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First published online May 30, 2008
Journal of Experimental Biology 211, 1903-1910 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.017558
Slow desiccation improves dehydration tolerance and accumulation of compatible osmolytes in earthworm cocoons (Dendrobaena octaedra Savigny)
1 National Environmental Research Institute, University of Aarhus, Department of
Terrestrial Ecology, Vejlsøvej 25, 8600 Silkeborg, Denmark
2 Department of Zoophysiology, University of Aarhus, 8000 Aarhus C,
Denmark
3 Center for Insoluble Protein Structures (inSPIN), Interdisciplinary
Nanoscience Center (iNANO) and Department of Chemistry, University of Aarhus,
8000 Aarhus C, Denmark
* Author for correspondence (e-mail: martin.holmstrup{at}dmu.dk)
Accepted 2 April 2008
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Summary |
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2 mol l–1 and 1 mol
l–1 total osmolytes, respectively. However, in addition to
osmolyte accumulation, the gradually desiccated cocoons also tolerated a
higher degree of water loss, demonstrating that gradually dehydrated
D. octaedra cocoons are able to survive loss of 95% of
the original water content. Although D. octaedra embryos can
probably not be categorized as a truly anhydrobiotic organism we propose that
they belong in a transition zone between the desiccation sensitive and the
truly anhydrobiotic organisms. Clearly, these earthworm embryos share many
physiological traits with anhydrobiotic organisms.
Key words: anhydrobiosis, betaine, dehydration, sorbitol, water loss
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). If soils become too dry, earthworms often move to deeper
soil layers where moisture conditions are more favourable because even small
decreases in soil water potential may cause a quick loss of body water.
Earthworm egg capsules (`cocoons') are, however, mostly situated in the upper
soil layers (Gerard, 1967) and
must therefore depend on tolerance of desiccation during dry periods. This is
in particular the case for surface dwelling species that place their cocoons
in leaf litter on the soil surface
(Rundgren, 1975).
The moss worm, Dendrobaena octaedra, lives in the litter layer of
the forest floor and under moss or lichens. It is a widely distributed species
and is found in most of the European forest zone and the European tundra,
Eastern Siberia, North America and Greenland
(Stöp-Bowitz, 1969). The
cocoons of D. octaedra are often exposed to desiccating conditions
during dry periods because of their surface habitat choice. Previous studies
have shown that cocoons of D. octaedra are very tolerant to
desiccation in comparison with other earthworm species
(Holmstrup and Westh, 1995).
Thus, about 50% of cocoons from a Danish population tolerate exposure to 93%
relative humidity (RH) for 14 days
(Holmstrup and Westh, 1995).
The permanent wilting point of plants is by convention set at 98.9% RH so a RH
of 93% represents a severe level of desiccation. Because of their location in
the topsoil, however, it is likely that cocoons may need to tolerate even
lower humidities. The embryos within these cocoons have therefore evolved
physiological adaptations to meet this desiccation stress and they are able to
tolerate the loss of practically all osmotically active water (approximately
85% water loss) (Holmstrup and Westh,
1995). Dehydration can be harmful to the embryos because cellular
shrinkage is potentially damaging, with proteins becoming irreversibly
denatured in dehydrated cells, and because cellular membranes may lose their
normal conformation in a liquid crystalline state
(Crowe et al., 1992). However,
it is well known that protective substances such as sugars and polyhydric
alcohols, or other compatible osmolytes, may ameliorate these effects
(Crowe, 2002;
Yancey, 2005). In addition to
protein-protective effects, compatible osmolytes may also limit the water loss
of the embryo during desiccation by their osmotic effects even though examples
of this are rare in the literature (Bayley
and Holmstrup, 1999).
It is clear that the survival of dehydration by invertebrates depends not
only on degree of water loss, but also on the rate of dehydration. Previous
reports have shown that pre-acclimation to a relatively mild desiccation
stress can improve severe desiccation tolerance in other soil invertebrates
such as nematodes, Collembola and midge larvae
(Hayward et al., 2007;
Sjursen et al., 2001;
Womersley and Ching, 1989). In
natural environments, soil organisms will be subjected to gradually increasing
desiccation stress because drying of soils is a buffered process occurring
over periods of days or weeks rather than hours. Studies that explore the
water balance, production of compatible osmolytes and survival of soil
organisms within these time and moisture scales have therefore greater
ecological relevance than studies using acute exposure regimes. However, such
studies are rare and have not yet been performed on earthworm cocoons. The aim
of the present study was therefore to simulate naturally occurring desiccation
rates and investigate how rate affects the responses of D. octaedra
in terms of desiccation tolerance, survival and accumulation of compatible
osmolytes.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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).
