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First published online February 12, 2007
Journal of Experimental Biology 210, 836-844 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02714
Slow dehydration promotes desiccation and freeze tolerance in the Antarctic midge Belgica antarctica
1 Department of Entomology, Ohio State University, Columbus, OH 43210,
USA
2 School of Biological Sciences, Liverpool University, Liverpool L69 7ZB,
UK
3 Red River Valley Station, USDA-ARS, Fargo, ND 58105, USA
4 Department of Zoology, Miami University, Oxford, OH 45056, USA
* Author for correspondence (e-mail: salh{at}liv.ac.uk)
Accepted 11 January 2007
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30% loss of OAW, and dramatically increased the
freeze tolerance of larvae to –10 and –15°C. The supercooling
point of animals was not significantly altered by this desiccation treatment,
and all larvae were frozen at –10°C. This is the first evidence of
desiccation increasing the freeze tolerance of a polar terrestrial arthropod.
Maximum water loss and body fluid osmolality were recorded after 5 days at
98.2% RH, but osmolality values returned to predesiccated levels following
just 1 h of rehydration in water, well before all the water lost through
desiccation had been replenished. This suggests active removal of osmolytes
from the extracellular fluids during the desiccation process, presumably to
intracellular compartments. Heat-shock proteins appear not to contribute to
the desiccation tolerance we observed in B. antarctica. Instead, we
suggest that metabolite synthesis and membrane phospholipid adaptation are
likely to be the underpinning physiological mechanisms enhancing desiccation
and cold tolerance in this species.
Key words: desiccation, freezing tolerance, heat-shock proteins, Chironomidae, polar insects
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). Polar terrestrial environments are also effectively deserts
(Campbell and Claridge, 1987),
with adaptations to low moisture availability perhaps even more crucial than
cold resistance (Kennedy,
1993). For the resident invertebrate fauna, this combination of
conditions poses significant problems. As poikilotherms, their tissues, cells
and molecules are exposed to the full effects of temperature variation,
necessitating major adaptive responses to cold stress. Maintenance of water
balance is also a particularly acute problem for invertebrates, owing to their
high surface area to volume ratio (Hadley,
1994). Intriguingly, cold and desiccation response mechanisms
demonstrate many similarities, including the use of low molecular mass solutes
such as sugars and polyols (Young and
Block, 1980; Womersley and
Smith, 1981; Bayley and
Holmstrup, 1999), membrane lipid alterations
(Bennett et al., 1997;
Holmstrup et al., 2002a) and
the expression of molecular chaperones such as heat-shock proteins (Hsps)
(Rinehart et al., 2000;
Hayward et al., 2004). A
connection between cold and desiccation tolerance has been identified in
several invertebrate species, including nematodes
(Forge and MacGuidwin, 1992),
earthworms (Holmstrup and Zachariassen,
1996), tardigrades
(Sømme, 1996),
Collembola (Worland et al.,
1998) and insects (Ring and
Danks, 1994; Williams et al.,
2004). This has led to the conclusion that many of the
physiological and molecular responses to cold may have originally been
adaptations for desiccation stress (Ring
and Danks, 1994; Block,
1996; Danks,
2000).
For invertebrates with high integumental permeability, e.g. nematodes,
enchytraeids, euedaphic Collembola and chironomid larvae, the risk of
desiccation is particularly acute, yet these groups dominate the soil fauna in
both the Arctic and Antarctic (Peterson and Luxton, 1982;
Convey and Block, 1996;
Wharton, 2003). It has been
assumed that behavioural strategies of desiccation avoidance play a crucial
role in survival (Hayward et al.,
2000; Hayward et al.,
2001), but considering the fact that relative humidities (RHs) as
high as 98.2% RH can pose a significant desiccation risk
(Bayley and Holmstrup, 1999),
moist refuges with conditions near 100% RH may be scarce at high latitudes.
