Planarian regeneration in space: Persistent anatomical, behavioral, and bacteriological changes induced by space travel

Analysis using mass spectrometry revealed that the water samples contained many small molecules (Fig. S2) and several proteins (Tables 4 and S1). Additional analyses are needed to determine the exact molecular identity of the small organic molecules identified during the LC‐MS experiments and the reasons why (and the mechanism(s) by which) they are selectively produced by the space‐exposed planaria. It is interesting to note that others have reported that C16 and C18 fatty acids, like the ones identified in the positive ion mode of our LC‐MS experiment, can induce apoptosis (Ulloth, Casiano, & De Leon, 2003; Yan et al., 2016) and that hexadecanoic acid (also known as palmitic acid) can generate reactive oxygen species (Gao et al., 2014; Lambertucci et al., 2008). Since these worms, and not the Earth‐only control worms, experienced high g force during liftoff and landing, it is possible that the presence of these fatty acids directly caused cell death via apoptosis (leading to the release of cytoplasmic proteins discussed below) or were just released into the water from damaged tissue or ruptured cells on the surface of the worm.

Of the 11 proteins remaining on the list (Table 4) it was possible to find orthologs in S. mediterranea for nine of them, including a homolog of fibrillin, a putative cathepsin C homolog, a putative myosin heavy chain homolog, a putative tubulin beta homolog, and a homolog of calmodulin. While homologs of most of the proteins on this list are not known to be secreted into the extracellular medium (e.g., myosin, tubulin, and calmodulin), fibrillin (Jensen & Handford, 2016) and cathepsin C (Legowska et al., 2016) are bona fide extracellular proteins. How these proteins, or any of the proteins found on this list that are presumably intracellular proteins, ended up in the water that surrounded the space‐exposed worms is not yet clear. Since these worms, and not the Earth‐only control worms, experienced a high g force during liftoff and landing, it is possible that the proteins were released into the water from damaged tissue or ruptured cells on the surface of the worm. Alternative possibilities include novel secretion pathways activated by microgravity or altered GMF. Additional work is needed to determine what role, if any, these proteins play in the unusual ‘water shock’ behavior or in the behavioral experiments described earlier in this paper.

The worm microbiome was different between space‐exposed and Earth‐only worms (one‐way PERMANOVA F = 12.29, p < 0.001) (Fig. 7; Table 6). The density of Chryseobacterium colonies significantly increased in space‐exposed worms, and Variovorax, Herminiimonas, and the unknown Comamonadaceae decreased in space‐exposed worms (t test, p < 0.01). These shifts could be driven by the direct effects of microgravity on bacterial populations or indirect effects mediated through the planarian host. Bacteria and other microbes have recently been shown to impact the development of a variety of model organisms (Lee & Brey, 2013). There is limited work on planarian microbiomes, so it is currently difficult to know the causes and consequences of microbiome composition shifts for planarian regeneration and patterning. Recent work with the planarian S. mediterranea found similar bacterial types as we detected in D. japonica, and blooms of Proteobacteria in the S. mediterranea microbiome were associated with tissue degeneration, while high abundances of Bacteroidetes, including Chryseobacterium and Pedobacter, were associated with healthy animals (Arnold et al., 2016). Work is ongoing to analyze the functional significance of the D. japonica microbiome, but we predict that shifts in the ratio of Proteobacteria and Bacteroidetes may also impact D. japonica growth and development. Chryseobacterium, which was enriched in space‐exposed worms, is a widespread genus of bacteria, with some species being rare pathogens in humans (Mukerji, Kakarala, Smith, & Kusz, 2016) and others providing benefits to animal and plant hosts through improved growth and pathogen protection (Antwis, Preziosi, Harrison, & Garner, 2015; Coon, Vogel, Brown, & Strand, 2014). Future efforts will elucidate the functional consequences of Chryseobacterium and other D. japonica bacteria.

Our experiments illustrate a template for regeneration experiments in space, piloting many aspects of the crucial logistics of such research. Planaria are an excellent model for the investigation of physiology, host−microbe interactions, behavior, and anatomy of a complex species exposed to space travel. It is clear that exposure to these conditions induces a range of detectable and long‐lasting changes in these organisms. As spaceflight becomes more accessible, future work in this and other model organisms will surely uncover new details of the interactions between gravitational and geomagnetic fields and processes in living systems. Exciting opportunities for biomedical discoveries abound, not only in terms of learning to mitigate risk factors for human space travel, but also for the discovery of novel biophysical mechanisms that could be exploited both on Earth and in space in the regenerative medicine field. Finally, it should be pointed out that, in light of their remarkable self‐repair and complex behavioral capabilities, planaria themselves present an ideal design challenge for the next generation of space exploration robots.

