Trehaloseandanhydrobiosisintardigrades–evidence for
divergence inresponsesto dehydration
Steffen Hengherr
1,2
, Arnd G. Heyer
3
, Heinz-R. Ko
¨
hler
1
and Ralph O. Schill
2
1 Animal Physiological Ecology, Zoological Institute, University of Tu
¨
bingen, Germany
2 Department of Zoology, Biological Institute, Universita
¨
t of Stuttgart, Germany
3 Department of Botany, Biological Institute, Universita
¨
t of Stuttgart, Germany
Desiccation in general leads to severe damage of cellu-
lar structures, which commonly results in the death
of cells and the organism. However, a number of
so-called anhydrobiotic organisms have developed
remarkable mechanisms, allowing them to minimize or
avoid such damage and survive extreme dehydration in
a cryptobiotic state [1–6]. Several species of inverte-
brate taxa have this ability, including the embryonic
cysts of crustaceans, rotifers, insect larvae, nematodes
and tardigrades [2,3,7–12]. Additionally, many pro-
caryotes, such as bacteria and cyanobacteria [13,14],
and even plant seeds [15–19] and adult plants, for
example the resurrection lycopode Selaginella lepido-
phylla [5,20], demonstrate dehydration tolerance.
Although Antonin van Leuwenhoek described anhy-
drobiosis over 300 years ago [21], the underlying mech-
anisms are still not fully understood. However, over
the last three decades, researchers have come to recog-
nize the important role of polyhydroxy compounds
such as the non-reducing disaccharide trehalose [22–
24]. This sugar is found in high concentrations in a
wide variety of anhydrobiotic organisms, including
nematodes, embryonic cysts of crustaceans, and yeast.
Trehalose concentrations as high as 13–18% of the dry
weights have been reported for anhydrobiotic cysts of
the crustacean Artemia franciscana [25–27] whereas the
nematode Aphelenchus avenae can accumulate 10–15%
of its dry weight as trehalose during anhydrobiosis
[8,9]. Studies on the anhydrobiotic insect larvae Poly-
pedilum vanderplanki report up to 18% trehalosein the
dry body mass [11]. Significantly increased trehalose
levels also have been found in the Arctic collembolan
Onychiurus arcticus during partial desiccation, induced
by sub-zero temperatures [28]. The disaccharide
sucrose fulfils a similar role in plants and accumulates
in desiccation-tolerant plant seeds and resurrection
Keywords
cryptobiosis; desiccation tolerance;
metabolite; nonreducing disaccharide;
tardigrade
Correspondence
R. O. Schill, Department of Zoology,
Biological Institute, Universita
¨
t of Stuttgart,
Pfaffenwaldring 57, D-70569 Stuttgart,
Germany
Fax: +49 711 685 6 5096
Tel: +49 711 685 6 9143
E-mail: ralph.schill@bio.uni-stuttgart.de
(Received 9 August 2007, revised 31 Octo-
ber 2007, accepted 19 November 2007)
doi:10.1111/j.1742-4658.2007.06198.x
To withstand desiccation, many invertebrates such as rotifers, nematodes
and tardigrades enter a state known as anhydrobiosis, which is thought to
require accumulation of compatible osmolytes, such as the non-reducing
disaccharide trehaloseto protect against dehydration damage. The treha-
lose levels of eight tardigrade species comprising Heterotardigrada and
Eutardigrada were observed in five different states of hydration and dehy-
dration. Although many species accumulate trehalose during dehydration,
the data revealed significant differences between the species. Although tre-
halose accumulation was found in species of the order Parachela (Eutardi-
grada), it was not possible to detect any trehalosein the species
Milnesium tardigradum and no change in the trehalose level has been
observed in any species of Heterotardigrada so far investigated. These
results expand our current understanding of anhydrobiosisin tardigrades
and, for the first time, demonstrate the accumulation of trehalosein devel-
oping tardigrade embryos, which have been shown to have a high level of
desiccation tolerance.
Abbreviations
HPAEC, high-performance liquid anion exchange chromatography; RH, relative humidity.
