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First published online February 27, 2009
Journal of Experimental Biology 212, 802-807 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.025973
Freeze tolerance, supercooling points and ice formation: comparative studies on the subzero temperature survival of limno-terrestrial tardigrades
1 Universität Stuttgart, Biological Institute, Department of Zoology,
Pfaffenwaldring 57, 70569 Stuttgart, Germany
2 British Antarctic Survey, Natural Environment Research Council, High Cross,
Madingley Road, Cambridge CB3 0ET, UK
* Author for correspondence (e-mail: ralph.schill{at}bio.uni-stuttgart.de)
Accepted 6 January 2009
 |
Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: tardigrada, differential scanning calorimetry, DSC, supercooling point, SCP, cooling rate, cold tolerance
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). Many
limno-terrestrial tardigrade species are able to tolerate harsh environmental
conditions in any developmental stage
(Schill and Fritz, 2008) by
entering a cryptobiotic state which is induced by desiccation (anhydrobiosis),
lack of oxygen (anoxybiosis), osmotic pressure (osmobiosis) or low
temperatures (cryobiosis) (Keilin,
1959). In this state metabolism is undetectable and life comes to
a reversible standstill until activity is resumed under favourable conditions
(Keilin, 1959). In the
anhydrobiotic state, tardigrades and other cryptobiotes such as nematodes and
rotifers show extraordinary tolerance of physical extremes including high and
subzero temperatures, ionizing radiation, alcohols and high pressure
(Horikawa et al., 2006;
Jönsson et al., 2008;
Jönsson and Schill, 2007;
Ramløv and Westh, 2001;
Rebecchi et al., 2007;
Seki and Toyoshima, 1998).
To survive exposure to temperatures below the freezing point of their body
fluids, cold tolerant organisms mainly use either the freeze avoidance or
freeze tolerance strategy (Storey and
Storey, 1996). While freeze avoiding organisms depress the
temperature of spontaneous freezing (supercooling point, SCP) by using
antifreeze proteins and other cryoprotectants (e.g. sugars and polyols)
(Danks et al., 1994;
Duman, 2001), ice formation of
the extracellular body water is tolerated by freeze tolerant organisms and is
often triggered at high temperatures by ice nucleating proteins
(Lee and Costanzo, 1998;
Ramløv, 2000). In many
freeze tolerant organisms, polyols and sugars are accumulated to protect
membranes and proteins against phase transition and to control the ice
fraction size and minimum cell volume resulting from freeze concentration and
osmotic dehydration (Ramløv,
2000; Sinclair et al.,
2003; Zachariassen,
1985). Ice active proteins are also sometimes present and may act
as recrystallization inhibitors, which prevent the growth and redistribution
of ice crystals once these have formed
(Duman, 2001;
Wharton, 2003).
A third survival strategy termed cryoprotective dehydration has been
observed in recent studies on the arctic springtail Onychiurus
arcticus (Worland and Block,
2003; Worland et al.,
1998) and the Antarctic midge Belgica antarctica
(Elnitsky et al., 2008). In
this case, desiccation occurs due to the difference in water vapour pressure
between the animal's supercooled body fluids and ice in its surroundings
(Elnitsky et al., 2008;
Holmstrup et al., 2002;
Worland and Block, 2003;
Worland et al., 1998).
Some organisms including the Antarctic nematode Panagrolaimus
davidi even survive intracellular freezing
(Smith et al., 2008;
Wharton and Ferns, 1995;
Wharton et al., 2003). Freeze
tolerance in the tardigrade Richtersius (Adorybiotus)
coronifer, originating from polar regions, has been reported
previously by Westh and colleagues (Westh
and Kristensen, 1992), with survival of freezing of more than 80%
of the body water during exposure to subzero temperatures. However, whether
tardigrades also tolerate intracellular freezing or keep the cytoplasm in a
liquid state either by synthesizing cryoprotectants or by cryoprotective
dehydration is unknown.
