The Tardigrade Ramazzottius Varieornatus As a Model Animal for Astrobiological Studies

The Tardigrade Ramazzottius varieornatus as a Model Animal for Astrobiological Studies

Daiki D. Horikawa1, 2, 3†

1 NASA Ames Research Center, Moffett Field, CA 94035, USA 2 SETI Institute, 515 N. Whisman Road - Mountain View, CA 94043, USA 3 NASA Astrobiology Institute

Abstract Tardigrades are invertebrate animals, some of which can enter into an ametabolic desiccated state called anhydrobiosis. In this state, tardigrades are extremotolerant and able to survive open space environments characterized by severe vacuum and intense radiation. For these reasons, tardigrades are expected to serve as important model organisms for astrobiological studies, particularly those focusing on how multicellular organisms can withstand the extreme environments of space and the possibility of extraterrestrial multicellular life forms. To develop tardigrades as model organisms, we established a culture system for the tardigrade species Ramazzottius varieornatus, a species that is tolerant to several types of extreme physical and chemical environmental conditions while in an anhydrobiotic, or “tun”, state. Based on studies using this culture system and the extremotolerance of this tardigrade in connection with its anhydrobiotic capacity, R. varieornatus is a suitable model organism for astrobiological studies and will provide new insight into the mechanisms underlying the ability of multicellular organisms to tolerate extreme environments. Key words: tardigrades, Ramazzottius varieornatus, anhydrobiosis, astrobiology, extremophiles

Introduction probabilities of interplanetary transfer and the existence A major goal in the field of astrobiology is to of extraterrestrial multicellular organisms. determine whether life can be transferred from one Tardigrades are a classic example of extremotolerant planet to another or if life can potentially exist elsewhere multicellular organisms, exhibiting considerable in the universe. Evaluation of an organism’s survivability resistance to many environmental extremes (Rothschild after exposure to open-space environments is important and Mancinelli, 2001; Cavicchioli, 2002). Tardigrades to estimate the possibilities of transportation of life are small invertebrates (0.1–1.0 mm in body length) between planets and the existence of extraterrestrial found in environments ranging from the deep sea to high life. In contrast to unicellular organisms (Horneck et al., mountains all over the world. Even terrestrial tardigrades, 1994; Kobayashi et al., 2000), the use of multicellular however, require a film of water surrounding them for organisms for space exposure experiments has received activities such as feeding, growth, and reproduction. little attention, presumably because there are few species When the terrestrial species lack water, their body water with potential tolerance to actual space environmental characteristically decreases to between 1% to 3% wt/ parameters, including extremely high or low wt in association with body shrinkage (Crowe, 1972; temperatures, vacuum, and ultraviolet (UV) and ionizing Horikawa et al., 2006). The contracted dry animal, radiation. Some multicellular species, however, show referred to as a tun, shows no visible signs of life, but high resistance to extreme environmental conditions, can resume activity within 1 h (Horikawa and Higashi and therefore flight experiments with multicellular 2004) when placed in a drop of water. This ametabolic organisms are of great interest for estimating the state induced by dehydration is referred as anhydrobiosis existence of complex life forms on other planets, as (Keilin, 1959). Tardigrades in an anhydrobiotic state well as the existence of simple single-cell life-forms. exhibit extraordinary tolerance to a variety of extreme Such investigation might provide new insights into the environmental conditions, such as a wide range of temperatures from –273˚C (Becquerel, 1950) to 151˚C (Rahm, 1921); exposure to organic solvents such as –4 Article ID: 08220309 alcohols (Ramløv and Westh, 2001); vacuum (5 × 10 Pa) (Utsugi and Noda, 1995); high pressure (600 MPa) Review Article (Seki and Toyoshima, 1998); and thousands Gy of X-rays Received: December 17, 2008 (May et al., 1964), gamma-rays (Horikawa et al., 2006; Accepted: February 10, 2009 Jönsson, et al., 2005), and heavy ions (Horikawa, 2006). †To whom correspondence should be addressed: NASA Based on these characteristics, several investigators have Ames Research Center, Mail Stop 239-20, Bldg N239 Rm suggested that tardigrades have the potential to survive 377, Moffett Field, CA 94035, USA. Tel: +1-650-604- extraterrestrial environments (Copley, 1999). 6165; Fax: +1-650-604-1088; E-mail: Daiki.Horikawa-1@ nasa.gov The tardigrades Richtersius coronifer and Milnesium

