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 av
Trang 1divergence in responses to dehydration
Steffen Hengherr1,2, Arnd G Heyer3, Heinz-R Ko¨hler1 and Ralph O Schill2
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 vanderplankireport up to 18% trehalose in 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 NovemOcto-ber 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 trehalose to 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 trehalose in 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 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 level of desiccation tolerance
Abbreviations
HPAEC, high-performance liquid anion exchange chromatography; RH, relative humidity.
Trang 2plants 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 and to preserve the
integrity of membranes during dehydration in 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 evidence for 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 in tardigrades 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 anhydrobiosis in tardigrades (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 in trehalose 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
Trang 3Surprisingly, 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 in trehalose 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 ‘richtersigroup’ 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 Artemiacysts 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)1protein) 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 ).
(0.022 ± 0.019) (0.141 ± 0.081) (0.153 ± 0.027) (0.078 ± 0.025) (0.042 ± 0.011)
(0.003 ± 0.009) (0.096 ± 0.035) (0.172 ± 0.041) (0.077 ± 0.025) (0.040 ± 0.011)
(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)
(0.023 ± 0.045) (0.002 ± 0.004) (0.003 ± 0.009) (0.006 ± 0.008) (0.014 ± 0.018)
(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 rehydrarehydra-tion V) (A)
Macrobio-tus ‘richtersi group 2’ (B) Echiniscus granulaMacrobio-tus.
Trang 4However, 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 trehalose and the inducaccumula-tion 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 trehalose in 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 trehalose and 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 trehalose in 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 trehalose in 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
Macrobiotus ‘richtersi group’ 1 0.044*
*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 ).
Macrobiotus richtersi 1.16 ± 0.0003 1.32 ± 0.002
Macrobiotus ‘richtersi group’ 1 1.92 ± 0,021 4.32 ± 0.002
Macrobiotus ‘richtersi group’ 2 1.25 ± 0.002 1.30 ± 0.002
Trang 5that 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 trehalose and 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-emiacysts [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 tardigrades to 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 sapiensBinda & 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,
Trang 6Chlorogonium elongatum The colourless eggs of the
Macorbiotusspecies 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 MgCl2 (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 95C for
60 min the homogenate was centrifuged (20 000 g at 4C 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: T1= 0.4 s, T2= 0.02 s, T3= 0.01 s,
T4= 0.07 s, E1= 0.1 V, E2=)2.0 V, E3= 0.6 V,
E4=)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 KCH3CO2,
5 mm Mg(CH3CO2)2, 20 mm Hepes, 2% protease inhibitor cocktail (Sigma-Aldrich Chemie GmBH), pH 7.4] The homogenate was subsequently centrifuged (20 000 g at 4C 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, 20C) 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),
Trang 70.001 < 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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