Freezing survival and cryoprotective dehydration as cold tolerance mechanisms in the Antarctic nematode Panagrolaimus davidi
1 Department of Zoology, University of Otago, PO Box 56, Dunedin, New
Zealand
2 Department of Biochemistry, University of Otago, PO Box 56, Dunedin, New
Zealand
* Author for correspondence (e-mail: david.wharton{at}stonebow.otago.ac.nz)
Accepted 17 October 2002
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Summary |
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Key words: Antarctic, nematode, Panagrolaimus davidi, freezing, ice, nucleation, dehydration
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Introduction |
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Similar hazards are faced by earthworms in cold terrestrial environments.
Earthworm eggs are enclosed within a fibrous cocoon, which provides protection
against inoculative freezing (Holmstrup and
Zachariassen, 1996). The cocoon fluid can thus remain unfrozen
even though the cocoon is in close contact with ice. This results in a vapour
pressure difference between the unfrozen, supercooled cocoon fluids and the
surrounding ice. Earthworm cocoons are very permeable to water molecules and
thus lose water until they are in vapour pressure equilibrium with the ice.
This dehydrates the cocoons, to below 0.5 g water g-1 dry mass, so
that they cannot freeze. This cold tolerance mechanism has been called the
`protective dehydration mechanism'
(Holmstrup and Westh, 1994
),
although `cryoprotective dehydration' might be a more appropriate term (M.
Holmstrup, personal communication) and will be used here. A similar phenomenon
has been described in an Arctic collembolan, Onychiurus arcticus
(Holmstrup and Sømme,
1998
; Worland et al.,
1998
) and in three species of adult or preadult enchytraeid
oligochaetes (Sømme and Birkemoe,
1997
). It has been suggested that cryoprotective dehydration
occurs in nematodes and chironomid larvae
(Holmstrup et al., 2002
). A
shrunken appearance upon thawing suggests that dehydration may be occurring
(Forge and MacGuidwin, 1992
;
Scholander et al., 1953
). Both
nematodes and chironomid larvae have been shown also to tolerate freezing
(Scholander et al., 1953
;
Wharton, 2002
), so the
relative importance of freezing tolerance and cryoprotective dehydration in
these groups is unclear.
The rate of ice formation in the soil water surrounding the nematode is
dependent upon the volume of water and the temperature at which ice nucleation
occurs. In samples of P. davidi frozen in Eppendorf tubes in a
cooling block, survival depended upon the rate of freezing, which was
determined by the sample volume and the temperature at which ice nucleation
was initiated (Wharton et al.,
2002). If contact between the growing ice crystals in the soil
water and the nematode occurs at a temperature above the melting point of the
body fluids of the nematode, inoculative freezing cannot occur and the
nematode may undergo cryoprotective dehydration, which prevents them from
freezing even if exposed to lower temperatures. In this paper we determine how
nucleation temperature, cooling rate and the medium in which freezing occurs
affect the relative importance of cryoprotective dehydration and freezing
survival in this nematode.
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Materials and methods |
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Freezing of nematodes was observed as a sudden darkening, whilst those assumed to be unfrozen tended to shrink and remain clear. After melting the sample was recovered from the cold stage and transferred to 1 ml of ATW in a watchglass. Nematode survival was determined after 24 h at room temperature, following a mechanical stimulus (expelling the medium from a glass pipette several times), by counting the number of motile and non-motile individuals (a total of at least 100 being counted).
The effect of nucleation temperature on freezing and survival
Nematodes were transferred to the cold stage and cooled at 0.5°C
min-1 to the test temperature (-1, -2, -3, -4, -5, -6°C). The
sample was checked to ensure that freezing had not occurred and then freezing
was initiated by placing a small ice crystal in contact with the edge of the
coverslip. The sample was observed to ensure that freezing did occur and to
qualitatively monitor the rate of freezing of the sample. The test temperature
was maintained for a further 30 min and the proportion of frozen nematodes
determined. The sample was warmed to 0°C at 1°C min-1 and,
after melting, the sample was recovered and survival determined.