The dehydration tolerance of polar invertebrates has typically been assessed
under extreme conditions, e.g. 35% RH
(Worland and Block, 1986), and
generally these organisms are thought to have no physiological or metabolic
means of regulating water loss (Harrison et al., 1991). For euedaphic
invertebrates, however, Bayley and Holmstrup highlighted the importance of
performing desiccation experiments at more ecologically relevant RH values
(Bayley and Holmstrup, 1999),
and in particular those close to the wilting point of plants (98.9% RH).
Under conditions of 98.2% RH, they identified a physiological response in the
collembolan Folsomia candida, in which animals were able to actively
combat water loss by regulating their internal osmotic pressure through sugar
and polyol synthesis (Bayley and Holmstrup,
1999). Interestingly, this desiccation response also resulted in
an increased tolerance to further desiccation
(Sjursen et al., 2001) and
cold (Bayley et al., 2001)
stress. The capacity of polar terrestrial invertebrates to employ a similar
strategy of cross-tolerance has not been addressed, but it could play an
important role given that sugar and polyol synthesis represent perhaps the
most important biochemical adaptation to both cold and desiccation
(Storey, 1997).
The chironomid Belgica antarctica is the largest entirely
terrestrial animal that lives in Antarctica, and it has the most southerly
distribution of any free-living holometabolous insect
(Sugg et al., 1983;
Usher and Edwards, 1984).
Endemic to the Antarctic Peninsula and its islands, B. antarctica has
a two-year life cycle that includes four edaphic larval stages
(Sugg et al., 1983).
Overwintering can occur in any of the four larval instars, whereas the
wingless adults live for fewer than 10 days during the brief Antarctic summer
(Convey and Block, 1996).
Larvae survive extracellular freezing, making B. antarctica the only
freeze-tolerant insect on the continent, and can tolerate temperatures down to
approximately –20°C (Baust and
Lee, 1987; Lee et al.,
2006). This is a somewhat modest level of cold tolerance in
comparison with freeze-avoiding Antarctic species (see
Cannon and Block, 1988), but
it is more than sufficient for survival considering the thermal stability of
B. antarctica's snow-covered microhabitat, which typically remains
between 0 and –2°C and rarely falls below –7°C
(Baust and Lee, 1981). In
common with many Chironomidae, B. antarctica larvae are not
particularly resistant to water loss, but are highly desiccation tolerant
(Baust and Lee, 1987). Indeed,
B. antarctica appears highly resilient to a diverse range of
environmental stressors (Baust and Lee,
1987), but few detailed physiological studies of this unique
species have been undertaken.
This study is the first detailed assessment of the physiological response of an Antarctic terrestrial invertebrate to ecologically realistic desiccation stress. By investigating water loss at RH values specifically relevant to the soil environment, we have identified a level of desiccation tolerance in B. antarctica masked by more severe RH treatments. In addition, we tested the hypothesis that physiological adaptations to desiccation stress promote cross-tolerance to freezing in this species. We conclude that B. antarctica larvae can survive the loss of >75% of their osmotically active water (OAW) under gradual desiccation and rehydration, and the loss of as little as 30% of their OAW significantly increases their freeze tolerance. Heat-shock proteins (hsps) appear not to contribute to the desiccation response, and we predict that osmolyte and/or cryoprotectant synthesis and membrane phospholipid adaptation underpin the dramatic increase in freeze tolerance noted in desiccated larvae.
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Materials and methods |
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Desiccation treatments
Larvae were placed on nylon gauze netting (pore diameter 100 µm), which
was then placed across the top of 50 ml centrifuge tubes containing 35 ml
aqueous NaCl solutions. Saturated NaCl solutions provided 75% RH conditions,
whereas 98.2% RH was achieved using 31.6 g of NaCl l–1 water
(Bayley and Holmstrup, 1999).
Controls were maintained under 100% RH conditions using demineralized water.
The midge larvae (N=10 per tube) were thus suspended above the
aqueous solution on the nylon gauze, which was then secured in position by a
tightly fitting lid. The air in this small closed system rapidly equilibrated
with the solution following Raoult's law. All desiccation treatments were
performed in an environmental chamber that maintained a temperature of
4.0±0.2°C.
