J. Meeßen

Extremotolerance and Resistance of Lichens: Comparative Studies on Five Species Used in Astrobiological Research I. Morphological and Anatomical Characteristics

Lichens are symbioses of two organisms, a fungal mycobiont and a photoautotrophic photobiont. In nature, many lichens tolerate extreme environmental conditions and thus became valuable models in astrobiological research to fathom biological resistance towards non-terrestrial conditions; including space exposure, hypervelocity impact simulations as well as space and Martian parameter simulations. All studies demonstrated the high resistance towards non-terrestrial abiotic factors of selected extremotolerant lichens. Besides other adaptations, this study focuses on the morphological and anatomical traits by comparing five lichen species—Circinaria gyrosa, Rhizocarpon geographicum, Xanthoria elegans, Buellia frigida, Pleopsidium chlorophanum—used in present-day astrobiological research. Detailed investigation of thallus organization by microscopy methods allows to study the effect of morphology on lichen resistance and forms a basis for interpreting data of recent and future experiments. All investigated lichens reveal a common heteromerous thallus structure but diverging sets of morphological-anatomical traits, as intra-/extra-thalline mucilage matrices, cortices, algal arrangements, and hyphal strands. In B. frigida, R. geographicum, and X. elegans the combination of pigmented cortex, algal arrangement, and mucilage seems to enhance resistance, while subcortex and algal clustering seem to be crucial in C. gyrosa, as well as pigmented cortices and basal thallus protrusions in P. chlorophanum. Thus, generalizations on morphologically conferred resistance have to be avoided. Such differences might reflect the diverging evolutionary histories and are advantageous by adapting lichens to prevalent abiotic stressors. The peculiar lichen morphology demonstrates its remarkable stake in resisting extreme terrestrial conditions and may explain the high resistance of lichens found in astrobiological research.

UV-C tolerance of symbiotic Trebouxia sp. in the space-tested lichen species Rhizocarpon geographicum and Circinaria gyrosa : role of the hydration state and cortex/screening substances

Many experiments were carried out in order to evaluate the survival capacity of extremotolerant lichens when facing harsh conditions, including those of outer space or of simulated Martian environment. For further progress, a deeper study on the physiological mechanisms is needed that confer the unexpected levels of resistance detected on these symbiotic organisms. In this work, the response of the lichenized green algae Trebouxia sp. (a predominant lichen photobiont) to increasing doses of UV-C radiation is studied. UV-C (one of the most lethal factorsto be found in spacetogether with vacuum and cosmic-ionizing radiation with high atomic number and energy (HZE) particles) has been applied in the present experiments up to a maximum dose analogue to 67 days in Low Earth Orbit (LEO). For that purpose we selected two extremotolerant and space-tested lichen species in which Trebouxia sp. is the photosynthetic partner: the crustose lichenRhizocarpon geographicumand the fruticose lichenCircinaria gyrosa.In orderto evaluatethe effect of the physiological state of the lichen thallus (active when wet and dormant when dry) and of protective structures (cortex and photoprotective pigments) on the resistance of the photobiont to UV-C, four different experimental conditions were tested: (1) dry intact samples, (2) wet intact samples, (3) dry samples without cortex/acetone-rinsed and (4) wet samples without cortex/acetone-rinsed. After irradiation and a 72 hours period of recovery, the influence of UV-C on the two lichens photobiont under each experimental approach was assessed by two complimentary methods: (1) By determining the photosystem II (PSII) activity in three successive 24 hours intervals (Mini-PAM fluorometer) to investigate the overall state of the photosynthetic process and the resilience of Trebouxia sp. (2) By performing high performance liquid chromatography (HPLC)-quantification of four essential photosynthetic pigments (chlorophyll a, chlorophyll b, β-carotene and lutein) of one sample of each species and dose. Results indicate that the physiological state of the thallus is the most important factor impairing the tolerance of Trebouxia sp. to UV-C radiation in both lichen species. Desiccated thalli were demonstrated to be more resistant to UV-C. No clear influence of UV-C radiation on the carotenoid content was detected. Comparing the respective doses applied, the individuals of R. geographicum are more sensitive than C. gyrosa.