Tardigrades are well known to survive freezing in the dehydrated state
(Wright, 2001) but are also
reported to survive exposure to –196°C fully hydrated
(Ramløv and Westh,
1992; Sømme and Meier,
1995). However, there have been very few experimental studies
concerning the ability of tardigrades to survive natural freezing conditions
in a hydrated state (Ramløv and
Westh, 1992; Sømme and
Meier, 1995; Westh and Hvidt,
1990; Westh et al.,
1991). In fact, few detailed studies have been undertaken on cold
tolerance as a general feature in any limno-terrestrial organism.
This study presents new data on the effect of various cooling rates on the survival of nine different tardigrade species originating from polar, temperate and tropical areas. SCPs of individual tardigrades were obtained for the first time using differential scanning calorimetry (DSC) and the effects of gut content and acclimation to low temperatures on the SCP were investigated. While SCP measurements in previous studies were obtained using groups of tardigrades, which might be influenced by the nucleation activity of the surrounding water resulting in one single freezing event for the whole group, this study provides the first data on SCPs of individual tardigrades.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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).
Laboratory cultures of the eutardigrade species M. tardigradum, M.
tonollii, M. sapiens, P. richtersi, P. richtersi `group 1', P.
richtersi `group 2' and P. richtersi `group 3' were scaled up
for growth on agar plates (3%; peqGOLD Universal Agarose, peqLAB, Erlangen,
Germany) covered by a thin layer of Volvic® water (Danone Waters,
Wiesbaden, Germany) according to Schill et al.
(Schill et al., 2004) and
Hengherr et al. (Hengherr et al.,
2008). The bdelloid rotifer Philodina citrina, raised on
the green algae Chlorogonium elongatum, was provided as food. Freshly
hatched tardigrades were also fed with C. elongatum. In addition to
rotifers and green algae, adult tardigrades were also fed with the nematode
Panagrellus sp. As we have yet to establish a laboratory culture of the herbivorous heterotardigrade species Echiniscus granulatus Doyère 1840 and Echiniscus testudo Doyère 1840 (both from temperate habitats), we separated individuals directly from moss samples originating from Tübingen (E. granulatus) and Munich (E. testudo), Germany, using a pipette and a dissecting microsope. Both localities were considered to be temperate.
Subzero temperature treatment
Groups of 40 individuals of each species were transferred in a water
droplet into microtubes. Subsequently, the remaining water was reduced using a
micropipette, leaving a residual water volume of 1–2 µl. Constant
cooling and thawing rates were achieved using a Reichert AFS automatic freeze
substitution system (Leica, Munich, Germany). Laboratory studies have used a
variety of cooling rates, usually distributed around 1°C
min–1 to investigate cold hardiness in insects, though these
cooling rates have been criticized as too fast compared with natural cooling
rates (Sinclair et al., 2003).
Natural cooling rates around 3 and 6°C h–1 have been
recorded in ecological studies on e.g. Drosophila melanogaster
(Kelty and Lee, 1999). To
study the cold tolerance of tardigrades under ecologically relevant cooling
rates, we used cooling rates of 9, 7, 5, 3 and 1°C h–1.
The tardigrades were exposed to the cooling rates starting at room temperature
(RT, 20°C) down to –30°C, followed by a warming period at
10°C h–1 up to RT. Alive and dead animals were recorded
after thawing. Tardigrades were assumed to be dead if there was no movement
visible within 2 h of rehydration. Four replicates per species were used for
each cooling treatment (see Table
1).