Survival and water loss
Samples were removed from each desiccation regime at set intervals over a
12-day period. Survival was assessed after 24 and 48 h of rehydration in
water, and determined by the ability of larvae to move following gentle
tactile stimulation.
Groups of 10 animals were weighed prior to desiccation (fresh mass), upon removal from each desiccation treatment (desiccated mass), and after 24 and 48 h of rehydration (rehydrated mass). Samples were then dried to constant mass at 60°C and their dry mass (DM) noted. From these values, mean initial water content and percentage water loss or gain could be calculated. At least three replicates of 10 animals were used for each time point under each treatment for both water content and survival assessments.
Osmotically active water (OAW) content and body fluid osmotic pressure
OAW content and body fluid osmotic pressure were calculated for larvae
desiccated at 98.2% RH. Total water content was converted to osmotically
inactive water (OIW) content according to the formula given in Worland et al.
(Worland et al., 1998). In
undesiccated controls the mean OIW content was 0.49±0.01 g water
g–1 DM (N=5), equating to 16% of the total
water content. This value, generated for each sample, was then subtracted from
total water content to give OAW content, expressed as g OAW
g–1 DM. The osmolality of body fluids was determined using a
vapour pressure depression technique
(Holmstrup and Sømme,
1998). Groups of 10 larvae were placed in a sample holder and
quickly crushed with a Teflon rod to expose the body fluids. The sample was
then allowed to equilibrate for 30 min following placement within a C-52
sample chamber, which was connected to a Wescor HR 33T Dew Point
Microvoltometer operated in the dew-point mode (Wescor Inc., Logan, UT, USA).
This voltage reading was converted to osmotic pressure (bar) using van't
Hoff's equation (1 bar=100 kPa). At least three replicates were performed for
each time point during the desiccation and rehydration treatments.
Changes in osmolality and mass during rehydration
To assess the time frame of the rehydration process, and whether this
changed depending on the extent of desiccation, the following experiment was
performed. Samples were desiccated for either 24 h or 120 h (5 days) at 98.2%
RH and then transferred to Eppendorf tubes containing water. The mass and
osmolality of larvae was recorded before and after desiccation, and following
either 1 or 24 h of rehydration, with surface water removed by blotting with
filter paper. Three replicates (N=10 larvae) were performed for each
data point.
Survival of freezing
To determine whether prior desiccation enhances the freezing tolerance of
larvae, groups of 10 animals were transferred to 1.5 ml Eppendorf tubes and
placed at either –10 or –15°C after 48 h at 98.2% RH. Larvae
were removed at set intervals over a further 72 h period and transferred back
to 4°C. At least three replicates were performed for each treatment and
survival was assessed as previously described.
Supercooling point (SCP) measurements
Larvae desiccated for 48 h at 98.2% RH and controls (blotted dry with
tissue paper) were placed in direct contact with a thermocouple cooled from 4
to –25°C at a rate of 1°C min–1. The SCP was
taken as the lowest temperature reached prior to the release of the latent
heat of fusion as the result of freezing of the body water.
Assessing hsp expression by northern blot hybridization
Clones of the genes encoding Hsp70 (GenBank accession number DQ459546),
Hsp90 (DQAA459547), a small Hsp (DQ459548) and a 28s ribosomal RNA fragment
(DQ459549) used as a control were described previously from B.
antarctica (Rinehart et al.,
2006).
Total RNA for northern blot hybridization was isolated using Trizol reagent from larvae desiccated under the following conditions: 0% RH for 20 h; 75% RH for 6, 24 and 48 h; and 98.2% RH for 6, 24, 96 and 120 h (N=25 per treatment). Twenty micrograms of each sample was heat denatured and separated by electrophoresis on a 1.5% agarose, 0.41 mol l–1 formaldehyde gel, transferred to a charged nylon membrane (Osmonics, Inc.) using a Turbo-Blotter (Schleicher and Schuell) and crosslinked by UV irradiation. Clones (hsp70, hsp90 and 28s) were digoxigenin (DIG)-labeled using DIG-high prime solution (Roche Applied Sciences, Inc.) for use as probes in northern hybridization using the Dig High Prime DNA Labeling and Detection Starter Kit II (Roche Applied Sciences, Inc.) following standard protocol. BioMax Chemiluminescence film (Kodak, Inc.) was then exposed to the blot for signal detection. Equal loading of samples was confirmed by alkaline stripping the membrane using 0.2 mol l–1 NaOH, 0.1% sodium dodecyl sulfate (SDS) followed by reprobing with the 28s rRNA probe. All northern analyses were run in triplicate.
