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AST-2010-0588-Raggio_1P
Type: research-article
Research Article
ASTROBIOLOGY
Volume 11, Number 4, 2011
ª Mary Ann Liebert, Inc.
DOI: 10.1089/ast.2010.0588
Whole Lichen Thalli Survive Exposure to Space Conditions:
Results of Lithopanspermia Experiment
with Aspicilia fruticulosa
J. Raggio,1 A. Pintado,1 C. Ascaso,2 R. De La Torre,3 A. De Los Rı́os,2 J. Wierzchos,2
G. Horneck,4 and L.G. Sancho1
Abstract
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 (k 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 CO2 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 stressinduced 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. Key Words: Astrobiology—Lichens—Panspermia—Chlorophyll fluorescence—CO2 exchange—Ultrastructure. Astrobiology 11,
xxx–xxx.
it has occurred, the various steps required for the transfer of
organisms from one planet to another have been the focus of
experimental testing (Cockell, 2008). This technical availability has led to several studies, either under simulated space
conditions (Buecker and Horneck, 1970; Mancinelli and
Klovstad, 2000; De la Torre et al., 2003) or in spaceflight experiments (Horneck, 1993; Fajardo-Cavalzos et al., 2005;
Sancho et al., 2007; De los Rı́os et al., 2010; Horneck et al., 2010).
One of the main objectives of these astrobiological experiments has been to test whether different kinds of organisms can survive in the extremely hostile conditions of
1. Introduction
T
he development of space technology has opened the
gates to experiments in astrobiology, a relatively new and
interdisciplinary field of research that aims to achieve a better
understanding of the processes that led to the origin, evolution, and distribution of life on Earth or elsewhere in the
Universe (Horneck, 1995). Richter (1865) and Arrhenius (1903)
first proposed the panspermia theory, which speculates about
the transfer of life between planets. Although panspermia still
remains little more than an idea and there is no evidence that
1
Departamento Biologı́a Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain.
Instituto de Recursos Naturales, Centro de Ciencias Medioambientales, CSIC, Madrid, Spain.
3
Instituto Nacional de Técnica Aeroespacial (INTA), Madrid, Spain.
4
Deutsches Zentrum für Luft-und Raumfahrt, Institut für Luft-und Raumfahrtmedizin, Köln, Germany.
2
1
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interplanetary space with particular attention to the space
vacuum that causes dehydration of the samples and the high
intensity of cosmic rays and solar extraterrestrial UV radiation,
the latter being especially harmful to DNA. The survival capacity of exposed organisms is an interesting feature that can
indirectly support or deny the old panspermia theory and recent
revisions (Friedmann et al., 2001; Fajardo-Cavalzos et al., 2005).
In view of the harsh conditions of space, the choice of
suitable test organisms is of great importance. Some authors,
such as Horneck (1993), Horneck et al. (1994, 2001), and
Mancinelli et al. (1998), have worked with bacterial endospores and halophiles, respectively, because of their known
exceptionally high resistance to harsh terrestrial climatic
conditions, while, in recent years, lichens have also been
tested (Sancho et al., 2007, 2008; De la Torre et al., 2010).
Lichens are symbiotic organisms (green algae or cyanoAU1 c bacteria, or both, with a fungus) that are able to colonize a
wide range of habitats around the world (Kappen, 1988),
including harsh environments such as deserts (Lange et al.,
1994; Pintado et al., 2005), high mountains (Sancho and
Kappen, 1989; Reiter et al., 2007), and polar regions (Green
et al., 1998; Pannewitz et al., 2003). In Antarctica, they often
are the dominant organisms in terrestrial ecosystems and
have been reported as far south as 868290 S (Siple, 1938;
Øvstedal and Lewis Smith, 2001). Under particularly severe
conditions in continental Antarctica (De los Rı́os et al., 2005),
lichens survive inside rocks, where they form endolithic
communities. Lichens, as poikilohydric organisms, are only
active when wet; when dehydrated and inactive, they can be
highly tolerant to extreme conditions of light, temperature,
and drought (Kappen and Valladares, 2007). Because of these
characteristics, lichens have been successfully used in experiments under simulated space conditions (De la Torre
et al., 2003, 2007; De Vera et al., 2003, 2004), and they have
been launched into space in the experiment Lichens (Sancho
et al., 2007) and Lithopanspermia (Sancho et al., 2008; De la
Torre et al., 2010) in order to better understand the effects of
extreme space conditions on multicellular eukaryotic organisms.