A clonal planarian flatworm stain of D. japonica, cultured at 20˚C in commercial Poland Spring water (Poland, ME, U.S.A.) in the dark, was used.

As part of the SpaceX (Hawthorne, CA, U.S.A.) Commercial Resupply Service mission CRS‐5, also known as SpX‐5, live worms either whole or cut as indicated in Figures 1(D) and 2(A) were loaded into the SpaceX Dragon Spacecraft (Fig. 1G, I), which was launched into space on 10 January 2015 by the SpaceX Falcon 9 rocket from the Kennedy Space Center in Florida (Fig. 1H). The samples were exposed to the microgravity and micro‐geomagnetic field environment at the ISS approximately 78 h post‐amputation. The Dragon capsule returned to Earth into the Pacific Ocean off the coast of California approximately 32 days post‐amputation. Live worms were kept sealed inside 50 mL conical tubes and were secured inside the BRIC‐100VC (Biological Research in Canisters, see Fig. 1E, F) containers provided by NASA, with temperature data recorder attached, immediately prior to launch and throughout the mission.

Two sets of control worms were generated: one set of controls (‘concurrent’) consisted of live worms that had been sealed in Poland Spring water in the same manner as their space‐exposed counterparts and kept in full darkness at 20˚C for the same period of time as their space‐exposed counterparts. These worms were used as controls for all of the experiments described in this paper, except for the mass spectroscopy analysis. For the mass spectroscopy experiments, a second set of worms were generated after the space‐exposed worms returned to Earth that were ‘temperature‐matched’ (see Fig. S1), so that they experienced the same changes in temperature as the space‐exposed worms for the same duration.

For logistics on Earth between laboratories before launch and after splashdown, live worm samples were secured inside the proprietary battery operated precision‐temperature‐controlled shipping container iQ2 from Micro Q Technologies (Scottsdale, AZ, U.S.A., Fig. 1A–C), and FedEx Space Solutions (Memphis, TN, U.S.A.) was utilized for rapid shipment of the experimental worm group in the iQ2 container.

The initial CRS‐5 mission was expected to span a duration of approximately 30 days in space, starting from the Falcon 9 rocket launch out of Kennedy Space Center, the Dragon Spacecraft's docking with, berthed at, and then detaching from the ISS, until the Dragon capsule's return to Earth. The planarian worms were expected to survive within a sealed environment for a minimal 30‐day duration, without any water filtration/purification system. To determine the optimal air‐to‐water ratio of the sealed environment for a minimal 30‐day survival, 10 worms approximately 1 cm in length (average 0.25 g per worm) cultivated at 20˚C and starved for at least 1 week were sealed inside 50 mL conical tubes with different ratios of air to water and were maintained at either 10˚C or 20˚C in the dark. Aside from the worms with mostly no air, which survived for only 5 days, worms kept at 25%, 50%, or 75% air‐to‐water ratios survived for over 30 days, and up to 43 days after isolation. The water from the 75% air‐to‐water sample at 20˚C was transparent, but browner than water from the 25% air‐to‐water sample at 10˚C, which suggests that the water quality is declining more rapidly because of the higher air ratio and the higher culture temperature. This led us to determine the optimal condition for 30‐day survival of adding 25 mL of fresh Poland Spring water and air filling the rest of the 50 mL volume, at a 50% air‐to‐water ratio at 20˚C (to facilitate spontaneous fissioning while in space).

In parallel, we also tested 50 mL conical tubes equipped with air‐permeable waterproof membrane caps, with the hope that improved gas exchange would lead to higher worm survival. Although all worms survived up to 49 days, interestingly the worms kept inside membrane cap tubes were outlasted by worms sealed in non‐membrane tubes, while losing a small amount of water along the duration possibly due to evaporation. We concluded that an optimal experimental 30‐day condition in space was a maximum of 10 worms (1 cm in length), sealed inside a non‐membrane 50 mL conical tube, with 50% air‐to‐water ratio, maintained at 20˚C (to facilitate spontaneous fissioning while in space).

Since direct manipulation of the worms while in space was not an option for this mission, we next examined if this condition was suitable to facilitate spontaneous worm fissioning. We tested with different numbers of worms (4, 6, 8, or 10) sealed into a 50 mL conical tube with 50% air‐to‐water ratio, and this resulted in all worms fissioning within 1 week at 20˚C.

