Detection of new biohints on lichens with Raman spectroscopy after space- and Mars like conditions exposure: Mission Ground Reference (MGR) samples.

The extremophile lichen Circinaria gyrosa (C. gyrosa) is one of the selected species within the BIOMEX (Biology and Mars Experiment) experiment. Here we present the Raman study of a biohint found in this lichen, called whewellite (calcium oxalate monohydrate), and other organic compounds and mineral products of the biological activity of the astrobiologically relevant model system C. gyrosa. Samples were exposed to space- and simulated Mars-like conditions during the EXPOSE-R2 mission parallel ground reference experiment MGR performed at the space- and planetary chambers of DLR-Cologne to study Mars' habitability and resistance to real space conditions. In this work, we complete the information of natural C. gyrosa about the process of diagenesis by the identification of carbonate crystals in the inner medulla together with the biomineral whewellite. The analysis by Raman spectroscopy of simulated Space and Mars exposed samples confirm alterations and damages of the photobiont part of the lichen and changes related to the molecular structure of whewellite. The conclusions of this work will be important to understand what are the effects to consider when biological systems are exposed to space or Mars-like conditions and to expand our knowledge of how life survives in most extreme conditions that is a prerequisite in future planetary exploration projects.

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.

Ecophysiology and genetic structure of polar versus temperate populations of the lichen Cetraria aculeata.

We studied polar and temperate samples of the lichen Cetraria aculeata to investigate whether genetical differences between photobionts are correlated with physiological properties of the lichen holobiont. Net photosynthesis and dark respiration (DR) at different temperatures (from 0 to 30 °C) and photon flux densities (from 0 to 1,200 µmol m(-2) s(-1)) were studied for four populations of Cetraria aculeata. Samples were collected from maritime Antarctica, Svalbard, Germany and Spain, representing different climatic situations. Sequencing of the photobiont showed that the investigated samples fall in the polar and temperate clade described in Fernández-Mendoza et al. (Mol Ecol 20:1208-1232, 2011). Lichens with photobionts from these clades differ in their temperature optimum for photosynthesis, maximal net photosynthesis, maximal DR and chlorophyll content. Maximal net photosynthesis was much lower in Antarctica and Svalbard than in Germany and Spain. The difference was smaller when rates were expressed by chlorophyll content. The same is true for the temperature optima of polar (11 °C) and temperate (15 and 17 °C) lichens. Our results indicate that lichen mycobionts may adapt or acclimate to local environmental conditions either by selecting algae from regional pools or by regulating algal cell numbers (chlorophyll content) within the thallus.

Diversity of endosymbiotic Nostoc in Gunnera magellanica from Tierra del Fuego, Chile [corrected].

Global warming is causing ice retreat in glaciers worldwide, most visibly over the last few decades in some areas of the planet. One of the most affected areas is the region of Tierra del Fuego (southern South America). Vascular plant recolonisation of recently deglaciated areas in this region is initiated by Gunnera magellanica, which forms symbiotic associations with the cyanobacterial genus Nostoc, a trait that likely confers advantages in this colonisation process. This symbiotic association in the genus Gunnera is notable as it represents the only known symbiotic relationship between angiosperms and cyanobacteria. The aim of this work was to study the genetic diversity of the Nostoc symbionts in Gunnera at three different, nested scale levels: specimen, population and region. Three different genomic regions were examined in the study: a fragment of the small subunit ribosomal RNA gene (16S), the RuBisCO large subunit gene coupled with its promoter sequence and a chaperon-like protein (rbcLX) and the ribosomal internal transcribed spacer (ITS) region. The identity of Nostoc as the symbiont was confirmed in all the infected rhizome tissue analysed. Strains isolated in the present study were closely related to strains known to form symbioses with other organisms, such as lichen-forming fungi or bryophytes. We found 12 unique haplotypes in the 16S rRNA (small subunit) region analysis, 19 unique haplotypes in the ITS region analysis and 57 in the RuBisCO proteins region (rbcLX). No genetic variability was found among Nostoc symbionts within a single host plant while Nostoc populations among different host plants within a given sampling site revealed major differences. Noteworthy, interpopulation variation was also shown between recently deglaciated soils and more ancient ones, between eastern and western sites and between northern and southern slopes of Cordillera Darwin. The cell structure of the symbiotic relationship was observed with low-temperature scanning electron microscopy, showing changes in morphology of both cyanobiont cells (differentiate more heterocysts) and plant cells (increased size). Developmental stages of the symbiosis, including cell walls and membranes and EPS matrix states, were also observed.