Extremotolerance and Resistance of Lichens: Comparative Studies on Five Species Used in Astrobiological Research II. Secondary Lichen Compounds

Lichens, which are symbioses of a fungus and one or two photoautotrophs, frequently tolerate extreme environmental conditions. This makes them valuable model systems in astrobiological research to fathom the limits and limitations of eukaryotic symbioses. Various studies demonstrated the high resistance of selected extremotolerant lichens towards extreme, non-terrestrial abiotic factors including space exposure, hypervelocity impact simulations as well as space and Martian parameter simulations. This study focusses on the diverse set of secondary lichen compounds (SLCs) that act as photo- and UVR-protective substances. Five lichen species used in present-day astrobiological research were compared: Buellia frigida, Circinaria gyrosa, Rhizocarpon geographicum, Xanthoria elegans, and Pleopsidium chlorophanum. Detailed investigation of secondary substances including photosynthetic pigments was performed for whole lichen thalli but also for axenically cultivated mycobionts and photobionts by methods of UV/VIS-spectrophotometry and two types of high performance liquid chromatography (HPLC). Additionally, a set of chemical tests is presented to confirm the formation of melanic compounds in lichen and mycobiont samples. All investigated lichens reveal various sets of SLCs, except C. gyrosa where only melanin was putatively identified. Such studies will help to assess the contribution of SLCs on lichen extremotolerance, to understand the adaptation of lichens to prevalent abiotic stressors of the respective habitat, and to form a basis for interpreting recent and future astrobiological experiments. As most of the identified SLCs demonstrated a high capacity in absorbing UVR, they may also explain the high resistance of lichens towards non-terrestrial UVR.

Recognition mechanisms during the pre-contact state of lichens: I. Mycobiont-photobiont interactions of the mycobiont of Fulgensia bracteata

Lichens are an association of a photoautotrophic alga/cyanobacteria (photobiont) and a heterotrophic fungus (mycobiont) constituting the lichen thallus as a complex phenotype. Many mycobionts reproduce sexually and the ascospores are dispersed without the photobiont. For successful re-lichenization the specific photobiont must be recognized, contacted, and incorporated by the mycobiont. A so-called pre-contact stage has been postulated as the initial step of a gradual recognition process. In the present study, the effect of the specific Trebouxia photobiont, an unspecific Asterochloris photobiont and the non-lichenizing green alga Myrmecia bisecta on the development of the mycobiont Fulgensia bracteata was assessed by pre-contact assays. Three hypotheses were confirmed: (i) the pre-contact stage exists, (ii) it is characterized by morphological reactions in the development of the mycobiont, and (iii) the reactions depend on the interacting alga. Control conditions revealed a mycelial growth arrest but this effect was not observed in the presence of any of the three algae. Different algae induce distinct growth patterns with respect to hyphal length, morphological characteristics, and formation of mucilage. The specific Trebouxia photobiont had a positive impact on hyphal growth, branching frequency, and mucilage formation. These effects were less explicit with the non-specific Asterochloris photobiont. Myrmecia bisecta induced uncharacteristic growth patterns with pronounced hyphal growth and high numbers of aerial hyphae but less formation of mucilage. These results indicate that symbiont recognition mechanisms are established before physical contact. Pre-contact reactions may be an evolutionary advantage that supports the persistence of the mycobiont on newly colonized sites and improves the probability of re-lichenization.

Resistance of the Lichen Buellia frigida to Simulated Space Conditions during the Preflight Tests for BIOMEX—Viability Assay and Morphological Stability

