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Acta Physiol 2011, 202, 409–420

REVIEW
Survival in extreme environments – on the current
knowledge of adaptations in tardigrades

N. Møbjerg,1 K. A. Halberg,1 A. Jørgensen,2 D. Persson,1,2 M. Bjørn,3 H. Ramløv3 and

R. M. Kristensen2
1 Department of Biology, University of Copenhagen, Copenhagen, Denmark
2 Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark
3 Department of Science, Systems and Models, University of Roskilde, Roskilde, Denmark

Received 17 October 2010, Abstract

revision requested 13 November
2010,
revision received 6 January 2011,
accepted 10 January 2011
Correspondence: N. Møbjerg,
Department of Biology, August
Krogh Building, Universitetsparken
13, DK-2100 Copenhagen Ø,
Denmark.
E-mail: nmobjerg@bio.ku.dk
Tardigrades are microscopic animals found worldwide in aquatic as well as
terrestrial ecosystems. They belong to the invertebrate superclade Ecdysozoa,
as do the two major invertebrate model organisms: Caenorhabditis elegans
and Drosophila melanogaster. We present a brief description of the tardi-
grades and highlight species that are currently used as models for physio-
logical and molecular investigations. Tardigrades are uniquely adapted to a
range of environmental extremes. Cryptobiosis, currently referred to as a
reversible ametabolic state induced by e.g. desiccation, is common especially
among limno-terrestrial species. It has been shown that the entry and exit of
cryptobiosis may involve synthesis of bioprotectants in the form of selective
carbohydrates and proteins as well as high levels of antioxidant enzymes and
other free radical scavengers. However, at present a general scheme of
mechanisms explaining this phenomenon is lacking. Importantly, recent
research has shown that tardigrades even in their active states may be ex-
tremely tolerant to environmental stress, handling extreme levels of ionizing
radiation, large fluctuation in external salinity and avoiding freezing by
supercooling to below )20 C, presumably relying on efficient DNA repair
mechanisms and osmoregulation. This review summarizes the current
knowledge on adaptations found among tardigrades, and presents new data
on tardigrade cell numbers and osmoregulation.
Keywords cell numbers, cryptobiosis, evolution, osmoreglation, supercool-
ing, tardigrade.

Tardigrades, also known as water bears, are micro-
scopic metazoans (approx. 0.1–1.2 mm). They were
discovered in the 18th Century with the development of
early microscopes and were first described by the
German zoologist Goeze in 1773, who named them
‘kleiner Wasserbär’ or little water bear because of their
strong resemblance to a little bear (see e.g. Ramazzotti
& Maucci 1983, Nelson 2001, Schill 2010). Shortly
after in 1776, the current name, Tardigrada (from Latin
tardigradus, slow-moving), was given by the Italian
natural scientist Spallanzani (see e.g. Rebecchi et al.
2007). Tardigrades are exceptional among metazoans in
their adaptations to the most extreme environments. As
is also known from selected species of arthropods,
nematodes and rotifers, many species have the ability to
enter cryptobiosis; a state of suspended animation
believed to be ametabolic (Keilin 1959, Clegg 2001).
Corti already noted these adaptations in tardigrades in
1774, when he observed that these animals could be
revived after desiccation (Kinchin 1994). In 1962,
Tardigrada was recognized as a phylum by Ramazzotti
in Il Phylum Tardigrada (Ramazzotti 1962). There are

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Acta Physiologica  2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x 409
Adaptation to extreme environments in tardigrades Æ N. Møbjerg et al. Acta Physiol 2011, 202, 409–420

currently approx. 1000 described species (Guidetti &
Bertolani 2005, Degma et al. 2010), but this is likely far
from the real number of tardigrade species, as especially
the marine arthrotardigrades remain relatively unex-
plored.
Tardigrada belongs to the invertebrate superclade
Ecdysozoa, however; their precise phylogenetic position
is still debated and it is presently not clear whether the
group is more closely related to arthropods and
onychophorans or to the nematodes and nematomorphs
(Fig. 1) (Aguinaldo et al. 1997, Dunn et al. 2008,
Edgecombe 2010). In any case, as noticed in recent
papers, this group has a central position placed in
between the two major invertebrate model organisms –
the nematode Caenorhabditis elegans Maupas, 1900
and the arthropod Drosophila (Sophophora) melanog-
aster Meigen, 1830 (Goldstein & Blaxter 2002, Gabriel
& Goldstein 2007).
Little is known about the physiological mechanisms
underlying adaptations to extreme environmental con-
ditions in tardigrades. Past centuries of tardigrade
research have mainly focused on species descriptions
and morphological investigations related to phyloge-
netic analysis. In recent years, however, research in the
field has taken advantage of new molecular tools and an
increasing number of scientists find the group fasci-

Loricifera

Kinorhyncha

Priapulida

Nematomorpha
ECDYSOZOA

Nematoda*

?
Tardigrada*

Arthropoda*

Onychophora

PROTOSTOMIA

Brachiopoda

Annelida

LOPHOTROCHOZOA Mollusca

Rotifera*

Platyhelminthes

Chordata
DEUTEROSTOMIA
Echinodermata

Cnidaria

Porifera

Figure 1 Evolutionary position of tardigrades in the Animal Kingdom. Phylogeny of the Metazoa (animals) based on
Aguinaldo et al. (1997) and Dunn et al. (2008) showing selected phyla with emphasis on the position of the Tardigrada. The
inferred position of the tardigrades is based on EST sequences from Richtersius coronifer and Hypsibius dujardini. Ecdysozoa
includes all molting animals and is one of the two protostome superclades. The marked phyla have cryptobiotic species.

