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First published online August 17, 2006
Journal of Experimental Biology 209, 3469-3475 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02387
Freezing or supercooling: how does an aquatic subterranean crustacean survive exposures at subzero temperatures?
1 Ecologie des Hydrosystèmes Fluviaux, UMR CNRS 5023,
Université Claude Bernard Lyon 1, 69622 Villeurbanne cedex,
France
2 Laboratoire Souterrain de Moulis (CNRS), 09200 Moulis, France
3 Centre de Recherches sur les Très Basses Températures, CNRS,
BP 166, 38042, Grenoble cedex 9, France
4 Unité de Recherche en Résonance Magnétique
Médicale, UMR 8081 CNRS-Université Paris-Sud 91405 Orsay,
France
5 Ecosystèmes - Biodiversité - Evolution, UMR CNRS 6553,
Université Rennes 1, 35042 Rennes cedex, France
* Author for correspondence (e-mail: julien.issartel{at}univ-lyon1.fr)
Accepted 15 June 2006
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Key words: crustaceans, subterranean, epigean, freezing tolerance, bound water, crystallisation temperature, inoculative freezing, ice content, cold acclimation, glycogen
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;
Malard and Hervant, 1999), and
are also characterized by cool but highly buffered temperatures throughout the
year [11±1°C in French plains
(Ginet and Mathieu, 1968)].
Thus, most of the aquatic crustacean species living in these environments
should exhibit stenothermal characteristics
(Huey and Kingsolver, 1989).
However, we recently found that the hypogean amphipod Niphargus
rhenorhodanensis paradoxically survived -2°C for 60 days, whereas
they never normally endure such thermal conditions
(Issartel et al., 2005a). This
ability to survive low-temperature exposure may correspond to a relict
adaptation dating back to the European quaternary glaciations
(Issartel et al., 2005a;
Issartel et al., 2005b;
Lefébure, 2005). During
that cold palaeoclimate period, sub-zero temperatures presumably occurred,
perhaps even in sub-surface groundwater habitats
(Tweed et al., 2005). Thus if
organisms such as N. rhenorhodanensis did face sub-zero temperatures,
they had to avoid freezing by extensive supercooling or tolerate ice formation
in their tissues (Salt,
1961).
In freeze-avoiding species, the freezing temperature of body fluids, i.e.
the lowest temperature they can endure, is depressed (supercooled state)
particularly by accumulating large amounts of cryoprotectants [e.g. polyols,
sugars, free amino acids or antifreeze proteins (for reviews, see
Salt, 1961;
Storey, 1997;
Ramløv, 2000)]. By
their hydrophilic nature, cryoprotectants bind water molecules and reduce the
probability of them forming an ice embryo
(Ramløv, 2000). In
freezing-tolerant ectotherms, ice-nucleating agents are synthesized and
trigger nucleation at high sub-zero temperatures
(Lee and Costanzo, 1998). This
mechanism prevents the extensive supercooling of cells and thus reduces the
probability of lethal intracellular freezing
(Holmstrup and Zachariassen,
1996). Moreover, ice progression results in a strong increase of
the extracellular fluid concentration and causes a water loss from the cell to
the extracellular compartment
(Zachariassen and Kristiansen,
2000). In order to prevent the deleterious effects of `freezing'
dehydration (causing membrane and protein denaturation) in cells,
cryoprotective substances, such as glycerol, trehalose and free amino acids
may also be accumulated (Ramløv,
2000). In N. rhenorhodanensis, large accumulations of
trehalose and amino acids were found during low-temperature acclimation
(Issartel et al., 2005b); but
until now no studies have accurately investigated whether this subterranean
species is freeze tolerant or freeze avoiding.
In addition, some authors have studied the changes in the free water/bound
water ratio during low temperature acclimation in cold-hardy ectotherms. The
bound water is the water that is so closely associated with cellular or other
components in an organism that it is not available to participate in the
freezing processes (Hazelwood,
1977). The bound water content of the freeze-tolerant larvae of
Eurosta solidaginis increased with cold acclimation
(Storey et al., 1981), and
this was due to changes in water binding by cryoprotectants and macromolecules
(mainly glycogen and proteins). In freeze-tolerant species, bound water will
not participate in ice formation and this results in non-freezable shells of
water surrounding cellular components, protecting them from the denaturation
due to freezing dehydration (Storey et
al., 1981).