FEBS Journal 275 (2008) 281–288 ª 2007 The Authors Journal compilation ª 2007 FEBS 281
plants during drying [29–31]. It has been hypothesized
that non-reducing disaccharides have a protective role
during dehydration. Trehalose has been shown to sta-
bilize proteins in their native state andto preserve the
integrity of membranes during dehydrationin vitro
[4,32], Assuming a similar role in vivo, two models for
the mechanism of the protective role of trehalose have
been proposed that are not mutually exclusive: The
water replacement hypothesis [33] states that trehalose
forms hydrogen bonds with macromolecules and cellu-
lar structures in place of water during dehydration and
thus preserves native structures. In addition, the vitrifi-
cation hypothesis proposes the formation of amor-
phous sugar glasses during desiccation, which protects
proteins and membranes [23,32].
Despite evidencefor the protective role of trehalose
in animals and sucrose in plants, not all organisms
that undergo anhydrobiosis contain one of these sug-
ars or genes for its synthesis, as seen in bdelloid roti-
fers, which appear to lack trehalose synthesis entirely
[34,35].
Together with nematodes and rotifers, tardigrades
represent one of the three main invertebrate taxa [36],
where desiccation tolerance is widespread. Members of
these taxa display an exceptional ability to survive high
[7,37] and subfreezing temperatures [38–41] while in an
anhydrobiotic state. Very few studies have investigated
changes of trehalose levels intardigrades during entry
into anhydrobiosis. Crowe [42] compared the trehalose
level of hydrated and anhydrobiotic Macrobiotus areo-
latus and reported that trehalose accumulated in the
dehydrated state. Until now, the only quantitative
study to have shown a steady accumulation of treha-
lose during anhydrobiosisintardigrades (up to 2.3%
of the dry weight) is that by Westh and Ramløv [43]
on Adorybiotus coronifer.
To obtain more comprehensive data on whether tre-
halose may be important for desiccation tolerance in
tardigrades, we investigated eight different species, rep-
resenting the orders Parachela and Apochela of the
Eutardigrada, and Echiniscoidea of the Heterotardi-
grada. The present study represents the first extensive
quantitative measure on adult animals of different tar-
digrade species and the first observation of trehalose
accumulation in tardigrade embryos, using 12 800 indi-
viduals and 1440 embryos in total.
Results
Desiccation and protein level
Despite differences between species, none of the tardi-
grades investigated showed a significant change in total
protein concentration when dehydrated (Table 1).
Therefore, we chose total protein as reference parame-
ter for quantification of trehalose because of the high
sensitivity and accuracy of determination. However,
due to the sensitivity of the analysis technique for sam-
ple size and the small amount of protein present in tar-
digrade eggs, it was not possible to quantify the total
protein concentration in tardigrade eggs.
Trehalose levels during anhydrobiosis
Distinct changes in the level of trehalose during anhy-
drobiosis were found only in the species of the Macro-
biotidae (Table 2). Although there was a large
variation intrehalose concentrations between the Mac-
robiotus species, all showed a highly significant accu-
mulation of trehalose when passing through state II, in
which the animals draw in their legs but still perform
distinct movements. Trehalose accumulation reached a
maximum in the anhydrobiotic state (III), 48 h after
completion of tun formation (P < 0.001). Large dif-
ferences occurred in absolute trehalose concentration
and accumulation rate between the species. For exam-
ple, Macrobiotus tonollii accumulated trehalose by up
to sixfold (1.650 ± 0.291 ngÆlg
)1
protein; 0.153% of
dry weight) whereas Macrobiotus ‘richtersi group’ 2
displayed a 150-fold enrichment of trehalose (up to
7.415 ± 0.580 ngÆlg
)1
protein; 0.472% of dry weight)
(Fig. 1A and Table 2). Data recorded for the other
species are provided in Table 2. During the transitional
rehydration stage (IV), when the animals had pro-
truded their legs and the body again performed move-
ments, all Macrobiotidae showed a rapid decline in
trehalose concentration, which was further reduced
over the next 4 h until the completely re-active state V
was reached.
Table 1. Total protein content of the hydrated and dehydrated
state and dry weight per animal for the different tardigrade species.
Data are shown as the mean ± SD.