 2011 The Authors

410 Acta Physiologica  2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x
Acta Physiol 2011, 202, 409–420 N. Møbjerg et al.
nating. In particular, the mechanisms underlying the
ability to enter cryptobiosis have attracted considerable
scientific interest. This interest is no doubt associated
with the suspected translational output related to a
detailed understanding of the complex stress physiology
of tardigrades, i.e. in connection with cryopreservation
and dehydration of biological material. Noticeably, the
phenomenon cryptobiosis touches upon our conception
of life and death; one of the largest enigmas being how
metabolism is restarted after years of suspension. This
review puts focus on physiological and molecular
adaptations to extreme conditions found among tardi-
grades. We additionally present a brief description of
the phylum and highlight species that are currently used
as models for these investigations.
Phylum Tardigrada
Tardigrade phylogeny and evolution
About 35 extant animal groups have body plans and
genes that are distinct enough to warrant elevation to
phylum status (Nielsen 2001). The tardigrades, com-
prising the phylum Tardigrada, are one of these groups
(Fig. 1). Tardigrades are microscopic invertebrates with
a well developed organization including brain and
sensory organs, muscles, a complex feeding apparatus
and alimentary tract, reproductive and osmoregulatory
organs (see e.g. Rebecchi & Bertolani 1994, Dewel &
Æ Adaptation to extreme environments in tardigrades
Dewel 1996, Møbjerg & Dahl 1996, Eibye-Jacobsen
1997, Jørgensen et al. 1999, Greven 2007, Halberg
et al. 2009a). They are found worldwide in aquatic as
well as terrestrial environments, but depend on free
water to be in their active, reproducing state. It has been
suggested that tardigrades, like e.g. nematodes, have
eutely, but detailed studies on the subject are still
lacking. Cell counts based on nuclear staining with
DAPI (4¢,6-diamidino-2-phenylindole) in four active
stage adults of the marine eutardigrade Halobiotus
crispae Kristensen, 1982 revealed a total cell number of
around 1060 cells, when excluding gametes (Fig. 2).
This number could represent a slight underestimate of
the total somatic cell number as body cavity cells (also
known as storage cells) may have escaped two of the
specimens, which were punctured prior to the staining
(Fig. 2). We did not observe cell divisions (mitosis)
during the counts. Mitosis has, however, previously
been reported in post-embryonic eutardigrades (Berto-
lani 1970a,b, 1982).
There are two main evolutionary lines within the
tardigrades, represented by the classes Eutardigrada and
Heterotardigrada (Fig. 3) (see e.g. Jørgensen & Kris-
tensen 2004). The validity of a third class, Mesotardi-
grada, is currently uncertain. Mesotardigrada only
contains a single species, Thermozodium esakii Rahm,
1937 originally found in a hot spring in Japan. The type
specimens of T. esakii no longer exist and the type
locality was apparently destroyed in an earthquake

A P

br pb eo go
mg
gI gII
c.gl. gIII
c.gl.
c.gl. gIV
Leg 1 Leg 4
Leg 2 c.gl.
Leg 3

Figure 2 Cell numbers in Halobiotus crispae. 3-D reconstruction of cell arrangement in the eutardigrade H. crispae based on
confocal laser scanning microscopy of a DAPI stained specimen. In order to obtain an estimate of somatic cell numbers in this
species, specimens were relaxed in CO2-enriched water, fixed in 4% paraformaldehyde and ultrasonicated. Two of the four
specimens were additionally delicately punctured with a fine needle. The specimens were subsequently incubated with DAPI and
thoroughly rinsed before mounting. Image acquisition was performed using a Leica DM RXE 6 TL microscope equipped with a
Leica TCS SP2 AOBS confocal laser scanning unit. Cell counts and image processing were performed using the software program
Imaris (Bitplane, Zurich, Switzerland). The total number of somatic cells in H. crispae was estimated at approx. 1060, based on
stainings of four male specimens (mean  SD: 1058  53). This number could represent a slight underestimate of the total somatic
cell number as so-called body cavity cells may have escaped the two specimens that were punctured prior to staining (cell counts for
the punctured specimens: 998 and 1088; counts for the non-punctured specimens: 1036 and 1112). The largest number of cells is
clearly present in the anterior part of the animal containing the brain and buccopharyngeal apparatus. A, anterior; P, posterior; br,
brain; c.gl., claw gland; eo, esophagus; gI–IV, ventral ganglia I–IV; mg, midgut; go, gonad; pb, pharyngeal bulb. Scale bar: 50 lm.

