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Survivability of Moss and Fungal Spores in Tests Simulating Biological Sciences in Space, Vol.25 No.2-4, 83-92,Takahashi, 2011 Y. et al. Original Paper Survivability of Moss and Fungal spores irradiated for 30 min. On the other hand, a colony did not develop if the spores were Spores in Tests Simulating taken off the beads and irradiated for 10 min. Conditions of the ISS Outer Wall This indicates that UV does not penetrate to the other side of the beads, and so the spores on Yuichi Takahashi1*, Hirofumi Hashimoto2, that side can be protected from UV radiation. 3 1 ©2011 Jpn. Soc. Biol. Sci. Space; Article ID: Takuo Nakagawa and Shinpei Shibata 112502011 1Department of Astrophysics, Interactive Symbio- sphere Science, Yamagata University Graduate Key words; fungi, moss, spore, survivability, thermal cycle treatment, UV irradiation School of Science and Engineering, Kojirakawa- machi 1-4-12, Yamagata-City, Yamagata 990-8560, Introduction Japan 2 Institute of Space and Astronautical Science, There are many factors that can influence the survivability JAXA, Yoshinodai 3-1-1 Chuo-ku, Sagamihara- of terrestrial life (TL) in interstellar and interplanetary space, City, Kanagawa 252-5210, Japan for example full-spectrum electromagnetic radiation from 3Inspection room, Kojirakawa-Shiseidoh Hospital, the sun, temperature fluctuations, cosmic particle radiation, Higashihara-machi 1-12-26, Yamagata-City, Ya- vacuum, and microgravity. However, space conditions can magata 990-0034, Japan only be partially simulated in ground experiments. Recently, several experiments have investigated the possibility of TL Abstract survival in space, for example EXPOSE-E (ESA Human Space Flight (2010) Expose, http:// smsc.cnes.fr/EXPOSE/), BIORISK (Baranow et al., 2009; Novikova et al., 2010) and To investigate whether terrestrial life (TL) can BIOPAN (De la Torre Noetzel et al., 2007; Devara et al., survive in interstellar and interplanetary space, 2010), while another one, TANPOPO, will take place in the an experiment was performed to simulate near future. In the BIORISK experiment, 31 kinds of TL were conditions in the Earth’s orbit. There are many examined such as higher plants, insects, crustaceans, fungi factors which influence survivability. Among and bacteria. A high survival rate was observed in some them, the effects of temperature fluctuations samples of plant seeds (barley, rice) and chironomid larvae, and UV irradiations were addressed in this whereas a low survival rate was observed in samples of paper. Six species of moss spores and 3 bacteria and fungi. The plastic dishes of bacteria and fungi species of fungal spores were selected as melted during the 2nd experiment (Baranow et al., 2009; target TL. Temperature was fluctuated between Novikova et al., 2010), and after the experiment, the melting 80˚C and -80˚C once every 90 min, whereas UV 2 temperature of the plastic dishes was determined to be at 254nm was irradiated for 10 min (471mJ/cm ) 2 95˚C. So they were unable to reach a reliable conclusion and 30 min (1,413mJ/cm ). The moss spores of about the obtained results. Funaria hygrometrica (exp 1) and Pogonatum In this paper, a thermal cycle experiment and an inflexum (exp 2) were the most tolerable species ultraviolet ray (UV) irradiation experiment were carried to the thermal cycle treatment, with germination out as a ground experiment under defined conditions rates of 3.3±1.2% (n=5) and 7.9±3.2% (n=6), simulating the Earth’s orbit. Our purpose is to seek a respectively, after 3 weeks of treatment. candidate species for the TANPOPO and other space Germination occurred even after the spores had experiment such as BIORISK, and hopefully to participate been UV irradiated for 30 min (0.7% to 23.5%). in the space experiment itself. Furthermore, we aim Only a slight difference in the germination rate to test whether cellular tissues of living organisms are was observed using different culture media. damaged physically and their functions stopped due The treated spores were transferred to soil to the repetition of short-time temperature fluctuations where they grew into gametophytes, then through thermal cycle treatment and UV irradiation. sporophytes, and finally formed new capsules This paper describes a pilot study using moss and after 7-8 months. Two species of fungal spores fungal spores as potential candidates for the TANPOPO were allowed to adsorb onto beads before the project (Yamagishi et al., 2008), which proposes to beads were directly irradiated for 10 min and analyse the interplanetary migration of microbes and 30 min, respectively. Colonies developed the organic compounds and survivability of microbes at the International Space Station (ISS), located ca. 400 km Received: May 2, 2011; Accepted: November 8, 2011 above the Earth’s surface. As a part of this project, they *To whom correspondence should