The experiment Lithopanspermia included for the first
time the globoid lichen Aspicilia fruticulosa (Eversm.) Flagey
F1 c (Fig. 1A) from Guadalajara steppes, central Spain, a habitat
characterized by high temperature contrasts, high frost incidence, and very dry summers (Crespo and Barreno, 1978).
The species was suggested for this space study because it is
described as a good example of morphological adaptation to
harsh climate conditions (Sancho et al., 2000). In addition, its
size (average diameter of the samples 6.2 mm) is small enough to be accommodated in the exposure compartments of
the Lithopanspermia hardware. These size characteristics of
the samples provided the opportunity to expose, for the first
time, a complete individual lichen thallus to selected space
conditions as opposed to using fragments.
A few attempts have been made to better understand the
physiological parameters involved in the morphological
variation of A. fruticulosa. Weber (1967) thought that the
globoid structure of the Aspicilia genus species was an adaptation to hard climate conditions, and Kunkel (1980) observed a relationship between morphology and microhabitat
conditions for A. desertorum. Sancho et al. (2000) proposed an
ecophysiological explanation of the globular growth of A.
fruticulosa as a strategy for ameliorating water loss due to the
high evaporation characteristic of the habitat, with large
RAGGIO ET AL.
temperature contrasts and long periods of dryness. In the
same work, the authors showed low-temperature scanning
electron microscope (LTSEM) images of the anatomy of A. b AU2
fruticulosa, and they described the cortex as a two-layer
structure, one upper of 20–25 mm thickness and another below of 80–210 mm made up of strongly conglutinated hyphae.
In the previous Lichens experiment, the two crustose lichens (the alpine species Rhizocarpon geographicum (L.) DC.
and Xanthoria elegans (Link) Th. Fr. were studied with chlorophyll a fluorescence and microscopy analysis (Sancho et al.,
2007). Here, we not only used the same techniques but also
added CO2 exchange measurements for the first time. We
were able to measure accurately the physiological resistance
of the algal cells in terms of CO2 assimilation rates, and for
the first time we also used dark respiration to evaluate the
fungal physiological state after exposure. The combination of
chlorophyll a fluorescence, fungal and algal cellular ultrastructure by microscopy analysis, and CO2 exchange measurements provided additional information about the
survival capacity of the lichen symbiosis in space.
2. Material and Methods
2.1. Experimental design
Thalli of the globoid lichen A. fruticulosa growing on claylike red soil were collected near Zaorejas (Guadalajara,
Spain) at 1240 m above sea level (Fig. 1B). Intact lichen
samples 6–7 mm in diameter were mounted in the hardware
of the space experiment Lithopanspermia (designed by
INTA, Spanish Aerospace Establishment, Madrid, Spain),
which was included in the Biopan facility of ESA (Demets
et al., 2005). The experiment Lithopanspermia was part of the
Biopan-6 mission, during which the samples were exposed to
selected conditions in space for 10 days. The hardware
characteristics and space conditions are described in detail in
De la Torre et al. (2010).
During the 10-day spaceflight all samples were fully exposed to the space vacuum (about 106 Pa) and cosmic ionizing radiation (4–100 mGy, depending on mass shielding).
In addition, some samples (flight sun-exposed samples) were
exposed to solar extraterrestrial electromagnetic radiation,
whereas others (flight dark samples) were protected from
this radiation by a shield but otherwise still exposed to space.
Another set of samples (Earth controls) was kept on Earth
and stored at room temperature under dry and dark conditions. The flight sun-exposed samples were exposed beneath
an optical filter system that provided different UV radiation
environments. One sample was exposed to the full spectrum
of solar extraterrestrial UV and visible radiation (l 110 nm)
by use of a MgF2 filter; a second sample had a SQO synthetic
quartz filter that allowed transmission of wavelengths at
l 200 nm. A third sample had a long-pass filter that allowed exposure to l 290 nm and simulated Earth’s UV
climate conditions. A fourth sample was exposed to radiation of wavelengths longer than 400 nm, the zero-UV radiation treatment. Limitations of the hardware in space meant
that the number of samples for each flight sun-exposure
condition was n ¼ 1. However, because the different radiation exposures (for flight sun-exposed samples) showed no
apparent differences in their physiological response, we
consider the number of samples exposed to space conditions
for each experiment (sun exposed and flight dark) as n ¼ 4.
