Research ArticleSpace Flight Effects on Antioxidant Molecules inDry Tardigrades The TARDIKISS Experiment
Angela Maria Rizzo1 Tiziana Altiero2 Paola Antonia Corsetto1 Gigliola Montorfano1
Roberto Guidetti3 and Lorena Rebecchi3
1Department of Pharmacological and Biomolecular Sciences Universita degli Studi di MilanoVia D Trentacoste 2 20134 Milano Italy2Department of Education and Human Sciences University of Modena and Reggio Emilia Via A Allegri 9 42121 Reggio Emilia Italy3Department of Life Sciences University of Modena and Reggio Emilia Via G Campi 213D 41125 Modena Italy
Correspondence should be addressed to Angela Maria Rizzo angelamariarizzounimiitand Lorena Rebecchi lorenarebecchiunimoreit
Received 10 July 2014 Accepted 22 September 2014
Academic Editor Monica Monici
Copyright copy 2015 Angela Maria Rizzo et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
The TARDIKISS (Tardigrades in Space) experiment was part of the Biokon in Space (BIOKIS) payload a set of multidisciplinaryexperiments performed during the DAMA (Dark Matter) mission organized by Italian Space Agency and Italian Air Force in2011 This mission supported the execution of experiments in short duration (16 days) taking the advantage of the microgravityenvironment on board of the Space Shuttle Endeavour (its last mission STS-134) docked to the International Space StationTARDIKISS was composed of three sample sets one flight sample and two ground control samples These samples provided thebiological material used to test as space stressors includingmicrogravity affected animal survivability life cycle DNA integrity andpathways of molecules working as antioxidants In this paper we compared the molecular pathways of some antioxidant moleculesthiobarbituric acid reactive substances and fatty acid composition between flight and control samples in two tardigrade speciesnamely Paramacrobiotus richtersi and Ramazzottius oberhaeuseri In both species the activities of ROS scavenging enzymes thetotal content of glutathione and the fatty acids composition between flight and control samples showed few significant differencesTARDIKISS experiment together with a previous space experiment (TARSE) further confirms that both desiccated and hydratedtardigrades represent useful animal tool for space research
1 Introduction
As the interest in space exploration grows it becomes ofgreat importance to predict and know the response of uni-and multicellular organisms to unfavourable space condi-tions including microgravity This allows us to elaboratethe opportune countermeasures to avoid the risks imposedby space environmental stressors To date many studiesfor understanding physiological biochemical and molecu-lar mechanisms against space stressors are performed onunicellular organisms or cultivated cells of multicellularorganisms [1] Although the experiments on cell culturesare useful it is equally clear that cell cultures representonly the first level of life organization and they cannot be
compared to the response of an entire multicellular livingorganism The use of animals in space research allows us toconduct experiments with organisms characterized by a highlevel of hierarchical biological complexity and physiologicalprocesses comparable to those of humans [2]
Even though animals could be useful models in spaceresearch their use is often limited by the fact that many ofthem need specific rearing bioreactors of large volume [1 3]Tardigrades or water bears are little known and neglectedanimals that allow overcoming this problem Their use inspace research is supported by several reasons (i) they areminiaturized animals (from 200 to 1000120583m in length) thatcan be kept and reared in small facilitiesbioreactors (ii)while having tissues and organs they are simpler than several
Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 167642 7 pageshttpdxdoiorg1011552015167642
2 BioMed Research International
other animals having a limited cell number (about 1000)(iii) they can be easily reared under lab conditions (iv)many of them are parthenogenetic often apomictic so clonallineages can be obtained [1 2] Although all tardigrades areaquatic animals they thrive in terrestrial habitats subjectedto periodic desiccation thanks to their ability to enter ahighly stable state of suspended metabolic activity calledanhydrobiosis [4] Entering in this physiological state tardi-grades lose up to 97 of their body water and shrivel into adesiccated structure about one-third of its original sizeWhenrehydrated tardigrades can return to their active metabolicstate in a few minutes to a few hours [4 5] Desiccatedtardigrades can persist in anhydrobiosis for several years anda remarkable resilience to physical and chemical extremeshas been documented [4ndash6] By possessing the abilities towithstand complete desiccation severe cold microgravityvacuum and high levels of ionizing and UV radiationsanhydrobiotic tardigrades fulfill the most important criteriafor tolerating exposure to natural space conditions includingopen space [2]
Tardigrades have already been exposed to space stressorson Low Earth Orbit during the FOTON-M3 mission in2007 with different projects (TARDIS [7] TARSE [1 8]RoTaRad [9]) With the TARSE (Tardigrade Resistance toSpace Effect) project we analyzed the responses of bothdesiccated and hydrated physiological state of the tardigradeParamacrobiotus richtersi to spaceflight conditions withinthe spacecraft [1 8] Microgravity and radiation had noeffect on animal survival and life history traits even thougha higher number of laid eggs a shorter egg developmenttime and a higher number of flight-born juveniles wererecordedwith respect to tardigrades reared onEarth [1 10] Inaddition spaceflight induced in active tardigrades an increaseof glutathione content an increase of glutathione peroxidaseactivity and a decrease of catalase superoxide dismutase andglutathione reductase activities [1] Lastly no change in thio-barbituric acid reactive substances was detected On the basisof these results we developed the new project TARDIKISS(Tardigrades in Space) with the aim to deepen the study ofsurvivorship life history traits and regulation of antioxidantdefences on alive desiccated tardigrades under space stressorsincluding microgravity exposure The flight tardigrades ofthe project TARDIKISS have had a very high survival (morethan 91) and females laid eggs which were able to hatchproducing normal newborns able to reproduce in adulthood[11] In this paper we compared the molecular pathwaysof molecules with antioxidant activity thiobarbituric acidreactive substances and fatty acid composition between flighttardigrades and ground control ones with the final aim toprovide news about the biochemical mechanisms underlyingresistance to space stress conditions
2 Material and Methods
21 TARDIKISS Project The TARDIKISS project was partof the BIOKIS (Biokon in Space) payload a set of multidis-ciplinary experiments in the field of biology and dosimetryperformed in microgravity condition during the DAMA(Dark Matter) mission organized by Italian Space Agency
(ASI) and Italian Air Force in 2011 This mission sup-ported the execution of experiments in short duration (16days) taking the advantage of the microgravity environmenton board of the last mission (STS-134) of Space ShuttleEndeavour docked to the International Space Station (ISS)[11]
TARDIKISSwas composed of three sample sets one flightsample (F) and two ground control samples The formercontrol (temperature control TC) was a postflight controlin which samples were exposed to the temperature profileexperienced by tardigrades the days immediately beforeduring and just after the flight mission the latter (laboratorycontrol LC) was maintained in Modena laboratory for theduration of the flight at constant temperature These samplesprovided the biological material used to test as space stres-sors including microgravity affected animal survivabilitylife cycle DNA integrity and changes of the pathways ofmolecules working as antioxidants
Two anhydrobiotic eutardigrade species were consid-ered namely Paramacrobiotus richtersi (Murray 1911) (Mac-robiotidae) and Ramazzottius oberhaeuseri (Doyere 1840)(Ramazzottiidae) Paramacrobiotus richtersi is the modelspecies already used in the FOTON mission [1] P richtersiwas extracted fromahazel leaf litter (sample codeC3499) it iscarnivorous white in colour and the population here consid-ered is bisexual and amphimictic R oberhaeuseri (Figure 1)was extracted from the lichen Xanthoria parietina (L) ThFr (1860) (sample code C3282) it is herbivorous brownredin colour and the population considered in this study isunisexual and parthenogenetic To extract tardigrades fromtheir substrates leaf litter and lichen were sprinkled with tapwater and after 15min submerged in water for 15min at roomtemperature Later each substrate was sieved (mesh size ofsieves 250 120583mand 37 120583m)under runningwater then animalswere picked up from the sieved sediments with a glass pipetteunder a stereomicroscope
For both tardigrade species animals in desiccated (anhy-drobiotic) physiological state were used To obtain desiccatedspecimens tardigrades were dehydrated in lab under con-trolled air relative humidity (RH) and temperature Afterextraction from their substrates tardigrades were kept inwater for 24 h at 15∘C without any food source Then theywere forced into anhydrobiosis by placing groups of animalson a square (1 cm2) blotting paper with natural mineral water(30 120583L) The paper with tardigrades was initially exposedto 80 RH and 18∘C for 4 h then to 50 RH at 18∘C for4 h in a climatic chamber and finally to 0ndash3 RH at roomtemperature for 12 h [1]
Papers with desiccated tardigrades were stored in twelvesmall plastic Petri dishes (18 cm times 10 cm) enveloped withparafilm and integrated within the Biokon facility (KayserItalia) where a radiation dosimeter for neutrons and i-buttondata logger recorded temperature were also present [11]During the entire flight mission the temperature profile wasrelatively constant ranging from 21∘C to 25∘C [11] while thedose equivalent rates due to space radiation exposure were320 120583Sv (measured by TLD 100 and TLD 700) and 360 120583Sv(measured by TLD 600) [11]
BioMed Research International 3
(a) (b)
Figure 1 Micrographs by scanning electronmicroscopy of the tardigrade Ramazzottius oberhaeuseri showing its two physiological states (a)Hydrated and metabolically active specimen (b) Desiccated and metabolically inactive specimen Bars a = 10 120583m b = 5 120583m
22 Biochemical Assays Biochemical assays were performedon desiccated tardigrades comparing F samples with TCsamples
The activities of the enzymes superoxide dismutase(SOD total activity) catalase (CAT) glutathione peroxidase(GPx) and glutathione reductase (GR) were evaluated Thetotal glutathione (GSH) content thiobarbituric acid reactivesubstances (TBARS) and fatty acid composition were alsodetermined as previously described [12]
Substrates and reagents for enzyme determinations wereNAD(P)H DTNB GSH GSSG glutathione reductase andtert-butyl hydroperoxide all of them were purchased fromSigma-Aldrich (St Louis Missouri USA) For each sampleset and each species 6 or 8 (with the exception of SOD)replicates each made up by 10 in toto tardigrades werehomogenized in water on ice with potter using 3 cycles of30 sec each The homogenate was assayed for protein content(according to [13]) and used for enzyme determination Foreach enzyme homogenates were analyzed in duplicate
Briefly the activity of the enzyme superoxide dismutasewas assayed using the method based on NAD(P)H oxidationinhibition (according to [14]) the inhibition of NADPHoxidation by superoxide which was chemically generatedwas measured at 340 nm for 20min in the presence of tissueextracts The incubation mixture included 213 120583L of TDB(triethanolaminediethanolamine 100mM pH 74) 10 120583L ofNADPH 75 120583M 7 120583L of EDTA-MnCl
2(100mMndash50mM)
and 20 120583L of sample or blank One unit of SOD activity wasdefined as the amount of enzyme required to inhibit the rateof NADPH oxidation by 50
To evaluate the activity of catalase samples were assayedby measuring the consumption of H
2O2(according to [15])
Consumption of hydrogen peroxide by the tissue extracts wasdetermined at 240 nm for 1min at 30∘CThe incubation mix-ture included 10 120583L of H
2O2200mM 20 120583L of homogenate
and 170 120583L of Na-phosphate buffer (50mM pH 70) One unitofCATactivitywas defined as the amount of enzyme requiredto catalyze the decomposition of 1mmol of H
2O2minminus1
The activity of the glutathione reductase was assayedfollowing the oxidation of NADPH (according to [16])Briefly GSSG reduction and NADPH consumption werefollowed at 340 nmThe incubation mixture included 5 120583L ofGSSG 125mM 3120583L of NADPH 11mM animal homogenatefrom 20 to 50 120583L and K-phosphate buffer (100mM pH 70)to reach a final volume of 025mL One unit of GR activity
was defined as the amount of enzyme required to catalyze theoxidation of 1 120583mol NADPHminminus1
To evaluate the activity of selenium-dependent glu-tathione peroxidase the enzyme activitywas assayed (accord-ing to [17]) following the decrease in the absorbance at340 nm for 3min which corresponds to the rate of GSHoxidation to GSSG in the presence of NADPH and glu-tathione reductase The incubation mixture included 5 120583L ofGSH 100mM 3 120583L of NADPH 22mM GR 1 unit 5 120583L oftert-butyl hydroperoxide 20mM from 20 to 50 120583L of animalhomogenate and EDTA-K phosphate buffer (3mMndash100mMpH 70) to reach a final volume of 025mL One unit of GPxactivity was defined as the amount of enzyme required tocatalyze the oxidation of 1 120583mol of NADPHminminus1