The data dealing with freezing survival in invertebrates are overwhelming,
but very few studies have investigated the problem of freezing in aquatic
invertebrates (Moore and Lee,
1991; Frisbie and Lee,
1997; Lencioni,
2004). When water from aquatic environments freezes, the physical
constraints differ significantly from terrestrial ones: aquatic invertebrates
may be subjected to anoxia or mechanical stress due to external ice
(Frisbie and Lee, 1997;
Lencioni, 2004). Moreover,
contact with external ice may trigger ice growth inside the body, which
strongly increases the probability of freezing occurring. As a result,
supercooling in aquatic invertebrates may not be a likely strategy
(Frisbie and Lee, 1997).
In this study, we compared the responses of two freshwater amphipod crustaceans, the hypogean (i.e. subterranean) N. rhenorhodanensis and the morphologically close epigean (i.e. surface-dwelling) Gammarus fossarum when exposed to subzero temperatures. Thus, in both species we investigated the influence of cold acclimation on (1) the supercooling point, (2) the freezing resistance by inoculation, (3) the ice contents and (4) biophysical parameters such as the bound water content determined by a non-invasive method.
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;
Hervant et al., 1997b). The
tanks with N. rhenorhodanensis contained clay and stones. Tanks with
G. fossarum contained leaves. Both sets of organisms were fed once a
week. They were first maintained at 12°C for 15 days. Then they were
separated into three groups: the first group was kept at 12°C for 6
months, the second group was acclimated to 3°C for 6 months. The third
group was first acclimated to 3°C for 6 months and next acclimated to
-2°C for 2 weeks as described by Issartel et al.
(Issartel et al., 2005b). For
acclimation at -2°C, crustaceans were individually put in 6 ml plastic
tubes containing 3 ml of filtered rearing tank water. For all acclimation
groups, water was changed twice a week and no physico-chemical parameters
changed during acclimation. Food was removed from experimental tanks 1 week
before the measurements to ensure that the presence of food in the gut would
not affect the results.
Glycogen and protein assays
The glycogen content was determined by standard enzymatic methods as
described by Hervant et al. (Hervant et
al., 1995; Hervant et al.,
1996). Total proteins were extracted according to published
methods (Elendt, 1989;
Barclay et al., 1983), and then
determined using specific test-combinations. All assays were performed in a
recording spectrophotometer (Beckman DU-6) at 25°C. Enzymes, coenzymes and
test-combination substrates used for enzymatic assays were purchased from
Boehringer (Mannheim, Germany) and Sigma Co. (St Louis, USA).
Cryobiological experiments
Supercooling point measurement
To determine the crystallisation temperature (Tc) of
body fluids, we used a differential scanning calorimeter DSC7
system (Perkin-Elmer). The experiments were conducted using standard
hermetically sealed aluminium pans (Perkin-Elmer, 0219-0062) designed for
volatile samples. In order to check the insulation of the sealed pans, their
masses were measured at the end of the experiments and compared with the
masses obtained before the DSC7 measurements. We used a
microbalance (Sartorius, type 1712 001; accuracy ±0.1 mg). A single
individual was removed from the rearing tanks and placed briefly on a filter
paper to remove excess water as described elsewhere
(McAllen and Block, 1997). It
was then placed in the pan before sealing and weighing. The pan was placed in
the DSC7 oven manually. Each sample was run against an empty sealed
aluminium pan for reference. Temperature and heat flow DSC7
calibration were evaluated from the melting of the ice of deionized water
(T=0°C and latent heat of fusion of ice 
H=333.88 J
g-1) and from the crystallographic transition of cyclohexane to its
solid state (T=-87.1°C). Temperature values were found to be
reproducible within ±0.5°C. The samples were cooled from 20°C
to -15°C at a rate of 1°C min-1. Thermograms were recorded
on a computer (Pentium II), and Tc was obtained using the
Pyris 3.7.A software.
