Beneficial microbes mitigate molecular stress responses and accelerate developmental pathways in host animals during spaceflight

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Koch, Ana Conesa, Timothy J. Garrett, Rachel Ormsby, Ryan Bohl, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7103661/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract As humans continue the manned exploration of space, it is critical to understand the impact of this harsh environment on the beneficial microbes that interact with their bodies. Here, we explore whether the onset of symbiotic associations between microbes and animals are impacted during spaceflight. We used the association between the bobtail squid Euprymna scolopes and its beneficial bacterium Vibrio fischeri as an animal model system to examine how spaceflight affects symbiotic interactions at the transcriptomic, metabolomic, and lipidomic levels over time. Our results suggest that in the spaceflight environment, symbiotic microbes can mitigate molecular stress responses of the host animal and accelerate normal developmental pathways, such as neurogenesis and tissue morphogenesis. Overall, this work provides evidence that beneficial microbes can effectively colonize nascent host epithelial tissues in microgravity and play a critical role in shaping the host tissue environment to promote stability of symbiosis during spaceflight. Biological sciences/Cell biology Biological sciences/Microbiology Biological sciences/Molecular biology spaceflight host-microbe interactions symbiosis animal development Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Spaceflight exerts numerous environmental and physiological challenges on life. Whether it be the reduction of gravity, increased exposure to radiation, or the mental health challenges of being in a confining and isolating environment 1 – 3 , working and living in space presents unique challenges to not only plant and animal hosts but also their associated microbiomes 4 , 5 . Eukaryotes live in a microbial world, and space travel is no exception. Microbes are transported and exchanged with the crew, food supplies, experimental cargo, flight hardware, and the spacecraft itself; however, the processes by which interdomain communication is happening in the spaceflight ecosystem are not fully understood and represent an important area of research for on-going human spaceflight activities. Recently, there have been numerous studies on how the dynamics and diversity of the microbiome change within and amongst astronauts as well as the exchange between the crew and the built environment of the International Space Station (ISS) 6 – 16 . The results of these studies have been varied and appear to be highly dependent on the location within the body, with increased taxonomic diversity even after short exposures to space in the gut and saliva microbiomes, whereas other areas maintain homeostasis over time, such as the skin microbiome 5 . The changes in microbiome diversity and associated metabolism can be heavily influenced by additional modifiers, such as diet, age, medications, and flight durations 6 , 7 , 9 . Although efforts are underway to regularly track and monitor the microbiomes of crew and spacecraft to improve the overall understanding of what is a healthy microbiome during spaceflight 17 , 18 , there are numerous unresolved questions including what drives the variation of the host microbiome in space and how resilient the microbiome is to the many hazards of spaceflight. Additionally, due to the overall complexity of the microbiome, fundamental questions persist regarding whether spaceflight alters the normal communication between host animal tissues and their associated microbes and how beneficial microbes facilitate and mediate the normal development of animals in environmentally stressful conditions. One approach to address these fundamental questions of how microbes, particularly beneficial microbes, impact the normal health and development of animals during spaceflight is to use simplified model systems. As in Earth-based research, it is necessary to use multiple model systems in space biology research to generate a robust understanding of the impact that spaceflight has on animal-microbe communication and physiology. No single model system can address all questions regarding the impact of spaceflight on animal and human health 19 , 20 . Although there has been significant progress regarding the use of tissue and organoid chips for spaceflight experiments 21 – 23 , there are still significant limitations for examining complex organismal-level functions throughout the body as well as temporal and spatial variations between microbes and their hosts 24 , therefore using experimentally tractable animal-bacterial models that have short time frames of initiation and development are pivotal for space life sciences research. In this study, we examined how microbes colonize nascent animal epithelial tissues under the physiological stress of spaceflight using the binary symbiosis between the Hawaiian bobtail squid Euprymna scolopes and its bioluminescent bacterial partner Vibrio fischeri . The squid-vibrio symbiosis has served as a valuable model to help untangle the dynamic interplay between environmentally acquired bacteria, the