Briefly, the earthworms were kept at 15°C in 1 l plastic pots (10 worms in
each) with soil and moist cow dung as substrate. Cocoons were harvested
approximately every third week by washing and sieving, after which they were
placed on moist filter paper in Petri dishes. The cocoons were then incubated
at 20°C until they had developed to an early differentiated stage
characterized by the embryos having a worm-like shape but with no visible
internal structures or blood pigment
(Holmstrup, 1994). Once
cocoons had developed to this stage they were placed at 1°C to prevent
them from developing further until the experiments were carried out.
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). The
gradually desiccated cocoons were exposed to a series of decreasing humidities
by replacing the salt solutions of the desiccation chambers daily over a 10
day period (Fig. 1A). After
this period the cocoons were left at the final humidity (RH 91%) for 4 days
(short exposure) or 14 days (long exposure), respectively, at which time five
replicates of ten cocoons were sampled for survival assays. For the gradually
desiccated cocoons, samples for measurements of water content were taken daily
for the first 14 days and also on day 24. Samples for osmolyte measurements
were taken after 0, 2, 4, 8, 14 and 24 days
(Fig. 1A). Acutely exposed cocoons were exposed directly to a RH of 91% for either 4 or 14 days, after which survival was determined. Water content was determined after 2, 4, 8, 16 and 24 h during the first day of exposure, and subsequently after 2, 4 and 14 days of exposure (Fig. 1B). Samples for measurements of osmolytes were only taken after 4 days of exposure.
To further explore the relationship between pre-treatment, water content
and survival, a second experiment was conducted in which gradually and acutely
desiccated cocoons were exposed to a series of harsh desiccation treatments
after their initial exposure (Fig.
1C,D). The gradually desiccated cocoons were pre-acclimated for 14
days using the same desiccation protocol as in the first experiment after
which they were exposed to a final 4 day exposure to desiccation at RHs from
91% to 78% (Fig. 1C). For
comparison, the acutely desiccated cocoons were exposed directly to their
final desiccation treatment between 91% and 78% RH
(Fig. 1D). To obtain the nine
levels of dehydration, NaCl solutions were used to create RHs down to 83% as
described, whereas saturated solutions of
(NH4)2SO4 (ammonium sulphate) and
Na2S2O3·5H2O (sodium
thiosulphate) were used to create 81 and 78% RH, respectively
(Weast, 1989). For each
desiccation treatment, six replicates of one cocoon were used to establish the
water content, and survival was scored from ten replicates of five
cocoons.
Survival and water content
Cocoons used for determination of survival were placed in Petri dishes with
tap water at 20°C and checked every week for 6 weeks. Survival was scored
from the number of juveniles that emerged successfully during this period (one
juvenile emerges from each cocoon). Water content of single cocoons was
determined gravimetrically by weighing the cocoon immediately after sampling
and after drying at 60°C for 24 h. Weight measurements were performed with
a Cahn 4700 automatic eletrobalance, precise to 0.01 mg. Each sample for
osmolyte measurements consisted of five cocoons. Six samples were obtained for
each round of sampling. These samples were then stored at –80°C
until extraction.
Identification of osmolytes
Samples for osmolyte identification were dried at 60°C for 24 h after
which the dry mass was determined. The dried cocoons were then crushed and
extracted in 0.25 ml ethanol using a rotating glass rod in a 0.5 ml Eppendorf
tube. The sample was centrifuged at 20 000 g for 5 min at
4°C and the supernatant saved. This procedure was repeated three times,
after which the pooled supernatants were dried at 60°C for 24 h. The dry
sample was then stored at –80°C until further quantitative
analysis.
