Survival of Tardigrades in Extreme Environments: A Model Animal for Astrobiology
Tardigrades, which are tiny invertebrate animals, have been considered as an appropriate model for astrobiological studies based on their high survival ability under various types of environmental stresses. So far, researches have shown that tardigrades have high tolerance to ionizing radiation, wide ranges of temperatures, vacuum, and high pressures in anhydrobiosis, a state that organisms lack free water in the body, and they resume activity when water is added. In addition, recently, a short-term flight experiment demonstrated that tardigrades in an anhydrobiotic state survived open space environments at low Earth orbit. Results from those exposure experiments indicate that tardigrades are well tolerant of extremely low temperatures, vacuum, and high pressures. On the other hand, ionizing radiation, UV radiation, and high temperatures could be the critical factors to limit habitable environments for tardigrades. Future astrobiological research on tardigrades, such as long-term exposure experiments, might provide important insight into the possibilities of existence of animal-like life forms or interplanetary transfer of multicellular organisms in an anhydrobiotic state.
Among abiotic environmental factors, desiccation is considered to be the most detrimental stress to organisms. To cope with severe desiccation, some organisms have evolved a survival strategy called anhydrobiosis in which organisms are tolerant of extreme dehydration for an extended period. Organisms showing anhydrobiotic ability are distributed in many taxonomic groups including bacteria, fungi, algae, plants, protozoans, and invertebrate animals such as tardigrades, nematodes, rotifers, and arthropodes (Alpert 2006; Watanabe 2006). Anhydrobiotic organisms have been thought as appropriate model organisms for astrobiological studies (Horneck 2003) because those which fulfill conditions for surviving open space environments must require ability to be tolerant of extreme desiccation induced by space vacuum (Jönsson 2007) and of other extraterrestrial environmental parameters, including extremely high or low temperatures, vacuum, and ultraviolet (UV) and ionizing radiation. Particularly, anhydrobiotic animals can be considered as interesting model organisms for astrobiological research since they could provide an estimate for the probabilities of existence of animal-like extraterrestrial life forms by both ground and flight experiments. Unlike unicellular organisms, anhydrobiotic animals have complicated body structures such as tissues and organs, possibly providing new insight into survival strategies for organisms under extreme environmental conditions.
2 Anhydrobiosis in Tardigrades
Anhydrobiosis is defined as an ametabolic state induced by dehydration and followed by resurrection when rehydrated (Keilin 1959). When terrestrial tardigrades enter into anhydrobiosis, their body water decreases to between 1% and 3% wt./wt. in association with body contraction (Crowe 1972; Horikawa et al. 2006, 2008). The contracted anhydrobiotic animal, referred to as a tun (Fig. 1b), shows no visible signs of life but can resume activity when placed in a drop of water. Tardigrades have the ability to enter anhydrobiosis at any developmental stages (Horikawa et al. 2008). Pigon and Weglarska (1955) measured oxygen consumption of the tardigrade Macrobiotus hufelandi and demonstrated that metabolism in this species became almost completely arrested in the anhydrobiotic state under low humidities (below 48%). Although tardigrades are believed to be ametabolic during anhydrobiosis, tardigrade anhydrobiotes could not survive more than 10 years at room temperature under aerobic conditions (Guidetti and Jönsson 2002). It suggests that the accumulation of oxidative stress causes critical damage to the anhydrobiotic animals under such conditions.
There is a critical desiccation rate for tardigrades to enter anhydrobiosis (Wright 1989; Horikawa and Higashi 2004). The upper critical desiccation rate varies among tardigrade species and reflects moisture conditions in their microhabitat and a degree of their desiccation tolerance. For instance, a species inhabiting a microenvironment in which water evaporates quickly can enter anhydrobiosis at rapid desiccation rate while one inhabiting a relatively moist habitat needs slow desiccation rate to enter into anhydrobiosis successfully.