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Acta Physiologica  2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x 411
Adaptation to extreme environments in tardigrades
Echiniscoides Echiniscus Milnesium* Halobiotus
Echiniscoidae Echiniscidae Milnesiidae Isohypsibioidea
Arthrotardigrada Echiniscoidea Apochela
marine limno-terrestrial limno-terrestrial
non-cryptobionts Intertidal Cryptobionts
Cryptobionts
Heterotardigrada Mesotardigrada (?) Eutardigrada
Tardigrada
(Nelson 2002). However, a thorough re-sampling for
this species has to our knowledge not been performed.
Tardigrades most likely evolved within the marine
environment, and marine species are especially numer-
ous within the heterotardigrade order Arthrotardigrada
(Renaud-Mornant 1982, Maas & Waloszek 2001,
Jørgensen et al. 2010). Arthrotardigrades are present
in all oceans from intertidal zones to abyssal depths,
inhabiting different sediment types. In addition, marine
species are found within the other main heterotardi-
grade order, Echiniscoidea, represented by the intertidal
Echiniscoides sigismundi (M. Schultze, 1865). This
species may very well be the toughest creature on
Earth, having to endure periods of desiccation and low
oxygen tension as well as large perturbations in salinity
and freezing (Kristensen & Hallas 1980). Nevertheless,
the exact range of this tardigrade’s tolerances remains
to be investigated. It may be hypothesized that an
Echiniscoides-like tardigrade invaded the freshwater/
terrestrial environment and gave rise to the almost
exclusively limno-terrestrial eutardigrades (Kinchin
1994). This is however currently not supported by
molecular data (Sands et al. 2008, Jørgensen et al.
2010; Jørgensen et al. 2011).
The eutardigrades are divided into two orders;
Apochela and Parachela. Two genera within the latter
order, Ramajendas represented by Ramajendas renaudi
(Ramazzotti 1972) in the Southern Hemisphere and
Halobiotus in the Northern Hemisphere, have second-
arily invaded the marine environment (Ramazzotti
1972, Kristensen 1982, Møbjerg et al. 2007). Although
cryptobiosis is common in most eutardigrades, our
recent findings in H. crispae suggest that among these
secondary marine species, adaptations are present that
are quite extraordinary (Halberg et al. 2009b). The
tardigrades stay active while experiencing large fluctu-
ations in abiotic factors, fluctuations that in other
tardigrades would induce cryptobiosis.
412 Acta Physiologica  2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x
Æ N. Møbjerg et al. Acta Physiol 2011, 202, 409–420
Richtersius Hypsibius*
Paramacrobiotus Ramazzottius*
Macrobiotoidea Hypsibioidea
Parachela
limno-terrestrial
cryptobionts
non-cryptobionts
Figure 3 Tardigrade phylogeny. Phylog-
eny of tardigrades showing major clades
and position of model/discussed species.
The phylogeny is based on Sands et al.
*Genome projects
(2008).
Tardigrade genomes
There is a huge variation in the genome size of
tardigrades ranging from about 75–100 Mb in Hypsi-
bius and Ramazzottius to 800 Mb in Bertolanius
(Gregory 2010, C-values converted from picograms to
base pairs using the conversion 1 pg = 978 Mb accord-
ing to Dolezel et al. (2003), Bertolanius was previously
named Amphibolus). For comparison, the genome sizes
of Caenorhabditis elegans and Drosophila melanogas-
ter are about 100 Mb and 175 Mb respectively (Greg-
ory 2010). The general diploid chromosome numbers of
the eutardigrades are 10–12 and 14 for the heterotar-
digrade Echiniscus (Bertolani 1982). Polyploidy with
up to 24 chromosomes is common in eutardigrades
(Bertolani 1982). Three major sequencing projects are
currently ongoing within Tardigrada. However, all
three projects are investigating eutardigrade species;
no data are presently available for the other main
tardigrade group – the heterotardigrades. The interna-
tional collaborative Ecdysozoan Sequencing Project is
assembling the genome of Hypsibius dujardini (Doyère,
1840) as part of an investigation into the ancestral
genome of the Ecdysozoa lineage. Prior to this project,
the Edinburgh based TardiBASE project, generated
more than 5000 EST sequences for H. dujardini
(GenBank 2010). The German based FUNCRYPTA
project was focused on investigating cryptobiosis in
Milnesium tardigradum Doyère, 1840 through studies
of gene and protein expression (Förster et al. 2009,
Mali et al. 2010, Schokraie et al. 2010). The project
had in 2010 generated approx. 7000 quality EST
sequences and aimed at advancing the basic understand-
ing of protein expression in tardigrades through tran-
scriptomic and proteomic studies (Mali et al. 2010). The
Japanese based Kumamushi Genome Project is assem-
bling the genome of Ramazzottius varieornatus Berto-
lani and Kinchin, 1993 and has preliminarily predicted
 2011 The Authors
Acta Physiol 2011, 202, 409–420 N. Møbjerg et al.
about 20 000 candidate genes in this species (Horikawa
et al. 2008, Katayama et al. 2009). The great challenge
in the years to come will be to correlate the description of
tardigrade genes with studies on the function of these
genes as nicely illustrated in a recent study on a
purinergic P2X receptor from H. dujardini (Bavan et al.
2009).
Adaptation to extreme environments
Tardigrades are extraordinary in their tolerance to
extremes, including limno-terrestrial habitats that fre-