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When water loss exceeded 50% larvae appeared shrivelled and
motionless, although they rapidly rehydrated upon transfer to water, returning
to their `normal' shape within 24 h. Survival did not differ significantly
between larvae rehydrated for 24 or 48 h, thus survival data are presented
only for larvae rehydrated for 24 h.
Larvae maintained at 100% RH and 4°C experienced no significant changes
in water content or mortality throughout the 12-day experimental period
(Fig. 1A,B). Water was lost
rapidly at 75% RH: within 2 days larvae lost >70% of their initial water
content, retaining less than 1 g water g–1 DM
(Fig. 1A). Survival also
declined sharply at 75% RH, and no larvae survived more than 5 days at this
relative humidity (Fig. 1B).
The rate of water loss was considerably slower at 98.2% RH: within 2 days
larvae still had 2.0 g water g–1 DM, equating to a loss
of less than 30% of their initial water content
(Fig. 1A), and survival was
close to 100% (Fig. 1B).
Maximum water loss at 98.2% RH (70%) occurred after 5 days, and was the
only point at which the water content of larvae dropped below 1 g water
g–1 DM at this humidity
(Fig. 1A). The relationship
between water content and survival differed between 75 and 98.2% RH
(Fig. 1C), with survival after
5 days at 98.2% RH (50% survival at 0.86 g water g–1 DM)
significantly higher than for the equivalent water content at 75% RH (30%
survival of 0.88 g water g–1 DM, two-tailed t-test;
P<0.05). Between 5 and 12 days at 98.2% RH, the water content
remained relatively stable (Fig.
1A), although survival declined slightly during this period
(Fig. 1B).
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Effect of desiccation on OAW and osmotic pressure
The mean OAW content of larvae declined more than fourfold, from 2.3 g
g–1 DM to 0.5 g g–1 DM during the first 5
days of desiccation at 98.2% (Fig.
1D). Thus, 50% of larvae survived the loss of more than 75%
of their OAW. The OAW content of larvae then remained stable between 5 and 12
days.
In accordance with the loss of OAW, a consistent and significant increase in the osmotic pressure and/or osmolality of body fluids was observed during the first 5 days of desiccation at 98.2% RH (Fig. 1E, two-tailed t-test; P<0.01). In fact, after 5 days at 98.2% RH, larvae reached an osmotic pressure exceeding the water potential of the environment, approximately –25 bar. Although a slight decrease in the osmolality of body fluids was noted between 5 and 7 days, the osmotic pressure did not decline significantly below that of ambient conditions. After 7 days at 98.2% RH the osmotic pressure of body fluids again increased and remained hyperosmotic in relation to the environment for the remainder of the experiment.
Influence of rehydration rate on survival
To assess the impact of rehydration rates following desiccation in B.
antarctica we compared survival of larvae that were rehydrated either by
submergence in water or by being transferred to 100% RH
(Fig. 2). After 3 days of
desiccation, corresponding to the loss of 40% of initial water content,
the difference in survivorship between the two rehydration regimes was not
significant (two-tailed t-test; P=0.24). The survivorship of
larvae rehydrated at 100% RH after 7 or 12 days of desiccation at 98.2% RH,
however, was significantly higher than that for larvae rehydrated in water
(two-tailed t-test; P<0.05 for both time points). Thus,
the rate of rehydration has a significant influence on desiccation
survival.