In addition to having the worms spontaneously undergo fission and regeneration while in space, we also included worms amputated on Earth just prior to the launch. Fifteen flatworms in total were amputated by hand (Fig. 1D) on a stack of wetted filter paper using a scalpel into three different fragments (head, pharynx, tail; see Fig. 2A) on Earth approximately 31 h before launch. Fifteen of each fragment were separated and sealed into individual 50 mL conical tubes, with 50% air‐to‐water ratio. This is approximately equivalent to five whole worms per tube. Also, 4, 5, 6, 8, or 10 whole worms were also sealed into individual 50 mL conical tubes with 50% air‐to‐water ratio. A total of eight 50 mL conical tubes (Table 1) were then secured by custom 3D‐printed retainers inside two sealed BRIC‐100VC containers provided by NASA (Fig. 1E, F), with a temperature data logger secured inside each BRIC. As controls on Earth, eight tubes of exactly the same number of whole worms and amputated fragments were sealed inside 50 mL tubes with 50% air‐to‐water ratio and kept in the laboratory at 20˚C in the dark. These samples are considered concurrent (constant temperature) controls. The time from amputating the worms to the worms reaching the ISS was approximately 78 h, based on the temperature data retrieved from the attached data logger (see Fig. S1). The optimal temperature range for worms’ long‐term survival is considered a minimum of 10˚C to a maximum of 25˚C, with a short‐term durable range from 0˚C to 30˚C. The data logger indicates that the worms were maintained within the optimal temperature range throughout the mission (Fig. S1), with the help of sophisticated temperature‐maintaining gel packs provided by NASA during transport to and from the ISS in the Dragon Spaceship, similar to commercial ice packs, and while situated in an incubator onboard the ISS.

The worm samples spent approximately 29 days at microgravity and in a micro‐geomagnetic field environment in the ISS. After return to Earth, the worm samples were received in our laboratory approximately 68 h after splashdown. The worms were immediately subjected to basic analysis in the laboratory. All space worms were alive inside the sealed tubes. Live worms were either photographed or had video movies taken with a Canon (Tokyo, Japan) Rebel T3i DSLR (digital single‐lens reflex) camera attached to a Zeiss (Oberkochen, Germany) Stemi V6 dissecting microscope. Water from the concurrent, Earth‐only control and space‐exposed worms was frozen immediately after return to Earth and stored at −20°C.

The water which the space‐exposed planaria and the Earth‐only planaria inhabited during the course of the experiment was thawed in cold water, and 4.0 mL aliquots of each sample were freeze‐dried using a lyophilizer. The samples were then re‐suspended in 100 μL of 60% acetonitrile. The sample was centrifuged at 14,000g to remove debris. 5 μL of this reconstituted sample was injected for each LC‐MS analysis. A Thermo q‐Exactive Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, U.S.A.) coupled to a Thermo Ultimate 3000 (Thermo Fisher Scientific) high performance liquid chromatograph (HPLC) was used to perform the LC‐MS analysis of metabolites in biological samples and authentic chemical standards in both positive and negative ion mode using polarity switching. Two separate data‐dependent MS/MS analyses were conducted in positive and negative ion mode using the top five ions using dynamic exclusion for 30 s. Electrospray source settings included a sheath gas flow rate set at 35 (arbitrary units), an auxiliary gas flow rate at 5 L/min, a capillary temperature of 250°C, and an auxiliary gas temperature of 300°C. A calibration of the m/z range used was performed using the Thermo LC‐MS calibration mix immediately prior to the analysis for both positive and negative ion mode. A scan range of 66.7−1000 m/z was used at a resolving power of 70,000 with alternating positive and negative ion mode scans. The chromatographic separation of metabolites was performed using hydrophillic interaction liquid chromatography (HILIC) on a SeQuant® ZIC®‐pHILIC column, 5 μm, polymer PEEK 150 mm × 2.1 mm column (EMD Millipore, Billerica, MA, U.S.A.) at a flow rate of 0.1 mL/min. Mobile phase A was 20 mM ammonium bicarbonate with 0.1% ammonium hydroxide, and mobile phase B was acetonitrile.

The mobile phase composition was started at 100% B and subsequently decreased to 40% B over 20 min. The column was then washed at 0% B for 5 min before re‐equilibration to 100% B over 15 min.