quently dry out, habitats that freeze and habitats that
experience large fluctuations in e.g. osmotic pressure
and oxygen tension. Cryptobiosis, referred to as a
reversible ametabolic state induced by unfavorable
environmental conditions, is a common adaptation
especially among limno-terrestrial tardigrade species
(see e.g. Wright 2001). Four cryptobiosis inducing
physical extremes are traditionally recognized: dehy-
dration (anhydrobiosis), extremely low temperatures
(cryobiosis), lack of oxygen (anoxybiosis) and high salt
concentration (osmobiosis) (Keilin 1959), with desicca-
tion induced anhydrobiosis and freezing induced cryo-
biosis being the most extensively studied states.
Anhydrobiosis and cryobiosis are not equivalent phe-
nomena and likely involve different mechanisms for
protection of cells and tissues (Crowe et al. 1990,
1992). Little is known of cryobiosis in tardigrades
(Westh et al. 1991, Ramløv & Westh 1992, Westh &
Kristensen 1992, Halberg et al. 2009b, Hengherr et al.
2009, 2010), whereas a great deal of attention has been
paid to anhydrobiosis (Wright et al. 1992, Wright
2001, Rebecchi et al. 2007, Schill 2010). The anhydro-
biotic state is characterized by the formation of a so-
called tun with withdrawn legs and a longitudinally
contracted body (see e.g. Bertolani et al. 2004). Tun
formation is also seen in bdelloid rotifers, whereas
desiccation tolerant nematodes coil into a tight spiral.
Obviously, the ability to pack internal organs during
tun formation is an important adaptation to desicca-
tion. Importantly, tun formation is an active, regulated
event and not merely an effect of water removal (Crowe
1972). Along this line, our unpublished data show that
when we expose the active state of the cryptobiotic
tardigrade Richtersius coronifer (Richters, 1903) to
water containing high levels of chemical substances, the
tardigrades will respond by contracting their bodies
initiating tun formation thus undergoing chemobiosis–a
cryptobiotic response to environmental toxins. The
formation of the tun is a critical and necessary event
for tardigrades entering anhydrobiosis. Much more
work needs to be done in order to understand the
processes undertaken during transformation to this
ametabolic state, from the molecular to whole organism
 2011 The Authors
Acta Physiologica  2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x
Æ Adaptation to extreme environments in tardigrades
level, anatomically as well as physiologically. In the tun
state tardigrades may not only stand long periods of
desiccation and exposure to toxic chemicals but also
very low subzero temperatures, vacuum, high pressure,
radiation, extreme pH, anoxia and to some extent high
temperature (see e.g. Wright 2001, Rebecchi et al.
2007). It has been suggested and to some degree shown
that adaptations to these extremes may involve synthe-
sis of bioprotectants in the form of selective carbohy-
drates and proteins, high levels of antioxidant enzymes
and other free radical scavengers, biological membranes
containing specific phospholipids as well as powerful
DNA repair mechanisms (Westh & Ramløv 1991, Schill
et al. 2004, Jönsson et al. 2005, Rizzo et al. 2010).
In addition to cryptobiotic tardigrade species, we also
find species that form cysts and enter diapause (see e.g.
Møbjerg et al. 2007, Guidetti et al. 2008). Importantly,
it has been shown that tardigrades even in their active
states may be extremely tolerant to environmental stress
(May et al. 1964, Jönsson et al. 2005, Horikawa et al.
2006, Halberg et al. 2009b). Virtually nothing is known
about the normal physiology of tardigrades, and
answers to how they tolerate these extremes may
actually be found here. We have recently shown that
the littoral eutardigrade Halobiotus crispae handles
large fluctuation in external salinity and avoids freezing
by supercooling to around )20 C in its active stage
(Halberg et al. 2009b). This species is characterized by
seasonal cyclic changes in morphology and physiology
known as cyclomorphosis, one of the cyclomorphic
stages being freeze tolerant (Kristensen 1982, Møbjerg
et al. 2007, Halberg et al. 2009b). The morphological
changes occurring during cyclomorphosis in H. crispae
in some respects resemble the formation of dauer larvae
in C. elegans (see e.g. Cassada & Russell 1975, Burnell
et al. 2005).
Cryptobiosis in tardigrades
Many experiments on tardigrade cryptobiosis have been
performed on the eutardigrade Richtersius coronifer.
R. coronifer, also known as the giant yellow water bear,
has a body length of up to 1 mm (Fig. 4a–c). Both males
and females are present in some populations, but, as
commonly found among eutardigrades, several popula-
tions reproduce by parthenogenesis. R. coronifer lives in
moss in alpine and arctic environments and is further-
more numerous in moss on carbonated bedrock in dry
Swedish lowland areas known as Alvar (see e.g. Westh
& Kristensen 1992, Jönsson et al. 2001). R. coronifer is
a true cryptobiont, tolerating extreme desiccation as
imposed by e.g. space vacuum conditions (Jönsson et al.
2008, Persson et al. 2011) and it additionally survives
exposures to very low temperatures as encountered by
transfers into liquid nitrogen (approx. )196 C) in the
413
Adaptation to extreme environments in tardigrades Æ N. Møbjerg et al. Acta Physiol 2011, 202, 409–420