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The mean SCP value for undesiccated control larvae (±s.e.m.) was –8.6±0.9°C (N=11; range=–3.8 to –11.9°C; two individuals recorded SCP values below –10°C). The mean (±s.e.m.) SCP value of desiccated larvae was –9.3±0.4°C (N=8; range=–7.9 to –11.3°C, with two individuals recording SCP values below –10°C). SCP values were not significantly different between treatments (two-tailed t-test; P=0.52).
Hsp expression
Transcripts encoding Hsp70, Hsp90 and a small Hsp were already upregulated
in the midge larvae, and desiccation failed to further upregulate these
transcripts, regardless of the severity of desiccation stress
(Fig. 5).
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).
Yet even recent studies of polar terrestrial invertebrates persist in
assessing desiccation tolerance under conditions below 5% RH (e.g.
Sinclair et al., 2006). The
ecological relevance of measuring desiccation tolerance under such extreme
conditions is at best questionable but, more importantly, is likely to mask
subtle physiological responses that may occur under buffered moisture
conditions typically found in the soil environment. Based on the data
presented here, B. antarctica exhibits the most extreme desiccation
tolerance ever recorded in a polar insect. However, given the preponderance of
soil organisms with high cuticular permeability at polar latitudes, similar
physiological attributes may be widespread when assessed under appropriate
humidity conditions.
The slow rate of desiccation recorded in B. antarctica at 98.2% RH
significantly increased survival rates above those of larvae desiccated at 75%
RH, and enabled more than 50% of animals to survive the loss of >75% of
their OAW. Survival was further enhanced by rehydration at 100% RH rather than
direct contact with water (Fig.
2). Under similar desiccating conditions, certain species of
Collembola accumulate polyols and sugars to actively combat water loss
(Bayley and Holmstrup, 1999;
Holmstrup et al., 2001), and
it seems reasonable to assume that similar metabolites contribute to the
enhanced survival of desiccated B. antarctica larvae. Furthermore, an
accumulation of metabolites under slow desiccation (this time acting as
cryoprotectants) could explain the dramatically increased freeze tolerance of
desiccated B. antarctica larvae at –10 and –15°C.
Interestingly, the initial OAW content of B. antarctica, 2.3 g
water g–1 DM (Fig.
1D), was more than double that recorded in F. candida
prior to desiccation (1 g water g–1 DM)
(Bayley and Holmstrup, 1999).
This, in itself, may represent a desiccation tolerance mechanism in the polar
insect, and perhaps explains why B. antarctica larvae were able to
lose such a significant proportion of their OAW with only limited mortality.
Increased body water content has been identified as a selected characteristic
in desiccation-tolerant lines of Drosophila melanogaster
(Gibbs, 2002), and although a
high OAW content may represent a risk to freeze-avoiding species at high
latitudes, it is unlikely to pose a problem to the freeze-tolerant B.
antarctica. Another possible adaptation to an arid environment was noted
in the coiling and aggregative behaviour of B. antarctica larvae
during desiccation; animals desiccated in groups of 10 tended to entangle into
a `ball' of larvae as they became more dehydrated, and even individual larvae
coiled their bodies upon desiccation, perhaps as a strategy to reduce surface
area to volume ratio. Interestingly, we also observed this strategy operating
in the field, where larvae were found in dense aggregations at dry sites but
were more evenly dispersed in wet habitats. One problem with desiccating
multiple larvae, however, is that subtle differences in this coiling behaviour
could produce slightly different rates of water loss. This could explain the
somewhat inconsistent slope of the desiccation curve noted at 98.2% RH
(Fig. 1A).
The osmotic pressure of B. antarctica body fluids after 12 h of
desiccation at 98.2% RH (Fig.
1D) was approximately –12.5 bar. After 120 h (5 days), when
they have four times less OAW, their water potential deficit should be
approximately –50 bar, but instead the value was –33 bar. As
relatively few cells were ruptured when determining osmolality, extracellular
fluids, especially the haemolymph, predominantly contribute to this value.