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FRUTICOSE LICHEN SURVIVED SPACE EXPOSURE
The number of Earth control samples was n ¼ 8. The experimental design, which is a compromise between achieving a
useful result and enough information to plan future research,
went through several reviews and was approved by ESA
before the flight.
2.2. Chlorophyll fluorescence analysis
All thalli were reactivated after the flight in a growth
chamber at 108C and 100 mmol photon m2 s1 photon flux
FIG. 1.
3
density (PFD) for 12/12 h dark/light photoperiod. They
were moistened twice daily by spraying with bottled mineral
water. Chlorophyll a fluorescence of the reactivated samples
was measured with a photosynthesis yield analyzer (MiniPAM, Walz Company, Germany) as described by Sancho
et al. (2007). The chosen parameter for the metabolic evaluation
was the potential maximum photosystem II quantum yield
(Fv/Fm; Schreiber et al., 1994), where Fm is the maximum
fluorescence after a saturation pulse of actinic light (photosynthetic active radiation, 400–700 nm) and Fv ¼ (Fm - Fo) is
(A) Aspicilia fruticulosa in the field. (B) Sampling locality, Guadalajara High Steppes.
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the variable fluorescence; Fo is the minimal fluorescence after
20 min dark adaptation measured with modulated low light
(red light, maximum 650 nm).
The fluorescence measurements were carried out after 3,
24, 48, and finally 72 h of reactivation. All samples were
measured prior to the spaceflight or storage on Earth and
then subsequent to the flight.
2.3. CO2 exchange
After an additional period of 72 h reactivation, CO2 exchange was measured at 208C and 400, 800, 1200 mmol
photon m2 s1 PFD with an open flow differential IRGA
system (GFS-3000, Walz Company, Germany). This IRGA is
a four-channel CO2/H2O absolute nondispersive infrared
analyzer. The cuvette of this new gas exchange system is
small (3 cm long and 4 cm width) and allows rapid and accurate (0.1%) measurements on single samples of low
biomass. Temperature and relative humidity inside the cuvette were controlled, and the air flow was regulated at
600 mL min1.
A standard procedure was used. Samples were first hydrated by 20 min of mineral water immersion to ensure
complete saturation. Samples were then placed in the cuvette
and dark respiration (DR) was measured. The samples were
then illuminated with a LED light source 3040-L at 400 mmol
photon m2 s1 PFD until the net photosynthetic rate (NP)
reached a constant value, and this was repeated at 800 and
1200 mmol photon m2 s1 for 5 min each. Net CO2 exchange
at the highest PFD was taken as the maximum CO2 net
assimilation value (Amax). Measurements were made over a
5-day period.
2.4. Statistical analysis
Comparison of means of controls with a significance level
of p 0.05 was performed by one-way analysis of variance.
Comparisons of the data of the flight dark samples and the
Earth controls were performed with Duncan’s multiple range
test ( p 0.05). Statgraphics version 5.1 was used.
RAGGIO ET AL.
2.5.2. Transmission electron microscopy. After the gas
exchange measurements were made, small lichen fragments
of A. fruticulosa were fixed in glutaraldehyde and osmium
tetroxide solutions, dehydrated in a graded ethanol series,
and embedded in Spurr’s resin following the protocol described by Ascaso and Galván (1976), Ascaso (1978), and De
los Rı́os and Ascaso (2002). Ultrathin sections were poststained with lead citrate (Reynolds, 1963) and observed in a
Zeiss EM910 transmission electron microscope operating at
80 kV.
2.6. Thin layer chromatography
A few fragments of some A. fruticulosa samples were taken
for thin layer chromatography (TLC) assays to look for lichen b AU3
substances. The extraction was made following Huneck and
Yoshimura (1996), and samples were run on Merck silica gel
60 F254 pre-coated glass-backed TLC plates (layer thickness
0.25 mm) 2020 cm. Solvent system C was (170 mL toluene/
30 mL acetic acid) prepared according to Lumbsch (2002).