(a) A (b) A (c) A

st
pb
P P P
eo

st
mg
pb mg

* *
*

(d) (e) (f)

st st

pb pb
st
pb eo eo

mg mg
mg

go

* * * *
* * * *
*

Figure 4 Cryptobiotic and non-cryptobiotic survival in extreme environments. (a–c): Microscopy of Richtersius coronifer from
Stora Alvar on the Swedish island Öland in the Baltic Sea. Richtersius coronifer has been a model tardigrade for investigations on
cryptobiotic survival. (a) Active state; (b) light microscopy and (c) scanning electron microscopy of the cryptobiotic tun state
induced by desiccation. In this state R. coronifer e.g. tolerates complete desiccation as experienced by vacuum condition and
freezing in liquid nitrogen. (d–f): Microscopy of Halobiotus crispae from the Danish population at Vellerup Vig, Denmark. This
tardigrade is one of a few species of eutardigrades that have secondarily invaded the marine environment. Halobiotus crispae in the
active stage (d) uniquely adapted to cope with profound changes in ambient salinity occurring in tidal and subtidal habitats. Upon
transfers to dilute salt water solutions, active stage H. crispae swell (e) and subsequently regulate their body volume to near control
conditions. Halobiotus crispae is characterized by appearing in different cyclomorphic stages. The pseudosimplex 1 stage (f) is freeze
tolerant. eo, esophagus; mg, midgut; pb, pharyngeal bulb; st, stylet. Asterisks mark the position of three Malpighian tubules
presumably involved in osmoregulation. Scale bars: 50 lm.

tun as well as active hydrated state, however,with the
tuns tolerating considerable longer time of exposure
(Ramløv & Westh 1992, Persson et al. 2011). The
anhydrobiotic state survives temperatures of up to
approx. 70 C for 1 h, but survival rapidly decreases
when the temperature exceeds 70 C, and no specimens
survives exposure to 100 C (Ramløv & Westh 2001).
The increase in life span offered by anhydrobiosis in this
species seems to be restricted to approx. 5 years (Westh
& Kristensen 1992). This is less than the cryptobiotic
life expansion observed for the heterotardigrade Echi-
niscus testudo (Doyère, 1840), for which we have
revived specimens from moss cushions dried for approx.
20 years (Jørgensen et al. 2007). What then sets the
limits for cryptobiotic survival? Probably accumulated
damage to DNA and other molecules as well as to
tissues and organs obtained during the ametabolic state.
This includes damage obtained through oxidative pro-
cesses as well as predation, bacterial and fungal
infections. A recent investigation in Paramacrobiotus
richtersi (Murray, 1911) has shown that regulation of
antioxidant metabolism likely plays an important role
in defense against the potential oxidative damage
associated with dehydration (Rizzo et al. 2010). It has
long been known that there is a positive correlation
between the time spent in the anhydrobiotic state and
the time required for recovering active life after rehy-
dration (Crowe & Higgins 1967). Recent investigations
indicate that the longer the time spent in anhydrobiosis,
the more damage is inflicted to DNA (Neumann et al.
2009, Rebecchi et al. 2009b), which would explain the
prolonged recovery time, indicating that considerable
repair of DNA (and other molecules) occurs in the post-
anhydrobiotic state.
Trehalose accumulation. Current knowledge suggests
that specific carbohydrates and proteins are important
in protecting the cells from damage encountered during

