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First published online February 27, 2009
Journal of Experimental Biology 212, 859-866 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.026864
Dual roles of glucose in the freeze-tolerant earthworm Dendrobaena octaedra: cryoprotection and fuel for metabolism
1 National Environmental Research Institute, University of Aarhus, Department of
Terrestrial Ecology, Vejlsøvej 25, PO Box 314, DK-8600 Silkeborg,
Denmark
2 Department of Zoophysiology, Institute of Biological Sciences, University of
Aarhus, Building 540, DK-8000 Aarhus, Denmark
3 NSM, Research Unit for Functional Biomaterials, Roskilde University, Roskilde,
Denmark
* Author for correspondence (e-mail: biojo{at}biology.au.dk)
Accepted 7 January 2009
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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90%) glucose was
metabolised anaerobically. Both estimates of metabolic rate demonstrated a
10-fold metabolic depression associated with freezing. Thus, in addition to
the suspected role of glucose as cryoprotectant, the present study
demonstrates that glucose accumulation is vital to ensure substrate for
long-term anaerobic metabolism in frozen worms. On the basis of the estimated
metabolite levels, we calculate that the combined effect of metabolic
depression and large glucose stores enables a projected 3 months survival of
freezing at –2°C of the `average' D. octaedra. Such
conditions are very likely to occur in the northern distribution ranges of
this stress-tolerant earthworm.
Key words: cryoprotectant, glucose, anoxia, anaerobic metabolism, freeze survival
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Storey and Storey, 1988). Most
freeze-tolerant species produce cryoprotectants in relation to frost as this
limits both the amount and speed of ice formation in the extracellular fluid,
and some species may also produce ice-nucleating agents that help to initiate
freezing of the extracellular fluid at relatively high temperatures where
freezing occurs slower and is therefore a more controlled process
(Zachariassen, 1985;
Storey and Storey, 1988).
In earthworms, freeze tolerance is particularly prominent in the two
northerly distributed species, Eisenia nordenskioldi and
Dendrobaena octaedra, and individuals of D. octaedra may
tolerate freezing to temperatures as low as –20°C
(Holmstrup and Overgaard,
2007). Extracellular freezing of these species is associated with
a rapid and substantial accumulation of glucose, derived from glycogen stores.
In some individuals of adult D. octaedra glucose can constitute more
than 20% of dry mass less than 24 h after freezing has commenced. Glucose
is the only cryoprotectant that accumulates in response to freezing in D.
octaedra (Bundy et al.,
2003; Rasmussen and Holmstrup,
2002; Holmstrup,
2003) and similarly glucose has also been found to be the most
predominant cryoprotectant in many freeze-tolerant amphibians
(Schmid, 1982;
Storey and Storey, 1992;
Storey and Storey, 1996). In
D. octaedra it seems that the rapid accumulation of glucose is
triggered by small temperature changes below 0°C
(Overgaard et al., 2007) but
accumulation is also induced by rapid changes in osmotic pressure such as
those following extracellular ice formation
(Rasmussen and Holmstrup,
2002; Holmstrup,
2003; Overgaard et al.,
2007).
Although it seems clear that accumulation of glucose is an essential
component of freeze survival in D. octaedra, previous experiments
have not conclusively demonstrated that high glucose accumulation correlates
with short-term freeze survival. There was, for example, no significant
correlation between the average amount of glucose mobilized and tolerance to
low temperature when this was examined in a common garden experiment comparing
freeze tolerance of different D. octaedra populations from Denmark,
Canada, Sweden, Finland, Poland and Greenland
(Holmstrup et al., 2007).
Instead, the study by Holmstrup et al.
