Tolerance of anhydrobiotic eggs of the Tardigrade Ramazzottius varieornatus to extreme environments.
ABSTRACT Tardigrades are tiny (less than 1 mm in length) invertebrate animals that have the potential to survive travel to other planets because of their tolerance to extreme environmental conditions by means of a dry ametabolic state called anhydrobiosis. While the tolerance of adult tardigrades to extreme environments has been reported, there are few reports on the tolerance of their eggs. We examined the ability of hydrated and anhydrobiotic eggs of the tardigrade Ramazzottius varieornatus to hatch after exposure to ionizing irradiation (helium ions), extremely low and high temperatures, and high vacuum. We previously reported that there was a similar pattern of tolerance against ionizing radiation between hydrated and anhydrobiotic adults. In contrast, anhydrobiotic eggs (50% lethal dose; 1690 Gy) were substantially more radioresistant than hydrated ones (50% lethal dose; 509 Gy). Anhydrobiotic eggs also have a broader temperature resistance compared with hydrated ones. Over 70% of the anhydrobiotic eggs treated at either -196°C or +50°C hatched successfully, but all the hydrated eggs failed to hatch. After exposure to high-vacuum conditions (5.3×10(-4) Pa to 6.2×10(-5) Pa), the hatchability of the anhydrobiotic eggs was comparable to that of untreated control eggs.
- SourceAvailable from: Eliana Beltrán-Pardo[Show abstract] [Hide abstract]
ABSTRACT: Tardigrades represent one of the most desiccation and radiation tolerant animals on Earth, and several studies have documented their tolerance in the adult stage. Studies on tolerance during embryological stages are rare, but differential effects of desiccation and freezing on different developmental stages have been reported, as well as dose-dependent effect of gamma irradiation on tardigrade embryos. Here, we report a study evaluating the tolerance of eggs from the eutardigrade Milnesium cf. tardigradum to three doses of gamma radiation (50, 200 and 500 Gy) at the early, middle, and late stage of development. We found that embryos of the middle and late developmental stages were tolerant to all doses, while eggs in the early developmental stage were tolerant only to a dose of 50 Gy, and showed a declining survival with higher dose. We also observed a delay in development of irradiated eggs, suggesting that periods of DNA repair might have taken place after irradiation induced damage. The delay was independent of dose for eggs irradiated in the middle and late stage, possibly indicating a fixed developmental schedule for repair after induced damage. These results show that the tolerance to radiation in tardigrade eggs changes in the course of their development. The mechanisms behind this pattern are unknown, but may relate to changes in mitotic activities over the embryogenesis and/or to activation of response mechanisms to damaged DNA in the course of development.PLoS ONE 01/2013; 8(9):e72098. · 3.53 Impact Factor
Tolerance of Anhydrobiotic Eggs of the Tardigrade
Ramazzottius varieornatus to Extreme Environments
Daiki D. Horikawa,1,2Ayami Yamaguchi,3,* Tetsuya Sakashita,4,* Daisuke Tanaka,5,*
Nobuyuki Hamada,6,* Fumiko Yukuhiro,7Hirokazu Kuwahara,3Takekazu Kunieda,3Masahiko Watanabe,5
Yuichi Nakahara,5Seiichi Wada,8Tomoo Funayama,4Chihiro Katagiri,9Seigo Higashi,10Shin-Ichi Yokobori,1 1
Mikinori Kuwabara,12Lynn J. Rothschild,1Takashi Okuda,5Hirofumi Hashimoto,13and Yasuhiko Kobayashi4, 14
Tardigrades are tiny (less than 1mm in length) invertebrate animals that have the potential to survive travel to
other planets because of their tolerance to extreme environmental conditions by means of a dry ametabolic state
called anhydrobiosis. While the tolerance of adult tardigrades to extreme environments has been reported, there
are few reports on the tolerance of their eggs. We examined the ability of hydrated and anhydrobiotic eggs of the
tardigrade Ramazzottius varieornatus to hatch after exposure to ionizing irradiation (helium ions), extremely low
and high temperatures, and high vacuum. We previously reported that there was a similar pattern of tolerance
against ionizing radiation between hydrated and anhydrobiotic adults. In contrast, anhydrobiotic eggs (50%
lethal dose; 1690 Gy) were substantially more radioresistant than hydrated ones (50% lethal dose; 509 Gy).