3. Results
3.1. Chlorophyll a fluorescence
Preflight Fv/Fm for all samples (flight sun-exposed samples, flight dark samples, and Earth controls) ranged between 636 and 753 (values are 1000 times actual yield; Figs. b F2 F6
2–6 and Table 1). These values were taken as the reference b T1
level for an intact healthy thallus (Demmig-Adams et al.,
1990). After spaceflight, the Fv/Fm value of all flight sunexposed samples that were measured subsequent to 3 h
reactivation was significantly reduced compared to the preflight data. The lowest value (49% of the preflight value) was
observed for the sample that was exposed to the full spectrum of extraterrestrial solar UV and visible radiation
(l 110 nm, Fig. 2). All other flight sun-exposed samples had
a less pronounced, but reduced, initial Fv/Fm value after 3 h
2.5. Microscopy analysis
2.5.1. Low-temperature scanning electron microscopy. Lichen thalli of A. fruticulosa were examined with a
LTSEM after the gas exchange measurements. Small lichen
fragments were fixed onto the specimen holder of the cryotransfer system (Oxford CT1500), plunged into liquid
nitrogen, and then transferred to the scanning electron
microscope (SEM) via an air-lock transfer device. The frozen specimens were cryo-fractured in the preparation unit
and transferred directly via a second air lock to the microscope cold stage, where they were etched for 2 min at
908C. The following beam conditions for etching were
used: acceleration potential of 2 kV, probe current of 300 Pa,
beam diameter of 70 nm, and aperture size of 120 mm. After
ice sublimation, the etched surfaces were gold sputter
coated with 200 Å thick layer in the preparation unit.
Samples were subsequently transferred onto the cold stage
of the SEM chamber. Fractured and etched surfaces were
observed under DSM960 Zeiss SEM at 1358C under 15 kV
acceleration potential, 10 mm working distance, 200 pA
probe current.
FIG. 2. Fv/Fm recovery of flight sample exposed to solar
radiation 110 nm. Preflight value is depicted on horizontal
line.
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FRUTICOSE LICHEN SURVIVED SPACE EXPOSURE
FIG. 3. Fv/Fm recovery of flight sample exposed to solar
radiation 200 nm. Preflight value is depicted on horizontal
line.
5
FIG. 5. Fv/Fm recovery of flight sample exposed to solar
radiation 400 nm. Preflight value is depicted on horizontal
line.
of reactivation (84% for the samples exposed to l 200 nm;
77% for those exposed to l 290 or l 400 nm of solar radiation) (Table 1 and Figs. 3–5). Fv/Fm values gradually
increased with longer reactivation periods until maximum
values were reached at 72 h, some of which were identical to
the preflight level, some nearly identical. For example, the
most affected sample, that is, the one exposed to l 110 nm,
reached an Fv/Fm value of 633, which is very close to the
preflight value of 636. This high capacity of recovery is an
indication that damage to the photosystems was reversible
within 3 days of reactivation. The flight dark samples
reached, after 3 h, a 90% of Fv/Fm in relation to the preflight
value. At 48 h it was 95%, and at 72 h it rose until 98% of the
preflight record (percentages are averages of the four replicates). The Earth control samples showed a small reduction
(to 90%) in their mean Fv/Fm value at 3 h of reactivation;
then the value dropped to 89% at 24 h, rose to 90% at 48 h,
and finally reached 95% of the preflight yield at 72 h of reactivation (Fig. 6). Differences between flight dark sample
data and Earth control data at each time of the recovery
period were not significant ( p 0.05). For both flight dark
samples and Earth controls no significant differences
FIG. 4. Fv/Fm recovery of flight sample exposed to solar
radiation 290 nm. Preflight value is depicted on horizontal
line.
FIG. 6. Fv/Fm recovery of flight dark samples and Earth
control samples. Preflight values are depicted on horizontal
lines.
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RAGGIO ET AL.
Table 1. Chlorophyll a Yield Fluorescence (Fv/Fm) before and after the Spaceflight of A. fruticulosa,
Measured after Different Reactivation Times
Preflight
3h
24 h
48 h
72 h
Flight l 110 nm
n¼1
Flight l 200 nm
n¼1
Flight l 290 nm
n¼1
Flight l 400 nm
n¼1
Flight dark
n¼4
Earth control
n¼8
636
312
410
548
633
726
611
680
711
726
753
579
687
710
719
735
563
644
662
710
694.0 38.0a
625.8 81.2c
640.0 42.2bc
661.8 32.7abc
683.8 25.3ab
688.0 44.1a
620.4 71.4b
609.6 65.6b
618.8 58.5b
654.3 55.6ab
Mean value standard deviation.
Different letters in the same column mean significant differences according to Duncan’s multiple range test ( p 0.05).
( p 0.05) were found between preflight Fv/Fm and that
subsequent to recovery at 72 h.