To measure the total glutathione tardigrades werehomogenized on ice in 5 metaphosphoric acid thehomogenatewas centrifuged at 5000timesg for 10min at 4∘C andthe supernatant was assayed (according to [18]) with someslight modifications Briefly the sulfhydryl group of GSHalso generated from GSSG by adding GR reacts with DTNB(551015840-dithiobis-2-nitrobenzoic acid) and produces a yellow-coloured 5-thio-2-nitrobenzoic acid (TNB) The rate of TNBproduction is directly proportional to this reaction whichin turn is directly proportional to the concentration of GSHin the sample The measurement of the absorbance of TNBat 412 nm provides an accurate estimation of the GSH levelpresent in the sample
To evaluate the thiobarbituric acid reactive substances(TBARS) tardigrade samples standards (from 25 to100 pmol TEP 11-33 tetraethoxypropane) and blanks wereassayed (according to [19]) both before and after inductionof lipid peroxidation by FeSO
4and ascorbic acid TBARS
were determined using a fluorescence spectrophotometer(Carly Eclipse Varian CA USA) at an excitation wavelengthof 517 nm and an emission wavelength of 550 nm For eachsample set (F and TC) and species (R oberhaeuseri and Prichtersi) 2 or 4 replicates were analyzed
To evaluate the fatty acid composition lipids wereextracted from groups of 10 desiccated tardigrades withchloroformmethanol (according to [20]) The total extractwas used for derivatization with sodium methoxide inmethanol 333wv to obtain the fatty acid methylesters(FAME) FAME were injected into a gas chromatograph(Agilent Technologies 6850 Series II) equipped with a flameionization detector (FID) under the following experimental
4 BioMed Research International
Table 1 Percentage of fatty acid composition in the tardigrades Paramacrobiotus richtersi and Ramazzottius oberhaeuseri
Fatty acid Paramacrobiotus richtersi Ramazzottius oberhaeuseriTC F TC F
conditions capillary column AT Silar length 30m filmthickness 025120583m gas carrier helium temperatures injector250∘C detector 275∘C oven 50∘C for 2min and rate of10∘Cminminus1 until 200∘C for 20min A standard mixture con-taining methyl ester fatty acids was injected for calibrationFor each sample set and species 2 or 4 replicates wereanalyzed
23 Statistical Analysis Data were analyzed with Mann-Whitney test and expressed as mean plusmn SD using the pro-gramme SPSS
3 Results
The results of the enzyme activities in the tardigradesParamacrobiotus richtersi and Ramazzottius oberhaeuseri arealways indicated in relation to 120583g of proteins It is worthnoting that R oberhaeuseri contains a lower amount ofproteins compared to P richtersi (Figure 2)
In both species the comparative analysis of the enzymeactivities and other antioxidant molecules between flight (F)and temperature control samples (TC) showed few significantdifferences (Figures 3 and 4) In particular a significantdecrease (119875 lt 005) of the glutathione reductase activitywas detected in R oberhaeuseri F samples with respect toTC samples (Figure 4(b)) Although not statistically sup-ported in this species a tendency to decrease catalasesuperoxide dismutase and glutathione peroxidase activityand in glutathione content was detected In P richtersi atendency to decrease catalase superoxide dismutase and glu-tathione reductase activities and to increase the glutathioneperoxidase activity was detected Noteworthy differenceswere recorded in the activities of ROS scavenging enzymesbetween the two species
The total percentage fatty acid composition of F and TCsamples is reported in Table 1 In R oberhaeuseri a significant
000
020
040
060
080
100
120
P richtersi R oberhaeuseri
TCFlight
(120583g
prot
eins
aa)
Figure 2 Total protein content in flight and ground temperaturecontrol (TC) samples in the tardigrades Paramacrobiotus richtersiand Ramazzottius oberhaeuseri The bars show the mean with SD
decrease (119875 lt 005) was recorded for the fatty acidC22-6 n-3 and polyunsaturated fatty acids (PUFA) in theF samples with respect to the TC samples Moreover Roberhaeuseri has significantly lower amount of C22-6 n-3compared to P richtersi The amount of thiobarbituric acidreactive substances (TBARS) in tardigrades both before andafter induction of peroxidation in vitro is also reported inTable 1 No differences were detected between F and TCsamples in both species for basal levels and after inductionof peroxidation
BioMed Research International 5
0
005
01
015
02
025
03
035
04
P richtersi R oberhaeuseri
Superoxide dismutase (SOD)
TCFlight
(Un
g pr
otei
ns)
(a)
0
10
20
30
40
50
60
70
80
P richtersi R oberhaeuseri
Catalase
TCFlight
(mU
120583 g
prot
eins
)
(b)
Figure 3 Superoxide dismutase (a) and catalase (b) activities in flight and ground temperature control (TC) samples in the tardigradesParamacrobiotus richtersi and Ramazzottius oberhaeuseri The bars show the mean with SD
0
05
1
15
2
25
3
P richtersi R oberhaeuseri
Glutathione peroxidase
TCFlight
(mU
120583g
prot
eins
)
(a)
0010203040506070809
1
P richtersi R oberhaeuseri
Glutathione reductase
lowast
TCFlight
(mU
120583g
prot
eins
)
(b)
0
02
04
06
08
1
P richtersi R oberhaeuseri
Glutathione (GSH)
TCFlight
(mm
oles
120583g
prot
eins
)
(c)
Figure 4 Glutathione peroxidase (a) glutathione reductase (b) and total glutathione content (c) in flight and ground temperature control(TC) samples in the tardigrades Paramacrobiotus richtersi and Ramazzottius oberhaeuseri The bars show the mean with SD lowast119875 lt 005
6 BioMed Research International
4 Discussion
Exposure to space stress conditions induces oxidative stressOxidative stress resulting from an imbalance between theexcessive production of reactive oxygen species (ROS) andlimited action of antioxidant defences is implicated inthe development of many important human pathologiesincluding atherosclerosis hypertension inflammation can-cer Parkinson and Alzheimer diseases [21] Oxidative stressmay be highly destructive also in anhydrobiotic organismseven if the lower cellular water content decreases the produc-tion of ROS [21 22] Under normal conditions antioxidantsystems minimize the adverse effects caused by ROS butdesiccation stress could cause the loss or reduction of thesedefence control mechanisms since the metabolic activity isabsent or reduced [21ndash24]
The ability of some animals tardigrades among themto survive extreme desiccation involves a complex arrayof factors working at structural physiological and molec-ular level From a molecularbiochemical point of viewanhydrobiotic organisms synthesize molecules working asbioprotectants during entering permanence and leaving ina desiccated state [25] For example trehalose and sucrosestabilise the biological membrane avoiding protein unfoldingand membrane disturbances late embryogenesis abundantproteins and heat shock proteins work as chaperone systemsrepairing or eliminating damaged molecules while antiox-idant molecules counteract the negative effects of oxidativestress [25]
Since it is known that both hydrated and desiccatedtardigrades have a good natural capability to overcomeoxidative stress [26] they have been used in TARDIKISSexperiments to evaluate the role of antioxidant defence inovercoming oxidative stress induced by exposure to spacestress conditions such as ionizing and UV radiations
The first space experiment (TARSE) conducted withhydrated starved specimens of the tardigrade P richtersidemonstrated that some of the enzymes involved in antiox-idant defences were significantly influenced by the flightstresses [1] In particular there was a significant decrease incatalase and SOD activities the more active enzymes in Prichtersi In addition the glutathione system the less activesystem in not stressed specimens of this species [26] wassignificantly induced during space flight [1] These resultscould be related to the stresses experienced by the hydratedand metabolically active animals (microgravity starvationand radiations) during the flight On the contrary the analysisof antioxidant defences in desiccated tardigrades of theTARDIKISS experiment showed fewer differences relatedto space flight even if the tendency was similar to thatrecorded in hydrated metabolically active animals of theTARSE experiment A similar trend between TARSE andTARDIKISS experiments was also detected in regard to tardi-grade survival since flight animals did not show significantdifferences in survival from temperature laboratory controlones [1 11] Only inR oberhaeuseri (TARDIKISS experiment)a significant decrease in survival rate was recorded between Fand TC samples the species in which a significant decrease ofthe C226 n-3 fatty acid and of glutathione reductase activity
and even though not significant of the activity of the otherROS scavenging enzymes were detected
In conclusion TARDIKISS experiment together withprevious space experiments using tardigrades [1 7ndash9] furtherconfirms that both desiccated and hydrated physiologicalstates of tardigrades represent useful animal tool for spaceresearch To further develop the space research using tardi-grades the setup of experimentswith the possibility to changethe exposition condition of metabolically hydrated animalsas well as the possibility to expose desiccated tardigrades toopen space is necessary Experiments under true space con-dition provide a realistic evaluation of the mechanisms thatcould allowmulticellular organisms including tardigrades tosurvive the combined and synergic effects of space stressorsNevertheless experiments on ground using simulators ofmicrogravity radiation temperature and other space stressesare an essential part of space research complementing exper-iments under true space conditions The comparisons of twodifferent sets of data (ground and space data) will allow betterunderstanding of the physiological and molecular pathwaysof living organisms under space environment
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are very grateful to the Italian Space Agency(ASI) and the Italian Air Force (AM) which funded theDAMA mission The authors are also very grateful to KayserItalia (KI) which developed and manufactured the hardwareinvolved in the BIOKIS payload They are grateful to anony-mous reviewers for their constructive suggestions
References
[1] L Rebecchi T Altiero R Guidetti et al ldquoTardigrade resistanceto space effects First results of experiments on the LIFE-TARSEMission onFOTON-M3 (September 2007)rdquoAstrobiology vol 9no 6 pp 581ndash591 2009
[2] R Guidetti A M Rizzo T Altiero and L Rebecchi ldquoWhatcan we learn from the toughest animals of the Earth Waterbears (tardigrades) asmulticellularmodel organisms in order toperform scientific preparations for lunar explorationrdquoPlanetaryand Space Science vol 74 no 1 pp 97ndash102 2012
[3] H Marthy ldquoDevelopmental biology of animal models undervaried gravity conditions a reviewrdquo Vie et Milieu vol 52 no4 pp 149ndash189 2002
[4] N Moslashbjerg K A Halberg A Joslashrgensen et al ldquoSurvival inextreme environmentsmdashon the current knowledge of adapta-tions in tardigradesrdquo Acta Physiologica vol 202 no 3 pp 409ndash420 2011
[5] R Guidetti T Altiero and L Rebecchi ldquoOn dormancy strate-gies in tardigradesrdquo Journal of Insect Physiology vol 57 no 5pp 567ndash576 2011
[6] T Altiero R Guidetti V Caselli M Cesari and L RebecchildquoUltraviolet radiation tolerance in hydrated and desiccated
BioMed Research International 7
eutardigradesrdquo Journal of Zoological Systematics and Evolution-ary Research vol 49 supplement 1 pp 104ndash110 2011
[7] K I Jonsson E Rabbow R O Schill M Harms-Ringdahl andP Rettberg ldquoTardigrades survive exposure to space in low Earthorbitrdquo Current Biology vol 18 no 17 pp R729ndashR731 2008
[8] L Rebecchi T Altiero M Cesari et al ldquoResistance of the anhy-drobiotic eutardigrade Paramacrobiotus richtersi to space flight(LIFE-TARSE mission on FOTON-M3)rdquo Journal of ZoologicalSystematics and Evolutionary Research vol 49 supplement 1 pp98ndash103 2011
[9] D Persson K A Halberg A Joslashrgensen C Ricci N Moslashbjergand R M Kristensen ldquoExtreme stress tolerance in tardigradessurviving space conditions in low earth orbitrdquo Journal of Zoolog-ical Systematics and Evolutionary Research vol 49 supplement1 pp 90ndash97 2011
[10] T Altiero L Rebecchi and R Bertolani ldquoPhenotypic varia-tions in the life history of two clones of Macrobiotus richtersi(Eutardigrada Macrobiotidae)rdquo Hydrobiologia vol 558 no 1pp 33ndash40 2006
[11] M Vukich P L Ganga D Cavalieri et al ldquoBIOKIS amodel payload for multisciplinary experiments in micrograv-ityrdquo Microgravity Science and Technology vol 24 pp 397ndash4092012
[12] A M Rizzo L Adorni G Montorfano F Rossi and B BerraldquoAntioxidant metabolism of Xenopus laevis embryos duringthe first days of developmentrdquo Comparative Biochemistry andPhysiologymdashB Biochemistry and Molecular Biology vol 146 no1 pp 94ndash100 2007
[13] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951
[14] F Paoletti and A Mocali ldquoDetermination of superoxide dis-mutase activity by purely chemical system based on NAD(P)HoxidationrdquoMethods in Enzymology vol 186 pp 209ndash220 1990
[15] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984
[16] M C Pinto A M Mata and J Lopez-barea ldquoReversible inacti-vation of Saccharomyces cerevisiae glutathione reductase underreducing conditionsrdquo Archives of Biochemistry and Biophysicsvol 228 no 1 pp 1ndash12 1984
[17] J R Prohaska and H E Ganther ldquoSelenium and glutathioneperoxidase in developing rat brainrdquo Journal of Neurochemistryvol 27 no 6 pp 1379ndash1387 1976
[18] O W Griffith ldquoGlutathione and glutathione disulphiderdquo inMethods of Enzymatic Analysis H U Bergmeyer Ed vol 3 pp521ndash529 Academic Press New York NY USA 1984
[19] H E Wey L Pyron and M Woolery ldquoEssential fatty acid defi-ciency in cultured human keratinocytes attenuates toxicity dueto lipid peroxidationrdquo Toxicology and Applied Pharmacologyvol 120 no 1 pp 72ndash79 1993
[20] J Folch M Lees and G H S Stanley ldquoA simple method for theisolation and purification of total lipides from animal tissuesrdquoThe Journal of Biological Chemistry vol 226 no 1 pp 497ndash5091957
