Pre‐launch preparation and logistics. For logistics on Earth, live worm samples were secured inside the battery powered refrigerated shipping container iQ2 from Micro Q Technologies (Scottsdale, AZ, U.S.A.), and FedEx Space Solutions (Memphis, TN, U.S.A.) was utilized for rapid shipment of the iQ2 container. (A) iQ2, the proprietary battery operated precision‐temperature‐controlled shipping container. (B), (C) iQ2 inside the protective shipping exterior. (D) Manual worm amputation at Kennedy Space Center prior to launch. (E), (F) 50 mL conical tubes (blue caps) containing live worms were sealed, then secured in 3D‐printed custom retainers (yellow and purple), and placed inside the BRIC‐100VC containers (red) provided by NASA. (G) SpX‐5 SpaceX Dragon Spacecraft on top of the Falcon 9 rocket at Cape Canaveral SLC‐40 launch pad. (H) SpX‐5 liftoff on 10 January 2015, at 09:47 UTC. (I) SpX‐5 SpaceX Dragon Spacecraft in orbit prior to berthing with the ISS on 12 January 2015. Images reprinted with permission from Micro Q Technologies (A) and of SpaceX (G–I)

Our study sought to determine how spaceflight and the conditions on the International Space Station (ISS) would affect planarian regeneration (Fig. 1 ). What effects would microgravity and micro‐geomagnetic fields produce, and might these effects be persistent after return to Earth? We used a panel of behavioral, microbiological, and morphological assays to understand how the total experience of spaceflight (including the stresses of take‐off and landing, as well as the weightless and 0 GMF conditions on the ISS itself) would affect this complex regenerative model system. This project was also designed to establish protocols for performing planarian research in space so as to determine proper transfer logistics and conditions for future missions. As humans transition towards becoming a space‐faring species, it is important that we deduce the impact of spaceflight on regenerative health for the sake of medicine and future space laboratory research.

If space travel environments can change cellular behavior and physiology, it is imperative to begin to understand how they can impact regeneration. Much of the previous work studying the impact of spaceflight on regeneration has been done in urodeles, in particular investigating limb and lens regeneration (Grigoryan, Mitashov, & Anton, 2002 ; Mitashov, Brushlinskaya, Grigoryan, Tuchkova, & Anton, 1996 ). Newts undergoing limb regeneration have shown increased regenerative rates on biosatellites as well as increased proliferation in limb blastemas in a synchronous manner. Lenses also showed increased regenerative ability. After landing, there was a two‐fold increase in the number of proliferative cells within the region that provides the cells for lens regeneration as well as other parts of the eye. Upon further investigation replicating these experiments in microgravity conditions on Earth, it was suggested that these effects occur due to weightlessness (Blaber, Sato, & Almeida, 2014a ; Grigoryan, Anton, & Mitashov, 1998 ). Conversely, tail regeneration experiments did not find this same advancement in regenerative ability; however, changes in the pigmentation of tail blastemas in spaceflight animals were observed (Grinfeld, Foulquier, Mitashov, Bruchlinskaia, & Duprat, 1996 ). In Schmidtea mediterranea planaria, one study using simulated microgravity observed lethality while hypergravity led to decreased proliferation rates (Adell, Salo, van Loon, & Auletta, 2014 ). In contrast, another study found no distinguishing effects on Girardia tigrina (Gorgiladze, 2008 ). We used the species Dugesia japonica , not previously explored in space travel, with a range of analysis methods, to examine the effects of spaceflight conditions.