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Calorimetry
A Mettler-Toledo differential scanning calorimeter (DSC820, Mettler-Toledo,
Leicester, UK) was used to measure the SCP (=temperature of crystallization,
Tc) and quantity of water freezing in tardigrades as they
were cooled at 10°C min–1 from 25°C to 5°C and at
1°Cmin–1 from 5°C to –30°C. To obtain
crystallization points for each individual the animals were placed in separate
single droplets of water in aluminium pans. Residual water around each
individual was removed with filter paper directly before the pans were
hermetically sealed and placed in the calorimeter. After the determinations,
the animals were checked visually for any indication of dehydration resulting
from a poorly sealed pan, which would have a large effect on the SCP; data for
such samples were not included in this study. The calorimeter was calibrated
using indium as an upper temperature and enthalpy standard (melting point
156.6°C, enthalpy 28.71 J g–1) and the melting point of
ice as a lower temperature check. A standard temperature program starting at
25°C and cooling rapidly (10°C min–1) to 5°C then
to –30°C at 1°C min–1 and returning to 5°C
at the same rate was used for all measurements. The quantity of water freezing
in the animals (osmotically active water) was calculated from the freeze
exotherm using an enthalpy value of 334.5 J g–1.
The total body water of the animals was determined by deducting the dry mass (after drying for 24 h at RT over silica gel) from the fresh mass. The measurements were made on groups (N=4) with 3–8 animals.
To obtain the SCP of tardigrades with empty guts, animals of all species were starved over 2 days before starting the calorimetric analysis, whereas individuals of P. richtersi `group 3' (N=15) and P. richtersi (N=15) were additionally fed before measurement to study the effect of food content in the gut on the SCP. To test the influence of cold acclimatization on the SCP, individuals of all species were cold adapted at 15°C for 24 h, followed by 48 h at 8°C and a further 48 h at 4°C before starting the DSC measurements. The resulting SCPs were compared with the SCPs of starved animals. SCPs were obtained for 15–31 animals per species in each treatment. The exact number of animals used (N) for each SCP determination is indicated in Table 2.
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Statistics
The statistical significance of differences in the SCPs and frozen body
water was tested using a Kruskal–Wallace one way ANOVA followed by an
all pairwise multiple comparison procedure (Dunn's method; SigmaStat 3.5,
Systat Software GmbH, Erkrath, Germany). The survival rate after different
cooling rates was tested on significant differences using a one way repeated
measures ANOVA and a Tukey test as an all pairwise multiple comparison
procedure. Significance levels were P>0.05 (not significant) and
P
0.05 (significant).
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Interspecific comparison of the survival rate after cooling at different rates revealed high variations. M. tardigradum showed the highest survival rate of 95.0±4.1% at 9°C h–1 (mean±s.d.) and the lowest survival rate of 71.3±20.2% at 3°C h–1. However, no significant differences in survival between the different cooling rates were observed. Although P. richtersi and P. richtersi `group 1' also showed high survival rates after cooling at 9°C h–1 (92.5±9.6% and 90.0±10.8%), their recovery ability dropped significantly to 55.0±14.7% and 32.5±11.9%, respectively, after cooling at 5°C h–1. While the survival ability of P. richtersi `group 1' decreased further to 16.3±14.9% (3°C h–1) and only increased significantly to 27.5±5.0% after cooling at 1°C h–1, the value of P. richtersi increased steadily with decreasing cooling rate to a survival of 72.5±6.5% after the 1°C h–1 treatment. Most of the other species showed a lower but generally similar pattern of survival. A total loss of the ability to survive after cooling rates of 5 and 3°C h–1 was observed in M. sapiens and after 7°C h–1 in M. tonollii.
Calorimetry
In the second experiment, SCPs (means±s.d.) of the individual
specimens, measured by DSC, ranged between –23.7±3.9°C for
M. sapiens and –11.5±2.2°C for E.
granulatus in starved animals. Fig.
1 illustrates a representative thermogram with each peak
indicating a freezing event of an individual specimen. Ice formation appears
to be a rapid process with the freezing exotherm lasting less than 30 s in all
species used in this study. Differences in the SCP of starved and fed animals
were only observed between the two heterotardigrades and the eutardigrades
tested in this study (Table 2).