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414 Acta Physiologica  2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x
Acta Physiol 2011, 202, 409–420 N. Møbjerg et al.
entry and exit of cryptobiosis. Bioprotectants may
interact directly with macromolecular structures such
as membranes, DNA and proteins, or they may act as
osmolytes during osmotic or dehydration stress. As has
been shown for other cryptobiotic invertebrates, notice-
ably in the cysts of the brine shrimp Artemia francis-
cana Kellogg, 1906, some, but not all, cryptobiotic
tardigrades accumulate the dissacharide trehalose dur-
ing desiccation (Clegg 1965, Westh & Ramløv 1991,
Hengherr et al. 2008, Jönsson & Persson 2010). Tre-
halose has been proposed to act as a molecular stabilizer
replacing water and to further stabilize cellular struc-
ture through the formation of amorphous glasses, a
process known as vitrification (Clegg 2001). The highest
trehalose concentrations measured in anhydrobiotic
tardigrades ranges from 2.3% d.w. in R. coronifer
and Macrobiotus krynauwi Dastych and Harris, 1995
to 2.9% d.w. in Macrobiotus islandicus Richters, 1904
(Westh & Ramløv 1991, Jönsson & Persson 2010).
These trehalose values are, however, relatively low
when compared with the above mentioned crustacean,
which accumulates the disaccharide in concentrations of
around 15% d.w. Moreover, trehalose levels are barely
measurable and do not increase during dehydration in
the cryptobiotic Milnesium tardigradum (see Hengherr
et al. 2008, Jönsson & Persson 2010). The latter
investigations, analysing trehalose contents in eutar-
digrades as well as heterotardigrades, show that
trehalose accumulation does not represent a universal
protective mechanism enabling tardigrades to undergo
cryptobiosis.
Expression of heat-shock proteins. Cryptobiosis has
been suggested to rely on the synthesis of molecular
chaperones such as heat-shock-proteins, which may
assist folding of newly synthesized proteins, control
their final intracellular location, as well as protect them
from stress-associated denaturation and aid in renatur-
ation (Clegg 2001). During entrance into anhydrobiosis
Ramløv & Westh (2001) described the upregulation of
a protein with a molecular weight of approx. 71 kDa in
R. coronifer and proposed that this protein might
belong to the heat-shock-protein (Hsp70) family. Schill
et al. (2004) subsequently investigated RNA expression
patterns of three Hsp70 isoforms in active and anhyd-
robiotic states of M. tardigradum as well as during
entrance into and exit out of anhydrobiosis. Only one
of these isoforms (isoform 2) was significantly induced
by the transition from the active to cryptobiotic state
and interestingly, it showed a considerable increase in
expression during the post-cryptobiotic phase, while
the other isoforms were down regulated during cryp-
tobiosis as well as in transitional states. Jönsson &
Schill (2007) used an immuno-westernblot method to
quantify the induction of Hsp70 in R. coronifer in
 2011 The Authors
Acta Physiologica  2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x
Æ Adaptation to extreme environments in tardigrades
response to desiccation, ionizing radiation and heating.
They reported elevated levels of Hsp70 following both
heat and radiation treatment as well as in tardigrades
rehydrated after a period of desiccation. Noticeably,
the authors found that tardigrades in the desiccated
state had reduced Hsp70 levels as compared to the non-
treated control group and accordingly suggested that
Hsp70 may be involved in the repair processes after
desiccation rather than acting as a biochemical stabi-
lizer in the dry state. Based on the M. tardigradum EST
library, several additional heat-shock proteins have
been identified, including two a-crystalline heat-shock
proteins, and the relative abundance of the transcripts
coding for these stress proteins have been investigated
during phases of dehydration and rehydration (Reuner
et al. 2010). The results obtained suggested a limited
role for heat-shock proteins in the desiccation tolerance
of M. tardigradum. The authors found a variable
pattern of expression with most of the candidate genes
being down regulated, and only one of the genes (Mt-
hsp90), being significantly upregulated in the dehy-
drated state (Reuner et al. 2010). Comparable studies
in Paramacrobiotus richtersi did not show evidence for
an increased expression of either Hsp70 or Hsp90
between hydrated and dehydrated animals (Rizzo et al.
2010). Additionally, specimens of P. richtersi sent into
space (Foton-M3 mission) revealed no significant
change as compared to ground controls in the expres-
sion of these heat-shock proteins (Rebecchi et al.
2009a). These contrasting results indicate that Hsp
expression is species specific. A general role in the
cryptobiotic survival of tardigrades can at present not
be attributed to these stress proteins. Nevertheless, a
synergistic effect between HSPs and other bioprotec-
tants such as trehalose or LEA (late-embryogenesis
abundant) proteins might exist (see e.g. Goyal et al.
2005). Notably, LEA proteins have been described in
many other desiccation tolerant organisms from plants
to arthropods and might analogously be functionally
important in tardigrades (see e.g. Schill 2010, Warner
et al. 2010). Indeed, a putative LEA protein has
recently been detected in M. tardigradum (Schokraie
et al. 2010). Consequently, future studies on these and
other bioprotectants might offer additional clues in our
search to understand the phenomenon cryptobiosis.
Obviously, cryptobiosis is far from understood and
much more research is needed in order to understand
the mechanisms underlying this state. Interestingly, a
preliminary study from the Kumamushi Genome
Project on R. varieornatus suggests that comparative
metabolome profiling of active and anhydrobiotic
states may provide a novel insight into anhydrobiosis
(Arakawa et al. 2009). Additionally, clues to cryptobi-
otic survival may be found in the extreme tolerance
seen in active state tardigrades.
415