Microbes are also impacted by space conditions. Classically, it was concluded that cells smaller than 10 μM, including bacteria, would be affected very minimally by weightlessness (Pollard, 1965 ); however, more recently, experiments observing microorganisms in space‐like environments have suggested otherwise (Horneck, Klaus, & Mancinelli, 2010 ). Moreover, microgravity conditions have been shown to increase bacterial growth kinetics, biofilm formation, and stress resistance (Kim, Matin, & Rhee, 2014 ; Rosenzweig et al., 2010 ). Microbes continue to maintain their adaptability in the changing environment and have been shown to change their secondary metabolite production, gene expression, and virulent capability (Leys, Hendrickx, De Boever, Baatout, & Mergeay, 2004 ; Nickerson et al., 2000 , 2003 ). Although it still remains to be determined what physical factors are contributing to these changes (such as whether they are due to microgravity or fluid dynamics), it is clear that spaceflight can reshape microbial communities and what they produce. Aside from the clear biological implications, this also poses questions regarding manned spaceflight and protection from microorganisms that may be encountered while away from Earth.

Biological systems also operate under the physical constraint of the Earth's gravity (Bizzarri, Cucina, Palombo, & Masiello, 2014 ). Therefore, an emergent question in recent years has concerned the behavior, cellular and otherwise, of organisms in microgravity conditions. It has since become clear that system level changes occur in microgravity fields (Crawford‐Young, 2003 ). More specifically, microgravity has been shown to affect cell morphology (Crawford‐Young, 2003 ; Testa et al., 2014 ), cytoskeletal organization (Masiello et al., 2014 ), early development (reviewed by Ogneva, 2015 ; see also Dournon, 2003 ), the likelihood of the open state of ion channels (Goldermann & Hanke, 2001 ), gene expression profiles (Pardo et al., 2005 ), differentiation (Pisanu et al., 2014 ), and apoptosis (Monici et al., 2006 ). Microgravity, in most cases so far, has been shown to be an inhibitor of tissue growth and regeneration in mammalian tissues (Blaber et al., 2014b ). Microgravity research, on top of revealing how cells behave in response to altered physical forces, has also led to the development of innovative techniques. As an example, it has been found that 3D cultured cells allow for an unrestricted growth environment which is promising for the future of cell culture application to human medicine (Grimm et al., 2014 ; Souza et al., 2010 ).

On Earth, biological systems are also subject to the naturally varying geomagnetic field (GMF) (Dubrov, 1978 ). This variation in geomagnetic disturbance has been shown to impact not only animal behavior (Beischer, 1971 ; Zamoshchina et al., 2012 ), but also medically relevant phenomena such as ciliary motion (Sandoze, Svanidze, & Didimova, 1995 ), stem cell function (Mo, Liu, Bartlett, & He, 2014 ), cardiovascular regulation (Cornelissen et al., 2001 ; Feigin et al., 2014 ; Gmitrov & Gmitrova, 2004 ; Stoupel, 2006 ; Stoupel et al., 2014 ), the autonomic nervous system (Baevsky, Petrov, & Chernikova, 1998 ), memory (Wang, Xu, Li, Li, & Jiang, 2003 ; Xiao, Wang, Xu, Jiang, & Li, 2009 ; Zhang et al., 2004 ), and the interactions between neurons (Shibib, Brock, & Gosztony, 1987 ). Magnetic field reversals may even have placed selective pressures on organisms that have contributed to subsequent extinction (Hays, 1971 ; Plotnick, 1980 ) and morphological change (Harrison & Funnel, 1964 ), and planaria have specifically been shown to be sensitive to weak magnetic fields (Brown, 1962b , 1966 ). These observations have been tested in recent decades by generating a near null or hypogeomagnetic field in order to understand the role of the Earth's natural magnetic field in numerous biological processes (Krylov, Bolotovskaya, & Osipova, 2013 ; Krylov et al., 2014 ; Zaporozhan, Nasibullin, Hozhenko, & Shapranov, 2002 ). The effects of exposure to a null magnetic field have included changes in immune response (Dorofteiu, Morariu, Marina, & Zirbo, 1995 ), axonal myelination (Shibib et al., 1987 ), and tubulin assembly (Wang, Wang, Xiao, Liu, & He, 2008 ), as well as developmental patterning (Asashima, Shimada, & Pfeiffer, 1991 ; Mo, Liu, Cooper, & He, 2012 ). The physiological mechanisms contributing to the influence of the GMF on biological events are currently unknown.