Species
Protein (lg)
Dry weight
(lg)
Hydrated Dehydrated
Milnesium tardigradum 3.53 ± 0.21 3.80 ± 0.23 6.11 ± 0.20
Macrobiotus tonollii 3.28 ± 0.21 3.41 ± 0.30 3.61 ± 0.15
Macrobiotus richtersi 3.20 ± 0.25 3.65 ± 0.34 5.75 ± 0.19
Macrobiotus sapiens 2.33 ± 0.10 2.15 ± 0.18 2.92 ± 0.14
Macrobiotus ‘richtersi
group’ 1
3.69 ± 0.24 3.67 ± 0.13 6.52 ± 0.27
Macrobiotus ‘richtersi
group’ 2
3.92 ± 0.34 4.42 ± 0.26 6.55 ± 0.17
Echiniscus granulatus 1.24 ± 0.53 1.60 ± 0.29 2.44 ± 0.11
Echiniscus testudo 0.74 ± 0.18 0.80 ± 0.14 2.14 ± 0.16
Trehalose andanhydrobiosisintardigrades S. Hengherr et al.
282 FEBS Journal 275 (2008) 281–288 ª 2007 The Authors Journal compilation ª 2007 FEBS
Surprisingly, we were unable to detect any trehalose
at all in Milnesium tardigradum (Fig. 2 and Table 2).
Although we detected low trehalose concentrations in
the two Heterotardigrade species (Echiniscus granulatus
and Echiniscus testudo), there was no significant
change intrehalose level during the anhydrobiotic
states (Table 2) as shown by E. granulatus (Fig. 2B)
(P = 0.213).
Carbohydrate analysis resulted in several other
unidentified peaks that differentiated the active state I
and the anhydrobiotic state III for M. tardigradum,
Macrobiotus richtersi, Macrobiotus ‘richtersi group’ 1
and M. tonollii, respectively, although the increase was
low (Table 3). Only one peak (2.233 min retention
time) demonstrated significant changes during desicca-
tion in more than one species (M. tardigradum and
M. ‘richtersi group’ 1).
Sugar analysis of tardigrade embryos clearly
revealed a low level of trehalose accumulation in
Macrobiotus richtersi and M. ‘richtersi group’ 1
(Table 4), although it was not possible to perform
statistical tests because of the small number of
replicates. No trehalose was detected in eggs or
embryos of M. tardigradum or M. tonollii.
Discussion
Analysis of soluble sugar accumulation in tardigrades
during transition to an anhydrobiotic state showed
that trehalose accumulates in species of the Macro-
biotidae, although the absolute level of trehalose is
low compared to other anhydrobitic organisms (e.g.
Artemia cysts or the nematode A. avenae), where treha-
lose levels can reach 13–18% and 10–15% of the dry
weight, respectively [9,25–27]. Westh and Ramløv [43]
reported that the Eutardigrade A. coronifer (Parachela)
shows a more than 20-fold accumulation of trehalose
during transition to anhydrobiosis. This is within the
concentration range of trehalose accumulation in
the Macrobiotidae investigated in the present study.
Table 2. Trehalose concentration (ngÆlg
)1
protein). Values in parenthesis represents the amount of trehalose as % of dry weight. Data are
shown as the mean ± standard deviation. ND, not detected (beyond detection limit of 0.05 l
M).