Anhydrobiotic eggs also have a broader temperature resistance compared with hydrated ones. Over 70% of the
anhydrobiotic eggs treated at either -196?C or +50?C hatched successfully, but all the hydrated eggs failed
to hatch. After exposure to high-vacuum conditions (5.3·10-4Pa to 6.2·10-5Pa), the hatchability of the
anhydrobiotic eggs was comparable to that of untreated control eggs. Key Words: Tardigrades—Ramazzottius
varieornatus—Anhydrobiosis—Radiation tolerance—Temperatures—Vacuum—Astrobiology. Astrobiology 12,
the Universe or have the capacity to travel via, for example,
impact events to other planets. Tardigrades (‘‘water bears’’)
have been proposed as potential organisms that may survive
ne of the goals of astrobiology is to ascertain whether
organisms, including multicellular organisms, exist in
extreme environmental stresses (Rothschild and Mancinelli,
2001; Jo ¨nsson, 2007; Horikawa et al., 2008). Tardigrades are
tiny invertebrates (0.1–1.0mm in length) that are distributed
in various habitats from polar to tropical regions throughout
the world. Some terrestrial tardigrade species enter ‘‘anhy-
drobiosis,’’ which is defined as the latent state of life induced
by dehydration (Keilin, 1959). Anhydrobiotic adult tardi-
grades are extraordinarily tolerant to a variety of extreme
1NASA Ames Research Center, Moffett Field, California, USA.
2NASA Astrobiology Institute.
3Graduate School of Science, The University of Tokyo, Tokyo, Japan.
4Microbeam Radiation Biology Group, Japan Atomic Energy Agency, Takasaki, Japan.
5Anhydrobiosis Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Japan.
6Radiation Safety Research Center, Nuclear Technology Research Laboratory, Central Research Institute of Electric Power Industry
(CRIEPI), Tokyo, Japan.
7Insect-Microbe Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Japan.
8Kitasato University School of Veterinary Medicine, Towada, Japan.
9Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan.
10Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, Japan.
11School of Life Science, Tokyo University of Pharmacy and Life Science, Hachioji, Japan.
12Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
13Institute of Space and Astronautical Science, JAXA, Sagamihara, Japan.
14Department of Quantum Biology, Gunma University Graduate School of Medicine, Maebashi, Japan.
*These authors contributed equally to this work.
Volume 12, Number 4, 2012
ª Mary Ann Liebert, Inc.
environmental conditions, such as temperatures that range
from -273?C (Becquerel, 1950) to +151?C (Rahm, 1921),
pressures up to 7.5 GPa (Ono et al., 2008), chemicals that in-
clude organic solvents (Ramløv and Westh, 2001; Horikawa
et al., 2008) and methyl bromide (Jo ¨nsson and Guidetti, 2001),
UV radiation at doses of more than 104J/m2(Altiero et al.,
2011), and ionizing radiation of the order of kilograys, in-
cluding low linear energy transfer (LET) X-rays (May et al.,
1964), gamma-rays (Jo ¨nsson et al., 2005; Horikawa et al.,
2006), protons (Nilsson et al., 2010), and high-LET heavy ions
(Horikawa et al., 2006, 2008). The extreme tolerance of tardi-
grades to severe environmental stress raises a controversial
question of whether tardigrades can survive in outer space
environments (Copley, 1999; Jo ¨nsson, 2007; Horikawa et al.,
Ultimately, if we are to assess the ability of tardigrades to
survive transfer among planets or to thrive in extreme en-
vironments, they must be able to reproduce. Thus, the
present study focuses on the hatchability of tardigrade eggs
after exposure to environmental extremes. In contrast to
considerable studies of the tolerance in adult tardigrades, the
survival of anhydrobiotic tardigrade eggs after exposure to
the vacuum of space, space UV radiation, and cosmic radi-
ation has only been investigated in two studies. Jo ¨nsson et al.
(2008) reported that desiccated adults of two tardigrade
species, Richtersius coronifer and Milnesium tardigradum, sur-
vived exposure to the vacuum of space, and a small portion
of the tested individuals survived the combined exposure to
the vacuum of space and UV radiation for 10 days in low
Earth orbit. In addition, they demonstrated that desiccated
eggs exposed to the vacuum of space alone hatched at the
same level as the controls (Jo ¨nsson et al., 2008), which sug-
gests that tardigrade eggs in anhydrobiosis tolerate space
environmental stresses. In another flight experiment, Persson
et al. (2011) demonstrated that eggs of M. tardigradum toler-
ated low levels of cosmic radiation (less than 4 Gy) during a
10-day flight in low Earth orbit. Also, eggs of Ramazzottius
oberhauseri in anhydrobiotic state survived for a longer
period of time, with a maximum of 9 years, than did adult
(Guidetti and Jo ¨nsson, 2002). Here, we set out to estimate the
reproductive potential of tardigrade eggs in possible extra-
terrestrial environments by measuring their hatchability after
exposure to ionizing radiation, temperature, and vacuum.