(Holmstrup et al., 2007)
demonstrated that the magnitude of the glycogen reserves prior to freezing
correlated with population freeze tolerance. Moreover, several studies have
found large individual differences in glucose accumulation that do not
necessarily relate to freezing survival of individuals. Thus, individuals that
accumulate relatively low amounts of glucose may also survive short-term
freeze-tolerance tests (Rasmussen and
Holmstrup, 2002; Holmstrup et
al., 2007; Overgaard et al.,
2007). A similar relationship is also seen when considering
interspecific differences since some species of earthworms, such as
Aporrectodea caliginosa, show some freeze tolerance although their
cryoprotectant accumulation response is limited
(Holmstrup and Overgaard,
2007). Given the lack of a straightforward relationship between
glucose concentration and freeze tolerance in D. octaedra we
hypothesized that another primary role of glucose accumulation is to serve as
a well-distributed energy resource during long-term frost.
On a broader comparative scale, freeze-tolerant species of earthworms share
a number of features with freeze-tolerant amphibians and, to some extent,
reptiles. Thus, all groups have representatives that tolerate freezing of more
than 50% of body water and they all primarily use glucose as cryoprotectant.
Species from these diverse groups also share the use of convective transport
to sustain gas exchange as well as the risk of inoculative freezing when
hibernating. However, in reptiles, when glucose levels are low, the duration
of freeze tolerance is rarely above 1 week and in anuran amphibians, where
glucose is accumulated to high but heterogeneous levels, there are no reports
of freezing survival of more than a month
(Storey and Storey, 1988;
Layne and Kefauver, 1997;
Storey, 1990;
Voituron et al., 2002a;
Costanzo et al., 2006). Thus,
from a comparative perspective it seems that D. octaedra is much more
tolerant to long-term freezing and this may be linked to the much larger and
more uniform accumulation of glucose in these animals (M. Tolarova, personal
communication) which enables sustained metabolism of all organs during
freezing.
In this study we test the hypothesis that glucose accumulation is beneficial for long-term cold tolerance because it provides a reliable energy resource during long periods in a frozen state. Under such conditions there will be no, or very limited, convective transport of fermentable energy resources in the extracellular fluid and sufficient energy resources must, therefore, be distributed prior to freezing or before the freezing process is completed. Owing to the impeded convective transport and because oxygen diffuses extremely slowly through ice it is probable that frozen worms rely primarily on anaerobic metabolism during the extended periods with subzero temperatures. Given the inefficiency of ATP production per glucose molecule under anaerobic conditions we hypothesize that long-term freezing must be associated with a substantial mobilization of glucose, but also with a considerable metabolic depression, reducing glucose depletion during the winter period in these worms.
Here we report, for the first time, that long-term freezing tolerance is an environmentally realistic phenomenon since adult D. octaedra from Greenland survived Arctic winter conditions in frozen soil for at least 3 months. The relationship between glucose mobilisation, metabolic rate and long-term freezing survival in D. octaedra was examined further using a similar and highly freeze-tolerant population of D. octaedra from Finland showing a gradual depletion of glucose stores during long-term frost. In addition, it was investigated how anaerobic metabolism and general metabolic depression contributes to the long-term freeze tolerance of D. octaedra.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Collection and maintenance of D. octaedra used for laboratory experiments
Specimens of D. octaedra were collected in Konnevesi, Finland
(latitude of 62 deg. N, longitude of 25 deg. E) in September 2006 and brought
to Denmark in plastic beakers with moist vegetation from the location. Here
the animals were maintained at 10±1°C, in 1 l plastic beakers each
containing 10 individuals and 600–700 g moist soil. The worms were fed
every 4 weeks by the addition of a soil/cow dung mix (40 g dry soil, 10 g cow
dung and 55 ml water). The animals used for experiments were adults or large
juveniles with a dry mass ranging from 5 to 75 mg. Worms used for long-term
frost exposure were placed individually in 10 ml plastic vials containing
moist soil and they were acclimated at 2±1°C for 4 weeks prior to
the start of the experiment in February 2007. Worms used for calorimetrical
measurements were placed singly in containers with soil and kept at 5°C
for 2 weeks prior to the start of the experiment in January 2008. Worms used
for these experiments were from the F1 generation that had developed under
similar conditions as mentioned above.
