EXPERIMENT “BIOPHOBOS/ANABYOSIS” IN PROJECT “PHOBOS-GRUNT”

1. EXPERIMENT “BIOPHOBOS/ANABYOSIS” IN PROJECT “PHOBOS-GRUNT” О.I.Orlov1 V.R.Alekseev2 Е.А.Vorobyeva3 V.F.Galchenko4 Е.А.Deshevaya1 А.V.Zakharov5 М.А.Levinskikh1 О.V.Muravenko6 N.D.Novikova1 S.V.Poddubko1 N.А.Polykarpov1 V.N.Sychev1 1 - SSC RF – Institute of Biomedical Problems RAS, Moscow 2 - Zoological Institute RAS, St.Petersburg 3 - Soil Science Faculty, Moscow State University, Moscow 4 - Institute of Microbiology RAS, Moscow 5 - Space Research Institute RAS, Moscow 6 - Institute of Molecular Biology RAS, Moscow

2. LONG-TERM ASTROBIOLOGICAL RESEARCH PROGRAM Пути достижения поставленной цели Long-term program of applied science research and planned experiments onboard RS ISS Experiments : Biorisk-MSV; Biorisk-MSN; Expose-R; Aquarium; Plants-Seeds MARS-500 Program Satellite experiments Project “PHOBOS-GRUNT” Experiment “Biophobos/Anabiosis”

3. AIMS OF THE ASTROBIOLOGICAL RESEARCH Elucidation of viability of resting forms of organisms of different taxonomical groups under conditions of spaceflight and continuous interplanetary flights for solving problems of planetary quarantine, astrobiology and bio-medical problems of manned cosmonautics.

4. THE STUDY OF DORMANT FORMS OF VARIOUS ORGANISMS UNDER SPACE FLIGHT CONDITIONS To expose dormant forms of various organisms on the external surface of the ISS, which include not only bacterial and fungal spores but also seeds and dormant embryos of lower crustaceans, as well as multi-cellular organism in the cryptobiotic state. Cooperation with: Moscow State University, Moscow, Russia; Zoological Institute of RAS, St. Petersburg, Russia; Institute of microbiology of RAS, Moscow, Russia; St. Petersburg St. State University, St. Petersburg, Russia; Space Dynamic Laboratory, Utah State University, Utah, USA; Research Institute for Bioresources, Okayama University, Okayama, Japan; National Institute of Agrobiological Sciences, Ibaraki, Japan. INSIDE: RASTENIYA-2 AQUARIUM BRADOS BIORISK - KM BIORISC - MSV OUTSIDE: BIORISK – MSN EXPOSE-R

5. EXPERIMENT “BIORISK-MSN-2” HIGHER PLANTS Air-dry seeds of mustard Brassica rapa, Improved Basic line Air-dry seeds of mustard Brassica rapa, High Anthocyanin line Air-dry seeds of mustard Brassica rapa, Anthocyaninless line Air-dry seeds of mustard Brassica rapa, Rosette line Air-dry seeds of mustard Brassica rapa, AstroPlants line Air-dry seeds of mustard Brassica rapa, Somatic Variegation line Air-dry seeds of mustard Brassica juncea, RG Air-dry seeds of Arabidopsis thaliana, K-310 line Air-dry seeds of Arabidopsis thaliana, Columbia line Air-dry seeds of weed Nicandra physaloides Air-dry seeds of tomatoes Lycopersicum esculentium, Micro-Tom var. Air-dry seeds of radishes Raphanus sativus, Cherry Bomb var. Air-dry seeds of barley Hordeum vulgare, Haruna Nijo cv Air-dry seeds of barley Hordeum vulgare, K-305 cv Air-dry seeds of rice Oryza sativa, Rsp-5 Air-dry seeds of rice Oryza sativa, Rsp-7 VERTEBRATES Dry eggs of fish Nothobranchius guenteri INSECTS Cryptobiotic larvae of chyronamide Polypedium vanderplankii LOWER CRUSTACEANS Dormant eggs of Daphnia magna Dormant eggs of Daphnia pullicaria Dormant eggs of Artemia salina Dormant eggs of Streptocephalus torvicornis Dormant eggs of Eucypris ornate MOLDS Spores of Aspergillus sedowii # 9-6 Spores of Aspergillus versicolor # 4-3-4 Spores of Penicillium aurantiogresium # 9-9 Spores of Penicillium expansum # 4-3-3 BACTERIA Spores of Bacillus subtilis # 20 Spores of Bacillus licheniformis # 24 Spores of Bacillus subtilis # 25 Spores of Bacillus subtilis # 2335\105 Duration of the exposure: The 1-st container – 13 months The 2-nd container - 18 months The 3-rd container – 31 months

6. EXPERIMENT “BIORISK-MSN” ONBOARD OF RUSSIAN SEGMENT OF THE INTERNATIONAL SPACE STATION Germinated “space” seeds of barley Hordeum vulgare cv. Haruna Nijo and plants of radish Raphanus sativus strain «Cherry Bomb», grown from “space” seeds “Biorisk-MSN” hardware and its location on “Pirs” Docking Compartment Larvae of chironomid Polypedilum vanderplanki revived after 13, 18 and 31 months of exposition on the outer side of RS of ISS Growth of Bacillus subtilis # 24 strain after 13-months exposure on the outer side of ISS Reactivation of “space” and control resting eggs of Artemia salina

