson and Raml0v,
1995). The size of the proteins determines at which
temperature they initiate ice formation. Biological
ice nucleation theory predicts that, for example,
an ice nucleator with a diameter of 300A will
initiate ice formation at a temperature of -3 to
-8°C, depending on the geometry and contact
Cold tolerance in ectotherms
angle 0 between the ice nucleator and the ice
embryo (Govindajaran and Lindow, 1988; Burke
and Lindow, 1990; Wilson, 1994; Vali, 1995).
The lipoprotein ice nucleator (LPIN) is well-
characterized, consisting of two apolipoproteins of
265 and 81 kDa respectively. In LPIN the lipid
component is phosphatidylinositol (PI). It has been
suggested by Warner (1962) that inositol can order
water molecules into an ice-like structure (Duman
et al, 1995). It was shown by Neven et al. (1989)
that the PI component of LPIN is essential for ice-
nucleation activity. Therefore it was speculated
that the PI component of LPIN forms the template
around which the water molecules orientate them-
selves into the embryonic ice crystal (Duman
et al, 1991b).
It has also been speculated that some INAs
aggregate and thereby form a more efficient ice
nucleator because more water molecules can be
arranged into ice-like clusters (Mueller et al,
1990). Duman et al (1992) showed that an increas-
ing concentration of LPIN increased in ice-nucleat-
ing activity, producing a maximal SCP of -6°C at
concentrations >1.7X10~7 mol/1. This indicates
that aggregation and cooperation of several LPIN
molecules is required to induce maximal nucle-
ation activity.
In a number of cold-tolerant animals there is a
difference between the SCP of the haemolymph
and the whole body SCP, e.g. in H.maori the SCP
of the haemolymph is about -7.5°C (Wilson and
Raml0v, 1995) whereas the whole body SCP is
about -4°C (Raml0v et al, 1992). This difference
can have several reasons: (i) possibly there are
INAs in the gut contents which induce the crystal-
lization at about -4°C; (ii) the INAs which induce
freezing at -4°C are associated with various tissues;
or (iii) the INAs in haemolymph are more active
in vivo than in vitro. Why then does H.maori have
INAs in the haemolymph? One reason could be
that the ice nucleators in the haemolymph are just
incidental ice nucleators, that is, some substance
(in this case a protein) which has ice-nucleating
activity but which serves a different function in
the animal. Another reason could be that H.maori
usually relies on ice nucleation from the gut
content, but in some cases the animals may be
starved and the ice nucleators in the haemolymph
37
H.Raml0v
then serve as a 'back-up' system. There is probably
a high selection pressure for ice nucleation as no
ice nucleation in a freeze-tolerant animal may
be lethal.
Antifreeze proteins
Another group of proteinaceous substances control-
ling ice formation are the antifreeze or thermal
hysteresis proteins. These proteins are found in
polar fishes that inhabit waters at or close to the
melting point of seawater (-1.9°C) year round or
seasonally (DeVries, 1982, 1986; Denstad et al,
1987; DeVries and Cheng, 1992; Goddard et al,
1992) and in invertebrates inhabiting areas where
they at some time during the year are exposed to
cold (Duman et al, 1991a, 1992, 1993). In contrast
to agents which initiate ice formation the antifreeze
proteins inhibit the growth of ice by interacting
with specific crystal planes on the ice crystal
(DeVries and Cheng, 1992) or possibly stabilize
the metastable supercooled state by recognizing
embryo ice crystals before they grow large enough
to initiate ice growth in the solution (Zachariassen
and Husby, 1982). Further, antifreeze proteins
have been shown to inhibit recrystallization of ice
(Ramsay, 1964; Knight and Duman, 1986; Knight
et al., 1995). There may also be some proteins that
do not show any 'antifreeze' activity but which
nevertheless inhibit recrystallization (Raml0v
et al, 1996).
Substances with 'antifreeze' effects were
described for the first time by Ramsay (1964). In
his comprehensive study of the cryptonephridial
rectal complex of the mealworm Tenebrio molitor,
Ramsay employed a melting point apparatus in the
study of osmolalities in fluid from the rectal
complex. During his study he observed that ice
crystals decreased in size when the temperature
was raised, but that they did not increase in size
when the temperature was lowered until a certain
temperature was reached at which the ice crystals
suddenly grew instantaneously and the whole
sample solidified (Ramsay, 1964). On some occa-
sions Ramsay observed that the ice crystals in the
fluid did not increase in size before the temperature
was lowered as much as 10°C. Antifreeze proteins
produce, by a non-colligative mechanism (Westh
et al., 1997), a separation of the melting point and
38
the temperature at which an ice crystal will grow.