Stages I II III IV V
Milnesium tardigradum ND ND ND ND ND
Macrobiotus tonollii 0.241 ± 0.202 1.515 ± 0.873 1.650 ± 0.291 0.837 ± 0.272 0.454 ± 0.122
(0.022 ± 0.019) (0.141 ± 0.081) (0.153 ± 0.027) (0.078 ± 0.025) (0.042 ± 0.011)
Macrobiotus richtersi 0.055 ± 0.154 1.609 ± 0.586 2.888 ± 0.689 1.291 ± 0.410 0.664 ± 0.190
(0.003 ± 0.009) (0.096 ± 0.035) (0.172 ± 0.041) (0.077 ± 0.025) (0.040 ± 0.011)
Macrobiotus sapiens 0.140 ± 0.197 1.193 ± 0.424 1.137 ± 0.263 1.337 ± 0.721 0.316 ± 0.338
(0.011 ± 0.015) (0.092 ± 0.056) (0.087 ± 0.020) (0.103 ± 0.056) (0.024 ± 0.026)
Macrobiotus ‘richtersi group’ 1 0.041 ± 0.079 2.950 ± 0.985 4.640 ± 0.677 2.641 ± 1.168 1.033 ± 0.431
(0.002 ± 0.004) (0.166 ± 0.056) (0.262 ± 0.038) (0.149 ± 0.066) (0.058 ± 0.024)
Macrobiotus ‘richtersi group’ 2 0.049 ± 0.091 4.228 ± 1.810 7.415 ± 0.580 3.319 ± 1.145 1.394 ± 0.508
(0.003 ± 0.006) (0.269 ± 0.012) (0.472 ± 0.037) (0.211 ± 0.073) (0.089 ± 0.032)
Echiniscus granulatus 0.395 ± 0.780 0.030 ± 0.060 0.054 ± 0.153 0.109 ± 0.131 1.394 ± 0.311
(0.023 ± 0.045) (0.002 ± 0.004) (0.003 ± 0.009) (0.006 ± 0.008) (0.014 ± 0.018)
Echiniscus testudo 0.158 ± 0.316 0.219 ± 0.437 0.583 ± 0.412 0.889 ± 0.088 1.064 ± 0.096
(0.006 ± 0.010) (0.008 ± 0.014) (0.021 ± 0.013) (0.032 ± 0.003) (0.038 ± 0.003)
A
B
Fig. 1. Alterations of the trehalose level during anhydrobiosis
(active stage I; dehydration stage II; anhydrobiotic stage III; rehydra-
tion stage IV; active stage, 4 h after rehydration V). (A) Macrobio-
tus ‘richtersi group 2’. (B) Echiniscus granulatus.
S. Hengherr et al. Trehaloseandanhydrobiosisin tardigrades
FEBS Journal 275 (2008) 281–288 ª 2007 The Authors Journal compilation ª 2007 FEBS 283
However, the absolute trehalose levels detected in the
Macrobiotidae are generally lower than those reported
for A. coronifer (2.3% of dry weight) [43].
There is a clear correlation between the accumula-
tion of trehaloseand the induction of anhydrobiosis,
indicating a possible protective role of the disaccharide
during desiccation stress at least in the five species of
the Macrobiotidae investigated in the present study.
The apparent absence of trehalosein M. tardigrad-
um, which was the only species of the order Apochela
tested here, as well as in bdelloid rotifers [34], which
all show desiccation tolerance indistinguishable from
that of trehalose accumulating tardigrades, indicates
that trehalose accumulation is not essential for desicca-
tion tolerance in these animals. Milnesium tardigradum
and possibly other Apochela species may have lost the
ability to synthesize trehaloseand may have replaced
its function by some other mechanism that resulted in
the unknown peak (2.233 min retention time).
It is not uncommon for groups within a taxon to
follow different adaptation strategies, as shown in roti-
fers by Caprioli et al. [35], who detected only small
amounts of trehalosein monogonont and none in
bdelloid species. The absence of changes in the level of
trehalose in the anhydrobiotic Heterotardigrades
E. granulatus and E. testudo and the complete lack
of trehalosein M. tardigradum strongly argues against
a universal protective role in anhydrobiosis. Using an
RNA interference technique, Ratnakunar and Tunnac-
liffe [44] confirmed that there is no consistent relation-
ship between trehalose accumulation and desiccation
tolerance in Saccharomyces cerevisiae. Considering
phylogenetic relationships, it would be interesting to
investigate whether marine tardigrades accumulate tre-
halose as in the Macrobiotidae tested here. Because
the selective pressure for desiccation tolerance should
be low in a marine habitat, the presence of trehalose in
marine tardigrades would allow diagnosis of the direct
effects of trehalose on desiccation tolerance in animals
Fig. 2. High-performance liquid anion
exchange chromatograms of soluble sugars
extracted from 40 animals each of seven
tardigrade species dried down do
anhydrobiotic statge III. Species are:
Echiniscus granulatus (EG), E. testudo
(ET), Macrobiotus ‘richtersi group’ 1 (CL),
M. ‘richtersi group’ 2 (N), M. spaiens
(MS), M. richtersi (MR), M. tonollii (MT) and
M. tardigradum (Mil). Sugars were eluted
isocratically by 150 m
M NaOH from an anion
exchange column. The retention time for
trehalose was 2.65 min. The y-axis shows
relative detector units from pulsed
chromatography.