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tardigradum in an anhydrobiotic state were exposed to To date, however, there have been few reports on open-space environments in a low Earth orbit for 10 d tardigrade culture conditions that will allow for stable within the Biopan 6 research facility operated by the and continuous tardigrade research. To further develop European Space Agency and a few individuals of the tardigrades as a model for astrobiological studies, we M. tardigradum survived exposure to a combination of recently established culture systems for the tardigrade space vacuum and the full spectral range of UV radiation species Ramazzottius varieornatus using a green alga, (Jönsson et al., 2008), indicating the potential of these Chlorella vulgaris, as food (Horikawa et al., 2008). Our multicellular organisms to survive unprotected space findings indicate that this species has high anhydrobiotic travel. These findings have prompted researchers to focus ability and is tolerant to several types of extreme on tardigrades for astrobiological studies. Elucidation environmental conditions (Horikawa et al., 2008). Here, of the mechanisms underlying the tardigrade’s tolerance I review the culture systems, life history, anhydrobiosis, to extreme environmental conditions may provide and environmental tolerance of R. varieornatus and information about potential survival strategies of propose that this species, particularly among tardigrades, multicellular organisms in extraterrestrial environments. is a suitable model animal for astrobiological studies.

Fig. 1. A natural habitat of . (A) The landscape of the bridge in Sapporo, Hokkaido, Japan, and (B) dry moss vegetation where inhabits the pavement of the bridge.

Fig. 2. Light micrographs of (A) an adult of and (B) adults reared on a culture plate with the green algae (green material). The arrowheads indicate eggs produced. Scale bar = 100 µm (A).