The effect of cooling rate on freezing and survival
Nematodes were transferred to the cold stage, cooled rapidly to 0°C and
then to -5°C at rates of 0.5, 0.2 or 0.1°C min-1. Freezing
was initiated with an ice crystal at -1°C and the proportion of frozen
nematodes counted at 1°C intervals. The sample was then held at -5°C
for 30 min, warmed to 0°C at 1°C min-1, the sample
recovered from the cold stage and survival determined as before.
The effect of media on freezing and survival
Nematodes were transferred to either ATW, deionized water (dH2O)
or 0.1 mol l-1 NaCl dissolved in ATW. The osmolalities of these
solutions, measured using a Knauer semi-micro osmometer, were: ATW, 7 mosmol
l-1; dH2O, 0 mosmol l-1; 0.1M NaCl dissolved
in ATW, 195 mosmol l-1. The sample was washed three times in the
test solution, transferred to the cold stage and cooled to -1°C. Freezing
of the sample was seeded with an ice crystal and the sample was held at
-1°C for either a further 5 min or 30 min. The sample was then cooled to
-5°C at 0.5°C min-1 and the proportion of frozen nematodes
determined at 1°C intervals. The sample was held at -5°C for a further
10 min, photographed, and then warmed to 0°C at 1°C min-1
before being retrieved and survival determined as before.
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Results |
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The effect of cooling rate on freezing and survival
When cooled at 0.5°C min-1, most nematodes froze during
cooling and after nucleation at -1°C. The majority of nematode nucleation
events occurred between -2 and -4°C. In samples cooled at rates of 0.2 or
0.1°C min-1 the freezing of nematodes during cooling was much
less (Fig. 2). Those nematodes
that did not freeze took on a shrunken appearance, with the amount of
shrinkage increasing as the temperature decreased. Cooling rate had a
significant effect on the proportion frozen at -5°C (factorial analysis of
variance, ANOVA, after arcsin transformation: F(2,6)=98.7,
P<0.05) but not on survival (F(2,6)=2.2,
P>0.05) (Fig.
3).
|
|
The effect of media on freezing and survival
In samples in ATW or dH2O held for 5 min at -1°C after ice
nucleation, the proportion of nematodes frozen increased during cooling to
-5°C. In samples in 0.1 mol l-1 NaCl dissolved in ATW, the
increase in freezing was much less (Fig.
4). The proportion freezing during cooling to -5°C was reduced
in samples held at -1°C for 30 min after ice nucleation, compared to those
held at -1°C for 5 min (Fig.
4). The time held at -1°C had a marked effect on the
proportion frozen at -5°C for samples in ATW or dH2O but not
for those in 0.1 mol l-1 NaCl dissolved in ATW. Survival was
similar in all treatments (Fig.
5). In ATW and dH2O, those nematodes that did not
freeze took on a shrunken appearance. The amount of shrinkage appeared to be
greater in nematodes immersed in dH2O than in those in ATW
(Fig. 6). The freezing process
was not completed in 0.1 mol l-1 NaCl dissolved in ATW at -1°C.
No shrinkage was observed in this medium, even during cooling to -5°C
(Fig. 6).
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The effect of test solution on the proportion frozen at -5°C was significant (factorial ANOVA after arcsin transformation: F(2,12)=16.4, P<0.05), as was the effect of time held at -1°C (F(1,12)=57.8, P<0.05) and the interaction between test solution and time held at -1°C (F(2,12)=10.6, P<0.05). There were no significant differences in the proportion frozen at -5°C between ATW and dH2O (Scheffe post hoc test: P>0.05, d.f.=12) but 0.1 mol l-1 NaCl dissolved in ATW produced significantly lower freezing than either ATW or dH2O (P<0.05, d.f.=12). The proportion frozen at -5°C after being held for 5 and 30 min at -1°C was significantly different in ATW and dH2O (P<0.05, d.f.=12) but not in 0.1 mol l-1 NaCl dissolved in ATW (P>0.05, d.f.=12). There was no significant effect on survival of either test solution (F(2,12)=0.33, P>0.05) or of time held at -1°C (F(1,12)=0.04, P>0.05).