Inoculative freezing
To emphasize the possible role of acclimation, the experiment was run on
both crustacean species acclimated to 12°C, 3°C and -2°C. The
animals were tested out of the water because of the unnatural mechanical
stress that ice-filled containers can produce on the organisms
(Frisbie and Lee, 1997). Thus,
according to the methods described by Frisbie and Lee, individuals were placed
in contact with a thermocouple connected to a Consort data logger. Then the
animal and the thermocouple were wrapped in a water-saturated strip of paper
towel. The wrapped animal was closely fitted into a 5 ml pipette cone. The
cone was then lowered into a 15 ml plastic tube immersed in an alcohol-filled
low temperature bath the temperature of which was adjusted to -2°C. Ice
crystal formation was initiated in the wet paper towel by contact with a metal
rod cooled in liquid nitrogen. The inoculative freezing of the water in the
paper was verified by observing an exotherm (heat release during the freezing
process). Once the temperature had again reached the set temperature (i.e. 2 h
after the onset of the exotherm), the structure containing the animal was
heated to 3°C. To check the effect of inoculative freezing, control
structures were not inoculated, organisms were cooled and reheated in the
low-temperature bath at the same time as the inoculated structures. Survival
of the organisms was noted 24 h after the end of the experiment.
Ice content
To determine the ice content of frozen individuals, we used the whole-body
calorimetry technique (Layne and Lee,
1987; Layne and Lee,
1991). The calorimeter consisted of an insulated flask that was
imbedded in a block of Styrofoam insulation and fitted with a Styrofoam plug
that fitted down into the flask, leaving a space of only about 200 µl at
the bottom of the flask. Thawing was done in a volume of 200 µl water for
all the animals. A thermocouple was positioned below the water surface and
connected to a digital thermometer. The change in water temperature caused by
thawing the crustacean was recorded. Calculations of body ice content used
experimentally determined values for our system which were: F factor for the
calorimeter=1.17, the percentage of body mass that is water for N.
rhenorodanensis and G. fossarum is 73.44±0.36% and
76.10±0.38%, respectively (values do not vary with acclimation),
specific heat of the dry mass measured by calorimetry (±
s.d.)=0.18±0.04 and the melting point of body fluids as estimated from
osmolality determinations= -0.54°C for both species. Ice content was
expressed in percentage of total body water.
Bound water contents using nuclear magnetic resonance spectroscopy
Nuclear magnetic resonance (NMR) measurements were performed on a 4.7 Tesla
horizontal bore MR scanner, controlled with a TECMAG sequencer (Apollo,
Tecmag, Houston, TX, USA), using a 16 mm-diameter parallel plate resonator
(Gonord and Kan, 1994) built
in the laboratory. All NMR measurements were performed at room temperature
(20°C), at the proton NMR frequency of 200 MHz. Each specimen was
transferred into a 5 mm-diameter Plexiglas sample holder placed at the centre
of the NMR probe. For each sample, the whole NMR measurement lasted about 10
min: first the radiofrequency (RF) power was adjusted within 1 dB to obtain
the required flip angles; then a multi-echo Carr Purcell Meiboom Gill sequence
(CPMG) (Meiboom and Gill,
1958) was run to obtain the transverse relaxation curve, with an
interecho time of 5 ms, a repetition time of 8 s, 100 to 400 echoes and 16
averages. The specimens were also weighed with an accuracy of ±0.1 mg.
The relaxation S(t) curves systematically diverged from
monoexponentials, and they were analysed as sums of exponential decays with a
Laplace inversion algorithm, following:
 |
where Ai represents the relative weight of the exponential decay
with the time constant Ti. Choosing 12 components with
times equally spaced in log scale between 2 and 500 ms gave excellent fits of
the data (
2 lower than 2x10-4) and relatively
robust distribution curves with a bimodal shape. The relative weight
p of the peak of the short-time constant thus reflects the relative
amount of bound water in the specimen. It was checked on one specimen that the
interecho time tcp chosen in the experiment did not influence the bimodal
aspect of the decay curve and the resulting value of P, as compared
to inter-individual variations.