Long-term freezing
After acclimation, the worms were split into a control group, which
remained at 2±1°C, and a frozen group, which was moved to a
programmable freezing cabinet for 24 h, in which the temperature could be
controlled and gradually lowered to –2°C (–0.042°C
h–1). A small piece of ice was added to each plastic vial
when the temperature reached –1°C to initiate freezing of soil and
worms; the worms were subsequently left at –2°C throughout the
remainder of the experiment. Previous experiments have shown that worms freeze
when using this procedure (Bindesbøl
et al., 2005). After 3, 6, 10, 13, 20, 26 and 47 days,
approximately 15 worms, respectively, were thawed at room temperature and
cleaned of soil and debris. The survival status of each individual worm was
noted, after which they were rapidly frozen at –80°C for later
assessment of glucose, lactate, succinate, alanine and glycogen content (see
below). Pilot studies showed that earthworms could be considered alive if they
responded to tactile stimuli, showed normal locomotive activity and had no
visible signs of freezing damage, and this status could usually be scored
within 2 h of thawing. To test for effects of time, untreated control worms
were taken from 2°C after 0, 13 and 47 days. Each control sampling
consisted of eight worms, which were quickly cleaned and frozen at
–80°C for subsequent biochemical analysis.
Measurements of glucose, glycogen, lactate, alanine and succinate
Worms were taken from –80°C and immediately freeze dried for 24 h
after which the dry mass of each individual was determined. Worms with a dry
mass exceeding 10 mg were cut in pieces and randomly split between two
Eppendorf tubes to allow for determination of glycogen and metabolites
(glucose, lactate, alanine and succinate), respectively. Glycogen was not
measured for worms smaller than 10 mg dry mass (dm). Glycogen was extracted
for 3 h at 80°C in 1 ml 1 mol l–1 NaOH after which the
extract was stored at –80°C. Samples for glucose, lactate, succinate
and alanine measurement were treated with 600 µl of 6% perchloric acid
(PCA) and homogenized on ice using an Ultra-Turrax T8 Homogenizor (IKA-Werke
Gmbh and Co. KG, Staufen, Germany). The samples were left on ice for 10 min
and then slowly neutralized by the addition of 160 µl 2 mol
l–1 K2CO3. The neutralised extract was
left for 15 min on ice before the samples were centrifuged at 10,000
g for 10 min, after which the supernatant was stored at
–80°C until further analysis.
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)
using a buffer solution consisting of Tris-HCl buffer (25 mmol
l–1 Tris base, 25 mmol l–1 Tris-HCl), NADH
(50–150 µmol l–1), 
-ketoglutarate (200
µmol l–1), lactic dehydrogenase (2.0 µgm
l–1) and glutamic-pyrovic transaminase (100 µgm
l–1). Glycogen measurements were made by adding 100 µl of
the glycogen extract to 900 µl acetate buffer (0.25 mol
l–1, pH 4.75) containing 40 mg l–1
amyloglucosidase (EC 3.2.1.3, Sigma-Aldrich Denmark A/S, Copenhagen). This
solution was kept at 25°C for 2 h until the amyloglucosidase had cleaved
all glycogen to glucose. Subsequently, glycogen content was determined using
the same procedure as for glucose measurements. Concentrations of glucose,
glycogen, alanine and succinate were all calculated relative to known
standards that had been through the same extraction procedure as the
samples. Lactate was measured using a YSI 1500 SPORT lactate analyzer (YSI, Yellow Springs, Ohio, USA) which was calibrated with a lactate standard of 5.0 mmol l–1.