[21] M B Franca A D Panek and E C A Eleutherio ldquoOxidativestress and its effects during dehydrationrdquoComparative Biochem-istry and PhysiologymdashA Molecular and Integrative Physiologyvol 146 no 4 pp 621ndash631 2007
[22] R Cruz de Carvalho M Catala J Marques da Silva CBranquinho and E Barreno ldquoThe impact of dehydration rateon the production and cellular location of reactive oxygen
species in an aquatic mossrdquo Annals of Botany vol 110 no 5 pp1007ndash1016 2012
[23] I Kranner and S Birtic ldquoA modulating role for antioxidants indesiccation tolerancerdquo Integrative and Comparative Biology vol45 no 5 pp 734ndash740 2005
[24] R Cornette and T Kikawada ldquoThe induction of anhydrobiosisin the sleeping chironomid current status of our knowledgerdquoIUBMB Life vol 63 no 6 pp 419ndash429 2011
[25] L Rebecchi ldquoDry up and survive the role of antioxidantdefences in anhydrobiotic organismsrdquo Journal of Limnology vol72 no 1 pp 62ndash72 2013
[26] A M Rizzo M Negroni T Altiero et al ldquoAntioxidant defencesin hydrated and desiccated states of the tardigrade Paramac-robiotus richtersirdquo Comparative Biochemistry and Physiology BBiochemistry and Molecular Biology vol 156 no 2 pp 115ndash1212010
other animals having a limited cell number (about 1000)(iii) they can be easily reared under lab conditions (iv)many of them are parthenogenetic often apomictic so clonallineages can be obtained [1 2] Although all tardigrades areaquatic animals they thrive in terrestrial habitats subjectedto periodic desiccation thanks to their ability to enter ahighly stable state of suspended metabolic activity calledanhydrobiosis [4] Entering in this physiological state tardi-grades lose up to 97 of their body water and shrivel into adesiccated structure about one-third of its original sizeWhenrehydrated tardigrades can return to their active metabolicstate in a few minutes to a few hours [4 5] Desiccatedtardigrades can persist in anhydrobiosis for several years anda remarkable resilience to physical and chemical extremeshas been documented [4ndash6] By possessing the abilities towithstand complete desiccation severe cold microgravityvacuum and high levels of ionizing and UV radiationsanhydrobiotic tardigrades fulfill the most important criteriafor tolerating exposure to natural space conditions includingopen space [2]
Tardigrades have already been exposed to space stressorson Low Earth Orbit during the FOTON-M3 mission in2007 with different projects (TARDIS [7] TARSE [1 8]RoTaRad [9]) With the TARSE (Tardigrade Resistance toSpace Effect) project we analyzed the responses of bothdesiccated and hydrated physiological state of the tardigradeParamacrobiotus richtersi to spaceflight conditions withinthe spacecraft [1 8] Microgravity and radiation had noeffect on animal survival and life history traits even thougha higher number of laid eggs a shorter egg developmenttime and a higher number of flight-born juveniles wererecordedwith respect to tardigrades reared onEarth [1 10] Inaddition spaceflight induced in active tardigrades an increaseof glutathione content an increase of glutathione peroxidaseactivity and a decrease of catalase superoxide dismutase andglutathione reductase activities [1] Lastly no change in thio-barbituric acid reactive substances was detected On the basisof these results we developed the new project TARDIKISS(Tardigrades in Space) with the aim to deepen the study ofsurvivorship life history traits and regulation of antioxidantdefences on alive desiccated tardigrades under space stressorsincluding microgravity exposure The flight tardigrades ofthe project TARDIKISS have had a very high survival (morethan 91) and females laid eggs which were able to hatchproducing normal newborns able to reproduce in adulthood[11] In this paper we compared the molecular pathwaysof molecules with antioxidant activity thiobarbituric acidreactive substances and fatty acid composition between flighttardigrades and ground control ones with the final aim toprovide news about the biochemical mechanisms underlyingresistance to space stress conditions
2 Material and Methods
21 TARDIKISS Project The TARDIKISS project was partof the BIOKIS (Biokon in Space) payload a set of multidis-ciplinary experiments in the field of biology and dosimetryperformed in microgravity condition during the DAMA(Dark Matter) mission organized by Italian Space Agency
(ASI) and Italian Air Force in 2011 This mission sup-ported the execution of experiments in short duration (16days) taking the advantage of the microgravity environmenton board of the last mission (STS-134) of Space ShuttleEndeavour docked to the International Space Station (ISS)[11]
TARDIKISSwas composed of three sample sets one flightsample (F) and two ground control samples The formercontrol (temperature control TC) was a postflight controlin which samples were exposed to the temperature profileexperienced by tardigrades the days immediately beforeduring and just after the flight mission the latter (laboratorycontrol LC) was maintained in Modena laboratory for theduration of the flight at constant temperature These samplesprovided the biological material used to test as space stres-sors including microgravity affected animal survivabilitylife cycle DNA integrity and changes of the pathways ofmolecules working as antioxidants
Two anhydrobiotic eutardigrade species were consid-ered namely Paramacrobiotus richtersi (Murray 1911) (Mac-robiotidae) and Ramazzottius oberhaeuseri (Doyere 1840)(Ramazzottiidae) Paramacrobiotus richtersi is the modelspecies already used in the FOTON mission [1] P richtersiwas extracted fromahazel leaf litter (sample codeC3499) it iscarnivorous white in colour and the population here consid-ered is bisexual and amphimictic R oberhaeuseri (Figure 1)was extracted from the lichen Xanthoria parietina (L) ThFr (1860) (sample code C3282) it is herbivorous brownredin colour and the population considered in this study isunisexual and parthenogenetic To extract tardigrades fromtheir substrates leaf litter and lichen were sprinkled with tapwater and after 15min submerged in water for 15min at roomtemperature Later each substrate was sieved (mesh size ofsieves 250 120583mand 37 120583m)under runningwater then animalswere picked up from the sieved sediments with a glass pipetteunder a stereomicroscope
For both tardigrade species animals in desiccated (anhy-drobiotic) physiological state were used To obtain desiccatedspecimens tardigrades were dehydrated in lab under con-trolled air relative humidity (RH) and temperature Afterextraction from their substrates tardigrades were kept inwater for 24 h at 15∘C without any food source Then theywere forced into anhydrobiosis by placing groups of animalson a square (1 cm2) blotting paper with natural mineral water(30 120583L) The paper with tardigrades was initially exposedto 80 RH and 18∘C for 4 h then to 50 RH at 18∘C for4 h in a climatic chamber and finally to 0ndash3 RH at roomtemperature for 12 h [1]
Papers with desiccated tardigrades were stored in twelvesmall plastic Petri dishes (18 cm times 10 cm) enveloped withparafilm and integrated within the Biokon facility (KayserItalia) where a radiation dosimeter for neutrons and i-buttondata logger recorded temperature were also present [11]During the entire flight mission the temperature profile wasrelatively constant ranging from 21∘C to 25∘C [11] while thedose equivalent rates due to space radiation exposure were320 120583Sv (measured by TLD 100 and TLD 700) and 360 120583Sv(measured by TLD 600) [11]
BioMed Research International 3
(a) (b)
Figure 1 Micrographs by scanning electronmicroscopy of the tardigrade Ramazzottius oberhaeuseri showing its two physiological states (a)Hydrated and metabolically active specimen (b) Desiccated and metabolically inactive specimen Bars a = 10 120583m b = 5 120583m
22 Biochemical Assays Biochemical assays were performedon desiccated tardigrades comparing F samples with TCsamples
The activities of the enzymes superoxide dismutase(SOD total activity) catalase (CAT) glutathione peroxidase(GPx) and glutathione reductase (GR) were evaluated Thetotal glutathione (GSH) content thiobarbituric acid reactivesubstances (TBARS) and fatty acid composition were alsodetermined as previously described [12]
Substrates and reagents for enzyme determinations wereNAD(P)H DTNB GSH GSSG glutathione reductase andtert-butyl hydroperoxide all of them were purchased fromSigma-Aldrich (St Louis Missouri USA) For each sampleset and each species 6 or 8 (with the exception of SOD)replicates each made up by 10 in toto tardigrades werehomogenized in water on ice with potter using 3 cycles of30 sec each The homogenate was assayed for protein content(according to [13]) and used for enzyme determination Foreach enzyme homogenates were analyzed in duplicate
Briefly the activity of the enzyme superoxide dismutasewas assayed using the method based on NAD(P)H oxidationinhibition (according to [14]) the inhibition of NADPHoxidation by superoxide which was chemically generatedwas measured at 340 nm for 20min in the presence of tissueextracts The incubation mixture included 213 120583L of TDB(triethanolaminediethanolamine 100mM pH 74) 10 120583L ofNADPH 75 120583M 7 120583L of EDTA-MnCl
2(100mMndash50mM)
and 20 120583L of sample or blank One unit of SOD activity wasdefined as the amount of enzyme required to inhibit the rateof NADPH oxidation by 50
To evaluate the activity of catalase samples were assayedby measuring the consumption of H
2O2(according to [15])
Consumption of hydrogen peroxide by the tissue extracts wasdetermined at 240 nm for 1min at 30∘CThe incubation mix-ture included 10 120583L of H
2O2200mM 20 120583L of homogenate
and 170 120583L of Na-phosphate buffer (50mM pH 70) One unitofCATactivitywas defined as the amount of enzyme requiredto catalyze the decomposition of 1mmol of H
2O2minminus1
The activity of the glutathione reductase was assayedfollowing the oxidation of NADPH (according to [16])Briefly GSSG reduction and NADPH consumption werefollowed at 340 nmThe incubation mixture included 5 120583L ofGSSG 125mM 3120583L of NADPH 11mM animal homogenatefrom 20 to 50 120583L and K-phosphate buffer (100mM pH 70)to reach a final volume of 025mL One unit of GR activity
was defined as the amount of enzyme required to catalyze theoxidation of 1 120583mol NADPHminminus1
To evaluate the activity of selenium-dependent glu-tathione peroxidase the enzyme activitywas assayed (accord-ing to [17]) following the decrease in the absorbance at340 nm for 3min which corresponds to the rate of GSHoxidation to GSSG in the presence of NADPH and glu-tathione reductase The incubation mixture included 5 120583L ofGSH 100mM 3 120583L of NADPH 22mM GR 1 unit 5 120583L oftert-butyl hydroperoxide 20mM from 20 to 50 120583L of animalhomogenate and EDTA-K phosphate buffer (3mMndash100mMpH 70) to reach a final volume of 025mL One unit of GPxactivity was defined as the amount of enzyme required tocatalyze the oxidation of 1 120583mol of NADPHminminus1
To measure the total glutathione tardigrades werehomogenized on ice in 5 metaphosphoric acid thehomogenatewas centrifuged at 5000timesg for 10min at 4∘C andthe supernatant was assayed (according to [18]) with someslight modifications Briefly the sulfhydryl group of GSHalso generated from GSSG by adding GR reacts with DTNB(551015840-dithiobis-2-nitrobenzoic acid) and produces a yellow-coloured 5-thio-2-nitrobenzoic acid (TNB) The rate of TNBproduction is directly proportional to this reaction whichin turn is directly proportional to the concentration of GSHin the sample The measurement of the absorbance of TNBat 412 nm provides an accurate estimation of the GSH levelpresent in the sample
To evaluate the thiobarbituric acid reactive substances(TBARS) tardigrade samples standards (from 25 to100 pmol TEP 11-33 tetraethoxypropane) and blanks wereassayed (according to [19]) both before and after inductionof lipid peroxidation by FeSO
4and ascorbic acid TBARS
were determined using a fluorescence spectrophotometer(Carly Eclipse Varian CA USA) at an excitation wavelengthof 517 nm and an emission wavelength of 550 nm For eachsample set (F and TC) and species (R oberhaeuseri and Prichtersi) 2 or 4 replicates were analyzed
To evaluate the fatty acid composition lipids wereextracted from groups of 10 desiccated tardigrades withchloroformmethanol (according to [20]) The total extractwas used for derivatization with sodium methoxide inmethanol 333wv to obtain the fatty acid methylesters(FAME) FAME were injected into a gas chromatograph(Agilent Technologies 6850 Series II) equipped with a flameionization detector (FID) under the following experimental
4 BioMed Research International
Table 1 Percentage of fatty acid composition in the tardigrades Paramacrobiotus richtersi and Ramazzottius oberhaeuseri
Fatty acid Paramacrobiotus richtersi Ramazzottius oberhaeuseriTC F TC F
conditions capillary column AT Silar length 30m filmthickness 025120583m gas carrier helium temperatures injector250∘C detector 275∘C oven 50∘C for 2min and rate of10∘Cminminus1 until 200∘C for 20min A standard mixture con-taining methyl ester fatty acids was injected for calibrationFor each sample set and species 2 or 4 replicates wereanalyzed
23 Statistical Analysis Data were analyzed with Mann-Whitney test and expressed as mean plusmn SD using the pro-gramme SPSS
3 Results
The results of the enzyme activities in the tardigradesParamacrobiotus richtersi and Ramazzottius oberhaeuseri arealways indicated in relation to 120583g of proteins It is worthnoting that R oberhaeuseri contains a lower amount ofproteins compared to P richtersi (Figure 2)