2. Materials and Methods
2.1. Animal culture and egg collection
The tardigrade species Ramazzottius varieornatus, which
was originally collected from moss samples in Hokkaido,
Japan (43?03¢48†N, 141?22¢25†E), was used in this study. The
life cycle of R. varieornatus was revealed in our previous
study (Horikawa et al., 2008). Juvenile R. varieornatus become
mature adults subsequent to the molting processes (ecdysis).
On average, the life span of R. varieornatus is 35 days, the
number of eggs produced by an animal is 7.85, and the
hatching time of eggs is 5.7 days when the animals are cul-
tured at 25?C (Horikawa et al., 2008). In the present study,
tardigrades were cultured at 22–25?C on 1.5% w/w agar
plates with distilled water and the green alga Chlorella vul-
garis (Chlorella Industry Co., Ltd., Tokyo, Japan) as food.
Culture dishes were checked at 1–2 day intervals, and newly
laid eggs were transferred onto fresh agar plates containing
distilled water but not algae. Dark-colored eggs that failed to
develop were excluded, and normally developing eggs (2–3
days post-deposition) were used for experiments.
2.2. Preparation of anhydrobiotic egg samples
for exposure experiments
Here, we employed several desiccation methods that are
slightly different among types of exposure experiments as
described below. This was due to a limited availability of
incubators with identical temperature, a difference in sample
stage in each apparatus among those exposure experiments,
and a requirement for sample transportation in the vacuum
Anhydrobiotic eggs for scanning electron microscopy
(SEM) were prepared by placing them on double-sided
plastic tape and desiccated at 25?C under 85% relative hu-
midity (RH) for 1 day, followed by 0% RH for more than 2
days. RH was controlled by glycerol (Invitrogen, Carlsbad,
CA, USA) in water according to Johnson (1940).
For the irradiation experiment, 15–20 hydrated eggs in 100
lL of distilled water were placed on filter paper (1.33g, di-
ameter 35mm) in a plastic Petri dish (diameter 35mm) and
desiccated at 25?C under 0% RH for 2–7 days.
For the temperature experiment, 15–30 hydrated eggs in
100 lL of distilled water were placed on filter paper (0.55g,
2·2cm) in a plastic Petri dish (diameter 35mm) and desic-
cated at 25?C under 33.8% RH for 1 day and then desiccated
at 25?C under 0% RH for 4–6 days in a desiccator.
For the vacuum tolerance experiments, 25–100 hydrated
eggs in 100 lL of distilled water were placed on filter paper
(0.55g, 2·2cm) in a plastic Petri dish (diameter 35mm) and
desiccated at 22?C under 33.8% RH for 5 days and then kept
at 22?C under 0% RH for 9 days.
was statistically insignificant (Tukey’s multiple comparison
test, p>0.05: 88.8–5.8% for SEM, 93.9–0.9% for irradiation
experiments, 89.7–8.4% for temperature experiments, and
87.6–7.6% for vacuum experiments; values are the means of
three independent experiments).
2.3. Scanning electron microscopy
We examined the morphology of hydrated or anhy-
drobiotic eggs of R. varieornatus with SEM. Hydrated eggs in
500 lL distilled water in a 1.5mL microtube were frozen at
-80?C in a freezer and then dried in a vacuum system (FDU-
810, EYELA, Tokyo, Japan). Anhydrobiotic eggs were pre-
pared as described above. Both hydrated and anhydrobiotic
egg samples were coated with gold or platinum palladium
and observed with the VE-7800 scanning electron micro-
scope system (Keyence, Osaka, Japan).