be addressed: plan to test the survivability of the microbes in space using Tel: +81-(0)23-628-4552; Fax: +81-(0)23-628-4567 ; a direct exposure experiment, which will take place in the E-mail: [email protected] Japanese exposure unit “Kibo” on board the ISS. «Can life migrate between planets?» is a question that many Abbreviations TL: terrestrial life, exp 1: experiment 1, exp 2: experiment 2 people have an interest in. Nicholson (2006) reviewed the potential interplanetary transport of microbes and − 83 − Spores in ISS outer hull simulation tests classified the following stages; surviving the launch from well as one kind used in exp 1 were exposed for 21 days. a donor planet, surviving the transit through space, and As for fungi, spores were adsorbed onto the surface surviving atmospheric entry and landing onto a recipient of MicrobankTM beads (Pro-Lab Diagnostics, 20 Mural planet. Thus, TANPOPO’s proposed experiment will test Street, Unit #4, Richmond Hill, ON and L4B1K3 Canada). Nicholson’s second stage (i.e., surviving space transit). The MicrobankTM system consists of sterile vials containing The exposure experiment of microbes in space has beads which act as carriers to support the fungi for a long been performed mainly using the spore-forming bacterium time. Each bead is 3.5 mm in outer diameter, 1.0 mm in Bacillus sp. (Nicholson et al., 2000). In TANPOPO, inner diameter and 2.5 mm in thickness. Each bead can various microbial cells including Deinococcus radiodurans support 103 to 104 microbes (Chandler, 1994). Aspergillus and the newly isolated Deinococcus sp. from high- oryzae spores were exposed for 26 days, Cladosporium sp. altitude (ca. 10 km) will be used for exposure experiments spores for 14 days and Aspergillus niger spores for 21 days. (Yamagishi et al., 2008). Deinococcus radiodurans is UV irradiation known to show extreme tolerance to UV-light and gamma A UVS-14 EL series UV lamp with an Hg-law lamp radiation. Some isolates of Deinococcus sp. showed was used for the UV irradiation treatment. The amount of higher tolerance to UV-light than D. radiodurans (Yang et UV at 254nm irradiated from 7.6cm distance is 0.785mW/ al., 2008; Yang et al., 2009). In addition to the microbes, 2 cm . The expected amount of UV was calculated to be we are examining candidate species of spores belonging 2 2 471mJ/cm for 10 min and 1,413 mJ/cm for 30 min. to eukaryotes, which are expected to be tolerant to the UV treatment of moss spores was performed after space environment. Our candidate moss spores can they were taken out from the capsules and placed into the tolerate being in a vacuum (Saegusa et al., 2008). Since culture medium. The spore concentration was adjusted they are autotrophic, if they can be brought to Mars, 6 to 1×10 spores/ml using a haemocytometer. Twenty μl they would be expected to contribute to future space of spores were put into each well of a 96-well microplate agriculture (http://surc.isas.ac.jp/space_agriculture/ (Nunc company, Roskilde, Denmark, non-treated; flat; Archive/PDF/Space_Agri_ 2008-a.pdf). Considering the well volume 0.4ml) and irradiated for 10 or 30 min. A flat Earth’s history, mosses were one of the first plants to and small size well (7mm in diameter) was used in order inhabit terrestrial habitats (Kenrick, 2003; Wellman et to irradiate the UV light uniformly. UV treated moss spores al., 2003), and have been playing an important role in preparing the soil for vascular plant growth. Fungal spores are found in spaceships such as ISS (Novikova, 2010; Vesper, 2008), where they can cause trouble because they are difficult to get rid of once the fungi propagates and some of them cause allergies. The survivability of spores was obtained from germination rates in the case of moss spores and from colony formation in the case of fungal spores. Detection of fungal spores in the air is especially suitable for us, because we have developed an immunochemical technique for detecting fungi such as Cladosporium sp. in the air (Suzuki et al., 2008). It is the right target species for the examination of the change in allergenicity when it is carried out in interstellar and interplanetary space. Materials and Method Thermal cycle treatment The thermal cycle test device of the Institute of Space and Astronautical Science, Japan Aerospace Exploration Agency (JAXA) was used for the treatment (Fig.1). This Fig. 1. Thermal cycle test device, (a) sample containers and (b) the plate which controls the temperature of the container. device is designed to reproduce a repeating change in The sample containers (a) were accommodated on the plate (b) temperature at short-time intervals. We use temperature shown in this figure. fluctuations between 80˚C and -80˚C once every 90 min, Capsules were put into a hole in the sample containers (the and the vacuum level of the device is less than 1Pa.
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