Thus, osmolytes must have been removed from these extracellular fluids. The
dry mass of samples did not significantly change during the desiccation
treatment, indicating that there was no overall gain or loss of metabolites
and/or osmolytes during the desiccation treatment. Instead, osmolytes may have
been redistributed, for example, to intracellular compartments. The
acquisition of sugars in the cytoplasm of cells is thought to be a fundamental
component of successful anhydrobiosis
(Crowe et al., 2002), and
presumably would also contribute to less extreme examples of desiccation
tolerance. The intracellular partitioning of these and other compounds may
also play a crucial role during desiccation
(Oliver et al., 2002).
For B. antarctica, a reduced concentration of osmolytes in the haemolymph would mean that, upon rehydration, less water is required than that lost to return the haemolymph osmolality to its predesiccated value – providing, of course, that rehydration occurred at a faster rate than osmolyte transfer back out of the cells. This idea appears to be supported by the data presented in Fig. 3, in which the osmolality of body fluids returns to predesiccated levels within 1 h of rehydration, despite the fact that larvae have not yet regained all the water lost from desiccation, i.e. their mass was significantly different from that prior to desiccation. These data, therefore, suggest that osmolytes may be redistributed during the desiccation process, and that intracellular rehydration occurs more slowly than extracellular rehydration.
The active removal of osmolytes from the haemolymph to intracellular
compartments could be advantageous in many respects. This strategy would
reduce the initial rate of rehydration upon contact with water, which is known
to affect survival (Fig. 2)
(Bayley and Holmstrup, 1999).
An increased concentration of sugars and/or polyols is also likely to enhance
cellular and membrane integrity during both desiccation and freezing
(Crowe et al., 1984;
Crowe et al., 1992;
Sano et al., 1999;
Takagi et al., 2000). This
could explain the increased cold tolerance of desiccated samples
(Fig. 4). Removal of osmolytes
from the haemolymph is also consistent with the idea that a freeze-tolerant
organism should not have a low SCP value. In this regard, it should be noted
that the SCP values in samples desiccated for 48 h were only slightly reduced,
compared with controls, an observation that is somewhat surprising given the
increase in osmotic pressure during this period. However, it is possible that
other mechanisms, possibly the synthesis of ice nucleators, contribute to
maintaining a high SCP; indeed, maintaining a high SCP would be made easier if
osmolytes and/or cryprotectants were removed.
A link between enhanced cold tolerance following desiccation has long been
established (Ring and Danks,
1994; Ring and Danks,
1998) and, in B. antarctica, 48 h at 98.2% RH resulted in
a dramatic increase in survival at –10 and –15°C, relative to
undesiccated controls (Fig. 4).
This result concurs with Bayley et al.
(Bayley et al., 2001), who
found that 7 days at 98.2% RH significantly increased the cold tolerance of
F. candida, although, unlike the collembolan, Hsps appear not to
contribute to this response. In B. antarctica larvae, hsp70,
hsp90 and a small hsp gene transcripts are constitutively
expressed and cannot be further upregulated by exposure to either high or low
temperature (Rinehart et al.,
2006). This pattern of expression is similar to that observed in
the overwintering diapause of some temperate-zone species, e.g. the flesh fly
Sarcophaga crassipalpis, in which hsp70 and small
hsps are expressed throughout pupal diapause, but remain unresponsive
to temperature stress (Yocum et al.,
1998; Rinehart et al.,
2000; Hayward et al.,
2005). Interestingly, these hsp transcripts are also
unresponsive to desiccation stress during diapause in S. crassipalpis
(Hayward et al., 2004). Thus,
Hsps appear to contribute to an underlying enhanced stress tolerance of the
midge larvae (Rinehart et al.,
2006), but are not further upregulated in response to cold or
desiccation.
The increased cold tolerance noted in desiccated B. antarctica
larvae was not the result of a reduced SCP, as these values were not
significantly different between desiccated samples
(–9.3±0.4°C) and controls (–8.6±0.9°C).