3.2. CO2 exchange
The CO2 exchange results of the flight samples are shown
F7 c in Fig. 7 as DR (negative values) and NP (positive values) at
increasing PFD. The horizontal bars represent the mean of
both Earth control and flight dark samples, which were not
significantly ( p 0.05) different. Therefore, they were
grouped together as ‘‘controls’’ with regard to sun exposure.
However, we recognize that the flight dark samples, unlike
the Earth controls, were exposed to cosmic rays, the space
vacuum, and temperature fluctuations during the spaceflight. The dashed line is the mean Amax of the control, and
the solid line is the mean DR of the control.
Table 2 shows the numerical results of the CO2 exchange b T2
measurements. The Amax of the flight sun-exposed samples
and flight dark samples ranged between 1.1 and 1.7 mmol
CO2 kg dw1 s1, and were close to the 1.6 0.6 mmol CO2
kg dw1 s1 of the Earth control samples. For net photosynthesis of the four flight sun-exposed samples, we obtained
a mean 1.4 0.2 mmol CO2 kg dw1 s1, which suggests little b AU4
effect due to the different radiation treatments. The standard
deviation of the flight sun-exposed samples is even lower
than the standard deviation of the other two treatments,
FIG. 7. Amax values (positive) and DR values for all flight samples.
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Table 2. CO2 Exchange of A. fruticulosa after the Spaceflight
Amax
DR
Flight l 110 nm
n¼1
Flight l 200 nm
n¼1
Flight l 290 nm
n¼1
Flight l 400 nm
n¼1
Flight dark
n¼4
Earth control
n¼4
1.5
0.6
1.4
1.7
1.7
1.5
1.1
0.9
1.2 0.4
0.9 0.4
1.6 0.6
0.7 0.2
Standard deviation is included where n > 1.
which again supports that the different radiation treatments
had no effect. The DR of the flight dark samples and Earth
controls were statistically identical (Table 2) and were also
identical to DR values of the flight sun-exposed samples
exposed to solar radiation of l 110 and l 400 nm. However, flight sun-exposed samples exposed to either l 200 or
l 290 nm showed slightly elevated DR of 1.7 or 1.5 mmol
CO2 kg dw1 s1, respectively. Except for those two examples, all other flight samples showed similar metabolic activity, without clear differences between the different space
exposure conditions.
3.3. Microscopy analysis
F8F9 c Figures 8 and 9 show the appearance of photobiont and
mycobiont cells of the Earth control lichens. The photobiont
chloroplast thylakoid membranes showed a normal appearance, but the pyrenoids (P) were rather poor regarding the
number of pyrenoglobuli (Pg). It seems that there is a tendency toward swelling and curling thylakoids. The appearance of cytoplasmic lipid storage bodies (Sb) and
mitochondria (m) was normal (Fig. 8). Vacuoles were not
observed in photobiont cells. The mycobiont cells (Fig. 9) had
well-preserved mitochondria (m) and concentric bodies (cc).
F10 c The photobiont cells of the flight dark samples (Fig. 10)
had more pyrenoglobuli than the Earth control. The presence
of dense bodies in the cytoplasm vacuoles (V) and between
the thylakoids (head of arrow) probably indicates stress or
some degree of senescence. Starch granules were present (s).
F11 c Mycobiont cells exhibited vacuoles (Fig. 11) containing irregular dense bodies (V).
Algal pyrenoids had dense matrix and numerous thylakoid membranes in flight samples exposed to solar radiation
F12 c at l 110 nm (Fig. 12). Electron-dense membranous complex-like lipidic structures with a very dense appearance
were frequently observed inside the chloroplast (head of
arrow). Vacuoles were seen in the algal cytoplasm (V), either
empty or with dense bodies (indicating stress). Mitochondria
did not appear well defined in these cells. In the mycobiont
F13 c cells (Fig. 13), we observed a lack of concentric bodies, and
the mitochondria seemed to be in rather good shape. All the
F14 c vacuoles (V) contained very electron-dense deposits. Figure
14, obtained by low-temperature scanning electron microsAU5 c copy, shows the integrity of the algal (white arrows) cell
walls in the flight sample exposed to l 110 nm solar radiation.
The algal cells of the flight samples exposed to solar raF15 c diation of l 200 (Fig. 15) had pyrenoids with dense matrix
and many thylakoid membranes. Starch was observed inside
the chloroplast. Vacuoles with electron-dense bodies were
frequent in the cytoplasm (V) of photobiont cells, which
probably indicates cellular stress.
