1. Effects of some space flight factors on survivorship in crustacean resting stages V.R. Alekseev*, M.A. Levinskih**, N.D. Novikova**, V.N.Sychev**, T. Okuda***, *Zoological Institute of RAS, St. Petersburg, Russia ** Institute for biomedical problems RAS, Moscow, Russia *** National Institute of Agrobiological Science, Tsukuba, Japan

2. In accordance with the Russian space research program, the study on cryptobiosis is important for: Planetary and space craft quarantine Planetary and space craft quarantine Ecosystem transportation outside The Earth Biosphere Ecosystem transportation outside The Earth Biosphere Extraterrestrial life searching (Alekseev, Sychev, Novikova 2007) Extraterrestrial life searching (Alekseev, Sychev, Novikova 2007)

3. During space flight organisms are affected by a set of factors different from Earth conditions 1. Microgravity 2. Space (corpuscular) radiation 3. Transformed magnetic field 4. Over gravity (during start) 5. Industrial factors (noise, vibration, electro-magnetic field) 6. Varies combinations of 1-5 with possible synergic effects

4. OBJECTIVES of this study: to find out if Factors of Space Flight (FSF) play a negative role in vital abilities of cryptobiotic stages to find out if Factors of Space Flight (FSF) play a negative role in vital abilities of cryptobiotic stages if YES, what kind of effects the FSF on dormant stages can we get than and how to overcome the effects by before-flight measures if YES, what kind of effects the FSF on dormant stages can we get than and how to overcome the effects by before-flight measures

5. Two types of space experiments were conducted

6. 1.“AQUARIUM” experiment: Cryptobiotic stages were transported to ISS (Russian segment) and exposed 1-8 months INSIDE station

7. Resting stages of two species were selected for this experiment in space A phyllopod crustacean Streptocephalus torvicornis A cladoceran Daphnia magna

8. Материал: D. magna и S. torvicornis собирались в дафниевых прудах осетрового рыбзавода в дельте Волги D. magna и S. torvicornis собирались в дафниевых прудах осетрового рыбзавода в дельте Волги В этих прудах на протяжении 30 лет формировалась микроэкосистема из организмов способных к длительному биологическому покою. В этих прудах на протяжении 30 лет формировалась микроэкосистема из организмов способных к длительному биологическому покою.

9. 2. “Biorisk” experiment: cryptobiotic stages were placed for 13-18 months OUTSIDE ISSS

11. 1. Hatching of resting egg exposed inside ISS Hatching started on the third day after moving from 10 o to 25 o C Hatching started on the third day after moving from 10 o to 25 o C The reactivation rate : in ISS 39.6 % (30.6) The reactivation rate : in ISS 39.6 % (30.6) In control 51,8 % (39.9) (t-test; p=0.035) In control 51,8 % (39.9) (t-test; p=0.035)

12. Average body mass in newborn Daphnia after ISS treatment within one month

13. Size and reactivation in Daphnia First Medium Post

14. Differences in time of reactivation among clones confirmed with PCR F - first group F - first group M – middle group M – middle group P – posterior group P – posterior group

15. Discriminative analyze based on 24 signs revealed that time of reactivation in 3 clones were genetically specified F - first group F - first group M – middle group M – middle group P – posterior group P – posterior group

16. 1. 2 week NR Hatching =16.3+17.29% 2. 4 week NR Hatching = 23.5+18.16% 3. Control Hatching = 35.5 +20.75% p1-3=0,1 p2-3=0.22

17. FSF also affected some life cycle parameters in D. magna. The first clutch size : The first clutch size : in ISS-treated (11+3.6 egg i -1 ) in ISS-treated (11+3.6 egg i -1 ) in control group (14.3+2.8 egg i -1 ) in control group (14.3+2.8 egg i -1 ) ( t-test; p=0.0477). ( t-test; p=0.0477). Maturation time: Maturation time: in ISS (9.95+0.284 day) in ISS (9.95+0.284 day) in control (10.31+0.372 day) in control (10.31+0.372 day) (Mann-Whitney test z adjust =2.09, p=0.033).

18. Males in D. magna in offspring. In ISS offspring 54.9+25.94% males In ISS offspring 54.9+25.94% males No a single male in offspring from control group! No a single male in offspring from control group!

19. S. torvicornis resting eggs were used to trace increasing of negative changes with time Resting eggs of this species were collected in the same ponds in density several times more than Daphnia ephippia Resting eggs of this species were collected in the same ponds in density several times more than Daphnia ephippia

21. Effect of resting egss exposing at ISS on S. torvicornis hatching 1 month exposing, hatching/150 cysts 8 month exposing, hatching/150 cysts Controlgroup42+853+13 Experimentalgroup21+611.2+1,5

22. 2. Nine species were selected to study effect of open space conditions Daphnia magna (winter dormancy) Daphnia magna (winter dormancy) Daphnia pulicaria (summer dormancy) Daphnia pulicaria (summer dormancy) Streptocephalus torvicornis Streptocephalus torvicornis Artemia salina Artemia salina Eucycpris ornata Eucycpris ornata Polypedium vanderplankii Polypedium vanderplankii Nothobranchius guenteri (eggs of African fish) Nothobranchius guenteri (eggs of African fish)

23. Sleeping under the Sun

24. Results of “Biorisk” treatments Daphnia magna Daphnia magna Daphnia pulicaria Daphnia pulicaria Streptocephalus torvicornis Streptocephalus torvicornis Eucycpris ornata Eucycpris ornata Nothobranchius guenteri Nothobranchius guenteri Polypedium vanderplankii Polypedium vanderplankii Artemia salina Artemia salina Survived Survived Died Died Survived Survived Died Died Survived Survived

25. Trehalose content in resting stages Species Code Trehalose, % Daphnia, large ephippia (our data)DSE24.951 Daphnia large ephippia (our data)DLE19.8677 Ostracoda ( our data) OST17.3826 Streptocephalus (large) our data SLC25.07 Streptocephalus (small) our data SSC8.5 Artemia (after Clegg, 1965)ART15 P. vanderplanki (after Kikawada et al., 2007)HIR20

27. Conclusions: Cryptobiotic stages of aquatic invertebrates are effected by FSF inside space station but also are able to overcome them outside station Cryptobiotic stages of aquatic invertebrates are effected by FSF inside space station but also are able to overcome them outside station Space radiation seems like responsible for main negative effect inside station exposing Space radiation seems like responsible for main negative effect inside station exposing Resistance to FSF in most species studied was related with trehalose content in their bodies that can help for before-flight selection/preparation the most resistant to FSF specimens Resistance to FSF in most species studied was related with trehalose content in their bodies that can help for before-flight selection/preparation the most resistant to FSF specimens

28. Acknowledgments: This study was supported by RFBR-Japan bilateral grants This study was supported by RFBR-Japan bilateral grants VA got a fellowship from Max-Planck-Society VA got a fellowship from Max-Planck-Society Drs. N. Abramson. T. Kikawada, O. Gusev and Prof. Vladimir Tzetlin are very much appreciated for significant help in experiments and productive discussion on their results Drs. N. Abramson. T. Kikawada, O. Gusev and Prof. Vladimir Tzetlin are very much appreciated for significant help in experiments and productive discussion on their results THANK YOU FOR ATTENTION!