Lichens are extremely resistant organisms that colonize harsh climatic areas, some of them defined as “Mars-analog sites.” There still remain many unsolved questions as to how lichens survive under such extreme conditions. Several studies have been performed to test the resistance of various lichen species under space and in simulated Mars-like conditions. The results led to the proposal that Circinaria gyrosa (Lecanoromycetes, Ascomycota) is one of the most durable astrobiological model lichens. However, although C. gyrosa has been exposed to Mars-like environmental conditions while in a latent state, it has not been exposed in its physiologically active mode. We hypothesize that the astrobiological test system “Circinaria gyrosa,” could be able to be physiologically active and to survive under Mars-like conditions in a simulation chamber, based on previous studies performed at dessicated-dormant stage under simulated Mars-like conditions, that showed a complete recover of the PSII activity (Sánchez et al., 2012). Epifluorescence and confocal laser scanning microscopy (CLSM) showed that living algal cells were more abundant in samples exposed to niche conditions, which simulated the conditions in micro-fissures and micro-caves close to the surface that have limited scattered or time-dependent light exposure, than in samples exposed to full UV radiation. The medulla was not structurally affected, suggesting that the niche exposure conditions did not disturb the lichen thalli structure and morphology as revealed by field emission scanning electron microscopy (FESEM). In addition, changes in the lichen thalli chemical composition were determined by analytical pyrolysis. The chromatograms resulting from analytical pyrolysis at 500°C revealed that lichen samples exposed to niche conditions and full UV radiation consisted primarily of glycosidic compounds, lipids, and sterols, which are typical constituents of the cell walls. However, specific differences could be detected and used as markers of the UV-induced damage to the lichen membranes. Based on its viability responses after rehydration, our study shows that the test lichen survived the 30-day incubation in the Mars chamber particularly under niche conditions. However, the photobiont was not able to photosynthesize under the Mars-like conditions, which indicates that the surface of Mars is not a habitable place for C. gyrosa.

The lichen Circinaria gyrosa is an astrobiological model defined by its high capacity of resistance to space conditions and to a simulated martian environment. Therefore, it became part of the currently operated BIOMEX experiment on board the International Space Station and the recent STARLIFE campaign to study the effects of four types of space-relevant ionizing radiation. The samples were irradiated with helium and iron ions at doses up to 2 kGy, with X-rays at doses up to 5 kGy and with γ rays at doses from 6 to 113 kGy. Results on C. gyrosa's resistance to simulated space ionizing radiation and its post-irradiation viability were obtained by (i) chlorophyll a fluorescence of photosystem II (PSII), (ii) epifluorescence microscopy, (iii) confocal laser scanning microscopy (CLSM), and (iv) field emission scanning electron microscopy (FESEM). Results of photosynthetic activity and epifluorescence show no significant changes up to a dose of 1 kGy (helium ions), 2 kGy (iron ions), 5 kGy (X-rays)—the maximum doses applied for those radiation qualities—as well as a dose of 6 kGy of γ irradiation, which was the lowest dose applied for this low linear energy transfer (LET) radiation. Significant damage in a dose-related manner was observed only at much higher doses of γ irradiation (up to 113 kGy). These data corroborate the findings of the parallel STARLIFE studies on the effects of ionizing radiation on the lichen Circinaria gyrosa, its isolated photobiont, and the lichen Xanthoria elegans. Key Words: Simulated space ionizing radiation—Gamma rays—Extremotolerance—Lichens—Circinaria gyrosa—Photosynthetic activity. Astrobiology 17, 145–153.

As part of the Biology and Mars Experiment (BIOMEX; ILSRA 2009-0834), samples of the lichen Circinaria gyrosa were placed on the exposure platform EXPOSE-R2, on the International Space Station (ISS) and exposed to space and to a Mars-simulated environment for 18 months (2014–2016) to study: (1) resistance to space and Mars-like conditions and (2) biomarkers for use in future space missions (Exo-Mars). When the experiment returned (June 2016), initial analysis showed rapid recovery of photosystem II activity in the samples exposed exclusively to space vacuum and a Mars-like atmosphere. Significantly reduced recovery levels were observed in Sun-exposed samples, and electron and fluorescence microscopy (transmission electron microscope and field emission scanning electron microscope) data indicated that this was attributable to the combined effects of space radiation and space vacuum, as unirradiated samples exhibited less marked morphological changes compared with Sun-exposed samples. Polymerase chain reaction analyses confirmed that there was DNA damage in lichen exposed to harsh space and Mars-like environmental conditions, with ultraviolet radiation combined with space vacuum causing the most damage. These findings contribute to the characterization of space- and Mars-resistant organisms that are relevant to Mars habitability.