Statistical analysis
All results are presented as mean ± s.e.m. The intra- and
inter-specific differences in metabolite concentrations,
Tc, and bound water contents were investigated by a
two-way ANOVA. When significant differences were found, the Tukey's HSD
post-hoc test was performed. Data were log or square-root transformed
to homogenize variances when homoscedasticity was not observed. Statistical
analyses were performed with Statitistica 6 (StatSoft Inc., Tulsa, USA).
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Crystallisation temperature
The Tc values are presented in
Table 1. The mean
crystallisation temperature was statistically lower in N.
rhenorhodanensis than in G. fossarum, whatever the acclimation
temperature (P<0.05).
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Cold acclimation induced a significant increase in the Tc in both species: the Tc rose by 37% (P<0.001) in N. rhenorhodanensis and by 40% (P<0.01) in G. fossarum.
No survival was observed in either species after thawing.
Inoculative freezing
Survival values are presented in Table
2. In G. fossarum, no survival was observed after
inoculation, whatever the acclimation temperature. In N.
rhenorhodanensis, no survival was observed after inoculation in
12°C-acclimated individuals. After acclimation at 3°C and -2°C
survival rose to 90% and 100%, respectively. No mortality was recorded in
control (non inoculated) individuals of either species.
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Ice content
The percentages of crustaceans' body water transformed into ice after
inoculation are presented in the Fig.
2. No variation of the ice content was apparent in G.
fossarum whatever the temperature (54.40±6.41, 58.52±3.80
and 61.25±7.59% at 12°C, 3°C and -2°C, respectively).
N. rhenorhodanensis had ice contents of 62.07±4.30,
52.72±4.32 when acclimated at 12°C and 3°C, respectively. The
percentage of ice is significantly lower in specimens acclimated at -2°C
(40.07±4.1%; P<0.05) than in the control group.
Bound water content
The relative bound water contents in N. rhenorhodanensis and
G. fossarum when acclimated to 12°C, 3°C and -2°C is
shown in Fig. 3. No variation
in bound water was observed in the epigean G. fossarum whatever the
acclimation temperature. After being acclimated to 3°C and -2°C, the
subterranean N. rhenorhodanensis showed a significant increase in its
bound water content (P<0.001).
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;
Vernon and Vannier, 2002).
Ectotherms are either freeze-tolerant or freeze-avoiding species, depending on
their ability to survive the formation of extracellular ice
(Bale, 1987;
Lee, 1989). In freeze-avoiding
species, temperature of crystallisation (Tc) is relatively
low, often below -10°C, whereas many freeze-tolerant animals have limited
abilities to supercool, i.e. ice formation occurs at relatively high
temperatures [Tc above -10°C
(Bale, 1996;
Sinclair, 1999;
Sømme, 1999)].
Moreover, cold acclimation generally leads to a Tc
decrease in freeze-avoiding species (Danks,
1978; Duman et al.,
1991). In our study, amphipods exhibited a relatively high
Tc, and cold acclimation induced a Tc
increase in both G. fossarum and N. rhenorhodanensis, but no
survival was observed after thawing whatever the acclimation temperature. A
higher Tc was also reported in animals acclimated at the
lowest temperature in the intertidal copepod Tigriopus brevicornis
(McAllen and Block, 1997). In
addition, although it was increased, the Tc remained very
low (about -20°C) when the animals were acclimated at 0 or 10°C. Such
a Tc elevation in G. fossarum and N.
rhenorhodanensis when cold acclimated is thus probably non-adaptive but
may result from endogenous or exogenous ice nucleating agents.