Extreme water loss causes critical damage on cells, and, therefore, tardigrades and other anhydrobiotic animals must require systems for protecting their cells during anhydrobiotic state. Some types of compatible solutes have been considered to protect cells in anhydrobiotic animals. Nonreducing disaccharide trehalose has been believed to be a protective compound which was found in high concentration (about 10–20% wt./dry wt.) in several anhydrobiotic animals, including nematodes (Madin and Crowe 1975), embryos of the crustacean Artemia salina (Clegg 1962), and the insect larvae Polypedilum vanderplanki (Watanabe et al. 2003). The role of trehalose is thought to maintain the structures of biomolecules during dehydration by acting as a water-replacement substitute and/or by forming a glassy state (vitrification) (Crowe et al. 1987; Franks et al. 1991). However, tardigrades contain relatively low amounts of trehalose (approximately 0.002–2.3%) in the anhydrobiotic state (Westh and Ramløv 1991; Horikawa et al. 2006; Hengherr et al. 2008). Furthermore, no trehalose was detected in the anhydrobiotic bdelloid rotifers (Lapinski and Tunnacliffe 2003), implying that trehalose is not an essential solute for successful anhydrobiosis. Another candidate molecule responsible for the cell protection against dehydration is late embryogenesis abundant (LEA) proteins which may work as molecular chaperons helping other proteins protected from denaturation (Wise and Tunnacliffe 2004). LEA proteins are expressed in nematodes (Browne et al. 2004), rotifers (Denekamp et al. 2009), tardigrades (Schokraie et al. 2010), Artemia cysts (Hand et al. 2007), and Po. vanderplanki (Kikawada et al. 2006), but evidence in vivo for supporting the theory has not been reported yet.
In addition to the compatible solutes, results from recent studies suggest that maintaining DNA integrity is important for successful anhydrobiotic survival in tardigrades (Rebecchi et al. 2009a, b; Neumann et al. 2009). Dehydration causes DNA double strand breaks (DSBs) (Mattimore and Battista 1996), which are critical damage leading to an organism’s death. Significant DNA degradation was not detected in anhydrobiotic tardigrade Paramacrobiotus richtersi immediately after induction into anhydrobiosis (Rebecchi et al. 2009a, b) but Neumann et al. (2009) observed that there is a positive correlation between DNA damage accumulation and a period of anhydrobiosis in the tardigrade Milnesium tardigradum. Survival rate of tardigrades decreases according to a period that they are stored in the anhydrobiotic state (Guidetti and Jönsson 2002), suggesting that DNA damage accumulation presumably caused by oxidation leads to the death of anhydrobiotic tardigrades.
3 Radiation Tolerance
LD50 + standard error at 2, 24, and 48 h after irradiation of gamma-rays (60Co) or 4He ions in Mi. tardigradum (Reproduced from Horikawa et al. (2006) with modification).
2 h survival
24 h survival
48 h survival
5,500 ± 400
5,200 ± 1,700
5,000 ± 1,900
5,500 ± 500
5,400 ± 600
4,400 ± 500
7,800 ± 2,500
6,300 ± 600
6,200 ± 1,200
7,900 ± 30,000
5,400 ± 2,200
5,200 ± 2,900
As described above, hydrated animals are more radiation tolerant than anhydrobiotic ones in both the Ri. coronifer and the Mi. tardigradum in terms of post-irradiation short-time survival ability. However, in other anhydrobiotic animals, such as embryos of A. salina (Iwasaki 1964) and larvae of Po. vanderplanki (Watanabe et al. 2006), anhydrobiotic individuals showed higher tolerance to gamma radiation than hydrated ones. Organisms in an anhydrobiotic state are supposed to be more tolerant of irradiation than in a hydrated state because anhydrobiotes which contain extremely low water contents could avoid damage to biomolecules caused by the indirect radiation action through water molecules compared with hydrated animals. As described above, A. salina and Po. vanderplanki accumulate a large amount of trehalose in the anhydrobiotic state (Clegg 1962; Watanabe et al. 2002), while anhydrobiotic individuals of Ri. coronifer and Mi. tardigradum contain relatively low amounts of trehalose (2.3%, <0.2%, wt./dry wt., respectively) (Westh and Ramløv 1991; Horikawa et al. 2006). Yoshinaga et al. (1997) suggest that trehalose plays a role in protecting biomolecules against radiation. Thus, the difference in the radiation tolerance pattern between tardigrades and the other anhydrobiotic animals may be derived from the difference in trehalose concentration in a body during anhydrobiosis. Tardigrades are also known to have less mitotic activity at an adult stage (Bertolani 1970), and it is thought that the absence of somatic cell division enhances radiation tolerance in organisms (Ducoff 1972). These theories can be supported from the results that eggs of the tardigrade Ramazzottius varieornatus showed higher tolerance, based on hatchability, to heavy ion irradiation in the anhydrobiotic state than the hydrated state (Horikawa et al. unpublished data). This is because the somatic cell division actively occurs at an embryonic stage in tardigrades (Suzuki 2003; Gabriel et al. 2007), resulting in less tolerance of eggs to radiation in the hydrated state than the anhydrobiotic state.