Table 3. Unidentified peaks of high-performance liquid anion
exchange chromatography chromatograms of the state III (dry
state), which show a distinct change compared to the state I
(active state).
Retention
time (min) Species P value
2.233 Milnesium tardigradum 0.037*
Macrobiotus ‘richtersi group’ 1 0.044*
3.100 Macrobiotus richtersi 0.026*
3.767 Macrobiotus ‘richtersi group’ 1 0.003**
8.300 Macrobiotus tonollii 0.043*
10.333 Macrobiotus richtersi 0.003**
*P £ 0.05 (weakly significant); **P £ 0.01 (significant).
Table 4. Trehalose concentration (ng) per embryo. Data are shown
as the mean ± standard deviation. ND, not detected (beyond detec-
tion limit of 0.05 l
M).
Species Hydrated Dehydrated
Milnesium tardigradum ND ND
Macrobiotus tonollii ND ND
Macrobiotus richtersi 1.16 ± 0.0003 1.32 ± 0.002
Macrobiotus sapiens 1.10 ± 0.003 1.08 ± 0.001
Macrobiotus ‘richtersi group’ 1 1.92 ± 0,021 4.32 ± 0.002
Macrobiotus ‘richtersi group’ 2 1.25 ± 0.002 1.30 ± 0.002
Trehalose andanhydrobiosisintardigrades S. Hengherr et al.
284 FEBS Journal 275 (2008) 281–288 ª 2007 The Authors Journal compilation ª 2007 FEBS
that probably have not evolved alternative adaptation
strategies for desiccation tolerance. However, to date,
no studies have been conducted on the anhydrobiotic
abilities of tardigrades from the tidal zone.
Besides the animals themselves, eggs of the tardi-
grades also show a good desiccation tolerance [45,
unpublished data] and, in a first non-quantitative car-
bohydrate analysis of eggs of the Macrobiotidae, we
found that dry embryos also show low levels of tre-
halose accumulation. However, embryos of M. tar-
digradum and M. tonollii contained no trehalose.
There appears to be no correlation between trehalose
content and morphology, colour or type of oviposi-
tion because there are no differences between species
that deposit eggs in the exuvium (Milnesium) and free
ovipositing species (M. tonolli). However, it is possible
that eggs of M. tardigradum are more protected due
to a slower dehydration inside the exuviae compared
to eggs of the free ovipositing species. No additional
studies concerning this issue are available. Although
more quantitative studies on eggs of different species
are required to prove a correlation between the accu-
mulation of trehaloseand desiccation tolerance in tar-
digrade embryos, it appears that the ability to
synthesise trehalose during desiccation is not just a
feature of fully developed animals. However, relation-
ships between the developmental stage and the ability
to accumulate trehalose cannot be established based
on the data available in the present study because of
the unknown age of the eggs and developmental
stages of the embryos used. In conclusion, our results
support the hypothesis that non-reducing disaccha-
rides such as trehalose may be involved in the desic-
cation tolerance of anhydrobiotic invertebrates
[4,12,32]. However, trehalose does not appear to be
essential.
The fact that some anhydrobiotic organisms do not
require trehalose does not argue against an important
function of this non-reducing disaccharide in organ-
isms where it does occur. Trehalose may play an
important primary role as an energy source, as demon-
strated for yeast [46]. Additionally, in the organisms
where it accumulates, trehalose could constitute a
general stress defensive molecule and minimize in-
activation of proteins by functioning as a ‘chemical
chaperone’. In this case, it may not have an immediate
effect on protein folding on its own, as is the case for
other protein chaperones [47,48], but could act as a
stabilizing agent. A small a-crystalline stress protein,
p26, which was discovered in desiccation tolerant Art-
emia cysts [49–52], was demonstrated to protect native
protein conformation in vitro synergistically with treha-
lose [53]. The disaccharide in turn acts synergistically
with heat shock proteins during protein folding [49].
Because of the synergistic mode of action, neither the
protein nor the sugar alone are sufficient for improved
survival.