If temperature falls below 0°C, N. rhenorhodanensis and G.
fossarum probably encounter external ice crystals and thus become
vulnerable to inoculative freezing (IF). In such cases, supercooling is not
likely to work as a strategy (Frisbie and
Lee, 1997). We observed distinct patterns in the two crustaceans:
after they were in contact with ice crystals, only the 3°C and -2°C
acclimated N. rhenorhodanensis survived whereas all others, including
the 3°C and -2°C acclimated G. fossarum, died. Specimens that
survived IF showed a recovery time of a few hours whereas all control
organisms (exposed at -2°C but non-inoculated) were immediately active
when reheated at 3°C. This survival is probably linked to the lower ice
contents endured by cold-acclimated N. rhenorhodanensis that does not
exceed 53% ice within body, unlike the other groups. Contact with external ice
induced inoculative freezing of body fluids as the external ice lattice can
propagate through a body orifice or directly through the cuticle
(Salt, 1963;
Lee and Hankinson, 2003). The
very thin cuticle characteristic of amphipod gills (allowing gas diffusion)
may be the preferential sites from which ice will propagate through the
body.
A number of terrestrial arthropods that live in wet habitats require IF in
order to survive extracellular ice formation, since if contact with external
ice is prevented, they will supercool and die when spontaneous freezing occurs
(Lee et al., 1996).
Cold-acclimated N. rhenorhodanensis survived freezing if nucleation
occurred after an inoculation at high subzero temperature. A similar feature
was previously reported in the centipede Lithobius forficatus: it
survived freezing only when nucleation was initiated at temperatures of almost
-1°C by inoculative freezing (Tursman
et al., 1994). In nature, if temperature drops to almost -1°C,
N. rhenorhodanensis will experience inoculative freezing before
reaching its Tc, as it will be surrounded by ice.
Consequently, the tolerance to inoculative freezing seems to be an adaptive
trait in these organisms (Tursman et al.,
1994).
The physiology of cold tolerance of many arthropods is based on water and
its activity at low temperatures. Water content influences the supercooling
capacity of freezing-susceptible species, and in freezing tolerant ones a
proportion of body water remains unfrozen in order to allow a low level basal
metabolism (Block, 2003). Thus,
one of the key features that has been rarely studied in arthropods is the
capacity to bind water molecules (Storey
et al., 1981; Storey,
1983). In our study, we used an original non-invasive protocol to
determine the relative bound water content in crustacean bodies by proton NMR
transverse relaxation measurements performed on the whole live organisms. The
hypogean N. rhenorhodanensis contained 25% more bound water when
cold-acclimated, whereas no changes occurred in the epigean G.
fossarum. Adaptations that increase the amount of bound water are used to
ensure that the lethal limit is not exceeded
(Storey and Storey, 1989). Our
results are in agreement with these findings, as ice contents decrease with
increasing bound water in N. rhenorhodanensis. Furthermore, the
present results confirm previous work showing that both low-molecular weight
compounds (LMWs; mainly polyols and sugars) and high-molecular weight
compounds (mainly glycogen and proteins) participate in this phenomenon
(Storey et al., 1981;
Storey, 1983). Indeed,
cold-acclimated N. rhenorhodanensis accumulate both glycogen (this
study) and amino-acids and trehalose
(Issartel et al., 2005b). An
increase in glycogen is rather paradoxical as numerous studies have reported a
decrease in glycogen during cold acclimation of ectotherms: glycogen being
generally used as a fuel for synthesis of polyols and sugars. In G.
fossarum, which exhibited no changes in the amount of bound water,
glycogen remained stable. Furthermore, glycogen levels are twice as high in
N. rhenorhodanensis as in G. fossarum, which may partly
explain the larger bound water content found in the former. However, even if
the increased glycogen in cold-acclimated N. rhenorhodanensis may be
partly responsible for the increased bound water (together with increase of
amino acids and trehalose), its function in the freeze tolerance adaptation
still remains unclear and needs further investigations.