Patterning during regeneration, development, and cancer suppression is subject to the influence of physical forces including electric fields, magnetic fields, electromagnetic fields (Chernet & Levin, 2013 ; Funk & Monsees, 2006 ; Funk, Monsees, & Ozkucur, 2009 ), as well as other biophysical inputs (reviewed by Adams, 2008 ; Adams & Levin, 2013 ; Levin, 2014b ; Lobikin, Chernet, Lobo, & Levin, 2012 ; Mustard & Levin, 2014 ; Stewart, Rojas‐Munoz, & Izpisua Belmonte, 2007 ). In planaria specifically, electric forces have been known to alter patterning information for decades (Bonaventure, 1957 ; Hyman, 1932 ; Lange & Steele, 1978 ; Marsh & Beams, 1952 ). More recently, bioelectric physiology has been implicated in the regulation of the cell cycle (Barghouth, Thiruvalluvan, & Oviedo, 2015 ), polarity (Beane, Morokuma, Adams, & Levin, 2011 ), and morphology (Beane, Morokuma, Lemire, & Levin, 2013 ; Emmons‐Bell et al., 2015 ) in the planarian as well. It is probable that physical forces, both internal and external, are modulated by the physical force of Earth's gravity, which probably influenced the way that the regenerative and developmental abilities of living organisms have evolved on Earth (Bizzarri & Cucina, 2014 ).

Planarian flatworms are known for their mastery of regeneration (Reddien & Sanchez Alvarado, 2004 ; Sanchez Alvarado, 2003 ; Sheiman & Kreshchenko, 2015 ). These bilaterians have the ability to completely recapitulate all body parts, including complex organs, from small pieces of the body, with high morphological and proportional fidelity (Hill & Petersen, 2015 ) in a vast variety of perturbations (Morgan, 1898 ). The complex organs include a full, centralized brain (Pagán, 2014 ; Sarnat, 1985 ) and central nervous system (Cebria, 2008 ) which has the ability to produce a continuous brain wave pattern (Aoki, Wake, Sasaki, & Agata, 2009 ) and complex behaviors (Corning, 1964 ; Inoue, Hoshino, Yamashita, Shimoyama, & Agata, 2015 ) with impressively variable sensory capabilities as inputs (Asano, Nakamura, Ishida, Azuma, & Shinozawa, 1998 ; Brown, 1962a , 1966 ; Brown & Park, 1964 ; Brown, Dustman, & Beck, 1966 ; Carpenter, Morita, & Best, 1974 ; Hyman, 1951 ; MacRae, 1967 ). Planaria exhibit complex learning, curiosity, and problem‐solving abilities (Best & Rubenstein, 1962 ; Corning & Freed, 1968 ; McConnell, 1965 ; Pagán, 2014 ; Wells, 1967 ). Moreover, they are able to repair and remodel three major polarity axes, dorsal/ventral, anterior/posterior, and medial/lateral, with outstanding accuracy (Gentile, Cebria, & Bartscherer, 2011 ; Gurley, Rink, & Alvarado, 2008 ; Kato, Orii, Watanabe, & Agata, 2001 ; Lange & Steele, 1978 ; Molina, Saló, & Cebrià, 2007 ; Orii & Watanabe, 2007 ; Owlarn & Bartscherer, 2016 ; Reddien, Bermange, Kicza, & Alvarado, 2007 ). These complex regenerative abilities are attractive for human regeneration research especially because planaria have more genomic similarities to vertebrates than do Drosophila melanogaster or Caenorhabditis elegans (Sánchez Alvarado, Newmark, Robb, & Juste, 2002 ). All of these properties make planaria a prime model for research in diverse areas of biomedicine, from stem cell biology to drug addiction (Rawls, Cavallo, Capasso, Ding, & Raffa, 2008a ; Rawls, Gerber, Ding, Roth, & Raffa, 2008b ; Rowlands & Pagan, 2008 ; Sacavage et al., 2008 ).