Whole lichen thalli survive exposure to space conditions: results of Lithopanspermia experiment with Aspicilia fruticulosa.

The Lithopanspermia space experiment was launched in 2007 with the European Biopan facility for a 10-day spaceflight on board a Russian Foton retrievable satellite. Lithopanspermia included for the first time the vagrant lichen species Aspicilia fruticulosa from Guadalajara steppic highlands (Central Spain), as well as other lichen species. During spaceflight, the samples were exposed to selected space conditions, that is, the space vacuum, cosmic radiation, and different spectral ranges of solar radiation (λ ≥ 110, ≥200, ≥290, or ≥400 nm, respectively). After retrieval, the algal and fungal metabolic integrity of the samples were evaluated in terms of chlorophyll a fluorescence, ultrastructure, and CO(2) exchange rates. Whereas the space vacuum and cosmic radiation did not impair the metabolic activity of the lichens, solar electromagnetic radiation, especially in the wavelength range between 100 and 200 nm, caused reduced chlorophyll a yield fluorescence; however, there was a complete recovery after 72 h of reactivation. All samples showed positive rates of net photosynthesis and dark respiration in the gas exchange experiment. Although the ultrastructure of all flight samples showed some probable stress-induced changes (such as the presence of electron-dense bodies in cytoplasmic vacuoles and between the chloroplast thylakoids in photobiont cells as well as in cytoplasmic vacuoles of the mycobiont cells), we concluded that A. fruticulosa was capable of repairing all space-induced damage. Due to size limitations within the Lithopanspermia hardware, the possibility for replication on the sun-exposed samples was limited, and these first results on the resistance of the lichen symbiosis A. fruticulosa to space conditions and, in particular, on the spectral effectiveness of solar extraterrestrial radiation must be considered preliminary. Further testing in space and under space-simulated conditions will be required. Results of this study indicate that the quest to discern the limits of lichen symbiosis resistance to extreme environmental conditions remains open.

Endolithic growth of two Lecidea lichens in granite from continental Antarctica detected by molecular and microscopy techniques.

Through the combined use of molecular and microscopy techniques, the endolithic lichens Lecidea cancriformis and Lecidea sp. were identified, even in the absence of fruiting bodies, and positioned under epilithic lichens. Cells of both algal and fungal symbionts were observed in fissures and cracks of the lithic substrate with no clear heteromerous structure. At the ultrastructural level, the two lichens differed in terms of their algal-fungal relationships. Only one genotype of Trebouxia ITS sequence was identified from specimens of Lecidea sp., Umbilicaria aprina and Buellia frigida from the same zone, which could be mainly determined by low availability of alga in these extreme environments. These lichens showed features typical of both chasmoendolithic and euendolithic microorganisms. Signs of biogeophysical and biogeochemical action on the substrate were detected close to fungal cells. This action seemed to be mainly conditioned by the local physico-chemical features of the substrate. Evidence for the biomobilization of elements by these endolithic lichens was found. L. cancriformis was observed to accumulate substantial amounts of calcium-rich biominerals. The combined approach proposed is useful for mapping the distribution of endolithic lichens and analysing the processes that occur in their microscopic environment.

Viability of endolithic micro-organisms in rocks from the McMurdo Dry Valleys of Antarctica established by confocal and fluorescence microscopy.