In the flight sample exposed to solar radiation of
l 290 nm (Fig. 16), the algal cells had thylakoid membranes b F16
stacked inside the pyrenoid and less starch than the flight
dark samples and flight samples exposed to l 200 nm. The
cytoplasm of these cells contained vacuoles, and the mitochondria appeared ultrastructurally disorganized. Algal cells
(Fig. 17) had compact nucleoli (n), which could indicate b F17
that the cells were less active. The fungi possessed welldeveloped concentric bodies and vacuoles with less electrondense bodies (Fig. 17).
Algal cells of the flight sample that was exposed to
l 400 nm solar radiation (Figs. 18, 19) had pyrenoids and b F18F19
pyrenoglobuli similar to those of the flight control. However,
electron-dense lipid structures between thylakoids were not
observed. The appearance of lipid storage bodies in the algal
cytoplasm was similar to that observed in cells from Earth
control samples. Mycobiont cells of this sample (Fig. 19)
contained vacuoles without electron-dense bodies and concentric bodies with normal appearance.
3.4. Thin layer chromatography
Two different and simple thalli of A. fruticulosa collected
from the same locality were analyzed by TLC, and no lichen
substances were found.
4. Discussion
In outer space, organisms are exposed to a complex matrix
of extreme environmental conditions, which consist of high
vacuum, extraterrestrial solar UV radiation, and a wide
range of temperatures (Nicholson et al., 2005). The high
vacuum conditions are extremely dehydrating, an effect that
has been considered to be one of the most lethal factors of the
space environment (Sancho et al., 2007). Whereas most organisms are severely damaged by this treatment, the flight
dark samples of A. fruticulosa that were exposed to the vacuum of space for 10 days did not show any physiological
impairment as indicated by unchanged chlorophyll a activity, respiration, and net photosynthesis. A comparable high
resistance to the desiccating conditions of the space vacuum
was observed in previous spaceflight experiments for the
lichens Rhizocarpon geographicum (L.) DC and Xanthoria elegans (Link) Th. (Sancho et al., 2007, 2008), for tardigrades
( Jönsson et al., 2008), as well as for spores of the bacterium
Bacillus subtilis (reviewed in Horneck et al., 2010).
Although the physiological state of the flight dark samples
did not differ from that of the Earth controls, they did show
some ultrastructural changes, such as the presence of dense
bodies in the cytoplasm of photobiont cells as well as in
vacuoles of the mycobiont cells, all probably a consequence
of the exposure to the space vacuum. We also saw more
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RAGGIO ET AL.
FIGS. 8–13. TEM images of photobiont (8, 10, 12) and mycobiont cells (9, 11, 13) from Earth control (8 and 9), flight dark
sample (10 and 11), and flight sample exposed to solar radiation at l 110 nm (12 and 13) treatments. Pg, pyrenoglobuli; P,
pyrenoid; Sb, algal cells cytoplasmic storage bodies; m, mitochondria; N, nucleus; cc, fungal cells concentric bodies; V,
vacuoles; s, starch granules.
pyrenoglobuli in the flight dark samples than in the Earth
control samples.
Transmission electron microscopy pictures show the consequences of dehydration to be a peripheral location of
pyrenoglobuli inside the pyrenoid (Ascaso and Galván, 1976;
Ascaso, 1978), a decrease in the number of pyrenoglobuli per
pyrenoid area (Ascaso et al., 1986), or a weak staining of
parts of the proteinaceous pyrenoid matrix (Ascaso, 1978;
Brown et al., 1987). The desiccation tolerance of lichen species
from arid habitats has been fully reviewed and discussed by
Kappen and Valladares (2007) and in other works (Lange
et al., 1997, 2006; Pintado et al., 2005; Del Prado and Sancho,
2007). The role of lichenic sugar alcohols in the extreme
desiccation tolerance of these organisms in outer space was
discussed also in Sancho et al. (2007).