Calorimetry
Calorimetry was used to assess cumulative anaerobic and aerobic metabolism
for unfrozen worms at 10, 6 and 2°C and for frozen worms at
–2°C. Frozen worms were placed at –2°C in a water bath in
glass cells 6 days prior to the measurements. A small piece of ice was added
to each cell to initiate freezing and ensure that the worms had been frozen
prior to measurements of heat production. After each test the survival status
was noted and only data for surviving worms were used. We applied a DSC 4207
(Hart Scientific, Provo, UT, USA) operated in the isothermal mode. Individual
animals were placed in hermetically closed 700 ml steel cells mounted in the
calorimeter and the heat flow was recorded over 3 h exposures to 10, 6, 2 and
–2°C, respectively. The equipment had one reference cell and three
cells for samples, and was therefore capable of simultaneous measurement of
three individuals' heat production. The baseline offset was measured in
separate trials using the same protocol and empty cells, and subtracted to
quantify the heat production of the worm.
Statistical analysis
Effects of exposure time were based on linear regression analysis
(SigmaStat 2.03) and an effect was considered significant at the
P<0.05 level. Differences in metabolite levels between treatment
groups (control, frozen survivors and frozen dead) were tested using a one-way
ANOVA on Ranks and a post-hoc Dunn's test to separate groups that
differed. All data are presented as mean ± s.e.m.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Mortality and recovery in laboratory experiment
There was only a slight and insignificant increase in freeze mortality over
the 47 day experimental freezing period
(Fig. 2; P=0.203).
Hence, 60–70% of the worms survived at the different times of
collection. Recovery time was noted for the individual worms, but this did not
change with the duration of frost exposure (data not shown). Thus, the average
time to regain activity was approximately 60 min throughout the experimental
period.
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Temporal changes in metabolite levels during freezing
The metabolite concentrations reported in
Table 1 are average values over
the entire experimental period. However, in the surviving frozen worms,
concentrations of the different metabolites change markedly over time whereas
there were no significant changes in any of the metabolites measured in the
untreated controls (Figs 3 and
4). In frozen worms that
survived there was a highly significant decrease in glucose
(Fig. 3A; P<0.001)
and also a tendency for a reduction in glycogen content although this was not
statistically significant (Fig.
3B; P=0.057). These changes were accompanied by increases
in lactate (Fig. 4A;
P<0.001) and alanine (Fig.
4B; P<0.001) but not succinate
(Fig. 4C; P=0.386). On
average the surviving worms metabolised more than half of their glucose
reserves during the 47 days in a frozen state. Thus, average glucose
concentrations of surviving earthworms was 124.1±24.3 µg
mg–1 dm 3 days into the frost period and this was reduced to
an average of only 49.1±13.4 µg mg–1 dm after 47
days of frost. The cumulative increase in alanine, lactate and succinate
between day 3 and day 47 was from 4.0 to 10.1 µg mg–1 dm
and this increase does, therefore, only account for 8.1% of the glucose
decrease.
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Calculations of metabolic rate
The working hypothesis of this study was that the metabolism of frozen
worms is entirely anaerobic. Using the declining slope of glucose
concentration in frozen worms (Fig.
3A) we calculated the ATP production rate to be 2.6 µmol ATP
g–1 wet mass (wm) day–1
(Fig. 5A) assuming a dry/wet
mass ratio of 0.16 (personal observations) and that 1 mol glucose results in
the generation of 2 mol ATP. If 1 mol glucose results in the formation of 6
mol ATP as would be expected from the formation of propionate (see
Discussion), we calculate the ATP consumption rate to be 7.9 µmol ATP
g–1 wm day–1. Finally if it is assumed that
only 8% of metabolism is anaerobic (corresponding with the observed increase
in lactate and alanine) and that 92% is aerobic (giving 36 mol ATP
mol–1 glucose) we estimate a metabolic rate of frozen worms
to be 44 µmol ATP g–1 wm day–1. These
values are all lower than the value for unfrozen worms at –2°C
estimated by simple Q10 effects, by factors of 90, 30 or
5.3, respectively (see Fig.
5A).