Adaptation to extreme environments in tardigrades Æ N. Møbjerg et al.
Non-cryptobiotic survival in tardigrades
While tardigrades are well known for their abilities to
cope with extreme environmental conditions by enter-
ing cryptobiosis, little focus has been on their ability to
sustain metabolism and remain active during fluctuating
external circumstances. In this context it is interesting to
note that tardigrades are genuine aquatic animals – they
are dependent on free water to be in their active feeding
and reproducing states. Truly terrestrial conditions are
only survived following entry into the cryptobiotic
state. Furthermore, limno-terrestrial tardigrades that
have the ability to enter the tun state may do so in
response to environmental challenges (e.g. exposure to
chemical substances) that in marine species would not
force the animals into cryptobiosis. In the following we
discuss what is currently known about the physiology
and stress tolerance of tardigrades in their active states.
Radiation tolerance. Investigations on radiation toler-
ance in Richtersius coronifer have revealed that exposure
to c-radiation at doses up to 1 kGy does not affect
survival of desiccated nor hydrated animals, with
hydrated animals tolerating doses of up to 5 kGy (Jöns-
son et al. 2005). Horikawa and co-workers revealed that
both hydrated and desiccated Milnesium tardigradum
survive doses of c-radiation of more than 5 kGy, and up to
8 kGy of heavy ion radiation (Horikawa et al. 2006).
These and previous studies indicate that hydrated animals
are just as good or even better at tolerating radiation. This
indicates that radiation tolerance is not due to biochem-
ical protectants associated with the cryptobiotic state, but
suggests that tardigrades rely on efficient and yet uniden-
tified mechanisms of DNA repair (Jönsson et al. 2005,
Horikawa et al. 2006). Several recent studies involved
with the BIOPAN 6/Foton-M3 mission in 2007, funded
by the European Space Agency, have investigated the
impact on survival of tardigrades exposed to space
conditions (Jönsson et al. 2008, Rebecchi et al. 2009a,
Persson et al. 2011). During this mission, cryptobiotic
tardigrades (as well as nematodes and rotifers) were sent
into low earth orbit and exposed to space vacuum and
cosmic radiation. The three studies revealed discrepan-
cies in survival rates likely reflecting differences between
tardigrade species and experimental setups, however;
they unanimously conclude that tardigrades can survive
the rigors of space, and that M. tardigradum is likely the
most resistant species with embryos as well as adults
tolerating space conditions (see discussion in Persson
et al. 2011). Detailed knowledge of the life history of this
predatory tardigrade is available as a result of a compre-
hensive study by Suzuki (2003).
Osmoregulation. Experiments on the marine eutardi-
grade Halobiotus crispae have revealed an extraordinary
416 Acta Physiologica  2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x
Acta Physiol 2011, 202, 409–420
tolerance to perturbations in body volume and osmotic
pressure (Halberg et al. 2009b). The tidal habitat in
which this tardigrade lives is characterized by large
fluctuations in abiotic factors; most noticeably, altera-
tions are seen in salinity and temperature (Møbjerg et al.
2007). One way of coping with these extremes would be
to enter cryptobiosis. However, H. crispae is not a
cryptobiont. In the active stage the tardigrade handles
extremes by expending energy on active regulatory
mechanisms. In addition, the so-called pseudosimplex 1
(P1) stage of this animal is freeze tolerant (Halberg et al.
2009b); it is not, however, a cryobiont as the P1 stage does
not tolerate gradual freezing to )80 C. Cryobiosis is
defined by the apparent absence of a lower lethal
temperature (Wright 2001). The P1 stage is distinctly
characterized by a double cuticle and closed mouth and
cloaca (Kristensen 1982, Møbjerg et al. 2007).
Halobiotus crispae kept at a salinity of 20 ppt have an
extensive ability to supercool (avoid freezing) down to
around )20 C, enabling active stage animals to with-
stand subzero temperatures without freezing (Halberg
et al. 2009b). Similar supercooling points have been
reported from limno-terrestrial eutardigrades (Macro-
biotus, Paramacrobiotus and Milnesium), whereas het-
erotardigades (Echiniscus) had slightly higher points
(Hengherr et al. 2009). Much higher supercooling
points of )6.7 C and )7.4 C were originally observed
in respectively Richtersius coronifer and Bertolanius
nebulosus (Westh & Kristensen 1992). These high
supercooling points are likely the result of the presence
of ice-nucleating agents initiating the freezing process
(Westh et al. 1991). Figure 5 shows thermograms of H.
crispae kept in seawater with a salinity of 20 ppt
(Fig. 5a) and distilled water (Fig. 5b) prior to differen-
tial scanning calorimetry (DSC). Water content in the
animals kept at 20 ppt and in distilled water was
respectively 73% and 81%. The latter group of tardi-
grades had a higher supercooling point as compared to
the group kept in 20 ppt saltwater, illustrating the
expected correlation between water content and crys-
tallization temperature in species without ice-nucleating
agents (see e.g. Hengherr et al. 2009). Thus, the extent
of supercooling and thereby the ability to avoid freez-
ing, is coupled to osmoregulation.
Halobiotus crispae has a large capacity to tolerate
perturbations in ambient salinity making it an ideal
model for the study of osmoregulation and volume
regulation in tardigrades. Experiments on this species
revealed tolerance to a wide range of salinities, with
specimens of the Greenlandic population remaining
active in solutions ranging from distilled water to
saltwater with osmolalities up to 2000 mOsm kg)1
(Halberg et al. 2009b). During experiments with trans-
fers to strong hypotonic solutions, active stage H.
crispae swell with up to 60% before regulating back to
 2011 The Authors
Acta Physiol 2011, 202, 409–420 N. Møbjerg et al. Æ Adaptation to extreme environments in tardigrades
(a) 15 (a) 1400