Hydrated eggs were placed on filter paper with 200 lL of
distilled water in a plastic Petri dish (diameter 35mm). The
Petri dishes were covered with a 7 lm thick polyimide
Kapton film (Dupont-Toray, Tokyo, Japan) to prevent air-
drying, and the film was sealed with parafilm (Pechiney
Plastic Packaging, Inc., Chicago, IL, USA) on the side of the
HORIKAWA ET AL.
dishes. Anhydrobiotic eggs were placed in a similar way
except without water. Both hydrated and anhydrobiotic egg
samples were irradiated with 250–2000 Gy of4He (50 MeV,
16.3keV/lm) delivered from the azimuthally varying field
cyclotron installed at the Takasaki Ion Accelerators for Ad-
vanced Radiation Application
Atomic Energy Agency, as described previously (Hamada
et al., 2006; Horikawa et al., 2006). The absorbed dose was
calculated as follows: Dose (Gy)=1.6·10-9·LET (keV/
lm)·fluence (particle/cm2). Within 1h after irradiation,
2mL of distilled water was added to the dishes, and then the
eggs were incubated at 22?C.
In this experiment, three replicates were considered. To
determine the 50% lethal dose (LD50), the linear regression
analysis was performed between the dose and the hatch-
ability. Spearman’s correlation coefficient by rank test was
used to test correlations between the dose and the hatch-
ability. Hatchability was compared between anhydrobiotic
eggs and hydrated ones by using a chi-square test with a
2.5. Temperature experiments
In eighteen 1.5mL microtubes, hydrated eggs were sus-
pended in 1mL of liquid paraffin (Wako, Tokyo, Japan). In a
second set of 18 microtubes, anhydrobiotic eggs were also
suspended in 1mL of liquid paraffin. Three microtubes from
each of the two sets were then exposed to a temperature of
-196?C, -80?C, +22?C, +50?C, +80?C, and +100?C, all for
a total of 1h. For exposure to -196?C, microtubes containing
the egg samples were instantaneously frozen in liquid ni-
trogen. For the samples to be exposed to -80?C, they were
placed in a freezer. Cooling rates were -140–6.1?C/min in
liquid nitrogen and -7.7–1.0?C/min in the freezer, as de-
termined by recording temperature inside a microtube with a
thermocouple element connected to a logger inserted in liq-
uid paraffin. In the case of +50?C, +80?C, and +100?C, the
microtubes were heated in a dry heat block. Samples kept at
+22?C (room temperature) were considered as controls.
The microtubes were then removed from the devices and
left in ambient atmosphere for up to 2h to set back to room
temperature. After being washed one time in distilled water,
the eggs were transferred to Petri dishes (diameter 35mm)
and then rehydrated with 2mL of distilled water. Three in-
dependent experiments were carried out for each tempera-
ture condition. A chi-square test with a Yates’ correction was
used to compare the survival between anhydrobiotic and
2.6. Vacuum experiments
Anhydrobiotic samples were placed in a vacuum chamber
where the atmosphere was decompressed from atmospheric
pressure with a turbo-molecular pump (PT-500, Mitsubishi
Heavy Industries, Ltd., Tokyo, Japan) connected to the
chamber. An ion gauge (GI-TL3, ULVAC, Inc., Kanagawa,
Japan) inserted in the chamber indicated that pressure in the
vacuum chamber was 5.3·10-4Pa and 6.2·10-5Pa at 1h
and 7 days, respectively, after starting decompression.
Control eggs were kept at room temperature under atmo-
spheric pressure at 0% RH for 7 days. After returning to
atmospheric pressure, egg samples were kept with silica gel
in a tightly enclosed plastic bag. The samples were rehy-
drated with 2mL of distilled water 7 days after shipment
from H. Hashimoto’s laboratory (Tsukuba University, Japan)
to L.J. Rothschild’s laboratory (NASA Ames Research Cen-
Three independent replicate experiments were performed.
The survival of eggs exposed to vacuum and those that
comprised the untreated control were compared by a chi-
square test with a Yates’ correction.
2.7. Egg hatching
After rehydration, egg samples were transferred with a
glass pipette onto agar plate dishes (diameter 35mm) con-
taining a small amount of distilled water that covered the
agar surface with a thin film. For the irradiation experiment,
eggs were observed under a stereomicroscope with trans-
mitted illumination (SZ60, Olympus, Tokyo, Japan, or STE-
MI 2000-C, Zeiss, Jena, Germany) every day. For the
temperature and vacuum experiments, they were viewed
under the stereomicroscope at 1–5 day intervals. Since the
maximum development time of R. varieornatus eggs that has
been reported is 9 days after deposition (Horikawa et al.,
2008), the egg hatching was observed up to 10 days after
rehydration. Hatched juveniles were removed for the con-
venience of counting newborns each day.