7. EXPERIMENT “EXPOSE-R” (Start March 10, 2009) At present Russian scientists participate ESA experiment EXPOSE-R onboard ISS. In contrast to Biorsik-MSN experiment, a part of biological samples in EXPOSE-R is being exposed to solar UV. BACTERIA Group 18, subgroup Bacillaceae, genera Bacillus, species: Bacillus subtilis-2335/105 ; Bacillus subtilis-20 ; Bacillus pumilus-25 ; Bacillus licheniformis-24. Gram-positive rods, 0,5-2,5 x 1,2-10 mkm, spore - 6 mkm FUNGI Taxon - Mitosporic fungi, genera Aspergillus, Penicillium, Geomyces species: Aspergillus sydowi - 9-6; Geomyces pannorum – 2241; Aspergillus versicolor -4-3-4; Penicillium aurantiogresium –9-9; Penicillium expansum – 4-3-3. Spore 3-10 mkm ANIMALS Dried encysted embryos (diameter of a single egg is 200 micron) Daphnia magna belongs to family Daphniidae, order Daphniiformes, class Crustacea. Dried encysted embryos (diameter of a single egg is 160 micron) Streptocephalus torvicornis belongs to family Streptocephalidae, order Anostraca, class Crustacea. Dried encysted embryos(diameter of a single egg is 180-200 micron) Artemia salina belongs to family Artemiidae, order Anostraca, class Branchiopoda, subphylum: Crustacea. Dried encysted embryos(diameter of a single egg is 180-200 micron) Eucypris species belongs to family Cyprididae, order Ostracoda, class Crustacea. Cryptobiotic dried larvae (length 5 mm  0,8 mm) of Polypedilum vanderplankibelongs to family Chironomidae, order Diptera, class Insecta. PLANTS Seeds ofLycopersicum esculentum(Tomato Micro-Tom) Seeds ofRaphanus sativus(Radish Cherry Bomb) Seeds ofArabidopsis thalianaline WT–2.

8. EXPERIMENT “EXPOSE-R”(Start March 10, 2009) The biological objects in “EXPOSE-R” experiment are placed in plastic film bags 7x7 mm. The bags are fixed in the cells of special sets assembled in trays. EXPOSE-R hardware contains three trays composed by 4 sets of the cells with biological samples. The biological samples arranged in three levels, and those of the top level are exposed to solar UV.

9. EXPERIMENT “BIOPHOBOS/ANABYOSIS” • For realization of biological research in “Phobos-Grunt” project, a Workgroup chaired by academician RAS O.I. Orlov was organized by decision of the section “Space biology and physiology” of RAS Council on March 19, 2007. The workgroup consists of representatives of GSC IMBP RAS, ISS RAS, ZIN RAS, MSU, IMB RAS, FSCP “SAO after S.A. Lavochkin”. The workgroup has defined the main aims of biological experiments (Experiment “BioPhobos/Anabiosis”) in “Phobos-Grunt” project: • investigation of survival of resting stages of bacteria, fungi, animals and plants under conditions of interplanetary flights for estimation of damaging effect of factors of space environment on cryptobiotic stages of organisms and further development of technology of long-term preservation of organisms in conditions of biological resting; • investigation of biological, biochemical and molecular-genetic changes in organisms after long-term spaceflight;

10. EXPERIMENT “BIOPHOBOS/ANABYOSIS” • estimation of risk related to possibility of transformation of microorganisms inhabiting the space object after their exposure to outer space with further return inside of the space object. • comparison of survival under conditions of the spaceflight of resting stages with different level of survivability, including that of artificially induced be mean of cultivation with biologically active substances, for developing new biotechnological methods of investigations of resting stages for continuous space transportation. • comparison of survival of resting stages of organisms under space flight conditions (BioPhobos/Anabiosis) and an orbital flight in control experiment on low Earth orbit on the outer side of RS of ISS (experiment “Biorisk-MSN”).

11. EXPERIMENT “BIOPHOBOS/ANABYOSIS” Spores of the following prokaryotes (bacteria) are included in the list of experiment “BioPhobos/Anabiosis”

12. EXPERIMENT “BIOPHOBOS/ANABYOSIS” Spores of the following eukaryotes (fungi) are included in the list of experiment “BioPhobos/Anabiosis”

13. EXPERIMENT “BIOPHOBOS/ANABYOSIS” Animals Resting forms of the following higher eukaryotes are included in the list of experiment “BioPhobos/Anabiosis” Plants

14. Capsule with Phobos’ grunt and biological samples to be returned to the Earth For placing of the biological objects in two titanium “Phobos-capsules” with the size of  11  35 mm, technology similar to that of “EXPOSE-R” experiment will be used. 2 “Phobos-capsule” with 122 (1010 mm) packs with different biological objects

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