Effects of some space Effects of some space flight factors on flight factors on survivorship in survivorship in

crustacean resting crustacean resting stagesstages

V.RV.R.. Alekseev*,  M.A Alekseev*,  M.A.. Levinskih**, N.D. Levinskih**, N.D. Novikova**, Novikova**,

V.N.Sychev**, T. Okuda***,V.N.Sychev**, T. Okuda***, *Zoological Institute of RAS, St. Petersburg, Russia*Zoological Institute of RAS, St. Petersburg, Russia

**** Institute for biomedical problems RAS, Moscow, RussiaInstitute for biomedical problems RAS, Moscow, Russia****** National Institute of Agrobiological Science, Tsukuba , Japan National Institute of Agrobiological Science, Tsukuba , Japan

In accordance with the Russian space In accordance with the Russian space research program, the study on research program, the study on cryptobiosis is important for:cryptobiosis is important for:• Planetary and space craft quarantinePlanetary and space craft quarantine• Ecosystem transportation outside Ecosystem transportation outside

The Earth BiosphereThe Earth Biosphere• Extraterrestrial life searching Extraterrestrial life searching

(Alekseev, Sychev, Novikova 2007) (Alekseev, Sychev, Novikova 2007)

During space flight organisms During space flight organisms are affected by a set of factors are affected by a set of factors different from Earth conditionsdifferent from Earth conditions1.1. MicrogravityMicrogravity2.2. Space (corpuscular) radiationSpace (corpuscular) radiation3.3. Transformed magnetic fieldTransformed magnetic field4.4. Over gravity (during start)Over gravity (during start)5.5. Industrial factors (noise, vibration, Industrial factors (noise, vibration,

electro-magnetic field)electro-magnetic field)6.6. Varies combinations of 1-5 with Varies combinations of 1-5 with

possible synergic effectspossible synergic effects

OBJECTIVES of this study:OBJECTIVES of this study:• to find out if Factors of Space Flight to find out if Factors of Space Flight

(FSF) play a negative role in vital (FSF) play a negative role in vital abilities of cryptobiotic stagesabilities of cryptobiotic stages

• if YES, what kind of effects the FSF if YES, what kind of effects the FSF on dormant stages can we get than on dormant stages can we get than and how to overcome the effects by and how to overcome the effects by before-flight measuresbefore-flight measures

Two types of space Two types of space experiments were experiments were

conducted conducted

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

Resting stages of two species Resting stages of two species were selected for this experiment were selected for this experiment in spacein space

A phyllopod crustacean Streptocephalus torvicornis A cladoceran Daphnia magna

МатериалМатериал::

• D. magnaD. magna ии S. torvicornis S. torvicornis собирались в собирались в дафниевых прудах осетрового дафниевых прудах осетрового рыбзавода в дельте Волгирыбзавода в дельте Волги

• В этих прудах на протяжении 30 лет В этих прудах на протяжении 30 лет формировалась микроэкосистема из формировалась микроэкосистема из организмов способных к длительному организмов способных к длительному биологическому покоюбиологическому покою. .

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

1. Hatching of resting egg 1. Hatching of resting egg exposed inside ISS exposed inside ISS

• Hatching started on the third day after Hatching started on the third day after moving from 10moving from 10oo to 25 to 25oo C 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)

Hatching of D. magna in control and after 1 month ISS treatment

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16Days after beginning of reactivation

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Hatching of D. magna in control and after 1 month ISS treatment

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Average body mass in newborn Average body mass in newborn Daphnia after ISS treatment within one Daphnia after ISS treatment within one

monthmonth

0

0.001

0.002

0.003

0.004

0.005

0.006

0.007 Сухой вес, мг

10/27 10/28 10/29 10/30 10/31 11/1 11/2 11/3 11/4 11/5 11/6 11/7 Даты

Серии

Size and reactivation in Size and reactivation in DaphniaDaphnia

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0.007 Сухой вес, мг

10/27 10/28 10/29 10/30 10/31 11/1 11/2 11/3 11/4 11/5 11/6 11/7 Даты

Серии

Hatching of D. magna in control and after 1 month ISS treatment

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16Days after beginning of reactivation

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First

Medium

Post

Differences in time of Differences in time of reactivation among clones reactivation among clones confirmed with PCRconfirmed with PCR

• F - first F - first groupgroup

• M – middle M – middle groupgroup

• P – P – posterior posterior groupgroup

Discriminative analyze based on Discriminative analyze based on 24 signs revealed that time of 24 signs revealed that time of reactivation in 3 clones were reactivation in 3 clones were genetically specifiedgenetically specifiedRoot 1 vs. Root 2

F M P-8 -6 -4 -2 0 2 4 6

Root 1

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-1

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• F - first F - first groupgroup

• M – M – middle middle groupgroup

• P – P – posterior posterior groupgroup

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24.04.2006 01.05.2006 08.05.2006 15.05.2006Dates

Effect of neutron radiation on D. magna hatching(%) from resting eggs

NR

Control

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

FSF also affected some life FSF also affected some life cycle parameters in cycle parameters in D. D. magnamagna..• The first clutch sizeThe first clutch size : : in ISS-treated (11+3.6 egg i in ISS-treated (11+3.6 egg i -1-1) ) in control group (14.3+2.8 egg iin control group (14.3+2.8 egg i-1-1) ) ( t-test; ( t-test; pp=0.0477).=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 (Mann-Whitney test zzadjustadjust=2.09, =2.09, pp=0.033). =0.033).