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;
Uvarov, 1998). In these
previous studies, oxygen consumption rates of D. octaedra were
determined to be an average of 83.5 µl O2 g–1
wm h–1 at 10°C and 59 µl O2
g–1 wm h–1 at 5°C. Assuming that 1 mol
O2 generates 6 mol ATP these oxygen consumptions can be
re-calculated to 536 and 379 µmol ATP g–1 wm
day–1 at 10 and 5°C, respectively. Since D.
octaedra does not show a critical thermal minimum and remains active
until freezing we used the Q10 from these respiration
measurements (Q10=2.43) to estimate the aerobic metabolism
of a supercooled worm at –2°C to be 233 µmol ATP
g–1 wm day–1
(Fig. 5A) which is a value
considerably higher than the estimates calculated from the glucose loss.
Calorimetry
In order to determine the metabolic rate of frozen worms we used
calorimetry, which gives a direct measurement of the heat produced by
metabolism. The calorimetric measurements were recalculated to ATP consumption
rates using a conversion of 2500 kJ mol–1 glucose and 36 mol
ATP mol–1 glucose (i.e. 70 kJ/mol ATP). Measurements at
10, 6 and 2°C showed a decrease in metabolism with temperatures
characterised by a Q10 of 3.76
(Fig. 5B). Using this
Q10 we estimated a value for metabolism of a supercooled
worm at –2°C to be 99 µmol ATP g–1 wm
day–1. The measured heat production of frozen worms at
–2°C corresponded to 10.0±2.8 µmol ATP
g–1 wm day–1 which is close to, but slightly
higher, than the values calculated assuming exclusively anaerobic metabolism
or mixed aerobic/anaerobic metabolism (compare
Fig. 5A and 5B).
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Irwin and Lee,
2002; Sinclair et al.,
2004). In earthworm species such as D. octaedra and
E. nordenskioldi it has previously been assumed that the rapid
accumulation of cryoprotectants serves to limit the extent and speed of ice
formation and hence reduce freeze-induced cell shrinking
(Holmstrup and Zachariassen,
1996; Overgaard et al.,
2007). However, it is also possible that glucose accumulation is
important for distributing fermentable energy resources before long-term
freezing events. Clearly this would help worms to maintain sufficient
anaerobic ATP production rates during extended periods in a frozen state. This
hypothesis is consistent with a number of observations. Generally,
osmolyte/cryoprotectant systems have converged to include a fairly limited
number of substances that are characterised by being compatible, even in high
concentrations, with sustained cellular and protein function
(Yancey et al., 1982).
However, earthworms and amphibians are the only animal groups that utilize
glucose as their primary cryoprotectant, although this osmolyte may have some
potential disadvantages. Thus, insects, bacteria and plants often use
osmolytes of lower molecular mass such as glycerol, betaine and TMAO
(trimethylamine N-oxide), which increases the number of osmotically
active particles, as compared to larger osmolytes such as glucose and
trehalose (Yancey et al.,
1982; Storey and Storey,
1996). The reasons for using glucose could relate to the natural history of freeze-tolerance in earthworms and amphibians. Both these animal groups overwinter in or near the soil surface and they cannot be expected to supercool to any considerable extent because their integuments are permeable to inoculative freezing from the external environment. Another general difference from more exposed cold hardy animals is that freeze tolerant amphibians and earthworms will probably experience relatively stable subzero temperatures that will rarely drop below –10°C and consequently they will be expected to experience few but long periods of freezing at relatively high subzero temperatures constraining these animals to anaerobic ATP production over long periods (see Fig. 1 for an example of soil temperature profiles from one of the most northerly distributions of D. octaedra). Clearly these animals cannot fully exploit the thermal reduction in metabolic rate that more cold exposed animals can, nor can they rely on occasional thaw periods because of the thermal inertia of the frozen soil in which they reside. The results of our study strongly support the hypothesis that D. octaedra relies on anaerobic metabolism of the accumulated glucose, as we found a highly significant gradual decline in glucose levels during the 47 days that worms were frozen (Fig. 3). When the slope of decreasing glucose concentration was recalculated to ATP consumption (assuming that all metabolism was anaerobic), the estimated metabolic rate was very similar to that measured empirically by calorimetry (Fig. 5).