Hemolymph osmolality (mOsm kg–1)

1200 H. crispae
Cooling
Differential heat flow (mW)

1000

Endotherm
800
Exotherm

7.5
600

400

Heating 200
Osmotic performance
Isoosmotic line
20 ppt. 0
0 0 200 400 600 800 1000 1200 1400
–40 –35 –30 –25 –20 –15 –10 –5 0 5
(b) 1400
(b) 15

Hemolymph osmolality (mOsm kg–1)

1200 R. coronifer
Cooling Endotherm
Differential heat flow (mW)

1000
Exotherm

800

7.5 600

Heating 400

0
–40 –35 –30 –25 –20 –15 –10
Temperature (°C)
Figure 5 Supercooling in Halobiotus crispae. (a) Supercooling
in H. crispae kept in seawater with a salinity of 20 ppt.
Thermogram modified from Halberg et al. (2009b). (b) Halo-
biotus crispae kept in distilled water. Cooling and subsequent
reheating of a sample containing 46 specimens of H. crispae
from Vellerup Vig, Denmark kept in distilled water for 45 min
prior to the transfer into pans for differential scanning calo-
rimetry (DSC). Freezing ()12.2 C) and melting ()1.2 C)
temperatures were estimated as onsets of peaks of respectively
exothermic and endothermic events using DSC7 software
(cooling rate 2 C min)1) (DSC7; Perkin Elmer Inc., Wellesley,
MA, USA).
near control values (Fig. 4e). Similarly, specimens
transferred into hypertonic solutions shrink and there-
after respond by regulating body volume. In a series of
experiments with transfers into saltwater solutions with
a salinity between 2 ppt (62 mOsm kg)1) and 40 ppt
(1245 mOsm kg)1) the active stage tardigrades hyper-
regulated at all times (Fig. 6a) (Halberg et al. 2009b).
Hyperregulation is likely a general feature of at least the
eutardigrades, as our data on R. coronifer reveal that
this species also keeps its body fluids hyperosmotic as
compared to the surroundings (Fig. 6b). Our data
indicate that this limno-terrestrial cryptobiont is less
tolerant of high salinity solutions than the littoral H.
crispae. When exposed to water with increasing salt
200
Osmotic performance
Isoosmotic line
0
0 ppt. 0 200 400 600 800 1000 1200 1400
External Osmolality (mOsm kg–1)
–5 0 5
Figure 6 Osmoregulation in eutardigrades. (a) Measured he-
molymph osmolality of active stage Halobiotus crispae from
Vellerup Vig, Denmark as a function of external osmolality.
Figure modified from Halberg et al. (2009b). (b) Hemolymph
osmolality of Richtersius coronifer from Öland, Sweden as a
function of external osmolality. External solutions with osmotic
concentrations of 100, 200, 300 and 500 mOsm kg)1 were
prepared from artificial seawater salt (Tropic Marin, Dr Biener
GmbH, Germany). Each point on the graph represents
mean  SD of hemolymph osmolality measurements (nanolitre
osmometry; Clifton Technical Physics, Hartford, NY, USA)
made in tardigrades kept in demineralized water and the differ-
ent salt water solution for 30 min. Five animals were used for
hemolymph determination at each of the experimental solutions.
Arrow indicates the upper lethal line for R. coronifer.
concentrations, R. coronifer will become inactive and
eventually die, exhibiting an upper lethal limit of
around 500 mOsm kg)1 (Fig. 6b). In solutions ranging
from demineralized water to salt water with an osmo-
lality of 500 mOsm kg)1, the tardigrade, however,
maintains a consistent osmotic gradient of around
170 mOsm kg)1 above that of the external environment
(Fig. 6b). In comparison, H. crispae exposed to the
same salinity range maintains an osmotic gradient of
around 300 mOsm kg)1 above that of the surroundings
(Fig. 6a). These data on two different species of
tardigrades indicate that eutardigrades have a relatively

 2011 The Authors

Acta Physiologica  2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02252.x 417
Adaptation to extreme environments in tardigrades Æ N. Møbjerg et al.
high water turnover, and that they excrete a hypo-
osmotic fluid – the likely organs involved in this
excretion being Malpighian tubules and the gut system
(Fig. 4) (Møbjerg & Dahl 1996, Halberg et al. 2009b).
At present the composition of tardigrade extracellular
fluids (as well as intracellular fluids) are unknown.
Obviously, information on the composition of the
hemolymph is much needed for our understanding of
tardigrade physiology and future discussions on how
tardigrades regulate their body fluids and especially
which osmolytes they are regulating. It is likely that the
osmotic pressure obtained through hyperregulation is
partly built by organic solutes, which in turn may act as
bioprotectants or may be important for e.g. the above
mentioned ability to supercool.
In summary, recent research has shown that tardi-
grades in their active state may tolerate large fluctua-
tions in abiotic factors and cope with environmental
alterations by staying active and maintaining high
metabolic rates. It would seem that these extraordinary
organisms have two ways of handling environmental
extremes; one way is entering a dormant state, i.e.
cryptobiosis or diapause/encystment and the other,
likely requiring a high metabolic rate, is relaying on
e.g. osmoregulation and DNA repair, while staying
active. The mechanisms enabling tardigrades to with-
stand environmental extremes in their active state may
provide clues to the much debated mechanism under-
lying the phenomenon of cryptobiosis. Intriguingly,
adaptations to some of the harshest environments on
Earth have given tardigrades the ability to survive
conditions that by far exceed the extremes presented by
the environment (e.g. extreme levels of ionizing radia-
tion and extremely low temperatures), presenting an
unresolved puzzle for contemporary biology.
Conflict of interest
There are no conflicts of interest.
We would like to thank Peter Westh for help in generating the
DSC data presented in Figure 5. Special thanks are due to the
organizers of the August Krogh Symposium 2010: Erik Hviid
Larsen, Ylva Hellsten and Jørgen Wojtaszewski. Funding came
from the 2008 Faculty of Science, University of Copenhagen
Freja-Programme and from the Carlsberg Foundation.
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