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Culture systems and life history of R. varieornatus There are several possible molecular protectants R. varieornatus, originally collected from the dry involved in stabilizing macromolecules and cells involved moss vegetation on a bridge in Sapporo, Japan, was in anhydrobiosis in R.varieornatus. The disaccharide cultured with the green alga C. vulgaris (Chlorella trehalose is considered to be a desiccation protectant that Industry Co., Ltd., Tokyo, Japan) as food (Horikawa generally accumulates during entry into anhydrobiosis et al., 2008)(Fig. 1A, B; Fig.2 A). Agar gel was coated in anhydrobiotic invertebrates (reviewed by Watanabe onto the bottom of a Petri dish (35 mm in diameter) as 2006), including several species of tardigrades (Westh a culture medium with a thin layer of water covering & Ramløv 1991, Hengherr et al., 2008), but whether the agar. A culture dish containing approximately 60 trehalose accumulates in R. varieornatus is unknown. individuals was covered with a lid to inhibit water The kinds of protectants responsible for successful evaporation and kept at 25˚C in the dark. Tardigrades anhydrobiosis in R. varieornatus is one of the most were transferred to new culture dishes every 3 to 6 d. interesting features to address in further tardigrade When tardigrades were transferred to new culture dishes, research. In view of their relatively high desiccation they were anesthetized by pouring CO2–dissolved water tolerance, this species is expected to contribute to studies into the culture dish, picked up using a pipette, and then of desiccation tolerance in multicellular organisms. transferred to new culture dishes. Fresh C. vulgaris was added to the culture dishes each time the tardigrades Tolerance of R. varieornatus to extreme were transferred. environmental conditions Under the culture conditions described, the average Previous evaluation of the tolerance of R. varieornatus life span of R. varieornatus was approximately 35 d, to extreme physical and chemical environmental and the maximum life span was 87 d (Horikawa et conditions indicated that high proportion (greater than al., 2008). The average number of eggs produced by a 85%) of anhydrobiotic adults of R. varieornatus resumed single individual was 7.85, and the first egg laying was activity after exposure to 90˚C, –196˚C, 4000 Gy of 4He observed 9 d after hatching (Horikawa et al., 2008). ions, or 99.8% acetonitrile (Horikawa et al., 2008)(Fig. 4). Of the eggs produced, 83% hatched, and the average While no hydrated individuals survived exposure to 90˚C embryonic stage was 5.7 d (Horikawa et al., 2008). R. and 99.8% acetonitrile, 22.3% of them survived freezing varieornatus has a shorter life span and embryonic stage at –196˚C for 15 min, demonstrating that even hydrated than other investigated terrestrial tardigrade species, R. varieornatus tolerates freezing at extremely low such as Macrobiotus richtersi (Altiero et al., 2001) temperatures (Horikawa et al., 2008)(Fig. 4). In addition, and Milnesium tardigradum (Suzuki, 2003), which is hydrated and anhydrobiotic R. varieornatus showed a advantageous for the analysis of life history traits after high survival rate (100%) after irradiation with 4000 Gy exposure experiments. R. varieornatus produced the next of 4He ions (Horikawa et al., 2008)(Fig. 4). This finding generation under solitarily cultured conditions, indicating is consistent with that of other reports that hydrated that this species is parthenogenic or hermaphroditic. To tardigrades Macrobiotus areolatus, R. coronifer, and M. advance this species as a useful experimental organism, tardigradum are tolerant to exposures to thousands of we isolated a standard strain named “YOKOZUNA-1” grays of ionizing radiation (May et al., 1964; Jönsson from a single egg (Horikawa et al., 2008).

Anhydrobiosis in R. varieornatus R. varieornatus in the egg, juvenile, and adult stages showed high anhydrobiotic capacity based on recovery rates (greater than 90%) after rehydration followed by desiccation at 25ºC under 85% relative humidity for 24 h and under 0% relative humidity for 10 d (Horikawa et al., 2008). The high capacity of R. varieornatus to tolerate dehydration seems to reflect its adaptation to its original microhabitat in which mosses inhabited by the tardigrade population are often exposed to prolonged drought. Anhydrobiotic adults contracted into the tun formation (Fig. 3), which is the characteristic form of anhydrobiotic tardigrades, and their body water content was reduced from 78.6% in the active state to 2.5% (Horikawa et al., 2008). This small amount of body water in the anhydrobiotic state is thought to be bound water that exists on the surface of macromolecules (Liltorp et Fig. 3. A scanning micrograph of an anhydrobiotic adult al., 2001) and therefore does not evaporate under room . Reproduced with permission from temperature conditions. Horikawa (2008).

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(A) Heat (90 ˚C) (B) Cold (-196 ˚C) ** * 100