The finding that glucose may be important as a metabolic fuel does not
diminish its role as a potentially important cryoprotectant/osmolyte. However,
glucose accumulation may have evolved primarily to support metabolic needs
during long-term freezing since the present study demonstrates that animals
that died during the long-term freeze test were characterised by fourfold
lower glucose and glycogen levels than those surviving
(Table 1). This suggests that
dead individuals succumbed because they had exhausted their energy reserves
during freezing. Mortality could obviously also be linked to the lower levels
of osmolytes/cryoprotectants in these individuals, or alternatively low
glucose levels could be a result of post-mortem microbial respiration.
However, previous studies using short-term freeze tests did not show mortality
linked to individual glucose levels of D. octaedra
(Overgaard et al., 2007), and
glucose levels are always highly variable between individuals of D.
octaedra, indicating that the low levels found in dead worms are not an
artefact of microbial respiration. Further support for this notion comes from
the vague interdependence of glucose accumulation and freeze survival in
D. octaedra populations from the northern hemisphere
(Holmstrup et al., 2007) and
among different freeze-tolerant earthworm species
(Holmstrup and Overgaard,
2007). Similarly, there was no clear relationship between the
ability to survive freezing and glucose accumulation when eight species of
hatchling freshwater turtles were compared with respect to freeze tolerance
(Costanzo et al., 2006).
Indeed, freeze-tolerant reptiles generally accumulate much lower levels of
putative cryoprotectants than freeze-tolerant anurans, insects or earthworms,
but freeze-tolerant reptiles are also only able to survive fairly short
episodes of freezing temperatures (Storey,
1990; Storey,
2006). It seems possible, therefore, that the level of glucose
accumulation in general may be more important for the ability to survive
freezing over long periods than for the actual absence/presence of freeze
tolerance in freeze-tolerant animals.
Anaerobic metabolism in frozen D. octaedra
In general, earthworms survive without oxygen for several hours or even
days at normal summer temperatures (Gruner
and Zebe, 1978). Under these conditions, energy will be generated
by anaerobic metabolism, although with poor efficiency, where the end product
can include a variety of compounds such as succinate, lactate, alanine,
valeric acid, propionate or acetate
(Hochachka et al., 1973;
Gruner and Zebe, 1978;
Pörtner et al., 1984;
Loomis et al., 1989;
Bundy et al., 2003).
It is often found that anoxia tolerance is an important physiological trait
favouring freeze tolerance and the working hypothesis of this study was that
metabolism of frozen D. octaedra is completely anaerobic. However the
evidence for facultative anaerobic metabolism in this study is confusing.
Thus, being frozen for 47 days at –2°C resulted in a small
accumulation of the anaerobic metabolic waste products, alanine and lactate,
in D. octaedra (Fig.
4). This increase only accounted for 8% of the decrease in glucose
levels and additional screenings of the entire metabolome by use of
1H-NMR spectroscopy [as described by Malmendal et al.
(Malmendal et al., 2006)]
showed no other obvious candidates for anaerobic waste products in the
extracts of frozen worms (data not shown). Although this may suggest that
worms were aerobic we found that the metabolic rate measured by calorimetry
was lower than that calculated from the glucose loss assuming a primarily
aerobic metabolism. Metabolic rates were only slightly higher than the values
calculated assuming that metabolism was exclusively anaerobic
(Fig. 5A) and the most probable
explanation for this discrepancy must be that the metabolism of frozen worms
was a mixture of aerobic and anaerobic metabolism. The anaerobic contribution
to total metabolism may be underestimated if only lactate and alanine are
taken into account as it is possible that additional waste products are lost
from the earthworm tissues, either in the frozen state during the experiment
or during the sample preparation. A number of facultatively anaerobic
invertebrates including several annelid species have the biochemical capacity
to metabolise succinate further to propionate during anoxia
(Surholt, 1977;
Gruner and Zebe, 1978;
Pörtner et al., 1984).
