1. National Aeronautics and Space Administration Fruit Fly Lab – 01 Op TIM Payload Team Kevin Martin Matthew Lera Sharmila Bhattacharya Zhe Lu Ops Lead Crissy Canerday Crew Rep Lisa Anderson Payload Team Kevin Martin Matthew Lera Sharmila Bhattacharya Zhe Lu Ops Lead Crissy Canerday Crew Rep Lisa Anderson 6/16/2013

2. This research addresses several areas outlined in the Decadal Survey:  AH14 & AH15 Both to address the mechanism(s) of the changes in the immune system and to develop measures to limit the changes, data from multiple organ/system-based studies need to be integrated.  AH16 Study the transmission of structural and functional changes induced by exposure to space across multiple generations.  CC8 Expand the use of animal studies to assess space radiation risks to humans from cancer, cardiovascular disease, neurologic dysfunction, degenerative disease It also addresses areas of NASA’s Fundamental Space Biology Plan 2010-2020 2.  Do changes in gravity affect the basic metabolic rate and metabolism of living systems? Is Lifespan affected? Are changes expressed in altered gravity environments reversible when returned to 1g?  Are the normal defense systems of organisms compromised at fractional/hyper gravity?  Do organisms that are raised in altered gravity environments develop normally? Are reproduction, lifespan and the aging processes affected? Why is this research needed?

3. Aim: Replicate and extend scientific return from FIT experiment, STS 121, 2006 (PI for FIT mission: Dr. Sharmila Bhattacharya, NASA ARC) and baseline future Drosophila spaceflight hardware capabilities. Science data obtained from FIT: Reduced immune function after spaceflight Developmental changes & slight increase in lethality (presumably at pupal stage) All assays conducted immediately after landing with live samples (genomics, phagocytosis cellular assays, clearance of gram negative E.coli infection postflight, counts of animals in each developmental stage to assess increased lethality) Data published in 2011 (PLoS One; 6(1): p. e15361, doi:10.1371/journal.pone.0015361) Science Data to be Obtained on FFL-01: Immunological, developmental, cellular, and molecular changes and lethality assessment following bacterial infection in-flight assessed via: Imaging of insects during flight Dissection of and examination of insects post-flight Protein expression during and post-flight 3 What will be accomplished?

4. What technology will be demonstrated? Support Drosophila studies on ISS by upgrading capabilities of existing hardware that supported successful immunology study on STS-121. Demonstrating reflight of FIT hardware on existing ISS centrifuge facilities to provide on-board 1g control experiments for the first time. Overcome increased sample handover time issues (compared to previous shuttle flights) thus demonstrating the science utility of SpaceX flights. Implement fly treatment technique to study immune response of organism to in-flight challenge. Gather behavioral data using a new camera system compatible with existing micro gravity and centrifuge hardware. Demonstrate ability of hardware to support multigenerational studies on-orbit. 4

5. What will be the impact of this research? Value to Agency (Space Benefit) More extensive knowledge of genetic responses to micro-gravity and effects on immunity, behavior, and reproduction in a complex organism that has been extensively used in labs as a model for humans. This is a capability that is lacking, but desired, by all of the international partners for on-orbit space biology research. A better understanding of the risks space-flight imposes, particularly posed during long-term missions, on astronaut health, which may lead to the development of future countermeasures. Value to Public (Earth Benefit) Microgravity exposure has unmasked genetic mechanisms in simpler organisms, and needs to be studied in more complex organisms. Strong potential for education outreach paired with the science. 5

6. Hardware / Facility Descriptions Fly Cassette with food tray partially inserted. 6 Changeout Platform with cassette. Food tray in right slot. Tethered T-bar inserted in left slot. µg rack and centrifugeDouble-volume Type-1 Container

7. Crew Operations Timeline 7 Launch FD ~5/6 new food tray provided to old cassettes. Old food trays frozen in new cassettes. FD ~12 old food trays transferred to new cassettes. Old cassettes frozen. FD ~20 treated food trays provided to ½ old cassettes. Old food trays frozen in new cassettes. FD ~1 Install 6 cassettes containing flies each on centrifuge and µg rack Op #1 Op #2 Op #3Op #4 FD ~26 subset of old food transferred to new cassettes. Both frozen. Another subset left alone. Op #5 FD ~28 all frozen cassettes transferred to Dragon. Live samples packed for ambient return. Op #6