The effect of freezing on survival
Using data from the previous experiments, survival was compared with that
predicted if each nematode that had frozen died
(Fig. 7A). Most points lie
above the prediction line, indicating that nematodes are surviving freezing.
There was a significant difference between survival and that predicted by
freezing (2=11561, P<0.0001, d.f.=48). There was a
significant negative correlation between freezing and survival
(r2=45.2%, d.f.=49, t=6.36,
P<0.0001). However, if only the data for samples that froze at
-1°C is considered (Fig.
7B), there is no correlation between freezing and survival
(r2=7.3%, d.f.=25, t=-1.1,
P>0.05).
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General observations on freezing and thawing
The spread of ice through the body of nematodes was faster the lower the
temperature at which inoculative freezing occurred. If inoculative freezing
occurred at a high temperature (such as -1°C), ice propagation through the
nematode was slow and appeared to be consistent with the ice being confined to
extracellular compartments. If inoculative freezing occurred at a low
temperature (such as -5°C), ice propagation was rapid and both
extracellular and intracellular compartments appeared to freeze.
Immediately upon thawing some nematodes had a shrunken appearance. Those that did not freeze but dehydrated appeared more shrunken, but shrinkage was also observed in nematodes that had frozen. Internal gas bubbles were observed in some samples. These effects were not quantified.
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Discussion |
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When freezing of the medium was completed at -1°C the nematodes did not
freeze, even though they were surrounded by ice, and instead appeared to
dehydrate. There are two possible mechanisms for the loss of water from the
nematodes during and after the freezing of the surrounding water. As water
freezes salts are excluded from the growing ice crystals and are concentrated
in the remaining unfrozen medium (Shepard
et al., 1976). This freeze concentration effect could dehydrate
the nematodes via the resulting osmotic stress. Wharton and To
(1996
), however, found that
inoculative freezing of P. davidi occurred in solutions of
osmolalities up to 1130 mosmol l-1 and that inoculative freezing
would still occur even if salt concentrations were raised by a factor of 120,
which is much higher than concentrations likely to be experienced in nature.
In the present experiments we found that dehydration occurs upon freezing in
deionized water, in which a freeze concentration effect is not possible. It
thus seems likely that dehydration is driven by the vapour pressure difference
between ice and supercooled water at the same temperature, i.e. the
cryoprotective dehydration mechanism
(Holmstrup and Westh, 1994
).
There could, however, be some interaction between a freeze-concentration
effect and dehydration due to vapour pressure differences. Inoculative
freezing was inhibited in 0.1 mol l-1 NaCl dissolved in ATW even
after freezing at -1°C for 5 min. Survival was greater in this solution
than in those of higher or lower osmolality during freezing to -15°C at
1°C min-1 (Wharton and To,
1996
).
The ability to resist inoculative freezing is limited, with nucleation of
the nematodes' body fluids occurring at -2 to -4°C, depending upon the
time held at a temperature higher than the nucleation temperature and the rate
of cooling. Shrinkage of nematodes whilst being held at -1°C and in
unfrozen nematodes during cooling from this temperature indicates that they
are losing water to the surrounding medium. If sufficient water is lost this
prevents freezing and the nematodes survive through cryoprotective
dehydration, the loss of water being driven by the vapour pressure difference
between the surrounding ice and the supercooled solution within the nematode.
These are separated by the cuticle of the nematode. The cuticle provides a
barrier to ice nucleation (Wharton and
Ferns, 1995). The distribution of ice within a frozen nematode can
be visualised using transmission electron microscopy and freeze-substitution
techniques. Although ice is formed throughout the cells of the nematode, there
is no ice in the cuticle (D. A. Wharton, unpublished observations). The
cuticle may thus form a vapour-filled space that separates the supercooled
solution within the nematode from the surrounding ice, mediating the transport
of water between them.