Nuclear magnetic resonance spectroscopy (NMR) was used to screen for
increases in osmolytes as a result of desiccation. Thus, NMR was performed on
a sample from untreated control cocoons, cocoons after 4 days of acute
desiccation and cocoons after 14 days of gradual desiccation; each sample
consisted of 15 cocoons. The samples were resuspended in 650 µl of 50 mmol
l–1 phosphate buffer made up in distilled H2O (pH
7.4). The samples were vortexed and 600 µl were transferred to a 5 mm NMR
tube. The buffer contained 50 mg l–1 of the chemical shift
reference 3-(trimethylsilyl)-propionic acid-D4, sodium salt (TSP). NMR
measurements were performed at 25°C on a Bruker Advance 400 spectrometer,
operating at a 1H frequency of 400.13 MHz, and equipped with a HX
inverse probe. 1H NMR spectra were acquired using a
single-90°-pulse experiment with a
Carr–Purcell–Meiboom–Gill (CPMG) delay added in order to
attenuate broad signals from high molecular mass components. The total CPMG
delay was 40 ms and the spin-echo delay was 200 µs. Water was suppressed by
presaturation during the relaxation delay of 1.5 s. A total of 256 transients
of 16 K data points spanning a spectral width of 24 p.p.m. were collected,
corresponding to a total experiment time of 10 min. For assignment purposes a
two-dimensional 1H–1H TOCSY spectrum with 80 ms
mixing was acquired. Signals were assigned using the TOCSY spectrum and by
comparison with known metabolite chemical shifts
(Fan, 1996;
Lindon et al., 1999;
Malmendal et al., 2006).
Quantification of osmolytes
The concentration of sorbitol, glucose, alanine, betaine, mannitol and
trehalose of cocoons was determined before and during dehydration. Sorbitol,
glucose, alanine, betaine and possibly mannitol were identified as candidate
osmolytes from the NMR spectra whereas trehalose was included because of its
well known role as a major inducible osmolyte in several other
dehydration-tolerant organisms. Sorbitol, glucose, trehalose and mannitol were
quantified spectrophotometrically using commercial enzymatic kits from
Megazyme International (Bray, Co. Wicklow, Ireland). Similarly, alanine was
measured spectrophotometrically as described previously
(Lowry and Passonneau, 1972).
For these measurements the extracts were rehydrated with 200 µl distilled
water and subsamples were used for measurements of the different osmolytes.
All measurements were performed at 25°C and concentrations were calculated
relative to known standards.
Betaine could not readily be measured with a similar method, and instead betaine concentration was quantified in the individual samples by use of NMR spectroscopy. The amount of betaine was assessed from the ratio of the TSP signal to the betaine signal, and the concentration was calculated relative to standards of known concentration. To perform these measurements using NMR, a sub-sample of the rehydrated sample was lyophilized and treated as described above in the NMR section.
Statistics
Student's t-tests were used to test for differences in survival
and water content between gradually and acutely exposed cocoons after both
short and long exposures to 91% RH. Mann–Whitney rank sum tests were
used in cases lacking normality. Differences in osmolyte content were analysed
using a one-way ANOVA for each osmolyte individually. Here a post hoc
Bonferroni test was used to separate groups that differed significantly from
the hydrated control. Similar tests were used to assess the estimated embryo
osmolyte concentrations. Again a post hoc Bonferroni test was used to
separate groups that differed significantly. A one-way ANOVA was also used to
test for differences in survival after the `harsh' dehydration treatments.
Differences in water content following the `harsh' dehydration treatments were
tested at each level of dehydration using a t-test, since unequal
variance did not allow for a two-way analysis of variance. Survival data were
arcsin transformed and all statistics were calculated using SigmaStat 2.03. An
effect was considered significant at the P<0.05 level and all data
are presented as means ± s.e.m.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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0.35 mg water mg–1 dry mass
(Fig. 2A). In the gradual
desiccation treatment, cocoon water content fell gradually for the first 10
days, by which time they had reached a stable level at 0.38 mg water
mg–1 dry mass (Fig.
2B). The water content of the gradually desiccated cocoons was
significantly higher (P<0.05) than the acutely exposed cocoons
when both groups were sampled after 4 days at 91% RH
(Fig. 2C). A similar tendency
existed after 24 days of gradual desiccation and 14 days acute desiccation,
although not statistically significant (P=0.064). There was only
minor mortality associated with 4 days of acute desiccation at 91% RH or 10
days of gradual desiccation followed by 4 days at 91% RH
(Fig. 2D). Here, the slightly
better survival of the gradually desiccated treatment group was not
significant after the short exposure (P=0.08; Mann-Whitney test).
However, there was a marked benefit of the gradual desiccation treatment when
the exposure time was extended to 14 and 24 days, for the acutely and
gradually exposed cocoons, respectively (P<0.001).