The SCP of E. granulatus was significantly different from the SCP of
all other tested species except E. testudo. For the SCP of E.
testudo significant differences from M. sapiens, M. tonollii and
M. tardigradum were observed. Neither acclimation to low temperatures
nor a well fed condition in P. richtersi (temperate) and P.
richtersi `group 3' (polar) affected the SCP significantly
(Table 2).
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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) that the cooling rate has a large influence on the survival
of tardigrades. In contrast, however, most of our tested species not only
showed an increased level of survival at the slowest cooling rate but also
often showed an even better survival at the fastest cooling rate (9°C
h–1). Although we cannot completely explain the reasons for
this effect, it might be due to the physical effects of rapid cooling and ice
crystal formation.
Several studies have reported that the capacity to supercool decreases with
increasing body mass (Johnston and Lee,
1990; Lee and Costanzo,
1998). Although our data partly support these results, with the
smallest eutardigrade in our study (M. sapiens) having the lowest
(though not significantly so) SCP of all tested eutardigrades, it may be
challenged by the presence of the much higher SCP of the equally small
heterotardigrades E. granulatus and E. testudo.
It has been documented that some arthropod species shed their mid-gut
during moulting, involving the complete evacuation of the gut contents, which
would otherwise initiate ice nucleation at a relatively high temperature. It
is known that this process decreases the SCP in some arthropods
(Lee and Costanzo, 1998;
Worland and Convey, 2008;
Worland et al., 2006). As it
was not possible to culture the two heterotardigrades E. granulatus
and E. testudo and therefore starvation could not be performed, it is
quite possible that some gut contents initiated ice nucleation, which may
explain the higher SCP. Interestingly, our data for the eutardigrades P.
richtersi and P. richtersi `group 3', where no difference in SCP
distribution of starved and well fed animals was observed, challenges an
involvement of the gut contents as ice nucleators in tardigrades. This is in
accordance with other studies made in insects where no effect of the gut
content on the SCP distribution has been observed, thus making the role of
feeding and nutritional condition as a SCP influencing factor contentious
(Klok and Chown, 1998;
Worland and Block, 1999).
However, an effect due to moulting, as observed in Collembola
(Worland et al., 2006), cannot
be excluded. Considering the fact that limno-terrestrial tardigrades live in
habitats where frequent freeze and thaw cycles may occur, it might be
energetically essential to retain gut contents during freezing periods and
freeze tolerance may be crucial due to the possible hazards of inoculation
from nucleators. However, it has been shown that tardigrades are capable of
surviving fairly long periods without food
(Ramazzotti and Maucci,
1983).
As no changes in either the melting point (data not shown) or the SCP
distribution were observed in either the tardigrades kept at room temperature
or those acclimatized to low temperatures, it is unlikely that low molecular
weight cryoprotectants such as polyols are synthesized as observed in freeze
tolerant insects (Worland,
2005; Zachariassen,
1985). The non-reducing disaccharide trehalose has been found in
tardigrades of the genus Richtersius, Macrobiotus, Paramacrobiotus
and Echiniscus (Hengherr et al.,
2008; Westh and Ramløv,
1991) but, interestingly, carbohydrate analysis did not detect
trehalose in active or anhydrobiotic M. tardigradum
(Hengherr et al., 2008) which
tend to show the highest survival. However, low concentrations of other small
carbohydrates may still be involved in freeze tolerance.
Cold acclimation at 4°C resulted in a decreased amount of water in all
tested tardigrade species of the present study as has been shown in winter
acclimatized animals of Richtersius coronifer and Amphibolus
nebulosus (Westh and Kristensen,
1992). This may reflect an increase in the amount of `bound' water
due to interactions between water and macromolecules, as investigated in
insect larvae (Storey et al.,
1981).
