Validation of the Rob-2 experiment at Biotesc
The teams of UNamur, Kayser Italia and ESA validated in January 2020 at Biotesc in Lucern (Switzerland) the Rob-2 experiment of rotifers.
Dried and irradiated (500Gy X-ray) Adineta vaga rotifer individuals were transported from LEGE laboratory UNamur on dry ice in the transport hardware designed by R. Coos (LARN engineer) to Biotesc and integrated in the Rob-2 modules from Kayser Italia with water and salad juice for future rehydration.
In total 20 Rob-2 modules were assembled and checked on quality control during 2 days at Biotesc, 16 Rob-2 modules were validated.
The space flight simulation was done at Biotesc:
1) cold storage of Rob-2 modules at 3°C (till 8°C),
2) transfer of Rob-2 to KUBIK module (@ 21°C) and automatic rehydration of Adineta vaga individuals within Rob-2 (21°C),
3) after 4 days of rehydration, samples transferred to -80°C to stop the experiment (samples used for the DNA repair study),
5) after 11 days of rehydration, samples transferred to -80°C (to study the genome structure to verify whether DNA repair took place in space).
Half of the Rob-2 modules were placed under microgravity, the other half on a centrifuge simulating earth gravity.
The launch to ISS from LEGE and LARN laboratories, UNamur
Within the Rob2 mission we investigate through ground-based experiments and space flight whether microgravity and cosmic radiation has an impact on the DNA repair processes of living organisms using the bdelloid rotifer Adineta vaga as model system.
During this Rob2 mission A. vaga individuals submitted to desiccation and 500 Gy X-ray or proton radiation (inducing DNA DSBs) will be sent to space. Their capacity to repair their broken genome will be compared in space and on earth. Indeed, desiccated and irradiated A. vagawill be rehydrated respectively at 1G and 0G microgravity on ISS, and on earth.
Survival, fertility and DNA damages at genomic level will be investigated as well as the repair kinetic and gene expression (RNAseq).
The first step is the desiccation of 1.800.000 A. vaga individuals , under controlled conditions, in the LEGE laboratory (UNamur). This was perfomed by technician J. Berthe and postdocs V. Moris and B. Hespeels on November 20th, 2020.
Irradiation at the LARN laboratory, UNamur
After desiccation on LMP agarose, the 1.800.000 dried A. vaga individuals were irradiated at the LARN laboratory (UNamur) by engineer Richard Coos, technician Jérémy Berthe and postdoc Boris Hespeels from LEGE on November 25th, 2020.
Patches of dried A. vaga individuals were exposed to 500Gy X-ray or 500Gy proton radiation in the facilities of LARN.
Both type of radiations induce high amounts of DNA damages without affecting the survival of A. vaga (at least on earth). At these doses most A. vaga individuals can also still reproduce, as demonstrated by the research team UNamur (Hespeels et al. 2020 – Frontiers in Microbiology). This is an extreme tolerance to ionizing radiation for a multicellular animal, one of the most resistant on earth.
Integration in the Rob-2 modules designed by Kayser Italia
Dried and irradiated Adineta vaga individuals were integrated in the Rob2 modules designed by Kayser Italia.
The integration takes place under the hood in the LEGE laboratory at URBE (UNamur). 16 Flights units were assembled (+ 2 spare ones): 8 units (+1) for the 0G microgravity experiment on ISS and 8 units (+1) for the 1G microgravity experiment on ISS. In addition, 8 Ground Reference units were assembled, as a comparison to the space experiments.
These units were assembled by the LEGE postdocs Victoria Moris and Boris Hespeels, LEGE technicians Jérémy Berthe and Lucie Bruneau, LARN technician Margot Cardinal and LARN engineer Richard Coos under the digital supervision of Kayser Italia and ESA on November 26th and 27th 2020 at UNamur.
Team work UNamur and Kayser Italia, launch from Kennedy Space Center
The construction of each Rob2 module by 3 masked persons took approximately 1 hour: while one person is doing the assembly, another one is preparing the equipment while the third person reads the guidelines, under the supervision of Kayser Italia through digital communication. A strong team work at UNamur, in continuous interaction with Kayser Italia and ESA, and Prof. Karine Van Doninck present at UNamur.
In total, 26 Rob2 modules were constructed for the ISS and ground-based experiments and send to Kayser Italia for quality control check (November 27th, 2020). The quality control at Kayser Italia in Italy was done on November 29th, 2020; all Rob2 modules were validated.
The 16 Flight unit Rob2 modules were send to Kennedy Space Center in Florida (USA) and arrived on December 1st, 2020. They were integrated on Falcon 9 Space X rocket on December 4th, 2020. The 8 Rob2 modules for the ground-based experiments were send back to LEGE (UNamur).
The Space X CRS21 was successfully launched on December 6th, 2020.
Evaluation of survival, fertility and genomic integrity of control samples at UNamur
In parallel to flight samples, several specimens were used as controls to evaluate survival, fertility and genomic integrity.
To this end, some samples were rehydrated with salad juice at UNamur, by the LEGE postdoc Boris Hespeels helped by Victoria Moris, to mimic the rehydration performed in the flight modules in the ISS.
Rehydrated rotifers were inspected a few hours after rehydration by the two postdocs with the microscope to evaluate the quality of the samples (numbers of rotifers, contaminations, morphology and activity of rotifers).
Activation of ground reference experiments at UNamur
The LEGE postdocs Boris Hespeels and Victoria Moris activate the 8 ground reference units at UNamur using a Kayser Italia hardware similar to the one on board of the ISS. Rotifers were rehydrated on Earth with the exact same time line as flight samples in the ISS.
To verify the hardware efficiency, each module was inspected after rehydration. The LEGE postdocs Boris Hespeels and Victoria Moris, and the LEGE technician Jérémy Berthe verified that the water was transferred to the compartment containing the rotifers.
PI team simulated astronaut activity by gently shaking each module after its activation to confirm its rehydration.
This activation step was performed at UNamur on 8 December 2020, at the same time as on the ISS.