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A second set of experiments were conducted to further explore the relationship between acute and gradual dehydration with regard to survival and water content. Here the cocoons were exposed to a series of harsh exposures from 91 to 78% RH (Fig. 3A). The acutely exposed cocoons showed significantly decreased survival at RHs below 89% (one way ANOVA; P<0.001) and below 85% RH acute desiccation caused 100% mortality. In the gradually exposed cocoons the survival was significantly affected at RHs below 83% (one way ANOVA; P<0.001), but even at these harsh RHs mortality did not rise above 40% (Fig. 3A). Water content of the cocoons decreased with lowered RH for both the acute and gradual exposures, but the gradually exposed cocoons had a significantly higher water content at practically all desiccation levels (Fig. 3B). To investigate the importance of water content for survival, we plotted these two parameters against each other for the two treatment groups (Fig. 3C). It is evident that gradual desiccation increases survival more than can be explained by water content alone.
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Calculation of osmotic contributions of osmolytes in embryos
Previous studies have shown that osmolytes only accumulate in the embryo of
the cocoon (Holmstrup, 1995).
To determine the concentration of osmolytes in the embryos it was necessary to
establish a general relationship between cocoon mass and embryo mass. This was
done by measuring the length and width of 10 cocoons and their embryos under a
stereomicroscope. The cocoons are ellipsoid in shape and the embryos are
cylindrical. The volume, V, was therefore calculated with the
formulas, V=4/3
abc (abc=length, height and
width) and V=r2h (r=radius;
h=height) for cocoon and embryo, respectively. The average volume of
a cocoon was 3.97 mm3 and the average volume of an embryo was 0.17
mm3, thus the average embryo mass was estimated to be 4.3% of
cocoon mass.
The osmotic activity of osmolytes depends of course as much on the water
content in dehydrated embryos as it does on their concentration on a dry mass
basis. The water content of the cocoons can be divided into two fractions;
osmotically inactive water (OIW) and osmotically active water (OAW). OAW is
readily removed during desiccation whereas OIW is bound by structures such as
membranes and proteins, and even during severe dehydration it is not readily
removed from the tissues (Holmstrup and
Westh, 1994). It is therefore necessary to calculate the OAW
fraction for a given water content. In the present study, OIW was estimated to
be 0.12 mg water mg–1 dry mass, which was the lowest observed
water content of cocoons after an acute dehydration (data not shown). This
results in a conservative estimate of the osmotic contributions of the
osmolytes when contents (µg mg–1 dry mass cocoon) are
converted to molal concentrations (mol kg–1 embryo OAW).
Osmolyte accumulation in embryos
The calculated concentrations of osmolytes in embryos show that sorbitol,
glucose, mannitol, alanine and betaine were elevated at specific times during
the gradual and acute dehydration (Table
1). Glucose concentration was slightly elevated at day 4, but
reached a maximum concentration of 0.635 mol kg–1 OAW at day
8 before falling to 0.114 mol kg–1 OAW at day 14. Glucose
concentration of the acutely desiccated cocoons rose to 0.779 mol
kg–1 OAW. The concentration of sorbitol reached a maximum of
1.539 mol kg–1 OAW at day 14 and had fallen to 0.950 mol
kg–1 OAW at day 24. Mannitol concentration rose to
approximately 0.12 mol kg–1 OAW at day 14 and remained at
this level until day 24. The rise in mannitol concentration was not due to
de novo synthesis but rather to a concentration effect from water
loss. Trehalose concentration was also slightly elevated because of the
reduced water content during both the gradual and acute exposure, but this was
not statistically significant. Alanine rose to a maximum of 0.167 mol
kg–1 OAW at day 14, whereas the concentration in embryos of
the acutely exposed cocoons was only 0.078 mol kg–1 OAW. The
highest concentration of betaine, 0.113 mol kg–1 OAW, was
found at day 8 and embryos of the acutely exposed cocoons contained almost as
much betaine, 0.086 mol kg–1 OAW as the gradually exposed
embryos. This estimated total osmolality of these measured compatible
osmolytes peaked at day 14 in the gradually exposed 91% RH group at a
concentration of 2.073 mol kg–1 OAW. This was almost twice
the maximum seen in the 91% RH acute group where the calculated osmolality
reached 1.052 mol kg–1 OAW after 4 days.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). The present
study confirmed the presence of sorbitol in D. octaedra, but also
revealed that other osmolytes were accumulated in response to desiccation at
91% RH. Glucose was elevated in the gradually desiccated cocoons but
apparently only transiently in the early phase of desiccation. When cocoons
were dehydrated to 0.5 mg water mg–1 dry mass, glucose
disappeared and sorbitol increased significantly. A plausible explanation for
this is that glucose was derived from glycogen via glucose
6-phosphate and further transformed to sorbitol by polyol dehydrogenase when
the water content was so low that oxygen supply to the embryo became a
limiting factor (Storey and Storey,
1991). When most OAW has been lost the embryo is encased in a
sheath of water-reduced albuminous fluids which may greatly hamper the normal
oxygen diffusion across the cocoon fluid–embryo surface interface.