This leads to the question as to what protective
mechanisms are employed by anhydrobiotic organisms
such as M. tardigradum or bdelloid rotifers, which do
not accumulate trehalose. Multiple strategies may exist
and complex mechanisms may be required where
changes in protein composition may be of greater
importance than changes in carbohydrate levels. We
could not identify other carbohydrate compounds that
specifically accumulated in non-trehalose tardigrade
species during drying. Molecular chaperones may be
particularly important in protecting proteins against
stress-induced denaturation [54]. In nematodes, as well
as in plants, the induction of late embryogenesis abun-
dant proteins [55] has been associated with dehydra-
tion tolerance [5,18,20,56]. Studies on anhydrobiotic
tardigrades also indicate that these peptides might be
involved in protective mechanisms (McGee B, Schill R
& Tunnacliffe A, unpublished data). Besides the induc-
tion of putative late embryogenesis abundant proteins,
higher expression of stress genes of the hsp70 family
have been detected in M. tardigradum undergoing an-
hydrobiosis [57], indicating a function within the multi-
ple strategies to withstand desiccation stress. Apart
from the function of protein chaperones in assisting
nascent and misfolded proteins to gain their correct
conformation, the exact role of heat shock proteins in
the protection of cells during dehydration has yet to
be addressed.
A continued search for additional mechanisms allow-
ing organisms such as tardigradesto survive almost
complete desiccation, as well as the further study of
such adaptations, should lead to a better understanding
of the remarkable phenomenon of anhydrobiosis.
Experimental procedures
Tardigrade culture
Individuals of six eutardigrade species and two heterotardi-
grade species were used to investigate changes of trehalose
concentrations during anhydrobiosis. For the investigation,
M. tardigradum Doye
`
re 1849 (order Apochela), M. tonollii
Ramazotti 1956, M. richtersi Murray 1911, Macrobio-
tus sapiens Binda & Pilato, 1984, M. ‘richtersi group’ 1 and
M. ‘richtersi group’ 2 (all order Parachela) were maintained
in laboratory cultures. The cultures were scaled up
for growth on agar plates (3%) covered with a thin layer of
water. Tardigrades were fed bdelloid rotifers, Philodina
citrina, which had been raised on the green algae,
S. Hengherr et al. Trehaloseandanhydrobiosisin tardigrades
FEBS Journal 275 (2008) 281–288 ª 2007 The Authors Journal compilation ª 2007 FEBS 285
Chlorogonium elongatum. The colourless eggs of the
Macorbiotus species are laid free and show the characteris-
tic truncate conical shape with projections. Animals of
Milnesium deposit the colourless or rosy, oval and smooth
eggs in exuvium. Because it was not possible to maintain
hatcheries of the heterotardigrades E. granulatus (Doye
`
re
1840) and E. testudo (Doye
`
re 1840), the animals were
manually isolated directly from mosses using a pipette and
a stereomicroscope.
Tardigrade sampling
Tardigrades were starved over 2 days before harvesting to
avoid contamination with food-organisms. After repeated
washing with clean water, animals were transferred into
microliter tubes (40 individuals per tube). Fertilized eggs of
the cultured species were isolated 4 days after fertilization
(only known for M. tardigradum) cleaned with water and
subsequently transferred into microliter tubes (60 eggs per
tube). The age of fertilized eggs of the other species was
not known. The remaining fluid in the tubes was reduced
to approximately 1–2 lL.
Stages of anhydrobiosis
Five different states according to Schill et al. [57] were
investigated. Microliter tubes containing animals repre-
senting the active state (I) were frozen directly in liquid
nitrogen. To produce different anhydrobiotic states, the
animals were dried in open microliter tubes at room tem-
perature, exposed to 85% relative humidity (RH) in a
chamber containing a saturated solution of KCL (Roth,
Karlsruhe, Germany). State II was defined as the state at
which the legs were drawn in but the body still per-
formed distinct movements. Animals in the anhydrobiotic
state (III) were sampled after completed tun formation
and further drying at 35% RH over 48 h in a chamber
containing a saturated solution of MgCl
2
(Roth), The
remaining animals were rehydrated. Animals were sam-
pled in this transitional state IV, that was defined as the
state at which the legs protruded and the body again per-
formed movements. This state was achieved after approxi-
mately 15–20 min. State V was an active one in which
the animals moved around 4 h after state IV. This
method of producing distinct anhydrobiotic states has
been tested for survival and no animals died during the
procedure. For all investigated states, eight batches
containing 40 animals each were used for each species
(n = 8). Eggs of the hydrated state were also frozen
directly in liquid nitrogen. Eggs of the dehydrated state
were treated like animals of stage III. Due to the small
size of the objects for investigating the embryos, we could
use only two batches for each hydrated and dehydrated
state per species (n = 2).