This is called the thermal hysteresis activity and
the temperature at which the ice crystal grows is
termed the hysteresis freezing point (DeVries,
1986). Although antifreeze activity was first
observed in an insect larva, antifreeze proteins
have been studied the most in teleost fishes (Feeney,
1974; DeVries and Cheng, 1992; Deng and
Laursen, 1998). Scholander and colleagues (1957)
were puzzled by the fact that teleost fishes in the
arctic, whose blood had a melting point of -0.5
to -0.8°C, live in close contact with ice in waters
at temperatures around -1.7 to -1.8°C, thus being
supercooled by almost 1°C but with no inoculation
of the body fluids occurring. Scholander et al.
(1957) did not identify the substances responsible
for the inhibition of inoculation and/or formation of
ice in the fishes. However, DeVries and Wohlschlag
(1969), who worked in the Antarctic, discovered
that a glycoprotein could explain 30% of the
freezing point depression in the serum obtained
from fishes in McMurdo Sound. Since then, a
number of different types of antifreeze proteins
have been discovered in polar fishes, all synthesized
in the liver and secreted into the blood (Cheng and
DeVries, 1991). Today five types are known, (i)
The glycoproteins found in Antarctic notothenioid
fishes (DeVries and Wohlschlag, 1969; DeVries
et al, 1970, 1971; DeVries, 1982, 1986; DeVries
and Cheng, 1992). These glycoproteins are found
in eight distinct sizes named antifreeze glyco-
protein (AFGP) 1-8 ranging from 2.6 to 34 kDa
(DeVries et al, 1971; Duman and DeVries, 1972;
Duman et al, 1993). They are composed of a
glycotripeptide unit alanyl-alanyl-threonine with a
disaccharide, Af-acetylgalactosamine and galac-
tose [P-D-galactopyranosyl( 1 —>3)2-acetamido-2-
deoxy-2-oc-D-galactopyranose] linked to the threon-
ines (DeVries, 1971; DeVries et al, 1971; Duman
et al, 1993). In the small AFGP 7 and 8, the
threonines are sometimes substituted with proline
(Lin et al, 1972; Kieran et al, 1980); these also
differ from the larger ones by having a somewhat
lower antifreeze activity. AFGP identical to those
in the notothenioid fishes are also found in the
unrelated species of gadoid fishes from the arctic,
e.g. in Boreogadus saida and Gadus ogac (Van
Voorhies et al, 1978), recognized as one of the

most important examples of convergent evolution
in biochemistry (Chen et al, 1997). (ii) The Type-
I antifreeze proteins are found in winter flounder,
Pseudopleuronectes americanus and other North-
ern flatfishes and sculpins (Duman and DeVries,
1976; Hew et al, 1985). These are alanine-rich,
amphipathic cc-helix peptides with mol. wt of 3300-
5000 Da and often contain 11 amino acid repeating
units (Raymond et al, 1975; Davies and Hew,
1990; Duman et al, 1993). (iii) The Type-II
antifreeze proteins are found in the sculpin sea
raven, Hemitripterus americanus. These do not
have an obvious repeat structure but are cysteine
rich globular proteins that have a significant amount
of (^-structures and mol. wt of -14 kDa (Slaugther
et al, 1981; Hayes et al, 1989). The Type-Ill
antifreeze proteins are found in eelpouts and zoar-
cid fishes. These peptides have no amino acid bias
but lack histidine and tryptophan. They also have
no obvious repeating sequences and lack helical
structures but contain compact (3-sheet structures
(Davies and Hew, 1990). They have a mol. wt of
-7000 Da (Li et al, 1985; Cheng and DeVries,
1989). In 1998, Deng and Laursen described a
new antifreeze protein (LS-12) from the long-horn
sculpin, Myoxocephalus octodecimspinosis (Deng
and Laursen, 1998). This antifreeze consists of
four amphiphatic a-helices of similar length, folded
into a four-helix bundle. This antifreeze may be
considered the first example of a new type (Type
IV) fish antifreeze protein.