Long-term survival and reproductive abilities of Ri. coronifer and Mi. tardigradum after gamma-irradiation were also examined. Ri. coronifer was maintained without any food sources (Jönsson et al. 2005) whereas Mi. tardigradum was cultured by supplying bdelloid rotifers as food (Horikawa et al. 2006). In both tardigrade species, post-irradiation life span in the hydrated and anhydrobiotic states decreased in a dose-dependent manner (Jönsson et al. 2005; Horikawa et al. 2006). Ri. coronifer laid eggs after irradiation with doses up to 5,000 Gy in the hydrated state and up to 2,000 Gy in the anhydrobiotic state. In Mi. tardigradum, egg laying was observed in one individual irradiated with 2,000 Gy in the hydrated state. No eggs produced by irradiated animals in both species hatched. Even though the high doses of gamma-irradiation inhibit normal embryonic development in tardigrades, they can be considered as the most radiation tolerant taxonomic group based on these results. The considerable tolerance of tardigrades to radiation suggests that they have effective systems for protecting DNA against radiation or repairing damaged DNA, because such high doses of ionizing radiation cause critical DNA damage, such as DSBs (Mattimore and Battista 1996). There also might be a protective system against radiation-induced oxidation in tardigrades, as protection to oxidative stress may be an essential factor for radiation tolerance in the bacterium Deinococcus radiodurans (Daly et al. 2007; Krisko and Radman 2010).
Mi. tardigradum also showed a high short-term survival rate after exposure to high linear energy transfer (high-LET) heavy ions (4He ions), with LD50 doses of 6.2 kGy in the hydrated state and 5.2 kGy in the anhydrobiotic one 48 h after irradiation (Table 1) (Horikawa et al. 2006). Further investigations on effects of high-LET heavy ions on tardigrades are necessary for estimating the possibilities for tardigrades to survive in extraterrestrial environments.
4 Tolerance to Low and High Temperatures
Tardigrades have been shown to tolerate extremely low and high temperatures. Several studies have reported that tardigrades survived extreme low temperature in both the hydrated and anhydrobiotic states. Becquerel (1950) demonstrated that the tardigrade Mi. tardigradum and Ra. oberhauseri survived after exposure to −273°C, near absolute zero temperature, in the anhydrobiotic state. In addition, even in the hydrated state, tardigrade Ri. coronifer survived −196°C (Ramløv and Westh 1992). However, remarkable decrease in survival rate was observed in hydrated Ri. coronifer when they were exposed to −196°C at rapid cooling rate (approximately 1,500°C min–1) (Ramløv and Westh 1992). Besides, Horikawa et al. (2008) reported that survival rate in Ra. varieornatus in the hydrated state was much lower than that in the anhydrobiotic state after direct exposure to −196°C, indicating that tardigrades are more tolerant of low temperature when they are in the anhydrobiotic state than the hydrated state.