3.1. Morphology of anhydrobiotic eggs
The typical shapes of hydrated and anhydrobiotic eggs
desiccated eggs showed an erythrocytic form with the projec-
tions bent onto the egg surface (Fig. 1A), while hydrated eggs
had a spherical shape with numerous projections (Fig. 1B).
3.2. Radiation tolerance of tardigrade eggs
The hatchability of anhydrobiotic eggs was over 80% at
doses up to 1000 Gy, whereas that of hydrated eggs was de-
decreased the hatchability of anhydrobiotic eggs to approxi-
mately30% at 2000 Gy.The hatchability ofanhydrobioticeggs
was significantly higher than that of hydrated ones at all ra-
diation doses tested (P<0.001), and the LD50values were 509
Gy in hydrated eggs and 1690 Gy in anhydrobiotic ones.
3.3. Temperature tolerance
The hydrated eggs hatched successfully at +22?C, while
only a very small proportion of eggs hatched after the +50?C
treatment (Fig. 3). In contrast, in anhydrobiotic eggs, the
hatchability was relatively high (>79.0%) over a broad
temperature range, from -196?C to +50?C, although it
markedly decreased at +80?C (Fig. 3). None of 100?C-treated
eggs hatched. Anhydrobiotic eggs hatched at a significantly
higher rate than hydrated ones at low (-196?C and -80?C)
and high (+50?C and +80?C) temperatures.
3.4. Vacuum tolerance
There was no significant difference (P=0.71) in the hatch-
ing percentage between vacuum treatment (85.9–13.1%) and
the control under atmospheric pressure (87.6–7.7%) (Fig. 4).
TOLERANCE OF TARDIGRADE EGGS285
Anhydrobiotic animals generally show morphological
changes when they enter into an anhydrobiotic state. For
example, while entering anhydrobiosis, juvenile and adult
tardigrades contract their body into a barrel-like shape (tun)
(Baumann, 1927; Wright, 1989), and nematodes transition
into a coiled shape (Crowe and Madin, 1975). Eggs of
R. varieornatus could hatch even after 10-day preservation
under extremely dried conditions that caused them to enter
into anhydrobiosis (Horikawa et al., 2008).
This study demonstrated that the anhydrobiotic eggs
showed cross-tolerance to various extreme environments, as
was the case for the adults. It should be noted, however, that a
slight difference in the drying conditions from those that were
used here could affect the speed of evaporation from the eggs
during desiccation. This could result in different degrees of
egg tolerant ability in extreme environments among the ex-
posure experiment groups; therefore, careful consideration
would be required for direct comparison of the survival
ability of the anhydrobiotic eggs among the various exposure
experiments. However, the eggs used in this study that un-
derwent dehydration in RH 0% are considered to have en-
tered into an anhydrobiotic state, judging from the desiccating
methods used in our previous study (Horikawa et al., 2006).
To overcome these issues in future studies, a universal
method for desiccating tardigrades should be established.
It is thought that removal of free water from an anhy-
drobiotic body may fundamentally confer on tardigrades
robustness against extreme environmental stressors (Seki
and Toyoshima, 1998). Solar and cosmic ionizing radiation is
an important potential risk for various organisms in outer
space. The irradiation experiments in the present study
showed that LD50values of eggs of R. varieornatus against
4He ions were 1690 Gy in anhydrobiotic eggs and 509 Gy in
hydrated ones, which indicates that hydrated eggs are more
radiosensitive than anhydrobiotic ones (Fig. 2). This result is
(250–2000 Gy) of anhydrobiotic and hydrated eggs of the
tardigrade R. varieornatus. Error bars indicate plus and minus
one standard deviation (N=3–4). Asterisks indicate signifi-
cant differences in hatchability between anhydrobiotic and
hydrated eggs (*P<0.001).