Carbohydrate analysis
Samples were homogenised with an ultrasonicator (SONO-
PULS, HD3100; Bandelin Electronic, Berlin, Germany) in
100 lL of distilled water. After incubating at 95 °C for
60 min the homogenate was centrifuged (20 000 g at 4 °C
for 15 min) and the supernatant was used for carbohydrate
analysis. Water-soluble carbohydrates were determined by
high-performance liquid anion exchange chromatography
(HPAEC) using a CarboPac PA-1 column on a Dionex
DX-500 gradient chromatography system coupled with
pulsed amperometric detection by a gold electrode (Dionex,
Sunnyvale, CA, USA). The detector settings for the deter-
minations were: T
1
= 0.4 s, T
2
= 0.02 s, T
3
= 0.01 s,
T
4
= 0.07 s, E
1
= 0.1 V, E
2
= )2.0 V, E
3
= 0.6 V,
E
4
= )0.1 V, sensitivity range = 0.1 lC and integration
range = 0.2–0.4 s. Eluents were A = 0.15 m NaOH,
B = 0.85 m sodium acetate in 0.15 m NaOH. Following
10 min of isocratic elution with A, a linear gradient to
100% B within 2 min and an isocratic step with B for
2 min was used for column cleaning, before the column
was again equilibrated with A for 5 min. Trehalose eluted
at a retention time of 2.6 min. Pure commercially available
trehalose (Sigma-Aldrich Chemie GmBH, Munich, Ger-
many) in the concentration range 1–100 lm was used for
calibration.
Protein quantification
Samples were homogenised in two batches of 40 animals
and 60 eggs for each active and cryptobiotic state by ultra-
sonication (SONOPULS, HD3100, Bandelin Electronic) in
30 lL of a buffered extraction solution [80 mm KCH
3
CO
2
,
5mm Mg(CH
3
CO
2
)
2
,20mm Hepes, 2% protease inhibitor
cocktail (Sigma-Aldrich Chemie GmBH), pH 7.4]. The
homogenate was subsequently centrifuged (20 000 g at 4 °C
for 5 min). The total protein concentration in each superna-
tant was determined by a protein quantification mini-assay
based on the method of Bradford using Coomassie Protein
Assay Reagent (Pierce, IL, USA).
Dry weight determination
For each tardigrade species, four samples of 20 individuals
each were dried (35% RH, 20 °C) over 4 days and weighed
on a Supermicro S4 2405 (Sartorius, Go
¨
ttingen, Germany).
Statistical analysis
The statistical significance of differences in the trehalose
and protein level between the samples was tested using
one-way analysis of variance and the Bonferroni t-test.
Significance levels were P £ 0.001 (highly significant),
Trehalose andanhydrobiosisintardigrades S. Hengherr et al.
286 FEBS Journal 275 (2008) 281–288 ª 2007 The Authors Journal compilation ª 2007 FEBS
0.001 < P £ 0.01 (significant) and 0.01 < P £ 0.05 (weakly
significant).
Acknowledgements
Many thanks to Roger Worland and James S. Clegg
for critical discussion and manuscript pre-review.
This study was supported by the German Research
Foundation (DFG), SCHI865 ⁄ 1-1, and enabled using
the equipments made available by the project
FUNCRYPTA (0313838A), funded by the German
Federal Ministry of Education and Research, BMBF.
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Trehalose andanhydrobiosisintardigrades S. Hengherr et al.
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. Trehalose and anhydrobiosis in tardigrades – evidence for divergence in responses to dehydration Steffen Hengherr 1,2 , Arnd G. Heyer 3 , Heinz-R. Ko ¨ hler 1 and Ralph O. Schill 2 1. assisting nascent and misfolded proteins to gain their correct conformation, the exact role of heat shock proteins in the protection of cells during dehydration has yet to be addressed. A continued. current understanding of anhydrobiosis in tardigrades and, for the first time, demonstrate the accumulation of trehalose in devel- oping tardigrade embryos, which have been shown to have a high