The space‐exposed planaria and the Earth‐only planaria samples were analyzed for differences using XCMS (Tautenhahn, Patti, Rinehart, & Siuzdak, 2012): significant differences between the two samples were observed in positive ion mode (Fig. S2A), but fewer differences were observed in negative ion mode (Fig. S2B).

The water in which the space‐exposed planaria and the later established ‘temperature‐matched’ Earth‐only control planaria inhabited during the course of the experiment was thawed in cold water, and 4.0 mL aliquots of each sample were freeze‐dried using a lyophilizer. The samples were then re‐suspended in 100 μL of tetraethylammonium bromide (TEAB), reduced with 20 mM tris‐(2‐carboxyethyl)phosphine (TCEP) in 25 mM TEAB at 37°C for 45 min, and alkylated with 10 mM iodoacetamide (Sigma) in 25 mM TEAB and kept in the dark, room temperature, for 45 min. Then 2 μg of trypsin/LysC (V5073, Promega, Fitchburg, WI, U.S.A.) was added overnight. 14 μL from a final volume of 140 μL was injected into the instrument after a hard spin and supernatant was removed to a new HPLC vial as there were particulates on the floor of each digestion tube. LC‐MS/MS was performed on an Orbitrap Fusion Lumos™ Tribrid™ (Thermo Fisher Scientific) mass spectrometer with the 100716L 90 min ID 150 nL KASIL trap 300 bar method to generate a list of proteins that were present in each of the samples.

Each sample was submitted for a single LC‐MS/MS experiment that was performed on an LTQ Orbitrap Elite (Thermo Fisher Scientific) equipped with a Waters (Milford, MA, U.S.A.) NanoAcquity HPLC pump. Peptides were separated onto a 100 μm inner diameter microcapillary trapping column packed first with approximately 5 cm of C18 Reprosil resin (5 μm, 100 Å, Dr Maisch GmbH, Germany) followed by an analytical column ∼20 cm of Reprosil resin (1.8 μm, 200 Å, Dr Maisch GmbH, Germany). Separation was achieved through applying a gradient from 5% to 27% acetonitrile in 0.1% formic acid over 90 min at 200 nL/min. Electrospray ionization was enabled through applying a voltage of 1.8 kV using a home‐made electrode junction at the end of the microcapillary column and sprayed from fused silica pico tips (New Objective, MA, U.S.A.). The LTQ Orbitrap Elite was operated in the data‐dependent mode for the mass spectrometry methods. The mass spectrometry survey scan was performed in the Orbitrap in the range 395–1800 m/z at a resolution of 6 × 104, followed by selection of the 20 most intense ions (TOP20) for CID‐MS2 fragmentation in the ion trap using a precursor isolation width window of 2 m/z, automatic gain control (AGC) setting of 10,000, and a maximum ion accumulation of 200 ms. Singly charged ion species were not subjected to CID fragmentation. Normalized collision energy was set to 35 V and an activation time of 10 ms, AGC was set to 50,000, the maximum ion time was 200 ms. Ions in a 10 ppm m/z window around ions selected for MS2 were excluded from further selection for fragmentation for 60 s.

Raw data were submitted for analysis in Proteome Discoverer 2.1.0.81 (Thermo Fisher Scientific) software. Assignment of MS/MS spectra was performed using the Sequest HT algorithm by searching the data against a protein sequence database including all entries from the Human Uniprot database (SwissProt 16,768 and TrEMBL 62,460; a total of 79,228 protein forms, 2015) and other known contaminants such as human keratins and common laboratory contaminants. Sequest HT searches were performed using a 20 ppm precursor ion tolerance and requiring each peptides’ N/C termini to adhere with trypsin protease specificity while allowing up to two missed cleavages. Cysteine carbamidomethyl (+57.021) was set as static modifications while methionine oxidation (+15.99492 Da) was set as variable modification. An MS2 spectra assignment false discovery rate of 1% on protein level was achieved by applying the target‐decoy database search. Filtering was performed using a Percolator (64 bit version, reference 6). For quantification, a 0.02 m/z window was centered on the theoretical m/z value of each of the six reporter ions and the intensity of the signal closest to the theoretical m/z value was recorded. Reporter ion intensities were exported in the result file of Proteome Discoverer 2.1 search engine as Excel tables. All fold changes were analyzed after normalization between samples based on total unique peptide ion signal.