Various genera of Proteobacteria ( Herminiimonas , Pseudomonas , and an unknown bacterium in the family Comamonadaceae) and Bacteroidetes ( Chryseobacterium , Variovorax , and Pedobacter ) were the main bacterial morphotypes detected with culture‐based approaches (Fig. 7 ; Table 6 ). There was a significant difference in the composition of the culture‐based microbiome profiles between Earth‐only and space‐exposed worms (one‐way PERMANOVA F = 12.29, p < 0.001). The number 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). We conclude that exposure to the conditions of space travel can alter bacterial community composition of D. japonica , and indeed does so in a manner that is still altered years afterward.

Space‐exposed worms demonstrate more variable photophobic behavior than Earth‐only worms. (A) Earth‐only and space‐exposed planaria were placed individually in an automated behavior device which recorded animal location, speed, and response to light. (B) Overhead illumination is provided by LEDs which illuminate half the arena with red light (invisible to planaria) and half with blue light. (C) Space‐exposed worms demonstrated significant variability in their photo‐aversive behavior compared to Earth‐only worms ( F test, p < 0.001). N = 6 for both treatments. Error bars indicate ± 1 SD. In dot plots in (C), the lateral positioning of the dots, within each of the two groups, is only to enable the separate data points to be distinguished from each other even when they occupy the same horizontal coordinate

The behavior of space‐exposed and Earth‐only animals was tested in an automated assay 20 months after return to Earth. Individuals from each group were placed in individual arenas, illuminated half with red light (beyond the planarian visual spectrum) and half with blue light, for 18 h with lighting conditions reversing hourly (Fig. 6 A, B). Movement rates for each individual were recorded across the trial using motion tracking cameras and background subtraction algorithms. There was no significant difference in the overall rate of motion between treatments (data not shown). We then scored the percentage of time each worm (of six, from control and space‐exposed groups) spent in the dark half of a Petri dish versus the blue light‐emitting diode (LED) illuminated half. The controls spent 95.5% of their time in the dark, as is normal for this negatively phototaxic species. In contrast, the worms that had experienced space travel spent only 70.5% of their time in the dark. While the difference in the two groups’ means was not statistically significant due to the small sample size ( t test, p = 0.17), the variance was significantly different ( F test, p < 0.001) between treatments: Figure 6 C shows that the space‐exposed worms exhibited a much less uniform (i.e., more variable) preference for light levels (see also Table 5 ).

Amputation of double‐headed worm from space results in double‐headed morphology. (A) Schematics of amputation of the double‐headed space worm. (B) Double‐headed space worm before amputation at the dotted line; note that this photograph is the same as the image that appears in Figure 4 B. (C) Double‐headed worm immediately after amputation of both heads. (D) Amputated double‐headed worm after 2 weeks of regeneration. Note that, while the two head fragments regenerated into two single‐headed worms like a normal worm, the head‐less fragment regenerated into a double‐headed worm. (E), (F) Close‐up images of each of the two regenerated heads of the re‐amputated double‐headed worm

We next amputated this specific double‐headed space‐exposed worm by making two decapitating cuts to remove both heads. Remarkably, the head‐less middle fragment regenerated into a double‐headed phenotype (Fig. 5 ), demonstrating that the major body‐plan modification that occurred in this animal is stable and persists for at least two rounds of cutting and subsequent regeneration after exposure to space travel. Given the long‐term alterations observed in these animals, we next asked whether two other aspects of their organismal physiology—behavior and microbiome composition—might also be permanently altered.

The most striking morphological change was observed with one of the 15 pharynx fragments from space which had been manually amputated on Earth prior to the launch. Figure 4 shows the unusual ‘double‐headed’ phenotype, which is extremely rare within a control population by spontaneous fissioning or even with manual amputation of a control worm. Although the sample number is low, the spontaneous occurrence of such a rare phenotype itself should be considered highly significant: in our own laboratory, we have not observed any spontaneous occurrences of double‐headedness in >18 person‐years of maintaining a colony of D. japonica . We estimate about 15,000 control worms in the last 5 years, without a single double‐headed animal arising from an untreated control fragment. Given this background, the Z score calculator for two population proportions gives a Z score of 31.6238 and a p value of <0.01 against chance.