Despite the various classes of antifreeze proteins
having few structures in common they all exhibit
inhibition of the growth of ice crystals by adsorp-
tion to or at least perturbation of different crystal
faces in the ice crystal (Raymond and DeVries,
1977). It is interesting that, unlike most other
molecules, AFGP and antifreeze proteins are
incorporated into the ice if a solution of these
molecules freezes (Cheng and DeVries, 1991).
Raymond and DeVries (1977) proposed that the
AFGP hydrogen binds to the ice crystals via the
hydrogen-bonding groups in the protein and the
water molecules in the ice lattice. This adsorption
inhibits the ice growth. Raymond and DeVries
(1977) noted that the growth habit of ice crystals
changed in the presence of antifreeze proteins.
When the antifreeze solutions reached the hyster-
Cold tolerance in ectotherms
esis freezing point the ice crystals grew as long,
thin, parallel needles (spicules) whose axes were
aligned with the ice c-axis. This is the thermodyn-
amically non-preferred direction of growth. Usu-
ally ice grows in the direction perpendicular to the
c-axis, i.e. parallel to the a-axes (on the prism
plane) (the thermodynamically preferred direction
of growth) (Cheng and DeVries, 1991). The
observed growth pattern indicated that the anti-
freeze proteins preferentially adsorbed to the crystal
faces parallel to the c-axis (the prism planes) and
inhibited growth in the direction perpendicular to
the c-axis (Raymond and DeVries, 1977; Raymond
et al, 1989). From these observations it can be
deduced that the spicular growth occurs when it
becomes thermodynamically preferable for the ice
crystals to grow along the c-axis, i.e. on the basal
plane. Later investigations have indicated that other
types of antifreeze proteins adsorb to or perturb
different crystal planes in the ice crystals, e.g.
primary prism planes and secondary prism planes
(Knight et al, 1991; Ewart et al, 1999).
Antifreeze proteins are not restricted to marine
teleost fishes, there are more than 30 known
species of terrestrial or tidal invertebrates in which
antifreeze activity has been shown in the body
fluid (Theede et al, 1976; Duman, 1979; Husby
and Zachariassen, 1980; Block and Duman, 1989;
Duman et al, 1991a). The functions of antifreeze
proteins in terrestrial invertebrates may be the
same as in fish, namely inhibition of ice growth,
which would be relevant in, for example, tidal
invertebrates which are moist and thus likely to be
inoculated from the surroundings.
Terrestrial arthropods are often exposed to
extremely low temperatures during winter e.g. -50°C
(Zachariassen, 1985). The occurrence of antifreeze
proteins in freeze-avoiding insects may therefore
be understood in one of two contexts. Usually
inoculation of insects is not considered of great
importance as these have a wax-coated hydro-
phobic cuticle that is likely to prevent inoculation.
However, S0mme (1982) showed that inoculation
occurs more often than usually thought. Therefore
antifreeze proteins may fortify the insects' defences
against inoculation (Gehrken, 1992). Another pos-
sibility is that antifreeze agents stabilize the meta-
stable supercooled state in freeze-avoiding insects.
39
H.Raml0v
Zachariassen and Husby (1982) have shown that
there is an inverse log-linear relationship between
the hysteresis activity and the size of the ice crystal
in the solution, that is, the smaller the size of the
ice crystal the larger the thermal hysteresis. If
these findings (Zachariassen and Husby, 1982) are
extrapolated below the lower limit of observation
of the ice crystal size used in the experiment, it
would appear that the antifreeze proteins may be
able to inhibit the growth of clusters of water
giving rise to potential embryo ice crystals. These
findings are still under investigation (Kristiansen
et al, 1999).
So far the antifreeze agents have primarily been
found in the extracellular fluid in freeze-avoiding
insects, but antifreeze agents from the intestinal
fluid (Olsen and Duman, 1997) and intracellularly
(Kristiansen et al., 1999) have been described.
Antifreeze proteins are usually associated with
freeze-avoiding animals, but surprisingly antifreeze
proteins have also been found in freeze-tolerant
insects (Duman et al., 1991a). In these animals the
antifreeze effect is of no obvious function as these
animals often promote freezing by the production
of INAs (see above); however, the effect of anti-
freeze proteins on inhibiting recrystallization of
ice already formed (Knight et al., 1995) may be
relevant. Recrystallization is the growth of large
ice crystals at the expense of smaller ones. For
a theoretical background on the grain boundary
migration involved in recrystallization see Knight
et al. (1995). This change in size distribution of
ice crystals over time may cause damage to the
tissues either during thawing or during changes
in ambient temperature. It has therefore been
suggested that the role of antifreeze proteins in
freeze-tolerant animals is recrystallization inhibi-
tion to inhibit lethal recrystallization (Knight and
Duman, 1986). Recently recrystallization inhibition
was described in relatively dilute homogenates of
the freeze-tolerant Antarctic nematode Panagro-
laimus davidi (Raml0v et al., 1996). As this
animal is capable of surviving intracellular freezing
(Wharton and Ferns, 1995) it may be of great
importance that changes in crystal size, with the
ensuing disruption of cell membranes, are inhibited
(Raml0v et al., 1996).