Production of propionate as the primary anaerobic waste product has previously
been demonstrated in different earthworm species such as Lumbricus
terrestris and L. rubellus
(Gruner and Zebe, 1978) and it
is possible that anaerobic D. octaedra produce propionate under
frozen conditions. The use of this metabolic pathway could favour long-term
anaerobic survival since the ATP yield per mole of glucose is higher
(Pörtner et al., 1984)
and it is possible that we missed this metabolite in our NMR measurements
since propionate is a highly volatile fatty acid. It has been shown that close
to 90% of this anaerobic waste product was lost to the surrounding water when
the marine annelid Arenicola marina was maintained anoxic for 48 h
(Surholt, 1977).
In this study the levels of anaerobic waste products were similar in
surviving and dead worms, and mortality was, therefore, not related to the
accumulation of anaerobic waste products
(Table 1). In fact, previous
studies have shown that the accumulation of some waste products such as
alanine may have a cryoprotective role
(Loomis et al., 1989;
Kukal et al., 1991). However,
further study of the biochemical pattern of anaerobic metabolism in D.
octaedra and in particular on the putative origin and fate of the
metabolic end products and their associated protons are needed to clarify the
potential effects of continued anaerobic metabolism. Thus, in vertebrates it
seems clear that the ability to tolerate long-term anoxia is tightly linked to
the handling of the ensuing acid load
(Lutz and Nilsson, 1997;
Jackson, 2002).
Metabolic depression and duration of freeze-tolerance in D. octaedra
The calorimetrically based estimates of ATP consumption rates generally
corresponded well with or were slightly lower than those calculated under
oxygenated and unfrozen conditions
(Uvarov, 1998) (compare
Fig. 5A and 5B). The
differences that there were may be partially attributed to differences between
the populations studied by Uvarov (Uvarov,
1998) and us, and/or experimental setup. Furthermore, the
calculated estimates are obviously very sensitive to the assumptions chosen to
calculate ATP turnover rate.
A major objective of measuring metabolic rate was to assess if a metabolic
depression occurred during freezing in D. octaedra and we
conclusively found this to be the case. Thus, regardless of the assumptions of
the mode of metabolism it seems clear that the worms undergo a marked
metabolic depression when frozen (see Fig.
5A). Furthermore, our results generally support the hypothesis
that metabolism of frozen worms is anaerobic although it is possible that
there is a slight contribution of aerobic metabolism. Thus, to arrive at the
same value as that measured calorimetrically it must be assumed that
84–94% of the glucose is metabolised anaerobically and only 6–16%
aerobically, depending on the assumptions regarding the anaerobic metabolic
pathway used. This assumption is in accordance with observations from the
freeze-tolerant lizard Lacerta vivipara where oxidative metabolism
persists, albeit at a lowered rate, even after ice equilibrium has been
reached (Voituron et al.,
2002b). Furthermore, different insect species also maintain some
degree of oxidative metabolism during extracellular freezing
(Irwin and Lee, 2002;
Sinclair et al., 2004) and our
results could suggest that frozen D. octaedra may also be able to
maintain a slight exchange of respiratory gasses and waste products. However,
this must be dependent on the proportion of frozen body water, which at
–2°C can be estimated to be vary between 40 and 80% depending on the
magnitude of the glucose accumulation
(Overgaard et al., 2007).
Moreover, it is possible that free-ranging earthworms become trapped in frozen
soil which would rapidly bring the oxygen supply to a minimum as has been
reported for other freeze-tolerant invertebrates
(Scholander et al., 1953;
Conradi-Larsen and Sømme,
1973; Sømme and
Conradi-Larsen, 1977).