In both species the comparative analysis of the enzymeactivities and other antioxidant molecules between flight (F)and temperature control samples (TC) showed few significantdifferences (Figures 3 and 4) In particular a significantdecrease (119875 lt 005) of the glutathione reductase activitywas detected in R oberhaeuseri F samples with respect toTC samples (Figure 4(b)) Although not statistically sup-ported in this species a tendency to decrease catalasesuperoxide dismutase and glutathione peroxidase activityand in glutathione content was detected In P richtersi atendency to decrease catalase superoxide dismutase and glu-tathione reductase activities and to increase the glutathioneperoxidase activity was detected Noteworthy differenceswere recorded in the activities of ROS scavenging enzymesbetween the two species
The total percentage fatty acid composition of F and TCsamples is reported in Table 1 In R oberhaeuseri a significant
000
020
040
060
080
100
120
P richtersi R oberhaeuseri
TCFlight
(120583g
prot
eins
aa)
Figure 2 Total protein content in flight and ground temperaturecontrol (TC) samples in the tardigrades Paramacrobiotus richtersiand Ramazzottius oberhaeuseri The bars show the mean with SD
decrease (119875 lt 005) was recorded for the fatty acidC22-6 n-3 and polyunsaturated fatty acids (PUFA) in theF samples with respect to the TC samples Moreover Roberhaeuseri has significantly lower amount of C22-6 n-3compared to P richtersi The amount of thiobarbituric acidreactive substances (TBARS) in tardigrades both before andafter induction of peroxidation in vitro is also reported inTable 1 No differences were detected between F and TCsamples in both species for basal levels and after inductionof peroxidation
BioMed Research International 5
0
005
01
015
02
025
03
035
04
P richtersi R oberhaeuseri
Superoxide dismutase (SOD)
TCFlight
(Un
g pr
otei
ns)
(a)
0
10
20
30
40
50
60
70
80
P richtersi R oberhaeuseri
Catalase
TCFlight
(mU
120583 g
prot
eins
)
(b)
Figure 3 Superoxide dismutase (a) and catalase (b) activities in flight and ground temperature control (TC) samples in the tardigradesParamacrobiotus richtersi and Ramazzottius oberhaeuseri The bars show the mean with SD
0
05
1
15
2
25
3
P richtersi R oberhaeuseri
Glutathione peroxidase
TCFlight
(mU
120583g
prot
eins
)
(a)
0010203040506070809
1
P richtersi R oberhaeuseri
Glutathione reductase
lowast
TCFlight
(mU
120583g
prot
eins
)
(b)
0
02
04
06
08
1
P richtersi R oberhaeuseri
Glutathione (GSH)
TCFlight
(mm
oles
120583g
prot
eins
)
(c)
Figure 4 Glutathione peroxidase (a) glutathione reductase (b) and total glutathione content (c) in flight and ground temperature control(TC) samples in the tardigrades Paramacrobiotus richtersi and Ramazzottius oberhaeuseri The bars show the mean with SD lowast119875 lt 005
6 BioMed Research International
4 Discussion
Exposure to space stress conditions induces oxidative stressOxidative stress resulting from an imbalance between theexcessive production of reactive oxygen species (ROS) andlimited action of antioxidant defences is implicated inthe development of many important human pathologiesincluding atherosclerosis hypertension inflammation can-cer Parkinson and Alzheimer diseases [21] Oxidative stressmay be highly destructive also in anhydrobiotic organismseven if the lower cellular water content decreases the produc-tion of ROS [21 22] Under normal conditions antioxidantsystems minimize the adverse effects caused by ROS butdesiccation stress could cause the loss or reduction of thesedefence control mechanisms since the metabolic activity isabsent or reduced [21ndash24]
The ability of some animals tardigrades among themto survive extreme desiccation involves a complex arrayof factors working at structural physiological and molec-ular level From a molecularbiochemical point of viewanhydrobiotic organisms synthesize molecules working asbioprotectants during entering permanence and leaving ina desiccated state [25] For example trehalose and sucrosestabilise the biological membrane avoiding protein unfoldingand membrane disturbances late embryogenesis abundantproteins and heat shock proteins work as chaperone systemsrepairing or eliminating damaged molecules while antiox-idant molecules counteract the negative effects of oxidativestress [25]
Since it is known that both hydrated and desiccatedtardigrades have a good natural capability to overcomeoxidative stress [26] they have been used in TARDIKISSexperiments to evaluate the role of antioxidant defence inovercoming oxidative stress induced by exposure to spacestress conditions such as ionizing and UV radiations
The first space experiment (TARSE) conducted withhydrated starved specimens of the tardigrade P richtersidemonstrated that some of the enzymes involved in antiox-idant defences were significantly influenced by the flightstresses [1] In particular there was a significant decrease incatalase and SOD activities the more active enzymes in Prichtersi In addition the glutathione system the less activesystem in not stressed specimens of this species [26] wassignificantly induced during space flight [1] These resultscould be related to the stresses experienced by the hydratedand metabolically active animals (microgravity starvationand radiations) during the flight On the contrary the analysisof antioxidant defences in desiccated tardigrades of theTARDIKISS experiment showed fewer differences relatedto space flight even if the tendency was similar to thatrecorded in hydrated metabolically active animals of theTARSE experiment A similar trend between TARSE andTARDIKISS experiments was also detected in regard to tardi-grade survival since flight animals did not show significantdifferences in survival from temperature laboratory controlones [1 11] Only inR oberhaeuseri (TARDIKISS experiment)a significant decrease in survival rate was recorded between Fand TC samples the species in which a significant decrease ofthe C226 n-3 fatty acid and of glutathione reductase activity
and even though not significant of the activity of the otherROS scavenging enzymes were detected
In conclusion TARDIKISS experiment together withprevious space experiments using tardigrades [1 7ndash9] furtherconfirms that both desiccated and hydrated physiologicalstates of tardigrades represent useful animal tool for spaceresearch To further develop the space research using tardi-grades the setup of experimentswith the possibility to changethe exposition condition of metabolically hydrated animalsas well as the possibility to expose desiccated tardigrades toopen space is necessary Experiments under true space con-dition provide a realistic evaluation of the mechanisms thatcould allowmulticellular organisms including tardigrades tosurvive the combined and synergic effects of space stressorsNevertheless experiments on ground using simulators ofmicrogravity radiation temperature and other space stressesare an essential part of space research complementing exper-iments under true space conditions The comparisons of twodifferent sets of data (ground and space data) will allow betterunderstanding of the physiological and molecular pathwaysof living organisms under space environment
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are very grateful to the Italian Space Agency(ASI) and the Italian Air Force (AM) which funded theDAMA mission The authors are also very grateful to KayserItalia (KI) which developed and manufactured the hardwareinvolved in the BIOKIS payload They are grateful to anony-mous reviewers for their constructive suggestions
References
[1] L Rebecchi T Altiero R Guidetti et al ldquoTardigrade resistanceto space effects First results of experiments on the LIFE-TARSEMission onFOTON-M3 (September 2007)rdquoAstrobiology vol 9no 6 pp 581ndash591 2009
[2] R Guidetti A M Rizzo T Altiero and L Rebecchi ldquoWhatcan we learn from the toughest animals of the Earth Waterbears (tardigrades) asmulticellularmodel organisms in order toperform scientific preparations for lunar explorationrdquoPlanetaryand Space Science vol 74 no 1 pp 97ndash102 2012
[3] H Marthy ldquoDevelopmental biology of animal models undervaried gravity conditions a reviewrdquo Vie et Milieu vol 52 no4 pp 149ndash189 2002
[4] N Moslashbjerg K A Halberg A Joslashrgensen et al ldquoSurvival inextreme environmentsmdashon the current knowledge of adapta-tions in tardigradesrdquo Acta Physiologica vol 202 no 3 pp 409ndash420 2011
[5] R Guidetti T Altiero and L Rebecchi ldquoOn dormancy strate-gies in tardigradesrdquo Journal of Insect Physiology vol 57 no 5pp 567ndash576 2011
[6] T Altiero R Guidetti V Caselli M Cesari and L RebecchildquoUltraviolet radiation tolerance in hydrated and desiccated
BioMed Research International 7
eutardigradesrdquo Journal of Zoological Systematics and Evolution-ary Research vol 49 supplement 1 pp 104ndash110 2011
[7] K I Jonsson E Rabbow R O Schill M Harms-Ringdahl andP Rettberg ldquoTardigrades survive exposure to space in low Earthorbitrdquo Current Biology vol 18 no 17 pp R729ndashR731 2008
[8] L Rebecchi T Altiero M Cesari et al ldquoResistance of the anhy-drobiotic eutardigrade Paramacrobiotus richtersi to space flight(LIFE-TARSE mission on FOTON-M3)rdquo Journal of ZoologicalSystematics and Evolutionary Research vol 49 supplement 1 pp98ndash103 2011
[9] D Persson K A Halberg A Joslashrgensen C Ricci N Moslashbjergand R M Kristensen ldquoExtreme stress tolerance in tardigradessurviving space conditions in low earth orbitrdquo Journal of Zoolog-ical Systematics and Evolutionary Research vol 49 supplement1 pp 90ndash97 2011
[10] T Altiero L Rebecchi and R Bertolani ldquoPhenotypic varia-tions in the life history of two clones of Macrobiotus richtersi(Eutardigrada Macrobiotidae)rdquo Hydrobiologia vol 558 no 1pp 33ndash40 2006
[11] M Vukich P L Ganga D Cavalieri et al ldquoBIOKIS amodel payload for multisciplinary experiments in micrograv-ityrdquo Microgravity Science and Technology vol 24 pp 397ndash4092012
[12] A M Rizzo L Adorni G Montorfano F Rossi and B BerraldquoAntioxidant metabolism of Xenopus laevis embryos duringthe first days of developmentrdquo Comparative Biochemistry andPhysiologymdashB Biochemistry and Molecular Biology vol 146 no1 pp 94ndash100 2007
[13] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951
[14] F Paoletti and A Mocali ldquoDetermination of superoxide dis-mutase activity by purely chemical system based on NAD(P)HoxidationrdquoMethods in Enzymology vol 186 pp 209ndash220 1990
[15] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984
[16] M C Pinto A M Mata and J Lopez-barea ldquoReversible inacti-vation of Saccharomyces cerevisiae glutathione reductase underreducing conditionsrdquo Archives of Biochemistry and Biophysicsvol 228 no 1 pp 1ndash12 1984
[17] J R Prohaska and H E Ganther ldquoSelenium and glutathioneperoxidase in developing rat brainrdquo Journal of Neurochemistryvol 27 no 6 pp 1379ndash1387 1976
[18] O W Griffith ldquoGlutathione and glutathione disulphiderdquo inMethods of Enzymatic Analysis H U Bergmeyer Ed vol 3 pp521ndash529 Academic Press New York NY USA 1984
[19] H E Wey L Pyron and M Woolery ldquoEssential fatty acid defi-ciency in cultured human keratinocytes attenuates toxicity dueto lipid peroxidationrdquo Toxicology and Applied Pharmacologyvol 120 no 1 pp 72ndash79 1993
[20] J Folch M Lees and G H S Stanley ldquoA simple method for theisolation and purification of total lipides from animal tissuesrdquoThe Journal of Biological Chemistry vol 226 no 1 pp 497ndash5091957
[21] M B Franca A D Panek and E C A Eleutherio ldquoOxidativestress and its effects during dehydrationrdquoComparative Biochem-istry and PhysiologymdashA Molecular and Integrative Physiologyvol 146 no 4 pp 621ndash631 2007
[22] R Cruz de Carvalho M Catala J Marques da Silva CBranquinho and E Barreno ldquoThe impact of dehydration rateon the production and cellular location of reactive oxygen
species in an aquatic mossrdquo Annals of Botany vol 110 no 5 pp1007ndash1016 2012
[23] I Kranner and S Birtic ldquoA modulating role for antioxidants indesiccation tolerancerdquo Integrative and Comparative Biology vol45 no 5 pp 734ndash740 2005
[24] R Cornette and T Kikawada ldquoThe induction of anhydrobiosisin the sleeping chironomid current status of our knowledgerdquoIUBMB Life vol 63 no 6 pp 419ndash429 2011
[25] L Rebecchi ldquoDry up and survive the role of antioxidantdefences in anhydrobiotic organismsrdquo Journal of Limnology vol72 no 1 pp 62ndash72 2013
[26] A M Rizzo M Negroni T Altiero et al ldquoAntioxidant defencesin hydrated and desiccated states of the tardigrade Paramac-robiotus richtersirdquo Comparative Biochemistry and Physiology BBiochemistry and Molecular Biology vol 156 no 2 pp 115ndash1212010
Figure 1 Micrographs by scanning electronmicroscopy of the tardigrade Ramazzottius oberhaeuseri showing its two physiological states (a)Hydrated and metabolically active specimen (b) Desiccated and metabolically inactive specimen Bars a = 10 120583m b = 5 120583m
22 Biochemical Assays Biochemical assays were performedon desiccated tardigrades comparing F samples with TCsamples
The activities of the enzymes superoxide dismutase(SOD total activity) catalase (CAT) glutathione peroxidase(GPx) and glutathione reductase (GR) were evaluated Thetotal glutathione (GSH) content thiobarbituric acid reactivesubstances (TBARS) and fatty acid composition were alsodetermined as previously described [12]
Substrates and reagents for enzyme determinations wereNAD(P)H DTNB GSH GSSG glutathione reductase andtert-butyl hydroperoxide all of them were purchased fromSigma-Aldrich (St Louis Missouri USA) For each sampleset and each species 6 or 8 (with the exception of SOD)replicates each made up by 10 in toto tardigrades werehomogenized in water on ice with potter using 3 cycles of30 sec each The homogenate was assayed for protein content(according to [13]) and used for enzyme determination Foreach enzyme homogenates were analyzed in duplicate