Samples of the extremotolerant Antarctic endemite lichen Buellia frigida are currently exposed to low-Earth orbit-space and simulated Mars conditions at the Biology and Mars Experiment (BIOMEX), which is part of the ESA mission EXPOSE-R2 on the International Space Station and was launched on 23 July 2014. In preparation for the mission, several preflight tests (Experimental and Scientific Verification Tests, EVT and SVT) assessed the sample preparation and hardware integration procedures as well as the resistance of the candidate organism toward the abiotic stressors experienced under space and Mars conditions. Therefore, we quantified the post-exposure viability with a live/dead staining technique utilizing FUN-1 and confocal laser scanning microscopy (CLSM). In addition, we used scanning electron microscopy (SEM) to investigate putative patterns of morphological-anatomical damage that lichens may suffer under the extreme exposure conditions. The present results demonstrate that Buellia frigida is capable of surviving the conditions tested in EVT and SVT. The mycobiont showed lower average impairment of its viability than the photobiont (viability rates of >83% and >69%, respectively), and the lichen thallus suffered no significant damage in terms of thalline integrity and symbiotic contact. These results will become essential to substantiate and validate the results prospectively obtained from the returning space mission. Moreover, they will help assess the limits and limitations of terrestrial organisms under space and Mars conditions as well as characterize the adaptive traits that confer lichen extremotolerance.

Recognition mechanisms during the pre-contact state of lichens: II. Influence of algal exudates and ribitol on the response of the mycobiont of Fulgensia bracteata

Successful re-lichenization between the two bionts of the lichen symbiosis, the fungal mycobiont and its specific photobiont, is a process that is not well understood yet. To assess potential signalling between the two bionts during initial pre-contact, exudates of the Trebouxia photobionts of Fulgensia bracteata, Fulgensia fulgens, and Xanthoria elegans, of the Asterochloris photobiont of Lecidea lurida, and of the non-lichenizing green alga Myrmecia bisecta were investigated. The compounds identified in these exudates were tested with respect to their influence on germination and early development of the Fulgensia bracteata mycobiont. Additionally, carbohydrates (glucose, sucrose, ribitol) were tested to appraise their effect on the mycobiont growth patterns. Three hypotheses were confirmed: (i) photobionts exude various substances, (ii) the photobiont exudation pattern varies with the identity of the photobiont, and (iii) a pre-contact influence induces changes in the early development of the mycobiont of F. bracteata. This study gives comparative insight to exudates of lichen photobionts. In vitro photobionts differentially release compounds belonging to several substance classes which include indole-3-carbaldehyde, two cyclic dipeptides, and rhamnose. Two compounds had inhibitory effects on germination and germ-tube growth of the mycobiont and one other enhanced spore germination. Additionally, ribitol was found to elicit a strong effect on the mycobiont’s growth. In general, photobiont-exudation, its effect on the mycobiont, and the response to ribitol suggest that complex pre-contact signalling has a crucial role in lichen biont recognition.

Effects of UVC 254 nm on the photosynthetic activity of photobionts from the astrobiologically relevant lichens Buellia frigida and Circinaria gyrosa

In the past decade, various astrobiological studies on different lichen species investigated the impairment of viability and photosynthetic activity by exposure to simulated or real space parameters (as vacuum, polychromatic ultraviolet (UV)-radiation and monochromatic UVC) and consistently found high post-exposure viability as well as low rates of photosynthetic impairment (de Vera et al. 2003, 2004a; 2004b; de la Torre et al. 2010; Onofri et al. 2012; Sanchez et al. 2012, 2014; Brandt et al. 2014). To achieve a better understanding of the basic mechanisms of resistance, the present study subdued isolated and metabolically active photobionts of two astrobiologically relevant lichens to UVC254 nm, examined its effect on photosynthetic activity by chlorophyll a fluorescence and characterized the UVC-induced damages by quantum yield reduction and measurements of non-photochemical quenching. The results indicate a strong impairment of photosynthetic activity, photoprotective mechanisms and overall photobiont vitality when being irradiated in the isolated and metabolically active state. In conclusion, the present study stresses the higher susceptibility of photobionts towards extreme environmental conditions as UVC-exposure, a stressor that does not occur on the Earth. By comparison with previous studies, the present results highlight the importance of protective mechanisms in lichens, such as morphological-anatomical traits (Meesen et al. 2013), secondary lichen compounds (Meesen et al. 2014) and the symbionts pivotal ability to pass into anhydrobiosis when desiccating. Received 7 May 2014, accepted 16 July 2014

Humidity interaction of lichens under astrobiological aspects: the impact of UVC exposure on their water retention properties