Worms that returned from space, together with the control worms on Earth, were then maintained separately in the laboratory under the same conditions, being fed organic calf liver paste every week for two additional months. After that time period, both populations were counted. We observed that the number of worms in the container that had gone to space was slightly less than the number of worms that remained on Earth (Fig. S3, Table S2). Likewise, in the worm fragments amputated prior to launch, the worm population exposed to space then grew more slowly than the Earth‐only controls.

LC‐MS/MS analysis revealed that the water samples contained several proteins. The list of identified proteins in the space‐exposed planaria water sample 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 ground 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 (Table S1).

Our analysis of the unique ions from the space‐exposed worm water sample that were observed in negative ion mode indicates that molecules with long hydrocarbon chains were observed as well. For example, a peak with an accurate m / z of 285.2072 is consistent with [M−H + ] − of C 16 H 30 O 4 , which could be hexadecanedioic acid [HO 2 C(CH 2 ) 14 CO 2 H], and a peak with an accurate m / z of 285.2072 is consistent with [M−H + ] − of C 19 H 37 NO 4 , which could be dodecanoylcarnitine, octanoylcarnitine n ‐butyl ester, or N ‐palmitoyl serine, all of which have long hydrocarbon chains.

Our analysis of the unique ions observed in the space‐exposed worm water sample using the positive ion mode of LC‐MS indicate that many of them correspond to long‐chain fatty acids or mono‐hydroxylated/di‐hydroxylated long‐chain fatty acids. For example, a peak with an accurate m / z of 274.2730 is consistent with [M+NH 4 ] + of C 16 H 32 O 2 , which could be one or more skeletal isomers of hexadecanoic acid (e.g., CH 3 (CH 2 ) 14 COOH or CH 3 (CH 2 ) 7 CH[(CH 2 ) 5 CH 3 ]CO 2 H). Another peak, with an accurate m / z of 290.2680, is consistent with [M+NH 4 ] + of C 16 H 32 O 3 , which could be one or more regioisomers of hydroxyhexadecanoic acid [e.g., CH 3 (CH 2 ) 13 CH(OH)CO 2 H or HO(CH 2 ) 15 CO 2 H], the peak that has an accurate m / z of 334.2943 is consistent with [M+NH 4 ] + of C 18 H 36 O 4 , which could be one or more regioisomers of dihydroxyoctadecanoic acid [e.g., CH 3 (CH 2 ) 14 CH(OH)CH(OH)CO 2 H or CH 3 CH 2 CH(OH)CH(OH)(CH 2 ) 13 CO 2 H], and the peak that has an accurate m / z of 374.3617 is consistent with [M+NH 4 ] + of C 22 H 44 O 3 , which could be one or more regioisomers of hydroxydocosanoic acid [e.g., CH 3 (CH 2 ) 19 CH(OH)CO 2 H or HO(CH 2 ) 21 CO 2 H].

Samples of the water in which the space‐exposed worms and the Earth‐only worms had been living were frozen and stored at −20˚C. Although the water temperature of the worms in space was recorded throughout the mission, the information was not relayed in real time to Earth. After obtaining the space‐exposed worm water temperature information, a new set of ‘temperature‐matched’ Earth‐only control worms were kept in the same isolated conditions as before, with the temperature manually adjusted to follow the same profile and time course that the space‐exposed worms experienced.

Whole worms sent into space were found to have fissioned spontaneously, but control whole worms on Earth had not (Table 2 ). Fission was not observed in either space‐exposed or Earth‐only worm fragments that had been manually amputated prior to launch (Table 3 ). It must be noted, however, that the control worms on Earth were kept at 20˚C at all times, while the worms in space unavoidably experienced somewhat higher temperatures at some time periods (Fig. S1). For this reason, the observed difference in spontaneous fission rate must be interpreted with caution.