Briefly the activity of the enzyme superoxide dismutasewas assayed using the method based on NAD(P)H oxidationinhibition (according to [14]) the inhibition of NADPHoxidation by superoxide which was chemically generatedwas measured at 340 nm for 20min in the presence of tissueextracts The incubation mixture included 213 120583L of TDB(triethanolaminediethanolamine 100mM pH 74) 10 120583L ofNADPH 75 120583M 7 120583L of EDTA-MnCl
2(100mMndash50mM)
and 20 120583L of sample or blank One unit of SOD activity wasdefined as the amount of enzyme required to inhibit the rateof NADPH oxidation by 50
To evaluate the activity of catalase samples were assayedby measuring the consumption of H
2O2(according to [15])
Consumption of hydrogen peroxide by the tissue extracts wasdetermined at 240 nm for 1min at 30∘CThe incubation mix-ture included 10 120583L of H
2O2200mM 20 120583L of homogenate
and 170 120583L of Na-phosphate buffer (50mM pH 70) One unitofCATactivitywas defined as the amount of enzyme requiredto catalyze the decomposition of 1mmol of H
2O2minminus1
The activity of the glutathione reductase was assayedfollowing the oxidation of NADPH (according to [16])Briefly GSSG reduction and NADPH consumption werefollowed at 340 nmThe incubation mixture included 5 120583L ofGSSG 125mM 3120583L of NADPH 11mM animal homogenatefrom 20 to 50 120583L and K-phosphate buffer (100mM pH 70)to reach a final volume of 025mL One unit of GR activity
was defined as the amount of enzyme required to catalyze theoxidation of 1 120583mol NADPHminminus1
To evaluate the activity of selenium-dependent glu-tathione peroxidase the enzyme activitywas assayed (accord-ing to [17]) following the decrease in the absorbance at340 nm for 3min which corresponds to the rate of GSHoxidation to GSSG in the presence of NADPH and glu-tathione reductase The incubation mixture included 5 120583L ofGSH 100mM 3 120583L of NADPH 22mM GR 1 unit 5 120583L oftert-butyl hydroperoxide 20mM from 20 to 50 120583L of animalhomogenate and EDTA-K phosphate buffer (3mMndash100mMpH 70) to reach a final volume of 025mL One unit of GPxactivity was defined as the amount of enzyme required tocatalyze the oxidation of 1 120583mol of NADPHminminus1
To measure the total glutathione tardigrades werehomogenized on ice in 5 metaphosphoric acid thehomogenatewas centrifuged at 5000timesg for 10min at 4∘C andthe supernatant was assayed (according to [18]) with someslight modifications Briefly the sulfhydryl group of GSHalso generated from GSSG by adding GR reacts with DTNB(551015840-dithiobis-2-nitrobenzoic acid) and produces a yellow-coloured 5-thio-2-nitrobenzoic acid (TNB) The rate of TNBproduction is directly proportional to this reaction whichin turn is directly proportional to the concentration of GSHin the sample The measurement of the absorbance of TNBat 412 nm provides an accurate estimation of the GSH levelpresent in the sample
To evaluate the thiobarbituric acid reactive substances(TBARS) tardigrade samples standards (from 25 to100 pmol TEP 11-33 tetraethoxypropane) and blanks wereassayed (according to [19]) both before and after inductionof lipid peroxidation by FeSO
4and ascorbic acid TBARS
were determined using a fluorescence spectrophotometer(Carly Eclipse Varian CA USA) at an excitation wavelengthof 517 nm and an emission wavelength of 550 nm For eachsample set (F and TC) and species (R oberhaeuseri and Prichtersi) 2 or 4 replicates were analyzed
To evaluate the fatty acid composition lipids wereextracted from groups of 10 desiccated tardigrades withchloroformmethanol (according to [20]) The total extractwas used for derivatization with sodium methoxide inmethanol 333wv to obtain the fatty acid methylesters(FAME) FAME were injected into a gas chromatograph(Agilent Technologies 6850 Series II) equipped with a flameionization detector (FID) under the following experimental
4 BioMed Research International
Table 1 Percentage of fatty acid composition in the tardigrades Paramacrobiotus richtersi and Ramazzottius oberhaeuseri
Fatty acid Paramacrobiotus richtersi Ramazzottius oberhaeuseriTC F TC F
conditions capillary column AT Silar length 30m filmthickness 025120583m gas carrier helium temperatures injector250∘C detector 275∘C oven 50∘C for 2min and rate of10∘Cminminus1 until 200∘C for 20min A standard mixture con-taining methyl ester fatty acids was injected for calibrationFor each sample set and species 2 or 4 replicates wereanalyzed
23 Statistical Analysis Data were analyzed with Mann-Whitney test and expressed as mean plusmn SD using the pro-gramme SPSS
3 Results
The results of the enzyme activities in the tardigradesParamacrobiotus richtersi and Ramazzottius oberhaeuseri arealways indicated in relation to 120583g of proteins It is worthnoting that R oberhaeuseri contains a lower amount ofproteins compared to P richtersi (Figure 2)
In both species the comparative analysis of the enzymeactivities and other antioxidant molecules between flight (F)and temperature control samples (TC) showed few significantdifferences (Figures 3 and 4) In particular a significantdecrease (119875 lt 005) of the glutathione reductase activitywas detected in R oberhaeuseri F samples with respect toTC samples (Figure 4(b)) Although not statistically sup-ported in this species a tendency to decrease catalasesuperoxide dismutase and glutathione peroxidase activityand in glutathione content was detected In P richtersi atendency to decrease catalase superoxide dismutase and glu-tathione reductase activities and to increase the glutathioneperoxidase activity was detected Noteworthy differenceswere recorded in the activities of ROS scavenging enzymesbetween the two species
The total percentage fatty acid composition of F and TCsamples is reported in Table 1 In R oberhaeuseri a significant
000
020
040
060
080
100
120
P richtersi R oberhaeuseri
TCFlight
(120583g
prot
eins
aa)
Figure 2 Total protein content in flight and ground temperaturecontrol (TC) samples in the tardigrades Paramacrobiotus richtersiand Ramazzottius oberhaeuseri The bars show the mean with SD
decrease (119875 lt 005) was recorded for the fatty acidC22-6 n-3 and polyunsaturated fatty acids (PUFA) in theF samples with respect to the TC samples Moreover Roberhaeuseri has significantly lower amount of C22-6 n-3compared to P richtersi The amount of thiobarbituric acidreactive substances (TBARS) in tardigrades both before andafter induction of peroxidation in vitro is also reported inTable 1 No differences were detected between F and TCsamples in both species for basal levels and after inductionof peroxidation
BioMed Research International 5
0
005
01
015
02
025
03
035
04
P richtersi R oberhaeuseri
Superoxide dismutase (SOD)
TCFlight
(Un
g pr
otei
ns)
(a)
0
10
20
30
40
50
60
70
80
P richtersi R oberhaeuseri
Catalase
TCFlight
(mU
120583 g
prot
eins
)
(b)
Figure 3 Superoxide dismutase (a) and catalase (b) activities in flight and ground temperature control (TC) samples in the tardigradesParamacrobiotus richtersi and Ramazzottius oberhaeuseri The bars show the mean with SD
0
05
1
15
2
25
3
P richtersi R oberhaeuseri
Glutathione peroxidase
TCFlight
(mU
120583g
prot
eins
)
(a)
0010203040506070809
1
P richtersi R oberhaeuseri
Glutathione reductase
lowast
TCFlight
(mU
120583g
prot
eins
)
(b)
0
02
04
06
08
1
P richtersi R oberhaeuseri
Glutathione (GSH)
TCFlight
(mm
oles
120583g
prot
eins
)
(c)
Figure 4 Glutathione peroxidase (a) glutathione reductase (b) and total glutathione content (c) in flight and ground temperature control(TC) samples in the tardigrades Paramacrobiotus richtersi and Ramazzottius oberhaeuseri The bars show the mean with SD lowast119875 lt 005
6 BioMed Research International
4 Discussion
Exposure to space stress conditions induces oxidative stressOxidative stress resulting from an imbalance between theexcessive production of reactive oxygen species (ROS) andlimited action of antioxidant defences is implicated inthe development of many important human pathologiesincluding atherosclerosis hypertension inflammation can-cer Parkinson and Alzheimer diseases [21] Oxidative stressmay be highly destructive also in anhydrobiotic organismseven if the lower cellular water content decreases the produc-tion of ROS [21 22] Under normal conditions antioxidantsystems minimize the adverse effects caused by ROS butdesiccation stress could cause the loss or reduction of thesedefence control mechanisms since the metabolic activity isabsent or reduced [21ndash24]
The ability of some animals tardigrades among themto survive extreme desiccation involves a complex arrayof factors working at structural physiological and molec-ular level From a molecularbiochemical point of viewanhydrobiotic organisms synthesize molecules working asbioprotectants during entering permanence and leaving ina desiccated state [25] For example trehalose and sucrosestabilise the biological membrane avoiding protein unfoldingand membrane disturbances late embryogenesis abundantproteins and heat shock proteins work as chaperone systemsrepairing or eliminating damaged molecules while antiox-idant molecules counteract the negative effects of oxidativestress [25]
Since it is known that both hydrated and desiccatedtardigrades have a good natural capability to overcomeoxidative stress [26] they have been used in TARDIKISSexperiments to evaluate the role of antioxidant defence inovercoming oxidative stress induced by exposure to spacestress conditions such as ionizing and UV radiations
The first space experiment (TARSE) conducted withhydrated starved specimens of the tardigrade P richtersidemonstrated that some of the enzymes involved in antiox-idant defences were significantly influenced by the flightstresses [1] In particular there was a significant decrease incatalase and SOD activities the more active enzymes in Prichtersi In addition the glutathione system the less activesystem in not stressed specimens of this species [26] wassignificantly induced during space flight [1] These resultscould be related to the stresses experienced by the hydratedand metabolically active animals (microgravity starvationand radiations) during the flight On the contrary the analysisof antioxidant defences in desiccated tardigrades of theTARDIKISS experiment showed fewer differences relatedto space flight even if the tendency was similar to thatrecorded in hydrated metabolically active animals of theTARSE experiment A similar trend between TARSE andTARDIKISS experiments was also detected in regard to tardi-grade survival since flight animals did not show significantdifferences in survival from temperature laboratory controlones [1 11] Only inR oberhaeuseri (TARDIKISS experiment)a significant decrease in survival rate was recorded between Fand TC samples the species in which a significant decrease ofthe C226 n-3 fatty acid and of glutathione reductase activity
and even though not significant of the activity of the otherROS scavenging enzymes were detected
In conclusion TARDIKISS experiment together withprevious space experiments using tardigrades [1 7ndash9] furtherconfirms that both desiccated and hydrated physiologicalstates of tardigrades represent useful animal tool for spaceresearch To further develop the space research using tardi-grades the setup of experimentswith the possibility to changethe exposition condition of metabolically hydrated animalsas well as the possibility to expose desiccated tardigrades toopen space is necessary Experiments under true space con-dition provide a realistic evaluation of the mechanisms thatcould allowmulticellular organisms including tardigrades tosurvive the combined and synergic effects of space stressorsNevertheless experiments on ground using simulators ofmicrogravity radiation temperature and other space stressesare an essential part of space research complementing exper-iments under true space conditions The comparisons of twodifferent sets of data (ground and space data) will allow betterunderstanding of the physiological and molecular pathwaysof living organisms under space environment
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are very grateful to the Italian Space Agency(ASI) and the Italian Air Force (AM) which funded theDAMA mission The authors are also very grateful to KayserItalia (KI) which developed and manufactured the hardwareinvolved in the BIOKIS payload They are grateful to anony-mous reviewers for their constructive suggestions
References
[1] L Rebecchi T Altiero R Guidetti et al ldquoTardigrade resistanceto space effects First results of experiments on the LIFE-TARSEMission onFOTON-M3 (September 2007)rdquoAstrobiology vol 9no 6 pp 581ndash591 2009
[2] R Guidetti A M Rizzo T Altiero and L Rebecchi ldquoWhatcan we learn from the toughest animals of the Earth Waterbears (tardigrades) asmulticellularmodel organisms in order toperform scientific preparations for lunar explorationrdquoPlanetaryand Space Science vol 74 no 1 pp 97ndash102 2012
[3] H Marthy ldquoDevelopmental biology of animal models undervaried gravity conditions a reviewrdquo Vie et Milieu vol 52 no4 pp 149ndash189 2002
[4] N Moslashbjerg K A Halberg A Joslashrgensen et al ldquoSurvival inextreme environmentsmdashon the current knowledge of adapta-tions in tardigradesrdquo Acta Physiologica vol 202 no 3 pp 409ndash420 2011
[5] R Guidetti T Altiero and L Rebecchi ldquoOn dormancy strate-gies in tardigradesrdquo Journal of Insect Physiology vol 57 no 5pp 567ndash576 2011
[6] T Altiero R Guidetti V Caselli M Cesari and L RebecchildquoUltraviolet radiation tolerance in hydrated and desiccated
BioMed Research International 7
eutardigradesrdquo Journal of Zoological Systematics and Evolution-ary Research vol 49 supplement 1 pp 104ndash110 2011
[7] K I Jonsson E Rabbow R O Schill M Harms-Ringdahl andP Rettberg ldquoTardigrades survive exposure to space in low Earthorbitrdquo Current Biology vol 18 no 17 pp R729ndashR731 2008