Sorbitol synthesis is not sensitive to oxygen limitation in the same way as
glucose synthesis (Storey and Storey,
1991), and the observed increase of alanine, a well-known
end-product of anaerobic metabolism
(Hochachka and Somero, 1984),
also suggests that desiccated embryos have experienced anaerobic or partly
anaerobic conditions when water contents became low. However, there was no
indication of increased lactate levels, although this is an anaerobic
end-product in adult D. octaedra (S. Calderon, personal
communication). The reduced rate of water loss in the gradually exposed
cocoons resulted in a different pattern of osmolyte production than in cocoons
exposed acutely to 91% RH. Apparently gradual desiccation gave the embryos
sufficient time to accumulate sorbitol in high concentrations, which the
acutely desiccated cocoons did not have the time to produce, or alternatively,
the biochemical pathways were completely changed.
The accumulation of glucose in high concentrations is known in
post-embryonic stages of D. octaedra subjected to freezing,
desiccation or osmotic shock (Overgaard et
al., 2007; Rasmussen and
Holmstrup, 2002), and thus the occurrence of this carbohydrate was
to be expected in dehydrated embryos. However, mannitol, trehalose, alanine
and perhaps most interesting, betaine, were also found. No previous reports
have shown the presence of these osmolytes in earthworms. Betaine is known
from many organisms including marine animals and thought to have the potential
to counteract the perturbing effects on protein structure of solutes such as
Na+ that may build up under osmotic stress
(Yancey, 2005). Although
betaine was not particularly elevated on a dry mass basis, it did occur in
significant concentrations (>100 mOsm) in dehydrated embryos where also the
original salts (Na+, K+ and Cl–) must
be highly concentrated and thus potentially damaging. It is therefore
suggested that protection against protein destabilization may be offered by
betaine and possibly other observed osmolytes under these circumstances.
Although other adaptations are also necessary, accumulation of trehalose in
high concentrations has often been associated with tolerance of extreme
desiccation in anhydrobiotic organisms such as tardigrades, nematodes and
yeast (Crowe et al., 2002;
Madin and Crowe, 1975;
Westh and Ramløv,
1991). In D. octaedra, trehalose was only detected in
negligible concentrations and did not respond to dehydration, whereas sorbitol
was the primary osmolyte in the gradually dehydrated cocoons. The accumulation
of sorbitol is restricted to the embryo and not found in the albuminous fluid
of the cocoons, which should be taken into account when estimating the
internal concentration (Holmstrup,
1995). Considering this, the total concentrations of osmolytes in
the gradually desiccated embryos reached a level of 2 mol
kg–1 OAW with sorbitol accounting for 75% of this estimate.
The higher concentration of some of the osmolytes such as mannitol and
trehalose was only caused by the lower water content and were not the result
of a higher accumulation. However, the concentrations of osmolytes in the
gradually exposed embryos were twice that in those acutely exposed (1 mol
kg–1 OAW).
Effects of acute and gradual desiccation on survival
The ability to survive both short and prolonged periods of desiccation is
an ecologically important trait for many soil invertebrates, especially those
inhabiting the top layer of the soil surface. In the present study we clearly
demonstrate that dehydration-tolerant cocoons of D. octaedra survive
much better when exposed to a gradual dehydration compared to an acute
dehydration. Thus, slow dehydration conferred a 70% survival in the gradually
exposed cocoons at the lowest humidity used (78% RH), compared to no survival
in the acutely exposed cocoons. Previous studies have also shown that slow
dehydration may confer improved tolerance of a given desiccation level as
compared to acute exposure to the same level of desiccation
(Crowe and Madin, 1975;
Hayward et al., 2007;
Sjursen et al., 2001;
Womersley and Ching, 1989).
However, many studies of invertebrate desiccation tolerance use acute exposure
regimes and only very few have used a gradual desiccation protocol similar to
the present study. Given the marked difference in dehydration tolerance found
between our two treatment groups we conclude that acute tolerance assays are
likely to underestimate the actual dehydration tolerance of many species as
the soil water is usually not readily removed over short timescales.