In addition to the dehydrating effect of the space vacuum,
organisms in space must cope with exposure to cosmic radiation and intense solar UV radiation. From the recovery
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FIGS. 14–19. LTSEM image of photobiont and mycobiont cells from flight sample exposed to solar radiation at l 110 nm
treatment (14). TEM images of photobiont and mycobiont cells from flight sample exposed to solar radiation at l 200 nm
(15), flight sample exposed to solar radiation at l 290 nm (16, 17), and flight sample exposed to solar radiation at
l 400 nm (18, 19) treatments. P, pyrenoid; V, vacuoles; N, nucleus; s, starch granules; cc, fungal cells concentric bodies;
n, compact nucleolus; Sb, algal cells cytoplasmic storage bodies; m, mitochondria.
data of chlorophyll a activity (Figs. 2–6), we concluded that
(i) the interval between 100 and 200 nm of solar UV radiation
was probably the most harmful part of the solar spectrum for
the samples, and (ii) the photosystem II activity of all UV- or
visible-exposed specimens fully recovered at the end of the
reactivation period of 3 days and reached values identical or
very close to those of the preflight samples, which shows that
their metabolism was not irreversibly damaged. The lack of
metabolic damage in the flight dark samples (Fig. 6) also
confirmed that solar extraterrestrial UV radiation was the
most harmful parameter in space rather than vacuum, cosmic radiation, or extreme temperatures.
Three days of reactivation were sufficient for the full recovery of the photosystem II system of space-exposed samples and showed that solar extraterrestrial UV and cosmic
radiation were not limiting factors for the photosynthetic
performance of A. fruticulosa after 10 days of spaceflight. This
observation was further confirmed by the CO2 exchange
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measurements. After the recovery period of 72 h and an
additional 72 h reactivation, the UV-exposed flight samples
had Amax values that were almost identical to those of the
Earth controls (Table 2).
Many authors (Millanes and Vicente, 2003; Solhaug et al.,
2003) have suggested that the lichen substances are a source
of effective protection against UV radiation. The lack of
these substances in A. fruticulosa points to other mechanisms in the lichen morphology and anatomy for protection
under UV radiation. This could be the structure of the upper cortex (Gauslaa and Solhaug, 2001). The role of the thick
and dense fungal cortex in lichens as a protective factor to
high UV exposures was demonstrated in field measurements by De la Torre et al. (2002). It seems that algae populations inside the lichens are extremely well protected by
this compact layer that works as a protective screen. Although different ultrastructural changes have been observed in the experiment, it appears that the photosystems
are well protected as one of the most important structures
inside the lichen photobionts.
Vacuoles with dense electron bodies were frequently observed in the cytoplasm of photobiont cells of thalli exposed
to wavelengths of l 110, 200, and 290 nm, but not in the
thalli exposed to a wavelength of l 400 nm. These electrondense bodies could possibly represent a lipid accumulation
as a consequence of the loss of lipid from the cellular membranes, which indicates some degree of senescence (Ascaso
et al., 1986). The integrity of algal cell walls can be seen as a
positive feature. The relationship between metabolic data
and ultrastructural analysis carried out by electron microscopy shows interesting features that must be carefully assessed. Disorganization of the thylakoid lamellae inside the
pyrenoids, which could be the result of the loss of lipids from
the thylacoidal membranes, was observed in response to
spaceflight. The majority of pyrenoid tubules became collapsed and their lumen almost entirely lost with the consecutive random relocalization of the pyrenoglobuli. The
collapse of the tubules could be due either to the withdrawal
of water from the lumen of the tubule or to a change of the
configuration of the dehydrated pyrenoid protein structure
(Ascaso et al., 1988). More affected pyrenoids were present in
cells from the flight dark samples and from the flight sunexposed sample that suffered the full solar spectrum
(l 110 nm), as well as those for the ranges of l 200 and
l 290 nm, where the thylakoid membranes had lost their
integrity. The presence of dense bodies between the thylakoid membranes in the flight dark samples and the sample
exposed to l 110 nm could be lipid loss from the thylakoid
membranes. In all these cases the next step could probably be
total disorganization of the pyrenoid matrix. Similar electrodense deposits were observed in R. geographicum after the
Lichens spaceflight (De los Rı́os et al., 2010). The appearance
of the thylakoid membranes inside the pyrenoid was normal
in the Earth controls and in the visible-exposed (l 400 nm)
flight sample, which showed pyrenoglobuli that were also
well attached to the thylakoid membranes. It appears that
ultrastructural changes were reversible or, at least, that the
cellular integrity was sufficiently high to maintain a properly
working lichen metabolism. Sancho et al. (2007) and De los
Rı́os et al. (2010) showed that ultrastructural damage on cells
of R. geographicum and X. elegans from high mountain environments did not necessarily indicate a metabolic failure.