Animals across a wide range of phyla lower their metabolic turnover when
stressed by various environmental factors and annelids have also previously
been shown to undergo metabolic depression when aestivating or when exposed to
low temperature and anoxia (Guppy and
Withers, 1999). Considering that frozen D. octaedra
undergo severe cellular hypoxia and dehydration when frozen it is not
unexpected that these worms also undergo metabolic depression under these
conditions. With regard to metabolic depression it is generally assumed that
freeze-tolerant animals have lower costs for maintenance of metabolic
functions than animals using a supercooling strategy
(Voituron et al., 2002a),
however, to our knowledge no previous study has estimated the long-term
metabolic rate in frozen annelids and records are generally scarce regarding
long-term studies of freeze-tolerant animals. In the present study, the most
reliable estimate of metabolic rate is that measured by calorimetry since this
measures the heat of metabolism directly without bias regarding assumptions of
the metabolic pathway. Using this method we found an estimated 90% reduction
of metabolism in frozen worms, which is well within the `normal' rate of
metabolic depression (Guppy and Withers,
1999) but slightly larger than the 50 to 70% depression found in
the frozen insects Eurosta solidaginis and Upis ceramboides,
respectively (Lundheim and Zachariassen,
1993; Irwin and Lee,
2002). A large decrease in metabolic rate was also found in the
sub-Antarctic caterpillar Pringleophaga marioni
(Sinclair et al., 2004),
however, in this species the metabolic depression was related to the critical
thermal minimum rather than the process of freezing and it was suggested that
metabolic depression is linked with reduced Na+/K+ pump
activity and failure to maintain ion gradients.
In D. octaedra metabolic depression is probably a result of
several different mechanisms including reduced activity and the presumed
reduction in energy used on growth, reproduction, digestion and excretion.
Metabolic depression is also generally associated with a number of biochemical
changes including decreased pH, protein phosphorylation and prioritization of
some cellular function (Na+/K+ pump) over others
(protein synthesis) (Hand and Hardewig,
1996; Guppy and Withers,
1999; Cowan and Storey,
2003). Although frozen D. octaedra may conform to these
general trends, a preliminary study found that extracellular [K+]
increased markedly following frost exposure (Marie Rohde, personal
communication) suggesting a possible reduction in Na+/K+
pump activity. Other freeze-tolerant animals, such as the larvae of E.
solidaginis, have also been shown to suppress
Na+/K+ pump activity during winter acclimation
(McMullen and Storey, 2008),
and it was also hypothesised that Na+/K+ pump activity
becomes insufficient to maintain ion balance in the freeze-tolerant
caterpillar P. marioni (Sinclair
et al., 2004). It is, therefore, possible that a reduction in
Na+/K+ pump activity also contributes to the metabolic
depression of D. octaedra.
Most worms survived the 47 day freeze exposure used in this experiment and
given the estimated metabolism of 8.3 µmol glucose g–1 dm
day–1 (Fig. 3)
the `average' worm would be able to survive for 83 days before glucose
supplies were exhausted. This may be sufficient to survive winter in the
southerly distribution ranges of D. octaedra. However, as illustrated
in Fig. 1, winter may be
considerably longer for northerly distributed populations. D.
octaedra exhibit a large variation between individuals in the amount of
glucose accumulated and also large variation in the magnitude of the glycogen
stores of autumn-collected worms
(Rasmussen and Holmstrup,
2002; Holmstrup et al.,
2007; Overgaard et al.,
2007). Such variance may naturally affect the projected survival
time as some individuals may have glucose levels of 1100 µmol glucose
g–1 dm, and glycogen levels prior to frost have in a few
cases been measured at levels higher than 1500 µmol glucosyl units
g–1 dm. Populations with large glycogen reserves, such as
those found in Greenland, Finland, Poland and Canada, may also be more
tolerant to long-term freezing than populations from Denmark and Sweden, which
have smaller glycogen stores (Holmstrup et
al., 2007). The projected survival time will also depend on
external conditions such as temperature where a low soil temperature will
decrease the use of glucose reserves, and therefore possibly extend the
duration of freeze survival.
In conclusion, the main result of our study indicate a novel role of glucose in survival of long-term freezing, namely that glucose usage as fuel for metabolism is perhaps the most important function. Our results therefore could be the opening of a new view on the roles of so-called cryoprotectants for winter survival.
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Footnotes |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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