The higher survival was probably linked to the higher water content in the
gradually exposed cocoons which in turn may be linked to the accumulation of
osmolytes, which by their colligative properties would have reduced the water
loss and increased the water content at equilibrium of embryos. However,
survival in the gradually desiccated cocoons was also higher when compared at
the same level of dehydration (i.e. WC being the same;
Fig. 4C) suggesting that other
non-colligative protective mechanisms also play an important role. An
increased concentration of sugars and/or polyols is likely to enhance cellular
and membrane integrity during both desiccation and freezing by replacing the
primary water of hydration and through the formation of amorphous glasses
(vitrification), thus stabilizing the structure of macromolecules and
membranes (Crowe et al., 1992).
Trehalose is proposed to function as such a `water replacement' molecule and
sorbitol and other osmolytes in earthworm embryos may have a similar function
important to survival. However, research in anhydrobiosis has recently focused
on mechanisms other than the production of trehalose and other compatible
osmolytes; notably, desiccation-induced synthesis of chaperoning proteins such
as LEA proteins, which could be a mechanism in earthworm embryos
(Tunnacliffe and Lapinski,
2003).
The cocoons were transferred directly to water after the desiccation
treatments. This rehydration regime exposes embryo cells to hypo-osmotic shock
with the rapid influx of water. This recovery treatment was used because it is
probably the best reflection of natural conditions where dehydrated cocoons
will be abruptly surrounded by liquid water after rainfall. Owing to
dehydration the intracellular osmotic pressure is extremely high (78% RH
15
Osm) and the cells will quickly absorb water and swell. Indeed cellular
leakage has been reported for other desiccation-tolerant organisms and model
membrane vesicles during rapid rehydration
(Cacela and Hincha, 2006), and
problems caused by rehydration are arguably as important in the maintenance of
cellular integrity and enzyme activity as those incurred from desiccation.
However, both acutely and gradually desiccated cocoons are assumed to be in
osmotic equilibrium, and this hypo-osmotic shock is therefore similar for both
experimental groups.
Water loss and survival
The highest loss of water in the present study was seen at 78% RH where the
gradually and acutely exposed cocoons both lost more than 92% of their initial
water content, but where the final water content in the gradually desiccated
cocoons was 11% higher than that of the acutely exposed (0.215 and 0.190 mg
water mg–1 dry mass, respectively). This RH was not tolerated
by the acutely desiccated cocoons, but about 70% of the gradually desiccated
cocoons survived. Holmstrup and Westh
(Holmstrup and Westh, 1995)
suggested that the lower critical water content for survival in D.
octaedra cocoons was equal to the OIW content, thus coinciding with the
loss of most, if not all OAW. Using differential scanning calorimetry, OIW of
partly desiccated D. octaedra cocoons (with a total water content of
0.65 mg mg–1 dry mass) has been estimated as the
`unfreezable' fraction of water at –60°C to approximately 0.44 mg
water mg–1 dry mass
(Holmstrup and Westh, 1994)
and much higher than the 0.215 mg mg–1 dry mass, at which
gradually desiccated cocoons in the present study survived reasonably well.
However, OIW is probably not a `fixed' pool of water, and it has been
suggested that a portion of the OIW of fully hydrated cocoons is `released'
and becomes OAW in response to the degree of dehydration
(Holmstrup and Westh, 1994;
Wharton and Worland, 2001).
Inspection of data shown in Fig.
4B suggests that OIW of extremely dehydrated cocoons is somewhat
lower than estimated by Holmstrup and Westh
(Holmstrup and Westh, 1994)
stabilising at about 0.2 mg mg–1 dry mass. Extrapolation of
the data for gradually desiccated cocoons in
Fig. 3C predicts that 100%
mortality would occur at OIW levels just below 0.2 mg mg–1
dry mass suggesting that survival of D. octaedra cocoons is not
possible if water loss exceeds loss of all OAW. Although the cocoons are able
to survive loss of 95% of their original water content they can probably
not be categorised as a truly anhydrobiotic organism as this group of animals
often tolerate the loss of 99% of their normal water content
(Crowe et al., 1992).
Nevertheless D. octaedra cocoons have an extreme desiccation
tolerance and the present study shows that these cocoons have many
physiological similarities to anhydrobiotic organisms, particularly when the
desiccation occurs gradually as would be expected in a natural setting. We
therefore propose that D. octaedra cocoons belong in a transition
category between the desiccation sensitive and the truly anhydrobiotic
organisms.
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Acknowledgments |
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