RAGGIO ET AL.
Although microscopy images provide many examples of the
different types of treatments, it is difficult to know exactly
the percentage of cells affected because we cannot evaluate
the entire population. The analysis of the ultrastructural
damage observed by microscopy and their consequences in
lichen metabolism is, as mentioned earlier, revealing several
responses to the effects of space radiation exposure, While
the results presented here constitute a good first approach,
these new observations should be investigated in detail.
5. Conclusions and Implications
The main advantage of A. fruticulosa with respect to other
lichen species previously included in space experiments is its
unattached and compact morphotype, which allows individual thalli to be studied rather than fragments attached to
the substrate. This also affords the opportunity to compare
the physiological performance of selected thalli within a
larger population, which clearly increases our current
knowledge in relation to lichen symbiosis resistance to outer
space conditions.
That we had only one experimental measurement of the
flight sun-exposed samples exposed to different UV ranges
of extraterrestrial solar radiation encourages future simulated and real flight experiments that will complement
promising preliminary results presented here. More work
with simulated space conditions on A. fruticulosa is currently underway with the intent to reinforce these results. If
we consider our experiment to have been one of survival/
nonsurvival, however, it should be viewed as a success in
that all the flight sun-exposed samples survived space
conditions.
The new experiment BIOMEX (Biology and Mars Experiment, ESA-ILSRA 2009-0834), which is coordinated by DLR
(German Aerospace Research Center, Berlin) and will take
place on the Expose-2R facility of the International Space
Station, has as its main objective study of the resistance and
survival of A. fruticulosa as a test system, in comparison with
other selected organisms (lichens, fungi, algae, bryophytes,
bacteria, biofilms, cyanobacteria, archaea), when exposed to
real space and simulated martian conditions. After a longterm exposure (December 2011 to May 2013), degradation of
the cell surfaces of A. fruticulosa, both in contact with terrestrial, lunar, and Mars analog minerals and as a complete
system, before and after the flight, will be studied by INTA
in coordination with UCM and other institutions. We hope to
obtain information about which type of basic traces should
be of significance when searching for signs of life in extraterrestrial habitats—in particular on the Moon and on Mars.
Whether the panspermia theory is a real possibility or just
speculation seems to be a more open question than ever. The
current availability of good experimental designs through
the development of suitable technology opens new gates to
researchers of many disciplines that are interested in this
issue. Lichens have proven to be exceptionally suitable organisms for experiments in astrobiology. The endurance of
different types of lichens from different habitats, the possibility of lichen propagules that resist atmospheric entry, and
long-term exposure experiments are the next challenges that
these amazingly resistant symbiotic organisms should afford
investigators in pursuit of answers to the remaining questions in radiation exposure biology.
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FRUTICOSE LICHEN SURVIVED SPACE EXPOSURE
Acknowledgments
The authors thank Drs. Allan Green and Inmaculada
Mateos-Aparicio for English revision and encouraging comments and Fernando Pinto, Sara Paniagua, Cesar Morcillo,
Teresa Carnota, and Gilberto Herrero for technical assistance. Financial Support was provided by ESA and Spanish
Ministerio de Ciencia e Innovación. Projects, CGL200612179-CO2-01, CGL2006-04658/BOS, CGL2007-62875/BOS,
and CTM2009-12838-CO4-01/03. J. Raggio acknowledges his
grant from Spanish Ministerio de Ciencia e Innovación.
Author Disclosure Statement
No competing financial interests exist for Jose Raggio, Ana
Pintado, Carmen Ascaso, Rosa De la Torre, Asunción De los
Rı́os, Jacek Wierzchos, Gerda Horneck, or Leopoldo G.
Sancho.
Abbreviations
DR, dark respiration; LTSEM, low-temperature scanning
electron microscope NP, net photosynthetic rate; PFD, photon flux density; SEM, scanning electron microscope; TLC,
thin layer chromatography.
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Address correspondence to:
J. Raggio
Departamento Biologı́a Vegetal II
Facultad de Farmacia
Universidad Complutense de Madrid
28040 Madrid
Spain
E-mail: [email protected]
Submitted 1 June 2010
Accepted 2 February 2011
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AUTHOR QUERY FOR AST-2010-0588-RAGGIO_1P
AU1:
AU2:
AU3:
AU4:
AU5:
Sentence ok as written?
Defined LTSEM here Ok?
Assays correct as written?
Sentence correct as written?
Arrows correct as written?