Flatworm amputation and space‐exposed and Earth‐bound worm sample schematics. (A) Approximately a third of the anterior part of the worm was cut off to create the head (H) fragment; then the posterior half was cut in half to create the pharynx (P) and tail (T) fragments, respectively. A total of 15 flatworms were cut and collected into three separate 50 mL conical tubes per fragment. (B) An identical number of worm samples, both whole and amputated fragments, were either sent into space or left on Earth for 32 days. (C) Immediately upon return to Earth, both space‐exposed and Earth‐only control worms from each sample tube were transferred to a Petri dish containing fresh Poland Spring water individually to identify any phenotypic changes

Immediately upon return to Earth, worms from each sample tube were transferred to a Petri dish containing fresh Poland Spring water to identify any phenotypic changes under the microscope (Fig. 2 ). The size of the worms did not differ appreciably between the two groups, within the normal variation of the length of D. japonica worms (data not shown). Surprisingly, only the sample containing 10 whole worms that had been launched into space showed immediate unusual behavior when introduced into fresh Poland Spring water: they curled up ventrally and were somewhat paralyzed and immobile (Fig. 3 , and Videos S1 and S2). There was no sign of immediate blistering of the worm's epidermis, which generally indicates acute toxicity. This shock‐like phenotype lasted for an hour; the worms then gradually started to flatten out on the surface, slowly regaining movement, and after 2 h they all returned to normal behavior and morphology. This indicates that the sample of 10 whole worms that had been launched into space in a single sealed tube modified their biological state to accommodate the environmental change; when reintroduced into fresh water, the environmental change back to standard living conditions resulted in severe shock because of their altered metabolic state. Water shock was not seen in the later established ‘temperature‐matched’ Earth‐only control worms.

Advances in regenerative medicine require an understanding of the remarkable mechanisms by which some organisms repair damage to their bodies. How these processes change when an organism is in outer space, in the absence of the normal gravitational and geomagnetic fields, is largely unknown. We undertook a series of experiments to understand the effects on organisms that spent an extended period of time in space. Planaria were either pre‐amputated or left as whole for spontaneous fission, and sealed into 50%/50% air/water tubes on Earth (Table 1 ). An identical set of worms were launched into space, spending over a month at the ISS under microgravity and micro‐geomagnetic force before returning to Earth. We evaluated these samples upon return, as well as after 20 months of maintenance in our laboratory; the latter time period was chosen as an optimal compromise between timely reporting of results so that they can contribute to the work of other groups and the ability to demonstrate truly long‐term consequencs of space travel.

3 DISCUSSION

Our study examined how the regenerative and physiological properties of planaria changed during a space mission. Conditions associated with space are impossible to fully replicate on Earth, and yet must be explored due to the inevitability of the presence of humans and other organisms in space. We analyzed morphological, behavioral, bacteriological, and biochemical endpoints, finding not only a number of differences immediately after return to Earth, but also ones that persisted for 20 months. These are the first data exploiting a unique opportunity—exposing a highly tractable regenerative model system to space travel—which our laboratory will build on in future trips to the ISS.

This experiment faced a number of unavoidable limitations, some of which will be addressed in future missions. Maintaining the temperature of control worms on Earth exactly the same as those samples that traveled to space during the entire space mission was more challenging than anticipated. Future missions will achieve more consistent temperature control for the experimental samples, as well as provide real‐time data back to Earth which can be used to alter the temperature of Earth‐only controls in real time. The biggest unknown is likely to be stress associated with liftoff and splashdown, which cannot be easily replicated on Earth; future experiments will mimic this by applying similar mechanical disturbances of the Earth‐only control organisms. Thus, we do not individually implicate microgravity, vibration of liftoff, or 0 GMF in the effects we describe—the differences between space‐exposed and Earth‐bound controls are consequences of the entire process of delivery to, and return from, a space environment. It should be noted, however, that this is not simply a confounder: since any actual space travel will by necessity include all of these aspects, the effects must be studied as a real component of space travel which living systems will experience. Given the results reported from recent work (Adell et al., 2014) using much longer exposures to g forces of similar magnitude to that experienced by our samples during liftoff (∼3g) and landing (∼7g), we do not think it likely that our results are due to the brief periods of higher gravity that our planaria experienced. Future work will explicitly dissociate mechanistically the individual effects of the various stresses from the microgravity and micro‐geomagnetic force exposure per se.