Changes in hatchability following4He irradiation
R. varieornatus. Scale bars=10 lm.
The representative scanning electron micrograph of anhydrobiotic eggs (A) and hydrated eggs (B) of the tardigrade
drated eggs of R. varieornatus following exposure to a series
of temperatures (-196?C to +100?C) for 1h. Eggs were ob-
served up to 10 days after exposure to temperatures, and
mean hatchability was determined for both anhydrobiotic
and hydrated groups. Error bars indicate plus and minus one
standard deviation (N=3). Asterisks indicate significant dif-
ferences in hatchability between anhydrobiotic and hydrated
eggs (chi-square test with a Yeats’ correction; *P<0.05,
Changes in hatchability of anhydrobiotic and hy-
286HORIKAWA ET AL.
consistent with the radiation sensitivity of Artemia salina
(brine shrimp) eggs, which, when fully hydrated, are less
tolerant to gamma-irradiation than desiccated ones (Engel
and Fluke, 1962). On the other hand, hydrated adult tardi-
grades have been shown to be highly radioresistant, as have
anhydrobiotic tardigrades (May et al., 1964; Jo ¨nsson et al.,
2005; Horikawa et al., 2006, 2008). It is known that cells of
tardigrades at the adult stage show less mitotic activity (Ber-
tolani, 1970), and the absence of somatic cell division may
lower the lethal effects of radiation (Ducoff, 1972). Tardigrade
eggs kept in an anhydrobiotic state for 9 years can successfully
hatch following rehydration (Guidetti and Jo ¨nsson, 2002), so it
is obvious that the cell cycle in these eggs is perturbed during
anhydrobiosis. Thus, the higher radiation tolerance anhy-
drobiotic eggs exhibited over that of hydrated eggs, as shown
in this study, might be the result of cell cycle arrest during
anhydrobiosis. Jo ¨nsson et al. (2008) reported that eggs of
R. coronifer and M. tardigradum that were exposed to 4.5 mGy of
cosmic radiation combined with the vacuum of space during a
10-day flight in low Earth orbit hatched, as did anhydrobiotic
eggs kept at normal pressures. Persson et al. (2011) demon-
strated that anhydrobiotic eggs of M. tardigradum that were
exposed to 4 Gy of cosmic radiation in low Earth orbit suc-
cessfully hatched as well. In addition, Rebecchi et al. (2009,
2011) reported that both hydrated and anhydrobiotic adults of
tardigrade Macrobiotus richtersi survived irradiation with 1.9
mGy of ionizing radiation inside the spacecraft Foton-M3
during a 12-day flight in low Earth orbit. Results of the present
study suggest that anhydrobiotic eggs of tardigrades tolerated
much higher doses of cosmic radiation than did the tardigrades
that were exposed in low Earth orbit in previous studies.
The present study is the first to evaluate the hatchability of
tardigrade eggs following exposure to extremely low and
high temperatures. As is the case for anhydrobiotic adults
(Becquerel, 1950; Ramløv and Westh, 2001; Horikawa et al.,
2008), eggs of R. varieornatus in an anhydrobiotic state tol-
erate low and high temperatures to a higher degree than do
hydrated ones (Fig. 3). Iwasaki (1973) reported that ap-
proximately 65% of a sum of dry eggs of A. salina hatched
after exposure to 100?C for 1h, whereas no R. varieornatus
eggs hatched with the same exposure (Fig. 3). Although this
result may suggest that brine shrimp have a higher tolerance
to a temperature of 100?C than do tardigrades, the difference
in the way the media in the present study was heated and the
way it was heated in the Iwasaki (1973) study might have
affected survivability. The liquid paraffin used in the present
study has a higher heat transfer efficiency compared to that
of air, which was what was used by Iwasaki (1973), who did
not utilize a special medium to suspend the heat-treated
Artemia eggs. The tardigrade eggs used in this study should,
therefore, have received more heat energy, which resulted in
lower survivability. It is likely that anhydrobiotic tardigrade
eggs are more tolerant to low temperature than they are to
high temperature (Fig. 3), as are adult anhydrobiotic tardi-
grades (Becquerel, 1950; Horikawa et al., 2008). Given this
capacity for anhydrobiotic eggs to tolerate a wide range of
temperatures, anhydrobiotic eggs could survive tempera-
tures at the martian surface, which range from -123?C to
25?C (Diaz and Schulze-Makuch, 2006). Moreover, the an-
hydrobiotic tardigrade eggs, juveniles, and adults could
survive temperatures below -269?C (Horneck, 1999) in in-
terplanetary space, since it is expected that even such ex-
tremely low temperatures would not change the structure of
biomolecules in anhydrobiotic organisms.