[8] L Rebecchi T Altiero M Cesari et al ldquoResistance of the anhy-drobiotic eutardigrade Paramacrobiotus richtersi to space flight(LIFE-TARSE mission on FOTON-M3)rdquo Journal of ZoologicalSystematics and Evolutionary Research vol 49 supplement 1 pp98ndash103 2011
[9] D Persson K A Halberg A Joslashrgensen C Ricci N Moslashbjergand R M Kristensen ldquoExtreme stress tolerance in tardigradessurviving space conditions in low earth orbitrdquo Journal of Zoolog-ical Systematics and Evolutionary Research vol 49 supplement1 pp 90ndash97 2011
[10] T Altiero L Rebecchi and R Bertolani ldquoPhenotypic varia-tions in the life history of two clones of Macrobiotus richtersi(Eutardigrada Macrobiotidae)rdquo Hydrobiologia vol 558 no 1pp 33ndash40 2006
[11] M Vukich P L Ganga D Cavalieri et al ldquoBIOKIS amodel payload for multisciplinary experiments in micrograv-ityrdquo Microgravity Science and Technology vol 24 pp 397ndash4092012
[12] A M Rizzo L Adorni G Montorfano F Rossi and B BerraldquoAntioxidant metabolism of Xenopus laevis embryos duringthe first days of developmentrdquo Comparative Biochemistry andPhysiologymdashB Biochemistry and Molecular Biology vol 146 no1 pp 94ndash100 2007
[13] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951
[14] F Paoletti and A Mocali ldquoDetermination of superoxide dis-mutase activity by purely chemical system based on NAD(P)HoxidationrdquoMethods in Enzymology vol 186 pp 209ndash220 1990
[15] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984
[16] M C Pinto A M Mata and J Lopez-barea ldquoReversible inacti-vation of Saccharomyces cerevisiae glutathione reductase underreducing conditionsrdquo Archives of Biochemistry and Biophysicsvol 228 no 1 pp 1ndash12 1984
[17] J R Prohaska and H E Ganther ldquoSelenium and glutathioneperoxidase in developing rat brainrdquo Journal of Neurochemistryvol 27 no 6 pp 1379ndash1387 1976
[18] O W Griffith ldquoGlutathione and glutathione disulphiderdquo inMethods of Enzymatic Analysis H U Bergmeyer Ed vol 3 pp521ndash529 Academic Press New York NY USA 1984
[19] H E Wey L Pyron and M Woolery ldquoEssential fatty acid defi-ciency in cultured human keratinocytes attenuates toxicity dueto lipid peroxidationrdquo Toxicology and Applied Pharmacologyvol 120 no 1 pp 72ndash79 1993
[20] J Folch M Lees and G H S Stanley ldquoA simple method for theisolation and purification of total lipides from animal tissuesrdquoThe Journal of Biological Chemistry vol 226 no 1 pp 497ndash5091957
[21] M B Franca A D Panek and E C A Eleutherio ldquoOxidativestress and its effects during dehydrationrdquoComparative Biochem-istry and PhysiologymdashA Molecular and Integrative Physiologyvol 146 no 4 pp 621ndash631 2007
[22] R Cruz de Carvalho M Catala J Marques da Silva CBranquinho and E Barreno ldquoThe impact of dehydration rateon the production and cellular location of reactive oxygen
species in an aquatic mossrdquo Annals of Botany vol 110 no 5 pp1007ndash1016 2012
[23] I Kranner and S Birtic ldquoA modulating role for antioxidants indesiccation tolerancerdquo Integrative and Comparative Biology vol45 no 5 pp 734ndash740 2005
[24] R Cornette and T Kikawada ldquoThe induction of anhydrobiosisin the sleeping chironomid current status of our knowledgerdquoIUBMB Life vol 63 no 6 pp 419ndash429 2011
[25] L Rebecchi ldquoDry up and survive the role of antioxidantdefences in anhydrobiotic organismsrdquo Journal of Limnology vol72 no 1 pp 62ndash72 2013
[26] A M Rizzo M Negroni T Altiero et al ldquoAntioxidant defencesin hydrated and desiccated states of the tardigrade Paramac-robiotus richtersirdquo Comparative Biochemistry and Physiology BBiochemistry and Molecular Biology vol 156 no 2 pp 115ndash1212010
conditions capillary column AT Silar length 30m filmthickness 025120583m gas carrier helium temperatures injector250∘C detector 275∘C oven 50∘C for 2min and rate of10∘Cminminus1 until 200∘C for 20min A standard mixture con-taining methyl ester fatty acids was injected for calibrationFor each sample set and species 2 or 4 replicates wereanalyzed
23 Statistical Analysis Data were analyzed with Mann-Whitney test and expressed as mean plusmn SD using the pro-gramme SPSS
3 Results
The results of the enzyme activities in the tardigradesParamacrobiotus richtersi and Ramazzottius oberhaeuseri arealways indicated in relation to 120583g of proteins It is worthnoting that R oberhaeuseri contains a lower amount ofproteins compared to P richtersi (Figure 2)
In both species the comparative analysis of the enzymeactivities and other antioxidant molecules between flight (F)and temperature control samples (TC) showed few significantdifferences (Figures 3 and 4) In particular a significantdecrease (119875 lt 005) of the glutathione reductase activitywas detected in R oberhaeuseri F samples with respect toTC samples (Figure 4(b)) Although not statistically sup-ported in this species a tendency to decrease catalasesuperoxide dismutase and glutathione peroxidase activityand in glutathione content was detected In P richtersi atendency to decrease catalase superoxide dismutase and glu-tathione reductase activities and to increase the glutathioneperoxidase activity was detected Noteworthy differenceswere recorded in the activities of ROS scavenging enzymesbetween the two species
The total percentage fatty acid composition of F and TCsamples is reported in Table 1 In R oberhaeuseri a significant
000
020
040
060
080
100
120
P richtersi R oberhaeuseri
TCFlight
(120583g
prot
eins
aa)
Figure 2 Total protein content in flight and ground temperaturecontrol (TC) samples in the tardigrades Paramacrobiotus richtersiand Ramazzottius oberhaeuseri The bars show the mean with SD
decrease (119875 lt 005) was recorded for the fatty acidC22-6 n-3 and polyunsaturated fatty acids (PUFA) in theF samples with respect to the TC samples Moreover Roberhaeuseri has significantly lower amount of C22-6 n-3compared to P richtersi The amount of thiobarbituric acidreactive substances (TBARS) in tardigrades both before andafter induction of peroxidation in vitro is also reported inTable 1 No differences were detected between F and TCsamples in both species for basal levels and after inductionof peroxidation
BioMed Research International 5
0
005
01
015
02
025
03
035
04
P richtersi R oberhaeuseri
Superoxide dismutase (SOD)
TCFlight
(Un
g pr
otei
ns)
(a)
0
10
20
30
40
50
60
70
80
P richtersi R oberhaeuseri
Catalase
TCFlight
(mU
120583 g
prot
eins
)
(b)
Figure 3 Superoxide dismutase (a) and catalase (b) activities in flight and ground temperature control (TC) samples in the tardigradesParamacrobiotus richtersi and Ramazzottius oberhaeuseri The bars show the mean with SD
0
05
1
15
2
25
3
P richtersi R oberhaeuseri
Glutathione peroxidase
TCFlight
(mU
120583g
prot
eins
)
(a)
0010203040506070809
1
P richtersi R oberhaeuseri
Glutathione reductase
lowast
TCFlight
(mU
120583g
prot
eins
)
(b)
0
02
04
06
08
1
P richtersi R oberhaeuseri
Glutathione (GSH)
TCFlight
(mm
oles
120583g
prot
eins
)
(c)
Figure 4 Glutathione peroxidase (a) glutathione reductase (b) and total glutathione content (c) in flight and ground temperature control(TC) samples in the tardigrades Paramacrobiotus richtersi and Ramazzottius oberhaeuseri The bars show the mean with SD lowast119875 lt 005
6 BioMed Research International
4 Discussion
Exposure to space stress conditions induces oxidative stressOxidative stress resulting from an imbalance between theexcessive production of reactive oxygen species (ROS) andlimited action of antioxidant defences is implicated inthe development of many important human pathologiesincluding atherosclerosis hypertension inflammation can-cer Parkinson and Alzheimer diseases [21] Oxidative stressmay be highly destructive also in anhydrobiotic organismseven if the lower cellular water content decreases the produc-tion of ROS [21 22] Under normal conditions antioxidantsystems minimize the adverse effects caused by ROS butdesiccation stress could cause the loss or reduction of thesedefence control mechanisms since the metabolic activity isabsent or reduced [21ndash24]
The ability of some animals tardigrades among themto survive extreme desiccation involves a complex arrayof factors working at structural physiological and molec-ular level From a molecularbiochemical point of viewanhydrobiotic organisms synthesize molecules working asbioprotectants during entering permanence and leaving ina desiccated state [25] For example trehalose and sucrosestabilise the biological membrane avoiding protein unfoldingand membrane disturbances late embryogenesis abundantproteins and heat shock proteins work as chaperone systemsrepairing or eliminating damaged molecules while antiox-idant molecules counteract the negative effects of oxidativestress [25]
Since it is known that both hydrated and desiccatedtardigrades have a good natural capability to overcomeoxidative stress [26] they have been used in TARDIKISSexperiments to evaluate the role of antioxidant defence inovercoming oxidative stress induced by exposure to spacestress conditions such as ionizing and UV radiations
The first space experiment (TARSE) conducted withhydrated starved specimens of the tardigrade P richtersidemonstrated that some of the enzymes involved in antiox-idant defences were significantly influenced by the flightstresses [1] In particular there was a significant decrease incatalase and SOD activities the more active enzymes in Prichtersi In addition the glutathione system the less activesystem in not stressed specimens of this species [26] wassignificantly induced during space flight [1] These resultscould be related to the stresses experienced by the hydratedand metabolically active animals (microgravity starvationand radiations) during the flight On the contrary the analysisof antioxidant defences in desiccated tardigrades of theTARDIKISS experiment showed fewer differences relatedto space flight even if the tendency was similar to thatrecorded in hydrated metabolically active animals of theTARSE experiment A similar trend between TARSE andTARDIKISS experiments was also detected in regard to tardi-grade survival since flight animals did not show significantdifferences in survival from temperature laboratory controlones [1 11] Only inR oberhaeuseri (TARDIKISS experiment)a significant decrease in survival rate was recorded between Fand TC samples the species in which a significant decrease ofthe C226 n-3 fatty acid and of glutathione reductase activity
and even though not significant of the activity of the otherROS scavenging enzymes were detected
In conclusion TARDIKISS experiment together withprevious space experiments using tardigrades [1 7ndash9] furtherconfirms that both desiccated and hydrated physiologicalstates of tardigrades represent useful animal tool for spaceresearch To further develop the space research using tardi-grades the setup of experimentswith the possibility to changethe exposition condition of metabolically hydrated animalsas well as the possibility to expose desiccated tardigrades toopen space is necessary Experiments under true space con-dition provide a realistic evaluation of the mechanisms thatcould allowmulticellular organisms including tardigrades tosurvive the combined and synergic effects of space stressorsNevertheless experiments on ground using simulators ofmicrogravity radiation temperature and other space stressesare an essential part of space research complementing exper-iments under true space conditions The comparisons of twodifferent sets of data (ground and space data) will allow betterunderstanding of the physiological and molecular pathwaysof living organisms under space environment
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are very grateful to the Italian Space Agency(ASI) and the Italian Air Force (AM) which funded theDAMA mission The authors are also very grateful to KayserItalia (KI) which developed and manufactured the hardwareinvolved in the BIOKIS payload They are grateful to anony-mous reviewers for their constructive suggestions
References
[1] L Rebecchi T Altiero R Guidetti et al ldquoTardigrade resistanceto space effects First results of experiments on the LIFE-TARSEMission onFOTON-M3 (September 2007)rdquoAstrobiology vol 9no 6 pp 581ndash591 2009
[2] R Guidetti A M Rizzo T Altiero and L Rebecchi ldquoWhatcan we learn from the toughest animals of the Earth Waterbears (tardigrades) asmulticellularmodel organisms in order toperform scientific preparations for lunar explorationrdquoPlanetaryand Space Science vol 74 no 1 pp 97ndash102 2012
[3] H Marthy ldquoDevelopmental biology of animal models undervaried gravity conditions a reviewrdquo Vie et Milieu vol 52 no4 pp 149ndash189 2002
[4] N Moslashbjerg K A Halberg A Joslashrgensen et al ldquoSurvival inextreme environmentsmdashon the current knowledge of adapta-tions in tardigradesrdquo Acta Physiologica vol 202 no 3 pp 409ndash420 2011
[5] R Guidetti T Altiero and L Rebecchi ldquoOn dormancy strate-gies in tardigradesrdquo Journal of Insect Physiology vol 57 no 5pp 567ndash576 2011
[6] T Altiero R Guidetti V Caselli M Cesari and L RebecchildquoUltraviolet radiation tolerance in hydrated and desiccated
BioMed Research International 7
eutardigradesrdquo Journal of Zoological Systematics and Evolution-ary Research vol 49 supplement 1 pp 104ndash110 2011
[7] K I Jonsson E Rabbow R O Schill M Harms-Ringdahl andP Rettberg ldquoTardigrades survive exposure to space in low Earthorbitrdquo Current Biology vol 18 no 17 pp R729ndashR731 2008
[8] L Rebecchi T Altiero M Cesari et al ldquoResistance of the anhy-drobiotic eutardigrade Paramacrobiotus richtersi to space flight(LIFE-TARSE mission on FOTON-M3)rdquo Journal of ZoologicalSystematics and Evolutionary Research vol 49 supplement 1 pp98ndash103 2011
[9] D Persson K A Halberg A Joslashrgensen C Ricci N Moslashbjergand R M Kristensen ldquoExtreme stress tolerance in tardigradessurviving space conditions in low earth orbitrdquo Journal of Zoolog-ical Systematics and Evolutionary Research vol 49 supplement1 pp 90ndash97 2011