Furthermore, as values remained above –10°C, larvae presumably froze
at some point during the 3-day cold treatments. This work therefore represents
the first evidence of gradual desiccation increasing the freezing tolerance of
a polar arthropod. This strategy is quite different from cryoprotective
dehydration, employed by certain Collembola and earthworm cocoons
(Holmstrup and Westh, 1995;
Holmstrup and Sømme,
1998), in which water loss, and an associated drop in SCP,
continues until water potential equilibrium between the organism and ice is
attained (Zachariassen, 1991;
Lundheim and Zachariassen,
1993; Holmstrup et al.,
2002b), preventing the animal from freezing.
In the anhydrobiotic nematode Aphelenchus avenae, ice formation
does not occur below a water content of 0.3 g water g–1 DM
(Crowe et al., 1983), whereas
for Artemia cysts the value is 0.6 g water g–1 DM
(Crowe et al., 1981). After 48
h at 98.2% RH, the group for which cross-tolerance was assessed, the water
content of B. antarctica larvae was >2 g water
g–1 DM. The lowest water content recorded at this humidity
was 0.86 g water g–1 DM
(Fig. 1C), suggesting that even
the most desiccated larvae were still susceptible to freezing. Cross-tolerance
was not assessed for this level of water loss, however, as there would have
been compounding mortality factors resulting from both desiccation and cold
stress. Indeed, cross-tolerance data for F. candida
(Bayley and Holmstrup, 1999)
was complicated somewhat by the fact that the desiccation treatment used
resulted in some mortality, which may explain the relatively limited
cross-tolerance to cold noted in this species
(Bayley et al., 2001). That
aside, the synthesis of polyols and sugars
(Bayley and Holmstrup, 1999)
and alterations in membrane phospholipid composition
(Bayley et al., 2001) seem the
most likely physiological mechanisms contributing to increased cold tolerance
noted in F. candida.
It seems likely that similar mechanisms underpin the desiccation response
of B. antarctica, but in this instance facilitate enhanced freezing
tolerance. Limited data exists regarding the principal stress-response
metabolites of B. antarctica. However, a preliminary analysis of
desiccation-responsive metabolites, using Fourier Transform Infrared (FTIR)
spectroscopy and a discrimination function analysis, indicated that the
polysaccharide region of the spectra is altered significantly in response to
desiccation. A variety of polyhydric alcohols and sugars, including erythritol
and trehalose, have already been identified in larvae of this species
(Baust and Edwards, 1979;
Baust and Lee, 1983) and
represent excellent water replacement molecules that could facilitate
increased desiccation and freezing tolerance
(Holmstrup and Westh, 1995;
Worland et al., 1998;
Sano et al., 1999).
The capacity to tolerate prolonged periods of low moisture availability is of considerable adaptive significance to polar terrestrial organisms. Yet, despite the apparent harshness of high-latitude terrestrial habitats, the buffered moisture status of the soil substrate should not be disregarded. This study highlights the crucial importance of performing desiccation tolerance experiments under ecologically relevant humidity conditions. The extreme desiccation tolerance noted in B. antarctica at high relative humidities also suggests that this parameter should perhaps be reassessed in other polar terrestrial invertebrates. Our study lends yet further support to the idea that adaptations to desiccation stress promote enhanced cold tolerance, and provides the first evidence that gradual desiccation can enhance the lower limit of freeze tolerance in a polar arthropod. The slow rate of water loss, which occurs in permeable edaphic invertebrates at humidities approaching the wilting point of plants (e.g. 98.2% RH), is as relevant to polar species, as it is for temperate and tropical soil faunas. Furthermore, as demonstrated here, such conditions can facilitate the survival of extensive water loss, and permit the identification of more subtle desiccation and cold tolerance strategies.
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Acknowledgments |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Baust, J. G. and Edwards, J. S. (1979). Mechanisms of freezing tolerance in an Antarctic midge, Belgica antarctica. Physiol. Entomol. 4, 1-5.
Baust, J. G. and Lee, R. E., Jr (1981). Environmental "homeothermy" in an Antarctic insect. Antarct. J. US 15,170 -172.
Baust, J. G. and Lee, R. E., Jr (1983). Population differences in antifreeze/cryoprotectant accumulation patterns in an Antarctic insect. Oikos 40,120 -124.
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