40
Specific protection by low molecular weight
substances during cooling and freezing
Most cold-tolerant animals synthesize cryoprotec-
tants either as a response to freezing or because
of environmental cues during autumn. Apart from
controlling ice formation and supercooling points,
these substances may also interact directly with
various structures in the organisms.
Glycerol is the most commonly found low mol.
wt protective substance in cold-tolerant animals
(S0mme, 1982; Zachariassen, 1985; Storey and
Storey, 1992); however, both sugars and free amino
acids are common in cold-tolerant animals, especi-
ally trehalose and proline (Zachariassen, 1985;
Storey, 1997; Raml0v, 1999). These substances are
efficient stabilizers of membrane integrity and
protein structure during cooling and dehydration
due to ice formation (Gekko and Timasheff,
1981a,b; Rudolph and Crowe, 1985; Carpenter
et al., 1986; Rudolph et al., 1986, Strauss et al,
1986). The mechanism of stabilization of mem-
branes by trehalose and other sugars is by pre-
venting the formation of the gel phase in
membranes. This effect is caused by hydrogen
bonding of trehalose to the head group of the
phospholipids thereby spreading the monolayers
(Rudolph et al., 1986; Strauss et al., 1986). Proline
seems to intercalate between the phospholipid head
groups (Rudolph et al., 1986) but its cryoprotective
effect is less well understood.
Summary
In the present article some of the important adapta-
tions to low temperature have been reviewed,
including changes in membrane and protein struc-
ture during cooling and freezing, the two 'strat-
egies' employed by cold-tolerant ectothermic
animals — freeze avoidance and freeze toler-
ance — the control of ice formation via the
colligative effects of low mol. wt cryoprotectants
and non-colligative effects by INA and antifreeze
proteins as well as the non-colligative actions of
low mol. wt cryoprotectants in the stabilization of
membranes and proteins.
Although we know much about what we think
are the adaptations to low temperatures in ecto-
thermic animals, an animal not already
cold-adapted cannot be made cold tolerant. Cryo-

protectants may be injected to induce ice formation
via INA, but not cold tolerance (apart from embryos
and dispersed cells in a number of animals). This
indicates that other adaptations are necessary in
conjunction with those already investigated. It has
been shown that membranes change their lipid
composition during cold exposure (Cossins and
Lee, 1985). It could be speculated that a special
membrane lipid composition is necessary for the
other adaptations e.g. low mol. wt cryoprotectants,
to work optimally. Further, it has recently been
shown that antifreeze proteins also bind to biolo-
gical membranes and change their properties
(Ewart et al., 1999).
There are many subjects within the field of
adaptations to cold in ectothermic animals where
further investigations are necessary. It is unclear
why a single animal species may contain several
different sizes of antifreeze proteins. It could be
speculated that these may work in synergy or that
some antifreeze proteins 'prepare' the surfaces of
the ice crystals for the action of others. The
mechanism of action of antifreeze proteins is still
a matter of debate, whether by direct binding
to, or perturbation of, the ice surface. Also, the
stabilization of the supercooled state by antifreeze
proteins is still not elucidated. Few of the structures
of the antifreeze proteins and ice nucleators found
in terrestrial invertebrates have been investigated
and elucidated (Liou et al, 1999).
Both cold-tolerant ectothermic invertebrates and
vertebrates synthesize low mol. wt cryoprotectants,
but there are large differences both in the types of
cryoprotectants synthesized and the environmental
cues which induce them. There are also large
differences between the two groups concerning the
supercooling, the amount of ice, the ice formation
rate and cooling rates tolerated.
Today the mechanisms controlling the synthesis
of the various cryoprotective substances, photo-
period, temperature, circadian rhythms and hor-
monal control have only been investigated in few
cases leaving much to be learnt.
Acknowledgements
Charlotte Rasmussen is thanked for help in preparing
the manuscript.
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