Anhydrobiosis confers a high degree of tolerance to high temperatures (70°C and above) on tardigrades. Doyère (1842) reported that the tardigrade Ma. hufelandi was resurrected post-heating treatment at 120–125°C. Mi. tardigradum and Ra. oberhauseri survived 110–151°C for 35 min (Rahm 1921). Ramløv and Westh (2001) studied high temperature tolerance of Ri. coronifer and showed that this species could not survive 100°C for 1 h, with LD50 temperature of approximately 76°C. In Ra. varioeornatus, more than 90% of adult anhydrobiotic specimens survived 1 h exposure to 90°C (Horikawa et al. 2008), and approximately 15% of egg anhydrobiotes survived 80°C for 1 h (Horikawa et al. unpublished data). The cause of death of the anhydrobiotic tardigrades after heat treatment is probably because of loosing the glassy state, a state helping stabilize functional biological structures in an anhydrobiote, by critical upper temperature, as suggested in a study on Po. vanderplanki (Sakurai et al. 2008).
Judging from the tolerance ability of tardigrades against extremely low and high temperatures, they could survive temperatures on Mars where the temperature fluctuates between −123°C and 25°C (Diaz and Schulze-Makuch 2006). It is also possible that they survive nearly absolute zero temperature (Horneck 1999) in interplanetary space.
5 Tolerance to Low and High Pressures
Extreme vacuum must cause considerable desiccation and therefore is critical to organisms. In concert with the tardigrade anhydrobiotic ability, animals in the anhydrobiotic state can tolerate open space vacuum. Horikawa et al. (unpublished data) exposed anhydrobiotic eggs of Ra. varieornatus to low pressure at 5.3 × 10–4 Pa to 6.2 × 10–5 Pa for 7 days and demonstrated that experiment and demonstrated that 86% of the eggs exposed hatched. Moreover, adult individuals of Mi. tardigradum and Ri. coronifer tolerated 10-day exposure to vacuum in open space environments (Jönsson et al. 2008). These results strongly suggest that anhydrobiote tardigrades can survive even in anoxic environments in the universe.
Tardigrades have also been shown to exhibit extraordinary tolerance to extremely high pressures in the anhydrobiotic state. Seki and Toyoshima (1998) found tolerance of tardigrades to high pressure for the first time, reporting that two species of tardigrades Ma. occidentalis and Echiniscus japonicus in the anhydrobiotic state survived after 20 min exposure to high hydrostatic pressure up to 600 MPa when a water-free liquid, perfluoro-hydrocarbon, was used as a pressure medium. The tardigrade Mi. tardigradum survived exposure to high hydrostatic pressure at 1.2 GPa (Horikawa et al. 2009) and 7.5 GPa (Ono et al. 2008). Extremely high pressure generally causes physical changes in biomolecules such as DNA and proteins, leading to death of organisms (Abe et al. 1999). It is likely that removal of water from a tardigrade body avoids biomolecule damage by high hydrostatic pressure. Considering the high pressure tolerance in tardigrades, there might be some organisms that exist in the anhydrobiotic state under high pressure environments on extraterrestrial planets.
6 Exposure to Actual and Simulated Extraterrestrial Environments
Hitherto, there have been two flight experiments on tardigrades (Jönsson et al. 2008; Rebecchi et al. 2009a, b). Jönsson et al. (2008) conducted a flight experiment using tardigrades Ri. coronifer and Mi. tardigradum which were exposed to open space environments at low Earth orbit for 10 days. In this experiment, adult and egg specimens of both species in the anhydrobiotic state were set in the Biopan-6 experimental platform, where temperatures were controlled from 10°C to 39°C, provided by the European Space Agency (ESA) in the FOTON-M3 mission and exposed to three different conditions: space vacuum alone, space vacuum and UV-A and UV-B (UVAB, 280–400 nm spectral range) with a dose of 7,095 kJ m–2, and space vacuum and the full UV range from vacuum UV to UV-A (UVALL, 116.5–400 nm spectral range) with a dose of 7,577 kJ m–2. In both species, adult and egg specimens exposed to space vacuum alone showed comparable survival rate compared with control specimens which were kept under ground conditions. In addition, adult samples of both species retained normal reproductive activities after space vacuum exposure. On the other hand, space vacuum with UV radiation considerably decreased survival rate of the adult and egg samples in both species. In both species, no eggs exposed to UV radiation hatched. Although UV radiation had high negative effects on survival of anhydrobiotic tardigrades, a few specimens of adult Mi. tardigradum survived space vacuum with UVALL. Rebecchi et al. (2009a, b) examined effects of space environments including microgravity and moderate galactic cosmic radiation on the tardigrade Pa. richtersi in hydrated and anhydrobiotic states. Pa. richtersi was exposed to space environmental conditions for 12 days in the FOTON M-3 spacecraft. Temperature inside the experimental compartment was between around 18°C and 26°C. The authors found that the space flight did not decrease survival rate of the animals in both states, and hydrated active individuals produced next generation. Interestingly, tardigrades experienced the space flight showed higher antioxidant activity than ground control animals. Although doses of cosmic radiation that the tardigrade samples received in these two flight experiments were quite low (4.5–23.53 mGy in Jönsson et al. (2008) and 1.9 mGy in Rebecchi et al. (2009a, b)), those studies demonstrated that anhydrobiotic animals can survive simultaneous space environmental parameters, and thus can offer the possibilities of interplanetary transfer of anhydrobiotic tardigrades.