Males in Males in D. magna D. magna in in offspring.offspring.

• In ISS offspring In ISS offspring 54.9+25.94% 54.9+25.94% malesmales

• No a single male No a single male in offspring from in offspring from control group! control group!

S. torvicornisS. torvicornis resting eggs resting eggs were used to trace increasing were used to trace increasing of negative changes with timeof negative changes with time

• Resting eggs of Resting eggs of this species were this species were collected in the collected in the same ponds in same ponds in density several density several times more than times more than DaphniaDaphnia ephippia ephippia

Reactivation in S. torvicornis after ISS and in control

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Days after start

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Effect of resting egss exposing Effect of resting egss exposing at ISS on at ISS on S. torvicornisS. torvicornis hatchinghatching

1 month 1 month exposing,exposing,hatching/hatching/150 cysts150 cysts

8 month 8 month exposing, exposing, hatching/150 hatching/150 cystscysts

ControlControlgroupgroup

42+842+8 53+1353+13

ExperimentaExperimentallgroupgroup

21+621+6 11.2+1,511.2+1,5

2. Nine species were selected 2. Nine species were selected to study effect of open space to study effect of open space conditionsconditions• Daphnia magna (winter Daphnia magna (winter

dormancy)dormancy)• Daphnia pulicaria Daphnia pulicaria

(summer dormancy)(summer dormancy)• Streptocephalus Streptocephalus

torvicornistorvicornis• Artemia salinaArtemia salina• Eucycpris ornataEucycpris ornata• Polypedium Polypedium

vanderplankiivanderplankii• Nothobranchius Nothobranchius

guenteri (eggs of guenteri (eggs of African fish)African fish)

Sleeping under the SunSleeping under the Sun

Results of “Biorisk” Results of “Biorisk” treatmentstreatments• Daphnia magnaDaphnia magna• Daphnia pulicariaDaphnia pulicaria• Streptocephalus Streptocephalus

torvicornistorvicornis• Eucycpris ornataEucycpris ornata• Nothobranchius guenteriNothobranchius guenteri• Polypedium vanderplankiiPolypedium vanderplankii• Artemia salinaArtemia salina

• Survived Survived • Died Died • SurvivedSurvived• SurvivedSurvived• DiedDied• SurvivedSurvived• SurvivedSurvived

Trehalose content in resting Trehalose content in resting stages stages

Species Code

Trehalose, %

Daphnia, large ephippia (our data) DSE 24.951Daphnia large ephippia (our data) DLE 19.8677Ostracoda (our data) OST 17.3826Streptocephalus (large) our data SLC 25.07Streptocephalus (small) our data SSC 8.5Artemia (after Clegg, 1965) ART 15

P. vanderplanki (after Kikawada et al., 2007) HIR 20

Trehalose content and resting stage reactivation

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DSE DLE OST SLC SSC ART HIR

SPECIES

Treh

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Trehalose, %Reactivation, %

Conclusions:Conclusions:• Cryptobiotic stages of aquatic invertebrates Cryptobiotic stages of aquatic invertebrates

are effected by FSF inside space station but are effected by FSF inside space station but also are able to overcome them outside stationalso are able to overcome them outside station

• Space radiation seems like responsible for Space radiation seems like responsible for main negative effect inside station exposingmain negative effect inside station exposing

• Resistance to FSF in most species studied was Resistance to FSF in most species studied was related with trehalose content in their bodies related with trehalose content in their bodies that can help for before-flight that can help for before-flight selection/preparation the most resistant to FSF selection/preparation the most resistant to FSF specimensspecimens

Acknowledgments:Acknowledgments:• This study was supported by RFBR-Japan bilateral grantsThis study was supported by RFBR-Japan bilateral grants• VA got a fellowship from Max-Planck-SocietyVA got a fellowship from Max-Planck-Society• Drs. N. Abramson. T. Kikawada, O. Gusev and Prof. Vladimir Drs. N. Abramson. T. Kikawada, O. Gusev and Prof. Vladimir

Tzetlin are very much appreciated for significant help in Tzetlin are very much appreciated for significant help in experiments and productive discussion on their resultsexperiments and productive discussion on their results

THANK YOU FOR ATTENTION!THANK YOU FOR ATTENTION!