DSC studies on R. coronifer and A. nebulosus showed a SCP
between –6.7 and –7.4°C during slow cooling
(Westh and Kristensen, 1992;
Westh et al., 1991). In
subsequent experiments, Westh and colleagues
(Westh et al., 1991) provided
conclusive evidence for the existence of ice nucleating agents (INA) in R.
coronifer. INAs consist of proteins or lipoproteins which initiate
heterogeneous ice nucleation and freezing at temperatures typically between
–5 and –10°C (Duman,
2001; Wright,
2001). A decrease of the SCP to –16°C after previous
heating to 90°C and DSC analysis of gel filtered body fluids indicate that
the ice nucleating activity is composed of proteins
(Westh et al., 1991). In
contrast, our DSC measurements show a much lower SCP in all species. Plotting
the SCPs as a function of water volume together with available data on the
nucleation temperature of pure water samples and freeze avoiding insects
(Mackenzie et al., 1977;
Wilson et al., 2003;
Zachariassen et al., 2004)
(Fig. 2) shows that the SCPs of
Macrobiotus, Paramacrobiotus and M. tardigradum fit the
regression lines of the earlier studies, indicating a homogeneous nucleation
and that INAs are not present in these eutardigrades. However, an involvement
of INAs cannot be excluded in the case of E. granulatus and E.
testudo. A further possible reason for higher SCPs might be the higher
potential for inoculative freezing when using pooled animals with probably
more surrounding water, as in the earlier studies
(Westh and Hvidt, 1990;
Westh and Kristensen, 1992;
Westh et al., 1991), than when
measuring individual specimens, resulting in distinct crystallization events
for each individual with a low potential for inoculative freezing due to
surrounding animals or water.
|
Although the invariant SCPs, the fast ice formation and the fast cooling
rates tolerated by the tardigrades may lead to the assumption that they do not
require metabolic and biochemical preparation prior to subzero temperature
exposure, we must not exclude it. In fact, the increase in survival at slow
cooling rates down to 1°C h–1 presented by all species
may indicate that the animals synthesize e.g. ice active proteins or
cryoprotective compounds to increase their survival ability. Considering the
trend of all species to survive better after cooling at slow rates we may
postulate higher survival rates below 1°C h–1. With
slower rates, the tardigrades would have even more time to synthesize or
recruit protecting agents involved in regulating ice growth to conserve cell
structure. Environmental cooling rates of around 0.6°C
h–1 in temperate environments are not unusual
(Sinclair, 2001). However,
further detailed studies concerning cooling rates in typical field
microhabitats such as moss cushions are required.
Extracellular ice formation will subject the cells and tissues to freeze
dehydration. As a consequence of ice formation, intracellular solutes become
more concentrated and the cells become osmotically dehydrated. Fast ice growth
in tardigrades, which has also been reported by Westh and Kristensen
(Westh and Kristensen, 1992),
presents an enormous osmotic shock which, together with cell volume collapse,
is probably the most likely cause of mortality in unprotected cells
(Lee and Costanzo, 1998;
Storey and Storey, 1996).
Therefore tolerance to subzero temperatures in tardigrades may be related to
tolerance to extreme dehydration, which also requires the ability to deal with
wide variations in cell volume and osmolality of body fluids. Besides the
osmotic shock, rapid ice formation increases the likelihood of intracellular
freezing with its associated physical problems resulting in cellular damage
due to rapid changes in cell volume and damage from growing ice crystals
(Wright, 2001). Intracellular
freeze tolerance, if it occurs, could provide a successful strategy for
tardigrades to tolerate low temperatures and reduce transmembrane osmotic
stress during freezing as has been demonstrated in the Antarctic nematode
P. davidi (Smith et al.,
2008; Wharton and Ferns,
1995; Wharton et al.,
2003). However, studies concerning intracellular freezing in
tardigrades have yet to be performed. Molecular and metabolic investigations
at slow cooling rates as used in this experiment could provide an insight into
the remarkable phenomenon of cold tolerance in tardigrades.
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Footnotes |
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