[10] T Altiero L Rebecchi and R Bertolani ldquoPhenotypic varia-tions in the life history of two clones of Macrobiotus richtersi(Eutardigrada Macrobiotidae)rdquo Hydrobiologia vol 558 no 1pp 33ndash40 2006
[11] M Vukich P L Ganga D Cavalieri et al ldquoBIOKIS amodel payload for multisciplinary experiments in micrograv-ityrdquo Microgravity Science and Technology vol 24 pp 397ndash4092012
[12] A M Rizzo L Adorni G Montorfano F Rossi and B BerraldquoAntioxidant metabolism of Xenopus laevis embryos duringthe first days of developmentrdquo Comparative Biochemistry andPhysiologymdashB Biochemistry and Molecular Biology vol 146 no1 pp 94ndash100 2007
[13] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951
[14] F Paoletti and A Mocali ldquoDetermination of superoxide dis-mutase activity by purely chemical system based on NAD(P)HoxidationrdquoMethods in Enzymology vol 186 pp 209ndash220 1990
[15] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984
[16] M C Pinto A M Mata and J Lopez-barea ldquoReversible inacti-vation of Saccharomyces cerevisiae glutathione reductase underreducing conditionsrdquo Archives of Biochemistry and Biophysicsvol 228 no 1 pp 1ndash12 1984
[17] J R Prohaska and H E Ganther ldquoSelenium and glutathioneperoxidase in developing rat brainrdquo Journal of Neurochemistryvol 27 no 6 pp 1379ndash1387 1976
[18] O W Griffith ldquoGlutathione and glutathione disulphiderdquo inMethods of Enzymatic Analysis H U Bergmeyer Ed vol 3 pp521ndash529 Academic Press New York NY USA 1984
[19] H E Wey L Pyron and M Woolery ldquoEssential fatty acid defi-ciency in cultured human keratinocytes attenuates toxicity dueto lipid peroxidationrdquo Toxicology and Applied Pharmacologyvol 120 no 1 pp 72ndash79 1993
[20] J Folch M Lees and G H S Stanley ldquoA simple method for theisolation and purification of total lipides from animal tissuesrdquoThe Journal of Biological Chemistry vol 226 no 1 pp 497ndash5091957
[21] M B Franca A D Panek and E C A Eleutherio ldquoOxidativestress and its effects during dehydrationrdquoComparative Biochem-istry and PhysiologymdashA Molecular and Integrative Physiologyvol 146 no 4 pp 621ndash631 2007
[22] R Cruz de Carvalho M Catala J Marques da Silva CBranquinho and E Barreno ldquoThe impact of dehydration rateon the production and cellular location of reactive oxygen
species in an aquatic mossrdquo Annals of Botany vol 110 no 5 pp1007ndash1016 2012
[23] I Kranner and S Birtic ldquoA modulating role for antioxidants indesiccation tolerancerdquo Integrative and Comparative Biology vol45 no 5 pp 734ndash740 2005
[24] R Cornette and T Kikawada ldquoThe induction of anhydrobiosisin the sleeping chironomid current status of our knowledgerdquoIUBMB Life vol 63 no 6 pp 419ndash429 2011
[25] L Rebecchi ldquoDry up and survive the role of antioxidantdefences in anhydrobiotic organismsrdquo Journal of Limnology vol72 no 1 pp 62ndash72 2013
[26] A M Rizzo M Negroni T Altiero et al ldquoAntioxidant defencesin hydrated and desiccated states of the tardigrade Paramac-robiotus richtersirdquo Comparative Biochemistry and Physiology BBiochemistry and Molecular Biology vol 156 no 2 pp 115ndash1212010
Figure 3 Superoxide dismutase (a) and catalase (b) activities in flight and ground temperature control (TC) samples in the tardigradesParamacrobiotus richtersi and Ramazzottius oberhaeuseri The bars show the mean with SD
0
05
1
15
2
25
3
P richtersi R oberhaeuseri
Glutathione peroxidase
TCFlight
(mU
120583g
prot
eins
)
(a)
0010203040506070809
1
P richtersi R oberhaeuseri
Glutathione reductase
lowast
TCFlight
(mU
120583g
prot
eins
)
(b)
0
02
04
06
08
1
P richtersi R oberhaeuseri
Glutathione (GSH)
TCFlight
(mm
oles
120583g
prot
eins
)
(c)
Figure 4 Glutathione peroxidase (a) glutathione reductase (b) and total glutathione content (c) in flight and ground temperature control(TC) samples in the tardigrades Paramacrobiotus richtersi and Ramazzottius oberhaeuseri The bars show the mean with SD lowast119875 lt 005
6 BioMed Research International
4 Discussion
Exposure to space stress conditions induces oxidative stressOxidative stress resulting from an imbalance between theexcessive production of reactive oxygen species (ROS) andlimited action of antioxidant defences is implicated inthe development of many important human pathologiesincluding atherosclerosis hypertension inflammation can-cer Parkinson and Alzheimer diseases [21] Oxidative stressmay be highly destructive also in anhydrobiotic organismseven if the lower cellular water content decreases the produc-tion of ROS [21 22] Under normal conditions antioxidantsystems minimize the adverse effects caused by ROS butdesiccation stress could cause the loss or reduction of thesedefence control mechanisms since the metabolic activity isabsent or reduced [21ndash24]
The ability of some animals tardigrades among themto survive extreme desiccation involves a complex arrayof factors working at structural physiological and molec-ular level From a molecularbiochemical point of viewanhydrobiotic organisms synthesize molecules working asbioprotectants during entering permanence and leaving ina desiccated state [25] For example trehalose and sucrosestabilise the biological membrane avoiding protein unfoldingand membrane disturbances late embryogenesis abundantproteins and heat shock proteins work as chaperone systemsrepairing or eliminating damaged molecules while antiox-idant molecules counteract the negative effects of oxidativestress [25]
Since it is known that both hydrated and desiccatedtardigrades have a good natural capability to overcomeoxidative stress [26] they have been used in TARDIKISSexperiments to evaluate the role of antioxidant defence inovercoming oxidative stress induced by exposure to spacestress conditions such as ionizing and UV radiations
The first space experiment (TARSE) conducted withhydrated starved specimens of the tardigrade P richtersidemonstrated that some of the enzymes involved in antiox-idant defences were significantly influenced by the flightstresses [1] In particular there was a significant decrease incatalase and SOD activities the more active enzymes in Prichtersi In addition the glutathione system the less activesystem in not stressed specimens of this species [26] wassignificantly induced during space flight [1] These resultscould be related to the stresses experienced by the hydratedand metabolically active animals (microgravity starvationand radiations) during the flight On the contrary the analysisof antioxidant defences in desiccated tardigrades of theTARDIKISS experiment showed fewer differences relatedto space flight even if the tendency was similar to thatrecorded in hydrated metabolically active animals of theTARSE experiment A similar trend between TARSE andTARDIKISS experiments was also detected in regard to tardi-grade survival since flight animals did not show significantdifferences in survival from temperature laboratory controlones [1 11] Only inR oberhaeuseri (TARDIKISS experiment)a significant decrease in survival rate was recorded between Fand TC samples the species in which a significant decrease ofthe C226 n-3 fatty acid and of glutathione reductase activity
and even though not significant of the activity of the otherROS scavenging enzymes were detected
In conclusion TARDIKISS experiment together withprevious space experiments using tardigrades [1 7ndash9] furtherconfirms that both desiccated and hydrated physiologicalstates of tardigrades represent useful animal tool for spaceresearch To further develop the space research using tardi-grades the setup of experimentswith the possibility to changethe exposition condition of metabolically hydrated animalsas well as the possibility to expose desiccated tardigrades toopen space is necessary Experiments under true space con-dition provide a realistic evaluation of the mechanisms thatcould allowmulticellular organisms including tardigrades tosurvive the combined and synergic effects of space stressorsNevertheless experiments on ground using simulators ofmicrogravity radiation temperature and other space stressesare an essential part of space research complementing exper-iments under true space conditions The comparisons of twodifferent sets of data (ground and space data) will allow betterunderstanding of the physiological and molecular pathwaysof living organisms under space environment
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are very grateful to the Italian Space Agency(ASI) and the Italian Air Force (AM) which funded theDAMA mission The authors are also very grateful to KayserItalia (KI) which developed and manufactured the hardwareinvolved in the BIOKIS payload They are grateful to anony-mous reviewers for their constructive suggestions
References
[1] L Rebecchi T Altiero R Guidetti et al ldquoTardigrade resistanceto space effects First results of experiments on the LIFE-TARSEMission onFOTON-M3 (September 2007)rdquoAstrobiology vol 9no 6 pp 581ndash591 2009
[2] R Guidetti A M Rizzo T Altiero and L Rebecchi ldquoWhatcan we learn from the toughest animals of the Earth Waterbears (tardigrades) asmulticellularmodel organisms in order toperform scientific preparations for lunar explorationrdquoPlanetaryand Space Science vol 74 no 1 pp 97ndash102 2012
[3] H Marthy ldquoDevelopmental biology of animal models undervaried gravity conditions a reviewrdquo Vie et Milieu vol 52 no4 pp 149ndash189 2002
[4] N Moslashbjerg K A Halberg A Joslashrgensen et al ldquoSurvival inextreme environmentsmdashon the current knowledge of adapta-tions in tardigradesrdquo Acta Physiologica vol 202 no 3 pp 409ndash420 2011
[5] R Guidetti T Altiero and L Rebecchi ldquoOn dormancy strate-gies in tardigradesrdquo Journal of Insect Physiology vol 57 no 5pp 567ndash576 2011
[6] T Altiero R Guidetti V Caselli M Cesari and L RebecchildquoUltraviolet radiation tolerance in hydrated and desiccated
BioMed Research International 7
eutardigradesrdquo Journal of Zoological Systematics and Evolution-ary Research vol 49 supplement 1 pp 104ndash110 2011
[7] K I Jonsson E Rabbow R O Schill M Harms-Ringdahl andP Rettberg ldquoTardigrades survive exposure to space in low Earthorbitrdquo Current Biology vol 18 no 17 pp R729ndashR731 2008
[8] L Rebecchi T Altiero M Cesari et al ldquoResistance of the anhy-drobiotic eutardigrade Paramacrobiotus richtersi to space flight(LIFE-TARSE mission on FOTON-M3)rdquo Journal of ZoologicalSystematics and Evolutionary Research vol 49 supplement 1 pp98ndash103 2011
[9] D Persson K A Halberg A Joslashrgensen C Ricci N Moslashbjergand R M Kristensen ldquoExtreme stress tolerance in tardigradessurviving space conditions in low earth orbitrdquo Journal of Zoolog-ical Systematics and Evolutionary Research vol 49 supplement1 pp 90ndash97 2011
[10] T Altiero L Rebecchi and R Bertolani ldquoPhenotypic varia-tions in the life history of two clones of Macrobiotus richtersi(Eutardigrada Macrobiotidae)rdquo Hydrobiologia vol 558 no 1pp 33ndash40 2006
[11] M Vukich P L Ganga D Cavalieri et al ldquoBIOKIS amodel payload for multisciplinary experiments in micrograv-ityrdquo Microgravity Science and Technology vol 24 pp 397ndash4092012
[12] A M Rizzo L Adorni G Montorfano F Rossi and B BerraldquoAntioxidant metabolism of Xenopus laevis embryos duringthe first days of developmentrdquo Comparative Biochemistry andPhysiologymdashB Biochemistry and Molecular Biology vol 146 no1 pp 94ndash100 2007
[13] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951
[14] F Paoletti and A Mocali ldquoDetermination of superoxide dis-mutase activity by purely chemical system based on NAD(P)HoxidationrdquoMethods in Enzymology vol 186 pp 209ndash220 1990
[15] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984
[16] M C Pinto A M Mata and J Lopez-barea ldquoReversible inacti-vation of Saccharomyces cerevisiae glutathione reductase underreducing conditionsrdquo Archives of Biochemistry and Biophysicsvol 228 no 1 pp 1ndash12 1984
[17] J R Prohaska and H E Ganther ldquoSelenium and glutathioneperoxidase in developing rat brainrdquo Journal of Neurochemistryvol 27 no 6 pp 1379ndash1387 1976
[18] O W Griffith ldquoGlutathione and glutathione disulphiderdquo inMethods of Enzymatic Analysis H U Bergmeyer Ed vol 3 pp521ndash529 Academic Press New York NY USA 1984
[19] H E Wey L Pyron and M Woolery ldquoEssential fatty acid defi-ciency in cultured human keratinocytes attenuates toxicity dueto lipid peroxidationrdquo Toxicology and Applied Pharmacologyvol 120 no 1 pp 72ndash79 1993
[20] J Folch M Lees and G H S Stanley ldquoA simple method for theisolation and purification of total lipides from animal tissuesrdquoThe Journal of Biological Chemistry vol 226 no 1 pp 497ndash5091957
[21] M B Franca A D Panek and E C A Eleutherio ldquoOxidativestress and its effects during dehydrationrdquoComparative Biochem-istry and PhysiologymdashA Molecular and Integrative Physiologyvol 146 no 4 pp 621ndash631 2007
[22] R Cruz de Carvalho M Catala J Marques da Silva CBranquinho and E Barreno ldquoThe impact of dehydration rateon the production and cellular location of reactive oxygen
species in an aquatic mossrdquo Annals of Botany vol 110 no 5 pp1007ndash1016 2012
[23] I Kranner and S Birtic ldquoA modulating role for antioxidants indesiccation tolerancerdquo Integrative and Comparative Biology vol45 no 5 pp 734ndash740 2005
[24] R Cornette and T Kikawada ldquoThe induction of anhydrobiosisin the sleeping chironomid current status of our knowledgerdquoIUBMB Life vol 63 no 6 pp 419ndash429 2011
[25] L Rebecchi ldquoDry up and survive the role of antioxidantdefences in anhydrobiotic organismsrdquo Journal of Limnology vol72 no 1 pp 62ndash72 2013
[26] A M Rizzo M Negroni T Altiero et al ldquoAntioxidant defencesin hydrated and desiccated states of the tardigrade Paramac-robiotus richtersirdquo Comparative Biochemistry and Physiology BBiochemistry and Molecular Biology vol 156 no 2 pp 115ndash1212010