Johnson et al. (2011) examined the survival ability of the tardigrade Ra. varieornatus in the anhydrobiotic state at a burial depth of 5 mm in regolith after 40-day exposure to simulated Martian environments with temperatures ranging from −40.4°C to 24.0°C, 19.3 Wm–2 of UV flux (200–400 nm), 10–22 mbar of atmospheric pressure, and 95.3% CO2 concentration. Seventy percent of the Ra. varieornatus specimens survived after exposure to the simulated Martian environments, implying that tardigrade anhydrobiotes and other anhydrobiotic multicellular organisms can survive on Mars-like planets.
The results from the exposure experiments would imply that even animals in the anhydrobiotic state can be transported among planets and suggest a possibility that animal-like organisms thrive on other planets. In order to estimate those possibilities, additional astrobiological studies, such as long-term exposure experiments using tardigrades or other anhydrobiotic animals, seem to be required. A high proportion of the tardigrade anhydrobiotes survived short-term exposure to natural space level vacuum (Jönsson et al. 2008; Horikawa et al. unpublished data), but it is possible that prolonged (e.g., years) extreme vacuum exposure reduces tardigrade survivability because considerable desiccation by space vacuum is thought to cause accumulation of critical DNA damage (Horneck 2003). Although both flight experiments (Jönsson et al. 2008; Rebecchi et al. 2009a, b) did not evaluate effects of natural space temperatures on tardigrades, it seems that tardigrade survivability is not affected by extremely low temperatures based on cold temperature exposure experiments. Since any kinds of chemical reactions in anhydrobiotes can nearly be stopped under extremely low temperatures, it is expected that anhydrobiotic tardigrades may be able to live for prolonged period in interplanetary space environments where the temperature is extremely low. On the other hand, it is likely that tardigrades cannot survive high temperatures more than 150°C according to data from Rahm (1921), meaning that habitable environments for tardigrades can be largely limited by the upper survival temperature for tardigrades. UV radiation and galactic cosmic radiation are thought to be the most detrimental environmental factors for anhydrobiotic tardigrades. Thus, the possibility that tardigrades complete interplanetary travel is considered; in the future, we need to evaluate whether they can be protected against radiation if they are packed inside a meteorite-like material.
Although tardigrade research has not prevailed compared with extremophilic bacteria, such as D. radiodurans, largely due to difficulties of culturing tardigrades, recent methodology development of artificial rearing systems for several tardigrade species (Altiero and Rebecchi 2001; Suzuki 2003; Horikawa et al. 2008; Hengherr et al. 2008) makes it possible to conduct molecular and biochemical experiments and thus would accelerate future investigations on mechanisms behind the tardigrade tolerance to extreme environments. Such research might provide new insights into the probabilities of interplanetary transfer and the existence animal-like life forms in extraterrestrial environments.
I thank Lynn J. Rothschild and John Cumbers from NASA Ames Research Center for providing research advice on my studies. I also thank the NASA Astrobiology Institute Postdoctoral Program for supporting my research project at NASA Ames Research Center.
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