The biggest factor reducing our ability to identify significant new regenerative phenotypes is probably the fact that worms were only put into space after being cut on Earth. We reduced the time between amputation and liftoff to the smallest delay compatible with the liftoff process, but it was not feasible to eliminate it completely due to the numerous logistics that have to take place before takeoff. Ideally, a forthcoming experiment will involve cutting them while in space; this experiment is important as many of the key steps of regeneration (and cellular decision‐making with respect to head−tail commitment of the blastema) occur very soon after cutting. In order for us to undertake this particular experiment, we will need to identify an astronaut residing on the ISS who is willing to assist us and is able to manually manipulate and cut the worms with a scalpel in microgravity. Future missions will also record ambient GMF values as a function of time throughout the experiment.

The finding of a single double‐headed worm in a population of 15 worms, which we have not observed in >18 person‐years of maintaining a colony of D. japonica, was exciting, even though it represents an N = 1 observation. Even more remarkable is the persistence of the phenotype, which recurred following a second and third round of amputation of the worm in normal conditions on Earth, in plain water, revealing a stable change to the organism's regenerative anatomy. It should be mentioned that recurrence of a two‐head phenotype in water‐only regeneration has been previously reported (Levin, 2014a; Oviedo et al., 2010); thus, while the reprogramming to a two‐head state was induced by space travel, its persistence across rounds of regenerations may be a general feature of such stable heteromorphoses (however induced) and not specifically due to space conditions.

While the exact mechanism of the induction of the two‐headed state by space travel is unknown, we can propose several hypotheses. It is known that reduced GMF disrupts cytoskeletal structures (Wang et al., 2008). It has also been shown that pharmacological disruption of microtubules induces double‐headed phenotypes (Mcwhinnie, 1955; Mcwhinnie & Gleason, 1957). Thus, one possibility is that the observed double‐headed worm was induced by a reduced GMF‐mediated disruption of cytoskeletal signaling. Another important recent finding is that microgravity alters ion channel electrophysiology (Richard et al., 2012); as several studies have shown the importance of endogenous bioelectrical signaling in regenerative patterning in planaria (Beane et al., 2011, 2013; Chan et al., 2014; Zhang, Chan, Nogi, & Marchant, 2011) and many other model systems (Bates, 2015; Levin, 2007, 2014b; Levin & Stevenson, 2012; Sundelacruz, Levin, & Kaplan, 2009), it is possible that some of our observed effects are mediated by alterations of ion channel function. Other possibilities include the effects of the space travel environment upon Wnt pathway molecules (Petersen & Reddien, 2007; Yazawa, Umesono, Hayashi, Tarui, & Agata, 2009) or physiological connectivity via gap junctions (Emmons‐Bell et al., 2015; Nogi & Levin, 2005; Oviedo et al., 2010). Especially interesting with respect to the hypothesis of gap junctional involvement is the recent observation that microgravity reduced the expression of two gap junction genes in embryonic stem cells (Blaber et al., 2015). Given the importance of gap junctions in planarian regeneration (Nogi & Levin, 2005; Oviedo et al., 2010) and in the control of stem cell (including planarian neoblast) biology (Oviedo & Levin, 2007; Oviedo et al., 2010; Starich, Hall, & Greenstein, 2014; Tazuke et al., 2002), this is a mechanism that will be investigated further in subsequent work.

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.