The relative importance of freezing survival and cryoprotective dehydration
in P. davidi depends upon the nucleation temperature, the cooling
rate and the freezing rate of the surrounding medium. If nucleation occurs at
a high subzero temperature (-1°C) the nematode does not freeze. If the
temperature is then held above the nucleation temperature for a sufficient
time, or if the sample is cooled sufficiently slowly, enough water is lost by
cryoprotective dehydration to prevent freezing during further cooling. It
should be noted, however, that in all regimes tested some freezing occurred.
The minimum freezing in samples at -5°C recorded in any experiment was
21±2% frozen (in nematodes in ATW cooled to -5°C at 0.1°C
min-1, freezing seeded at -1°C). Nematodes were recorded as
frozen if obvious ice crystals were observed. Some nematodes that had a
shrunken appearance nevertheless froze, so shrinkage is not a reliable
criterion for the presence or absence of freezing. A relatively low
magnification was used for these observations (100x) and the amount of
freezing may thus be underestimated. The pattern of ice formation varied, with
those freezing at a high subzero temperature having a pattern that might be
consistent with extracellular, rather than intracellular, ice formation. The
use of an ultrastructural technique, such as freeze substitution
(Wharton, 2002), would confirm
the presence or absence and the location of ice in specimens.
There are some interesting parallels between our experiments and
observations on the freezing of living, mainly mammalian, cells for the
development of cryopreservation protocols. During slow freezing mammalian
cells dehydrate, but survive if they can withstand the resulting dehydration,
or are protected from its effects by cryoprotectants. During rapid freezing,
intracellular ice formation occurs and in the cryopreservation literature this
is considered always to be fatal (Pegg,
2001). In P. davidi, dehydration is favoured during slow
freezing of the surrounding medium, whilst during rapid freezing the nematodes
freeze but survive, including surviving intracellular freezing
(Wharton and Ferns, 1995
;
present study). A cooling rate of 1°C min-1 is considered a
slow rate by cryobiologists, but in our study this would be considered to be a
fast cooling rate, which results in nematodes freezing. Dehydration during
slow freezing of mammalian cells is interpreted as being due to the freeze
concentration effect, as salts are progressively excluded from the growing ice
crystals (Pegg, 2001
). Some of
the earlier cryopreservation literature, however, considered dehydration of
cells during slow freezing to be the result of vapour pressure differences
between ice and supercooled water (Mazur,
1966
).
The freezing and thawing of soil is a complex phenomenon, influenced by a
variety of processes, including snow cover, thermal conductivity and
diffusivity, water content, soil water and salt migration and freezing point
depression (Eitzinger et al.,
2000). Soil moisture content in coastal sites of the McMurdo Sound
area of Antarctica varied from 15% to <1% (w/w), with appreciable
differences over short distances (Campbell
et al., 1997
). Water content at the point across a transect that
had the greatest abundance of P. davidi at Cape Bird, Antarctica
decreased from 57.1% to 14.8% in just 3 days due to the freezing of the
adjacent snow bank, which had been providing liquid water to the site
(Wharton, 1998
). Environmental
cooling rates are probably slow, with a maximum rate of 0.021°C
min-1 recorded at Keble Valley, Cape Bird
(Sinclair and Sjursen, 2001
)
but with frequent freezethaw cycles during late spring and summer
(Sjursen and Sinclair, 2002
).
Conditions are thus variable with respect to both temperature and water
content. Given the likely presence of ice nucleators and the bulk of the soil,
the freezing of soil water is likely to be slow and to thus favour
cryoprotective dehydration. However, with their limited ability to resist
inoculative freezing and variable environment, P. davidi must also
have the ability to tolerate freezing. This nematode is also capable of
anhydrobiosis (Wharton and Barclay,
1993
), and thus has a variety of mechanisms for surviving the
harsh conditions of its terrestrial Antarctic habitat.
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Acknowledgments |
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