Exposure to space stress conditions induces oxidative stressOxidative stress resulting from an imbalance between theexcessive production of reactive oxygen species (ROS) andlimited action of antioxidant defences is implicated inthe development of many important human pathologiesincluding atherosclerosis hypertension inflammation can-cer Parkinson and Alzheimer diseases [21] Oxidative stressmay be highly destructive also in anhydrobiotic organismseven if the lower cellular water content decreases the produc-tion of ROS [21 22] Under normal conditions antioxidantsystems minimize the adverse effects caused by ROS butdesiccation stress could cause the loss or reduction of thesedefence control mechanisms since the metabolic activity isabsent or reduced [21ndash24]
The ability of some animals tardigrades among themto survive extreme desiccation involves a complex arrayof factors working at structural physiological and molec-ular level From a molecularbiochemical point of viewanhydrobiotic organisms synthesize molecules working asbioprotectants during entering permanence and leaving ina desiccated state [25] For example trehalose and sucrosestabilise the biological membrane avoiding protein unfoldingand membrane disturbances late embryogenesis abundantproteins and heat shock proteins work as chaperone systemsrepairing or eliminating damaged molecules while antiox-idant molecules counteract the negative effects of oxidativestress [25]
Since it is known that both hydrated and desiccatedtardigrades have a good natural capability to overcomeoxidative stress [26] they have been used in TARDIKISSexperiments to evaluate the role of antioxidant defence inovercoming oxidative stress induced by exposure to spacestress conditions such as ionizing and UV radiations
The first space experiment (TARSE) conducted withhydrated starved specimens of the tardigrade P richtersidemonstrated that some of the enzymes involved in antiox-idant defences were significantly influenced by the flightstresses [1] In particular there was a significant decrease incatalase and SOD activities the more active enzymes in Prichtersi In addition the glutathione system the less activesystem in not stressed specimens of this species [26] wassignificantly induced during space flight [1] These resultscould be related to the stresses experienced by the hydratedand metabolically active animals (microgravity starvationand radiations) during the flight On the contrary the analysisof antioxidant defences in desiccated tardigrades of theTARDIKISS experiment showed fewer differences relatedto space flight even if the tendency was similar to thatrecorded in hydrated metabolically active animals of theTARSE experiment A similar trend between TARSE andTARDIKISS experiments was also detected in regard to tardi-grade survival since flight animals did not show significantdifferences in survival from temperature laboratory controlones [1 11] Only inR oberhaeuseri (TARDIKISS experiment)a significant decrease in survival rate was recorded between Fand TC samples the species in which a significant decrease ofthe C226 n-3 fatty acid and of glutathione reductase activity
and even though not significant of the activity of the otherROS scavenging enzymes were detected
In conclusion TARDIKISS experiment together withprevious space experiments using tardigrades [1 7ndash9] furtherconfirms that both desiccated and hydrated physiologicalstates of tardigrades represent useful animal tool for spaceresearch To further develop the space research using tardi-grades the setup of experimentswith the possibility to changethe exposition condition of metabolically hydrated animalsas well as the possibility to expose desiccated tardigrades toopen space is necessary Experiments under true space con-dition provide a realistic evaluation of the mechanisms thatcould allowmulticellular organisms including tardigrades tosurvive the combined and synergic effects of space stressorsNevertheless experiments on ground using simulators ofmicrogravity radiation temperature and other space stressesare an essential part of space research complementing exper-iments under true space conditions The comparisons of twodifferent sets of data (ground and space data) will allow betterunderstanding of the physiological and molecular pathwaysof living organisms under space environment
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are very grateful to the Italian Space Agency(ASI) and the Italian Air Force (AM) which funded theDAMA mission The authors are also very grateful to KayserItalia (KI) which developed and manufactured the hardwareinvolved in the BIOKIS payload They are grateful to anony-mous reviewers for their constructive suggestions
References
[1] L Rebecchi T Altiero R Guidetti et al ldquoTardigrade resistanceto space effects First results of experiments on the LIFE-TARSEMission onFOTON-M3 (September 2007)rdquoAstrobiology vol 9no 6 pp 581ndash591 2009
[2] R Guidetti A M Rizzo T Altiero and L Rebecchi ldquoWhatcan we learn from the toughest animals of the Earth Waterbears (tardigrades) asmulticellularmodel organisms in order toperform scientific preparations for lunar explorationrdquoPlanetaryand Space Science vol 74 no 1 pp 97ndash102 2012
[3] H Marthy ldquoDevelopmental biology of animal models undervaried gravity conditions a reviewrdquo Vie et Milieu vol 52 no4 pp 149ndash189 2002
[4] N Moslashbjerg K A Halberg A Joslashrgensen et al ldquoSurvival inextreme environmentsmdashon the current knowledge of adapta-tions in tardigradesrdquo Acta Physiologica vol 202 no 3 pp 409ndash420 2011
[5] R Guidetti T Altiero and L Rebecchi ldquoOn dormancy strate-gies in tardigradesrdquo Journal of Insect Physiology vol 57 no 5pp 567ndash576 2011
[6] T Altiero R Guidetti V Caselli M Cesari and L RebecchildquoUltraviolet radiation tolerance in hydrated and desiccated
BioMed Research International 7
eutardigradesrdquo Journal of Zoological Systematics and Evolution-ary Research vol 49 supplement 1 pp 104ndash110 2011
[7] K I Jonsson E Rabbow R O Schill M Harms-Ringdahl andP Rettberg ldquoTardigrades survive exposure to space in low Earthorbitrdquo Current Biology vol 18 no 17 pp R729ndashR731 2008
[8] L Rebecchi T Altiero M Cesari et al ldquoResistance of the anhy-drobiotic eutardigrade Paramacrobiotus richtersi to space flight(LIFE-TARSE mission on FOTON-M3)rdquo Journal of ZoologicalSystematics and Evolutionary Research vol 49 supplement 1 pp98ndash103 2011
[9] D Persson K A Halberg A Joslashrgensen C Ricci N Moslashbjergand R M Kristensen ldquoExtreme stress tolerance in tardigradessurviving space conditions in low earth orbitrdquo Journal of Zoolog-ical Systematics and Evolutionary Research vol 49 supplement1 pp 90ndash97 2011
[10] T Altiero L Rebecchi and R Bertolani ldquoPhenotypic varia-tions in the life history of two clones of Macrobiotus richtersi(Eutardigrada Macrobiotidae)rdquo Hydrobiologia vol 558 no 1pp 33ndash40 2006
[11] M Vukich P L Ganga D Cavalieri et al ldquoBIOKIS amodel payload for multisciplinary experiments in micrograv-ityrdquo Microgravity Science and Technology vol 24 pp 397ndash4092012
[12] A M Rizzo L Adorni G Montorfano F Rossi and B BerraldquoAntioxidant metabolism of Xenopus laevis embryos duringthe first days of developmentrdquo Comparative Biochemistry andPhysiologymdashB Biochemistry and Molecular Biology vol 146 no1 pp 94ndash100 2007
[13] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951
[14] F Paoletti and A Mocali ldquoDetermination of superoxide dis-mutase activity by purely chemical system based on NAD(P)HoxidationrdquoMethods in Enzymology vol 186 pp 209ndash220 1990
[15] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984
[16] M C Pinto A M Mata and J Lopez-barea ldquoReversible inacti-vation of Saccharomyces cerevisiae glutathione reductase underreducing conditionsrdquo Archives of Biochemistry and Biophysicsvol 228 no 1 pp 1ndash12 1984
[17] J R Prohaska and H E Ganther ldquoSelenium and glutathioneperoxidase in developing rat brainrdquo Journal of Neurochemistryvol 27 no 6 pp 1379ndash1387 1976
[18] O W Griffith ldquoGlutathione and glutathione disulphiderdquo inMethods of Enzymatic Analysis H U Bergmeyer Ed vol 3 pp521ndash529 Academic Press New York NY USA 1984
[19] H E Wey L Pyron and M Woolery ldquoEssential fatty acid defi-ciency in cultured human keratinocytes attenuates toxicity dueto lipid peroxidationrdquo Toxicology and Applied Pharmacologyvol 120 no 1 pp 72ndash79 1993
[20] J Folch M Lees and G H S Stanley ldquoA simple method for theisolation and purification of total lipides from animal tissuesrdquoThe Journal of Biological Chemistry vol 226 no 1 pp 497ndash5091957
[21] M B Franca A D Panek and E C A Eleutherio ldquoOxidativestress and its effects during dehydrationrdquoComparative Biochem-istry and PhysiologymdashA Molecular and Integrative Physiologyvol 146 no 4 pp 621ndash631 2007
[22] R Cruz de Carvalho M Catala J Marques da Silva CBranquinho and E Barreno ldquoThe impact of dehydration rateon the production and cellular location of reactive oxygen
species in an aquatic mossrdquo Annals of Botany vol 110 no 5 pp1007ndash1016 2012
[23] I Kranner and S Birtic ldquoA modulating role for antioxidants indesiccation tolerancerdquo Integrative and Comparative Biology vol45 no 5 pp 734ndash740 2005
[24] R Cornette and T Kikawada ldquoThe induction of anhydrobiosisin the sleeping chironomid current status of our knowledgerdquoIUBMB Life vol 63 no 6 pp 419ndash429 2011
[25] L Rebecchi ldquoDry up and survive the role of antioxidantdefences in anhydrobiotic organismsrdquo Journal of Limnology vol72 no 1 pp 62ndash72 2013
[26] A M Rizzo M Negroni T Altiero et al ldquoAntioxidant defencesin hydrated and desiccated states of the tardigrade Paramac-robiotus richtersirdquo Comparative Biochemistry and Physiology BBiochemistry and Molecular Biology vol 156 no 2 pp 115ndash1212010
eutardigradesrdquo Journal of Zoological Systematics and Evolution-ary Research vol 49 supplement 1 pp 104ndash110 2011
[7] K I Jonsson E Rabbow R O Schill M Harms-Ringdahl andP Rettberg ldquoTardigrades survive exposure to space in low Earthorbitrdquo Current Biology vol 18 no 17 pp R729ndashR731 2008
[8] L Rebecchi T Altiero M Cesari et al ldquoResistance of the anhy-drobiotic eutardigrade Paramacrobiotus richtersi to space flight(LIFE-TARSE mission on FOTON-M3)rdquo Journal of ZoologicalSystematics and Evolutionary Research vol 49 supplement 1 pp98ndash103 2011
[9] D Persson K A Halberg A Joslashrgensen C Ricci N Moslashbjergand R M Kristensen ldquoExtreme stress tolerance in tardigradessurviving space conditions in low earth orbitrdquo Journal of Zoolog-ical Systematics and Evolutionary Research vol 49 supplement1 pp 90ndash97 2011
[10] T Altiero L Rebecchi and R Bertolani ldquoPhenotypic varia-tions in the life history of two clones of Macrobiotus richtersi(Eutardigrada Macrobiotidae)rdquo Hydrobiologia vol 558 no 1pp 33ndash40 2006
[11] M Vukich P L Ganga D Cavalieri et al ldquoBIOKIS amodel payload for multisciplinary experiments in micrograv-ityrdquo Microgravity Science and Technology vol 24 pp 397ndash4092012
[12] A M Rizzo L Adorni G Montorfano F Rossi and B BerraldquoAntioxidant metabolism of Xenopus laevis embryos duringthe first days of developmentrdquo Comparative Biochemistry andPhysiologymdashB Biochemistry and Molecular Biology vol 146 no1 pp 94ndash100 2007
[13] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951
[14] F Paoletti and A Mocali ldquoDetermination of superoxide dis-mutase activity by purely chemical system based on NAD(P)HoxidationrdquoMethods in Enzymology vol 186 pp 209ndash220 1990
[15] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984
[16] M C Pinto A M Mata and J Lopez-barea ldquoReversible inacti-vation of Saccharomyces cerevisiae glutathione reductase underreducing conditionsrdquo Archives of Biochemistry and Biophysicsvol 228 no 1 pp 1ndash12 1984
[17] J R Prohaska and H E Ganther ldquoSelenium and glutathioneperoxidase in developing rat brainrdquo Journal of Neurochemistryvol 27 no 6 pp 1379ndash1387 1976
[18] O W Griffith ldquoGlutathione and glutathione disulphiderdquo inMethods of Enzymatic Analysis H U Bergmeyer Ed vol 3 pp521ndash529 Academic Press New York NY USA 1984
[19] H E Wey L Pyron and M Woolery ldquoEssential fatty acid defi-ciency in cultured human keratinocytes attenuates toxicity dueto lipid peroxidationrdquo Toxicology and Applied Pharmacologyvol 120 no 1 pp 72ndash79 1993
[20] J Folch M Lees and G H S Stanley ldquoA simple method for theisolation and purification of total lipides from animal tissuesrdquoThe Journal of Biological Chemistry vol 226 no 1 pp 497ndash5091957
[21] M B Franca A D Panek and E C A Eleutherio ldquoOxidativestress and its effects during dehydrationrdquoComparative Biochem-istry and PhysiologymdashA Molecular and Integrative Physiologyvol 146 no 4 pp 621ndash631 2007
[22] R Cruz de Carvalho M Catala J Marques da Silva CBranquinho and E Barreno ldquoThe impact of dehydration rateon the production and cellular location of reactive oxygen
species in an aquatic mossrdquo Annals of Botany vol 110 no 5 pp1007ndash1016 2012
[23] I Kranner and S Birtic ldquoA modulating role for antioxidants indesiccation tolerancerdquo Integrative and Comparative Biology vol45 no 5 pp 734ndash740 2005
[24] R Cornette and T Kikawada ldquoThe induction of anhydrobiosisin the sleeping chironomid current status of our knowledgerdquoIUBMB Life vol 63 no 6 pp 419ndash429 2011
[25] L Rebecchi ldquoDry up and survive the role of antioxidantdefences in anhydrobiotic organismsrdquo Journal of Limnology vol72 no 1 pp 62ndash72 2013
[26] A M Rizzo M Negroni T Altiero et al ldquoAntioxidant defencesin hydrated and desiccated states of the tardigrade Paramac-robiotus richtersirdquo Comparative Biochemistry and Physiology BBiochemistry and Molecular Biology vol 156 no 2 pp 115ndash1212010