The vacuum level of open space causes extreme desiccation
and is therefore lethal for most organisms. In the present
study, the anhydrobiotic eggs of R. varieornatus tolerated high
vacuum for 7 days (Fig.4). This is consistent with the results
of Jo ¨nsson et al. (2008), who reported that anhydrobiotic eggs
of other tardigrade species hatched after exposure of 10 days
to the vacuum of space. For assessing the effects of high
vacuum on anhydrobiotic tardigrades, a longer period of
time would be needed to estimate survivability more pre-
cisely, since exposure to vacuum for several years is known to
reduce survivability, probably due to the accumulation of
DNA strand breaks, even in spores of Bacillus subtilis, one of
the known most vacuum-resistant bacteria (Horneck, 2003).
Jo ¨nsson et al. (2008) demonstrated that adults and eggs of
anhydrobiotic tardigrades can tolerate the space environment
and that UV radiation is the most severe environmental factor
to animals under their flight experiment conditions. In addi-
tion, Altiero et al. (2011) showed that exposure to more than
75kJ/m2of UV radiation dramatically reduced the survival
fraction of adult tardigrades in anhydrobiotic state. In this
study, we showed that anhydrobiotic eggs of tardigrades
survived extremely low temperatures and space-level vacuum.
Considering the critical effects of UV radiation on tardi-
grades, these animals may have the capacity to complete
interplanetary travel only when they are protected inside a
rock that serves as shielding against UV radiation. In fact,
environmental chamber, 70% of adult specimens of R. var-
ieornatus in anhydrobiotic state survived exposure to 3.7·107
J/m2of UVC radiation (Johnson et al., 2011), a dose that cor-
responds to more than 1000 times the UV dose that caused the
killing of all anhydrobiotic eggs of tardigrades in outer space
(Jo ¨nsson et al., 2008).
Extremely low temperatures do not seem to reduce survival
of anhydrobiotic R. varieornatus, as suggested by this study
and our previous study (Horikawa et al., 2008). Anhydrobiotic
tardigrades appear to survive longer at low temperature
(Sømme and Meier, 1996) than at room temperature (Rebecchi
et al., 2006). Tardigrades in the anhydrobiotic state survived up
to 9 years under room-temperature conditions (Guidetti and
to hatch after exposure to vacuum (5.3·10-4Pa and
6.2·10-5Pa) and kept under atmospheric pressure (control)
for 7 days. Error bars indicate plus and minus one standard
deviation (N=3). There was no significant difference in
hatchability between anhydrobiotic and hydrated eggs (chi-
square test with a Yeats’ correction, P>0.05).
The ability of anhydrobiotic eggs of R. varieornatus
TOLERANCE OF TARDIGRADE EGGS 287
Jo ¨nsson, 2002), but it is expected that they can survive much
longer at the extremely low temperatures found in outer space.
In addition, Gusev et al. (unpublished data) demonstrated that
anhydrobiotic larva of the insect Polypedilum vanderplanki
showed better survival in the vacuum of space than under
atmospheric pressure, which suggests that the vacuum of
space is a favorable environmental condition for anhydrobiotic
survival. In this regard, evaluation of the tolerance of tardi-
grades to prolonged exposure to space-level vacuum seems
particularly important. This warrants future experiments in
which tardigrades would be packed into a rocklike material
and exposed to prolonged open space environments. But the
survival of tardigrades in space is only of significance to
panspermia if they are able to reproduce once they reach a
favorable environment. This study provides support for the
possibility of successful transfer in the vacuum of space by
showing that indeed anhydrobiotic eggs were substantially
more resistant to radiation than hydrated eggs.
This work was supported by the REIMEI research re-
sources of JAERI (7) and TIARA Cooperative Research Pro-
gram (51047). This research was also supported by an
appointment to the NASA Astrobiology Institute NASA
Postdoctoral Program by Oak Ridge Associated Universities.
We thank T. Kubo of The University of Tokyo for daily as-
sistance and encouragement and K. Shimada of Hokkaido
University for assistance with recording temperatures inside
samples in the temperature experiment. We also thank
M. Yamashita of JAXA, A. Yamagishi of Tokyo University of
Pharmacy and Life Science, and K. Kobayashi of Yokohama
National University for arrangement of vacuum exposure
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Address correspondence to:
Daiki D. Horikawa
Institut National de la Sante ´ et de la Recherche Me ´dicale Unit 571
University Paris-Descartes Medical School
156 rue de Vaugirard
75015 Paris Cedex 15
Submitted 20 November 2011
Accepted 20 February 2012
TOLERANCE OF TARDIGRADE EGGS289