The rocks of the McMurdo Dry Valleys desert in Antarctica harbour endolithic communities of micro-organisms such as lichens, fungi, cyanobacteria and bacteria. Establishing the physiological status and viability of these microbial colonies in their natural microhabitat has far-reaching implications for understanding the microbial ecology of the harsh environment of this polar desert. Here we describe the use of confocal microscopy and a specific fluorescent probe (FUN-1) to evaluate the metabolic activity of fungal cells. Application of confocal microscopy also served to identify living and dead bacteria or cyanobacteria using the fluorescent assay reagents Live/Dead SYTO 9 and propidium iodide or SYTOX Green, respectively. In addition, through the use of epifluorescence microscopy, live/dead bacteria and cyanobacteria could be detected by estimating fluorescence from their cell components provoked by simultaneously staining with nucleic acids stains such as DAPI and SYTOX Green.

Prognostic factors for myasthenia gravis treated by thymectomy: review of 61 cases.

BACKGROUND: Medical treatment for myasthenia gravis (MG) involves the use of anticholinesterase agents, immunosuppressive drugs, plasmapheresis, and gamma-globulin. However, these agents result in a complete clinical remission rate as low as 15%. As a consequence, thymectomy, preferably by transsternal approach, has become increasingly accepted as an efficacious procedure for MG, with reported complete clinical remission rates as high as 80%. METHODS: We have the clinical records of 61 patients diagnosed with MG at La Paz University Hospital, Madrid, Spain, from January 1977 to December 1994. All patients underwent thymectomy. The purpose of this investigation was to determine the major prognostic factors predicting MG outcome after operation. RESULTS: Our results indicate that patients with a length of the disease from onset to operation shorter than 8 months have the best prognosis. Ossermann stages I and III are also associated with higher complete clinical remission rates. In contrast, neither age nor sex were found to be significantly related to MG outcome after thymectomy, although female patients have better prognosis than men, and the younger the patient the more likely is complete clinical remission. Pathologic findings after the operation were not found to be of prognostic value either. CONCLUSIONS: We conclude that thymectomy is a beneficial procedure for MG patients, with a complete clinical remission rate of 46% at 5 years postoperatively in our series. Therefore we advocate thymectomy for MG patients as early as possible in the course of disease because time elapsed from diagnosis to operation is the main determinant of the outcome.

Carbon dioxide exchange of Antarctic crustose lichens in situ measured with a CO2/H2O porometer.

Photosynthesis and respiration of crustose lichens in their natural situation were measured by means of a Walz porometer with a modified cuvette and a plexiglass ring. Habitat influence and the specific performance of three maritime Antarctic species were demonstrated by diurnal courses of microclimate and CO2 exchange during rainy days. In further field experiments the photosynthetic response to soaking with water was tested. Haematomma erythromma is rain-exposed at its natural habitat on coastal rocks but tends to dry out quickly. The photosynthetic efficiency of the chlorophyll of this photophilous species was high. Lecidea sciatrapha appears to be chionophilous and had a low light compensation point of its net photosynthesis. The photosynthetic rates per surface area of these two species were low compared with those of Caloplaca sublobulata. According to its habitat selection C. sublobulata is typified as an ombrophobous lichen. This characterization was confirmed experimentally by our gas exchange measurements.

Photosynthesis and water relations and the role of anatomy in Umbilicariaceae (lichenes) from Central Spain.

The response of net photosynthesis and dark respiration in eight species of Umbilicariaceae (lichenes) to temperature (-5, 0, 5, 10, 15, 20, 25, 30°C) and irradiance (55, 110, 220, 400, 620 µmol photons m-2 s-1 PAR) was studied. The samples were collected in montane and alpine localities of the Spanish Sistema Central. The species differed widely in their net photosynthetic rates. The optimal temperature for net photosynthesis in alpine species was significantly lower than in montane species. Montane species were more photophytic than alpine ones. Water saturation and water loss rate were dependent on morphology and particularly anatomy of the thallus. The physiological and structural data are useful in the interpretation of the ecology and altitudinal distribution of the Umbilicariaceae. No adaptation could be linked to particularities of the mediterranean climate.