(1) Background: Lichens, as an important part of the terrestrial ecosystem, attract the attention of various research disciplines. To elucidate their ultrastructure, transmission electron microscopy of resin-embedded samples is indispensable. Since most observations of lichen samples are generated via chemical fixation and processing at room temperature, they lack the rapid immobilization of live processes and are prone to preparation artefacts. To improve their preservation, cryoprocessing was tested in the past, but never widely implemented, not least because of an extremely lengthy protocol. (2) Methods: Here, we introduce an accelerated automated freeze substitution protocol with continuous agitation. Using the example of three lichen species, we demonstrate the preservation of the native state of algal photobionts and mycobionts in association with their extracellular matrix. (3) Results: We bring to attention the extent and the structural variability of the hyphae, the extracellular matrix and numerous crystallized metabolites. Our findings will encourage studies on transformation processes related to the compartmentation of lichen thalli. They include cryopreserved aspects of algal photobionts and observations of putative physiological relevance, such as the arrangement of numerous mitochondria within chloroplast pockets. (4) Conclusions: In summary, we present accelerated freeze substitution as a very useful tool for systematic studies of lichen ultrastructures.

The search for extraterrestrial life and finding habitable environments on other planets and satellites like Mars, Europa, Enceladus and Titan are a priority of NASA and ESA, since the last decade. To contribute to these highly significant challenges, research has been done with established exposure platforms like those on the Foton satellite and EXPOSE on the ISS expanding now to ESA’s platform Bartolomeo. These were used to expose samples to space vacuum and space radiation, but also to provide gas supply and selected planetary radiation environments. Results obtained by these experiments have allowed to get supplemental knowledge necessary for supporting future investigations to search for life in the universe. Several extremophile lichen species, have been exposed to extraterrestrial environments, i.e. space- and Mars like parameters, during short and long periods on board of ESA’s space missions (Foton M2 and M3, EXPOSE E and R2) to investigate the limits of terrestrial life. To maximize the scientific outcome of these experiments, LICHENS [1], LITHOPANSPERMIA [2], LIFE [3], and BIOMEX [4], a common elaboration and analysis of the results obtained on analogue field studies with results obtained in planetary simulation facilities was necessary to check the survival potential and vitality of the samples before flight. Tests and experiments at different simulation facilities at DLR, and at INTA, included the exposure to space vacuum, space UV radiation and space cosmic radiation, and to Mars-like environment, i.e. Mars atmospheric composition and pressure, as well as Mars UV radiation. Not microgravity or reduced gravity, which is present in space and on Mars, was tried. Here we show the results of the resistance of two extremophile vagrant lichen species, Xanthoparmelia hueana and Circinaria gyrosa, to simulated microgravity (rotation speed of clinostate: 1 rpm) using the UNZIP clinostate at CIB-CSIC (Centro Investigaciones Biológicas Margarita Salas). This is the first time that lichens will be exposed to weightlessness environment in an attempt to isolate the potential contribution of microgravity from other extraterrestrial factors (radiation, vacuum). Combinations of simulated spaceflight conditions, including microgravity, will be necessary to check how this parameter affects the biomolecular level of lichens and their microbiome.   References [1] Sancho, L.G., de la Torre, R., Horneck, G., Ascaso, C., de los Rios, A., Pintado, A., and Schuster, M. (2007) Lichens survive in space: results from the 2005 LICHENS experiment. Astrobiology 7:443–454. https://doi.org/10.1089/ast.2006.0046 [2] de la Torre, R., Sancho, L.G., Horneck, G., de los Ríos, A., Wierzchos, J., Olsson-Francis, K., and Ott, S. (2010) Survival of lichens and bacteria exposed to outer space conditions—results of the Lithopanspermia experiments. Icarus 208:735–748. https://doi.org/10.1016/j.icarus.2010.03.010 [3] Onofri, S., de la Torre, R., de Vera, J.P., Ott, S., Zucconi, L., Selbmann, L., Scalzi, G., Venkateswaran, K.J., Rabbow, E., Sánchez, F.J., and Horneck, G. (2012) Survival of rock-colonizing organisms after 1.5 year in outer space. Astrobiology 12: 508-516. https://doi.org/10.1089/ast.2011.0736 [4] de Vera, J.P., , and the BIOMEX-Team and associatesTeam (Ute Boettger, Rosa de la Torre Noetzel et al.) (2019) Limits of Life and the Habitability of Mars: The ESA Space Experiment BIOMEX on the ISS. Astrobiology, 19-2. https://doi.org/10.1089/ast.2018.1897.