8. Crew Operations Detail 8 Op #2Op #3Op #1 1.Remove from launch packaging 12 fly cassettes containing flies. 2.Insert 6 fly cassettes into double-volume type-1 containers on µg rack. 3.Insert remaining 6 fly cassettes into double-volume containers on centrifuge. 4.Set centrifuge to Earth gravity and start centrifuge. 5.Initiate video recording (TBD). 1.Retrieve from 4 °C cold stowage 12 changeout platforms containing food. 2.Remove 12 cassettes from µg rack and centrifuge. 3.Place cassette into center of changeout platform, aligning food trays in platform and cassette. 4.Using tethered T-bar, insert new food tray into cassette. 5.Remove cassette from platform and insert into double-volume type-1 container on µg rack or centrifuge. 6.Place new blank cassette into used platform and insert old food tray using tethered T-bar. 7.Place new cassette containing old food tray into MELFI. 8.Repeat for all 12 cassettes. 9.Restart centrifuge. 10.Initiate video recording (TBD). 1.Retrieve from ambient stowage 12 changeout platforms containing empty food trays. 2.Remove 12 cassettes from µg rack and centrifuge. 3.Place cassette into center of changeout platform, aligning food trays in platform and cassette. 4.Using tethered T-bar, insert empty food tray into cassette. 5.Remove old cassette from platform and place into MELFI. 6.Place new blank cassette into used platform and insert old food tray using tethered T-bar. 7.Remove new cassette containing old food tray from platform and insert into double-volume type-1 container on µg rack or centrifuge. 8.Repeat for al 12 cassettes. 9.Restart centrifuge. 10.Initiate video recording (TBD). 1 hour2 hours

9. Crew Operations Detail (cont.) 9 Op #5 Op #6 Op #4 1.Retrieve from 4°C cold stowage 6 changeout platforms containing food and 6 platforms containing treated food. 2.Remove 12 cassettes from µg rack and centrifuge. 3.Place cassette into center of changeout platform, aligning food trays in platform and cassette. 4.Using tethered T-bar, insert new food tray into cassette. 5.Remove cassette from platform and insert into double-volume type-1 container on µg rack or centrifuge. 6.Place new blank cassette into used platform and insert old food tray using tethered T-bar. 7.Place new cassette containing old food tray into MELFI. 8.Repeat for all cassettes. 3 food trays with treated food will be provided to cassettes in µg rack, and 3 to cassettes in centrifuge. 9.Restart centrifuge. 10.Initiate video recording (TBD). 1.Retrieve from ambient stowage 12 changeout platforms containing empty food trays (previously used in Op #3). 2.Remove 5 cassettes from µg rack and 5 cassettes from centrifuge. 3.Place cassette into center of changeout platform, aligning food trays in platform and cassette. 4.Using tethered T-bar, insert blank food tray into cassette. 5.Remove old cassette from platform and place into MELFI. 6.Place new blank cassette into used platform and insert old food tray using tethered T-bar. 7.Remove new cassette containing old food tray from platform and place into MELFI. 8.Repeat for TBD cassettes. 9.Stow remaining 2 cassettes for live sample return. 1.Transfer all hardware to appropriate stowage on Dragon to return. 2.TBD µg rack and centrifuge shut down. 2 hours 1 hour

10. Training Proposal 10 Training: FFL-01 Science and Hardware Operation: 4 hours, L- 12 months? Training Methodology--Video training with procedures –Will fly up and back on SpaceX-5 –There are numerous subtle, yet very important differences between crew operations that, if mixed up would result in significant loss of science. –There is some touch involved in the use of the changeout platform. Because of this, successful operation is more likely with previous hands-on experience. –One crew member will be trained, with a back-up if possible. –The training will familiarize crew with organism and hardware, review of procedure to highlight important subtle differences between operations, and training with the hardware to be used during flight. –0.5 days is required for training.

11. Milestone Schedule 11 OpNom BaselineASAP Procedure Delivery & Chosen AuthorI-8 months Procedure Validation I-6 months Lesson Plan/Courseware DeliveryX-9 weeks PTDRX-7 weeks Dataset Baseline – Curriculum/HoursX-6 weeks Training Data SetX-5 weeks Ready for TrainingX

12. Fixation The currently planned Fruit Fly Lab mission experiment does not incorporate the capability to do animal fixation on orbit. We have been asked to put together a proposal to add this capability. Multiple approaches will be investigated if fixation is implemented. No Crew Ops developed since the hardware and approach are TBD. The task for the operation will likely be to remove the food tray from the fly cassette and insert into another device to administer fixative. The Crew Op will likely utilize a Disposable Glove Bag for containment, which limits the dexterity of the user. 12

13. KSC Fixation Unit (KFT) Possible Benefits: Existing triple containment design. Can probably be used in a disposable glove bag. Fixed samples could be safely frozen within the unit. Challenges: Sample tube area is too small to accommodate existing fly cassette food trays. A second version of the Fit food tray could be designed consisting of two pieces capable of fitting into sample tube. KFT unit could possibly be scaled up in size and remanufactured. Relatively small increase in mass and volume over plan. Effective sample coating and removing bubbles is more visible, but no less difficult (bubbles visible). Designed for “liquid only” experiments.