We quantitatively studied the hydration and dehydration behaviour of the three astrobiological model lichens Xanthoria elegans, Buellia frigida and Circinaria gyrosa by thermoanalysis and gravimetric isotherm measurements under close-to-Martian environmental conditions in terms of low temperature and low pressure. Additionally, the impact of UVC exposure on the isolated symbionts of B. frigida and X. elegans was studied by thermoanalysis and mass spectrometry as well as by gravimetric isotherm measurements. The thermal analysis revealed whewellite as a component of C. gyrosa which was not found in B. frigida and X. elegans. Neither the water retention nor the thermal behaviour of symbionts changed when irradiated with UVC under dry conditions. On the other hand, UVC irradiation of the wet mycobiont of B. frigida had a distinct impact on the hydration/dehydration ability which was not observed for the mycobiont of X. elegans. Possibly the melanin of B. frigida’s mycobiont, that is not present in X. elegans, or a specifically damaged acetamido group of the chitin of B. frigida may be the sources of additional UVCinduced sorption sites for water associated with the UVC exposure.

Simulated Space Radiation: Impact of Four Different Types of High-Dose Ionizing Radiation on the Lichen Xanthoria elegans

This study addresses the viability of the lichen Xanthoria elegans after high-dose ionizing irradiation in the frame of the STARLIFE campaign. The first set of experiments was intended to resemble several types of galactic cosmic radiation (GCR) as present beyond the magnetic shield of Earth. In the second set of experiments, γ radiation up to 113 kGy was applied to test the limit of lichen resistance to ionizing radiation. Entire thalli of Xanthoria elegans were irradiated in the anhydrobiotic state. After STARLIFE 1, the metabolic activity of both symbionts was quantified by live/dead staining with confocal laser scanning microscopy. The photosynthetic activity was measured after the respective irradiation to assess the ability of the symbiotic green algae to restore photosynthesis after irradiation. The STARLIFE campaign complements the results of the LIFE experiments at the EXPOSE-E facility on the International Space Station by testing the model organism Xanthoria elegans on its resistance to hazardous radiation that might be accumulated during long-term space exposure. In addition, the photosynthetic activity of metabolically active lichen was investigated after X-ray irradiation up to 100 Gy (3.3 Gy/min). Since previous astrobiological experiments were mostly performed with anhydrobiotic lichen, these experiments will broaden our knowledge on the correlation of physiological state and astrobiological stressors. Key Words: Astrobiology-Extremotolerance-Gamma rays-Ionizing radiation-Lichens-Viability. Astrobiology 17, 136-144.

Desiccation and low temperature attenuate the effect of UVC254 nm in the photobiont of the astrobiologically relevant lichens Circinaria gyrosa and Buellia frigida

Several investigations on lichen photobionts (PBs) after exposure to simulated or real-space parameters consistently reported high viability and recovery of photosynthetic activity. These studies focused on PBs within lichen thalli, mostly exposed in a metabolically inactive state. In contrast, a recent study exposed isolated and metabolically active PBs to the non-terrestrial stressor UVC254 nm and found strong impairment of photosynthetic activity and photo-protective mechanisms (Meesen et al. in 2014b). Under space and Mars conditions, UVC is accompanied by other stressors as extreme desiccation and low temperatures. The present study exposed the PBs of Buellia frigida and Circinaria gyrosa, to UVC in combination with desiccation and subzero temperatures to gain better insight into the combined stressors’ effect and the PBs’ inherent potential of resistance. These effects were examined by chlorophyll a fluorescence which is a good indicator of photosynthetic activity (Luttge& Budel in 2010) and widely used to test the viability of PBs after (simulated) space exposure. The present results reveal fast recovery of photosynthetic activity after desiccation and subzero temperatures. Moreover, they demonstrate that desiccation and cold confer an additional protective effect on the investigated PBs and attenuate the PBs’ reaction to another stressor – even if it is a non-terrestrial one such as UVC. Besides other protective mechanisms (anhydrobiosis, morphological–anatomical traits and secondary lichen compounds), these findings may help to explain the high resistance of lichens observed in astrobiological studies.