The list of identified ‘space worm’ proteins was filtered for reagents used in the trypsin digestion step, known contaminants (e.g., human keratin), and proteins that were also identified (i.e., the presence of at least one peptide in the mass spectrum) in the Earth‐only control sample. The remaining proteins were further filtered so that every protein on the list was identified via two or more unique peptides in the mass spectrum.

The full DNA sequences of the identified proteins were obtained from a recently published D. japonica transcriptome (Chan et al., 2016) and translated into potential protein sequences in all three reading frames using ExPASy (http://web.expasy.org/translate/). These D. japonica putative protein sequences (Table S1) were then subjected to a BLASTP search against the Smed_unigenes_20150217.aa database housed at the S. mediterranea Genome Database website (http://smedgd.stowers.org/) (Robb, Gotting, Ross, & Sanchez Alvarado, 2015). The resulting S. mediterranea ortholog was then subjected to pairwise BLASTP search against the sequence identified during the original mass spectrometry experiments to determine how similar the sequences of the D. japonica and the S. mediterranea proteins were (Supplemental Data and Table S1).

Live whole worms that returned from space were maintained in fresh Poland Spring water and kept at 20˚C in the dark, with weekly feeding of organic calf liver paste.

Randomly chosen whole worms from the space‐exposed and Earth‐only colonies were tested, 20 months after return to Earth, in an automated behavior platform as described previously (Blackiston, Shomrat, Nicolas, Granata, & Levin, 2010; Shomrat & Levin, 2013). Briefly, the device consists of 12 individual arenas each containing a standard disposable 60 mm × 15 mm Petri dish filled with 15 mL of Poland Spring water. Above each arena, an illumination control head provides red or blue light independently, or in combination, to each quadrant of the dish in 12 even intensity steps via LED illumination (Opto Semiconductors GmbH, Regensburg, Germany: blue LED; 470 nm part no. LBW5SM, red LED, 635 nm part no. LRG6SP). Below each dish, a motion tracking camera (Insight‐Micro 1400, Cognex Corporation, Natick, MA, U.S.A.) records the position of each animal every 1000 ms via hardware background subtraction algorithms (this capture rate was chosen to minimize centroid bounce, which can artificially inflate movement rates of slow moving irregular objects). Planarian locations, and lighting conditions, are recorded as a log file, which can be analyzed to determine animal movement rates, color preference, preference for edge versus center of the arena, and rotation direction (clockwise vs. counterclockwise).

The behavior trial lasted 18 h and consisted of the following settings. Background illumination in all quadrants was 50 lm red light, which is undetectable to planarians given their photoreceptor profiles (Azuma, Iwasaki, & Ohtsu, 1999; Brown & Ogden, 1968). In addition, half of the arena is illuminated by 420 lm blue light, giving the animals a choice between “light” and “dark” halves of the environment. Every hour, the lighting conditions were inverted, causing animals in the dark portion of the arena to be exposed to light and vice versa. Light rotation served dual roles. First, as planaria are photophobic it promoted movement of animals across the trail, and allowed movement rates to be compared across treatments. Second, without rotation, animals that remained stationary during the course of the experiment would be scored as having a 100% preference for either light or dark, depending on their starting position. By inverting the light, stationary behavior would instead result in a 50/50 preference, and would thus not bias the averages towards either extreme.

To determine differences in bacterial community composition between space‐exposed and Earth‐only worms, we used culture‐based assessments of individual worm microbiomes. We chose to use culture‐based approaches because unpublished work from our laboratories and recent studies of the S. mediterranea microbiome (Arnold et al., 2016) suggest that dominant bacteria types in planarian microbiomes are culturable, and bacterial colony morphotypes of dominant bacterial genera can be easily distinguished.

Ten randomly chosen worms from each group were homogenized in 400 μL of phosphate‐buffered saline using a sterile micropestle. Homogenates were serially diluted in phosphate‐buffered saline and plated on brain heart infusion agar plates and were incubated for a week at 24°C. Representative morphotypes that grew were identified based on previous in‐house sequencing and identification of D. japonica bacterial isolates. To confirm the identities of these morphotypes, representative colonies were isolated, DNA was extracted using a PowerSoil® DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, U.S.A.), and the the 16S rRNA region was amplified using the polymerase chain reaction primers 27f‐1492r; resulting amplicons were sequenced using Sanger sequencing. Differences in total community composition for the culture‐based microbiome data were determined using PERMANOVA, and differences in relative abundances of individual bacterial groups were determined using t tests, with Bonferroni corrections for multiple comparisons.