0 Hyd Dry Hyd Dry

Survivorship 100

0 Hyd Dry Hyd Dry

Fig. 4. Survival rate of adult in hydrated (Hyd) and anhydrobiotic (Dry) states after exposure to extreme environmental conditions. (A) 90˚C for 1 h, (B) –196˚C for 15 min, (C) , 4000 Gy of irradiation with 4He, and (D) 99.8% acetonitrile for 1 h. Asterisks denote significant differences in survival between hydrated and anhydrobiotic groups (Welch’s t-test was used for (A) heat, (B) cold, and (C) irradiation; chi-square test with Yeats correction for (D) chemical; *p < 0.05; ** p < 0.01). Error bars indicate standard deviations of three experiments. Reproduced with permission from Horikawa et al. (2008). et al., 2005; Horikawa et al., 2006). The considerable be more likely than previously thought. Extremotolerant tolerance of hydrated or anhydrobiotic tardigrades to multicellular and unicellular organisms are also potential ionizing radiation suggests that these animals have novel forms of life seeds. Cysts of the brine shrimp Artemia systems to protect against or to repair DNA damage, salina were used for flight experiments in which they because such high doses of ionizing radiation cause were exposed to cosmic radiation and were able to be critical DNA damage, such as double-strand breaks revived after space flight (Gaubin et al., 1979), but post- (Mattimore and Battista, 1996). Further, anhydrobiotic flight fertility of this species has not been observed, adults and eggs of R. varieornatus survived exposure presumably because of the difficulties in culturing to nearly open space levels of low pressure for 7 d in a Artemia. The tardigrade species M. tardigradum not only vacuum chamber (Horikawa et al., unpublished data). survives exposure to extreme environmental conditions combined with space vacuum and full-range UV R. varieornatus as a model animal for radiation in low Earth orbit, but also produce offspring astrobiological studies from eggs exposed to space vacuum combined with 280 Panspermia is the hypothesis that microscopic forms to 400 nm range UV radiation (Jönsson et al., 2008). of seed life are transferred from one planet to another Thus, astrobiological studies focusing on multicellular (Horneck, 2003). This concept requires that organisms organisms may be extended using tardigrades. leave the host planet, survive the transit to<<< a new Analysis of DNA damage and repair systems environment, and then reproduce. Many space exposure in tardigrades will be important to understand how studies with microorganisms have been performed these animals can tolerate high doses of radiation and to determine if transit of organisms in space is even severe desiccation by vacuum, both of which induce possible. The results of these studies indicate that some DNA strand breaks. In the radiotolerant bacterium organisms in a dormant state, such as Bacillus subtilis Deinococcus radiodurans there is an efficient DNA spores, are capable of forming colonies after several years repair mechanism in which DNA fragments shattered of open-space travel (Horneck et al., 1994). Thus, the by irradiation are precisely reassembled (Zahradka existence of seeds of life in interstellar space appears to et al., 2006). In contrast, there are few reports of the mechanisms underlying radiation tolerance or desiccation tolerance of extremotolerant multicellular organisms,

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including tardigrades. Elucidation of the molecular basis M., Kikawada, T., Nakahara, Y., Hamada, N., Wada, S., of the extremotolerance of tardigrades to desiccation and Funayama, T., Higashi, S., Kobayashi, Y., Okuda, T. and radiation using R. varieornatus may provide key insights Kuwabara, M. (2006) Radiation tolerance in the tardigrade for understanding how multicellular organisms can cope Milnesium tardigradum. Int. J. Radiat. Biol., 82, 843-848. with space environments and the potential existence of Horikawa, D.D., Kunieda, T., Abe, W., Watanabe, M., extraterrestrial multicellular life-forms. Nakahara, Y., Yukuhiro, F., Sakashita, T., Hamada, N., Wada, S., Funayama, T., Katagiri, C., Kobayashi, Y., Higashi, S. and Okuda T. (2008) Establishment of a rearing system of Conclusions the extremotolerant tardigrade Ramazzottius varieornatus: We established a culture system for the tardigrade a new model animal for astrobiology. Astrobiology, 8, 549- R. varieornatus to assess the organism’s tolerance to 556. extreme environmental conditions, including high Horikawa, D.D. and Higashi, S. (2004) Desiccation tolerance of and low temperatures, vacuum, and ionizing radiation the tardigrade Milnesium tardigradum collected in Sapporo, with regard to its anhydrobiotic capacity. Based on our Japan, and Bogor, Indonesia. Zool. Sci., 21, 813–816. findings, we suggest that R. varieornatus is a useful Horneck, G., Bücker, H., and Reitz, G. (1994) Long-term model organism for astrobiological studies, which may survival of bacterial spores in space. Adv. Space Res., 14, help to elucidate the mechanisms underlying its tolerance 41-45. to extraterrestrial environments. Horneck, G. (1993) Responses of Bacillus subtilis spores to

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