Storey and Storey (Storey and Storey,
1989) reported that the most important mechanism for controlling
the freezing process is the accumulation of LMWs. Moreover, total levels of
polyols and sugars are usually significantly lower in freezing tolerant
species than in freeze-avoiding ones. In N. rhenorhodanensis, we
found a significant accumulation of the total free amino acids (from
58.93±3.88 to 98.63±6.89 µmol g-1 FM at 12°C
and -2°C, respectively) and trehalose [from 1.19±1.2 to
19.66±5.2 µmol g-1 FM at 12°C and -2°C,
respectively (Issartel et al.,
2005b)]. These findings may also explain the increased bound water
found in N. rhenorhodanensis and we may hypothesize that accumulated
LMWs are used for controlling the amount of ice in the body rather than for
supercooling. On the other hand, LMW concentrations measured in both species
are probably too small to involve a decrease of the glycogen content as it is
usually observed in cold hardy invertebrates. From an adaptive standpoint, the
amount of bound water has been found to vary in direct proportion to cold
hardiness (Danks, 1978;
Storey et al., 1981,
Ring, 1981). By increasing the
amount of water-binding micro and macromolecules, a greater fraction of
intracellular water can exist as bound water, and therefore the probability of
intracellular freezing (which is lethal for organisms) is strongly decreased
(Storey et al., 1981;
Ramløv, 2000). However,
the osmotic water loss from the cell during extracellular freezing exposes the
intracellular components to a dramatic dehydration stress
(Zachariassen and Kristiansen,
2000). It is hypothesised that unfrozen water shells surrounding
sub-cellular components could prevent irreversible protein denaturation due to
freezing desiccation and cold temperatures
(Hazelwood, 1977;
Storey et al., 1981).
Thus, the presence of such adaptations in the subterranean N. rhenorhodanensis may explain its survival capacity when exposed to inoculative freezing.
According to the data in the literature, Sinclair
(Sinclair, 1999) proposed that
freezing tolerance is divisible into four groups according to
Tc and lower lethal temperature: partially
freeze-tolerant, moderately freeze-tolerant, strongly freeze-tolerant and
freeze-tolerant. Partially-freezing tolerant species survive the conversion of
a small proportion of their body water into ice, but do not survive if ice
formation reaches an equilibrium at or above the Tc, which
is visually represented by the total completion of the exotherm at a given
temperature (Sinclair, 1999).
The epigean crustacean G. fossarum that does not survive nucleation
whatever its acclimation, is a freezing intolerant species; it belongs to the
chill-susceptible species (species that die after brief chilling to high
sub-zero temperatures). The subterranean crustacean N.
rhenorhodanensis exhibited responses to subzero temperatures similar to
those found in freeze-tolerant species that survive IF. However, it appears
from our results that N. rhenorhodanensis can neither be classified
as a partially freeze-tolerant nor as a moderately freeze-tolerant species
since: (i) survival was observed after the total completion of the exotherm
which is lethal in partially freeze-tolerant individuals
(Sinclair, 1999), and (ii) no
survival was observed after the animals reached the Tc,
whereas moderately freeze-tolerant species survive after
Tc is reached. Thus, like numerous arthropods showing
similar characteristics (see Lee et al.,
1996), N. rhenorhodanensis seems to belong to a still
indeterminate category.
The presence of such complex adaptations in an organism that currently
never endures cold during its life cycle seems at first very paradoxical.
However, from recent biogeographical and phylogenetic studies, there are now
several proofs that the subterranean amphipod N. rhenorhodanensis
survived the quaternary glaciations at the limit or within the nunataks, i.e.
the mountain tops surrounded by ice never covered by the glaciers
(Lefébure, 2005). Thus,
in such palaeo-environments, freshly-melted water coming from the glacier [at
temperatures near, or even just below 0°C
(Tweed et al., 2005)] may have
infiltrated the sediment and considerably influenced the subterranean
temperatures. As a result, the hypogean crustacean N.
rhenorhodanensis may have encountered sub-zero temperatures and ice, and
may thus have been subjected to inoculative freezing.
To conclude, our results agree with this evolutionary scenario, and the
possible `near-glacial' survival of N. rhenorhodanensis during that
period may explain (i) the cold-induced accumulation of cryoprotectants
(Issartel et al., 2005b), (ii)
the bound water increase, and the resulting inoculative freezing tolerance
(this study).
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Acknowledgments |
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