host epithelial barrier, and the innate immune system for more than 30 years 25 – 31 . The squid harbors a specialized organ that houses the symbiosis and is embryologically derived from the squid hindgut 32 . The light organ exhibits both ciliated and microvillous epithelia, which are the two most common types of polarized mucosal epithelial within the animal kingdom that interface with bacterial symbionts 33 , 34 . Upon colonization, the light organ exhibits a mucosal innate immune response that shares extensive similarity to mammalian systems 35 . Additionally, previous research has shown that under simulated microgravity conditions wild-type strains of V. fischeri do not exhibit noticeable changes to their growth rate compared to gravity-based controls 36 , 37 . Together, these features coupled with the small size of the hatchling squid (~ 3 mm, Fig. 1 a), short embryogenesis (~ 21 days), and rapid colonization and developmental timelines make this system ideal for spaceflight experimentation 38 . Within minutes of the squid hatching, microbes are recruited from the environment to the light organ surface (Fig. 1 b). Ciliated epithelial cells on the exterior of the light organ aggregate the bacteria into the vicinity of pores on the light organ surface that connect to six independent crypt spaces lined with mucosal epithelial cells (Fig. 1 c) 39 – 41 . Evidence suggests that regardless of inoculum size, typically only one V. fischeri cell is required to enter the pore and migrate to each of the six individual crypt spaces to initiate colonization 42 , 43 . Once symbiosis-competent V. fischeri enter the crypt spaces they begin to replicate clonally and interact with the host epithelium 44 , 45 . During these interactions, the bacteria release diffusible molecules into the crypt spaces that can influence and modulate the host responses as well as foster communication between V. fischeri symbionts in the other crypt spaces 46 , thereby rapidly inducing host light organ morphogenesis 47 – 52 . To explore how spaceflight impacts the onset of beneficial animal-microbe interactions, newly hatched squid were sent to the ISS aboard the SpaceX Commercial Resupply Services-22 mission (SpX-22) where, once in the microgravity environment, they were inoculated with mutualistic V. fischeri . The transcriptomes, metabolomes, and lipidomes of the juvenile squid, in the presence and absence of the beneficial microbes, were sampled over time to provide a broad overview of how spaceflight impacted the initiation of the symbiosis as well as the onset of bacteria-induced developmental pathways. The results of this study provide new insights into how the space environment alters the normal timeline of bacteria-induced developmental processes in host tissues and how microbes can potentially mitigate stress responses and promote the stability of symbiotic interactions following environmental perturbations, such as spaceflight. Results Spaceflight experimental overview and hardware performance validation The experiment entitled Understanding of Microgravity on Animal-Microbe Interactions (UMAMI) was designed to monitor the initiation and early developmental pathways of the host animal in the presence and absence of beneficial microbes in the spaceflight environment. The UMAMI experiment was completed in two fluid processing cassettes (Fig. 1 d), one with V. fischeri (i.e., symbiotic, SYM) and one without V. fischeri (i.e., aposymbiotic, APO), and integrated into the ADvanced Space Experiment Processor (ADSEP) where it was launched aboard SpX-22 from the Kennedy Space Center. A detailed overview of the experimental design is provided in the methods section and visualized in Fig. 1 and Supplemental Fig. S1 . Briefly, the automated experiment used a loop design where animals were housed within cell culture bags (i.e., aquariums; Fig. 1 e) and upon reaching microgravity motors pumped in either filtered-sterilized seawater containing V. fischeri inoculant as SYM or only filtered seawater as APO controls. Animals were then incubated as SYM or APO for 0, 2, 6, or 12 h and then were infiltrated with RNALater to terminate the experiment and the bags were later moved to -80°C aboard the ISS until their return to Earth 30 days later (Fig. 1 f). This entire procedure was repeated under normal gravity conditions as a ground control. A total of 16 replicate animals were recovered for each colonization treatment with two technical replicates for both the spaceflight experiment (n = 128) and ground controls (n = 128). Electrical current draws and temperature readings from the ADSEP hardware suggested motor and pumping operations for both the spaceflight and ground controls occurred as planned (Supplemental Fig. S2 ). The temperature for the spaceflight and ground incubations ranged from 22.78°C − 23.32°C for the APO cassettes and 22.80°C − 23.67°C for the SYM cassettes, indicating less than 1°C fluctuation for the duration of the UMAMI experiment. To confirm that animals were effectively inoculated using the ADSEP hardware, replicate experiments, including the 39-h hold simulating the launch pad experience, were conducted on the ground during the Experiment Verification Test (EVT), a NASA requirement to ensure spaceflight readiness. The entire inoculation and incubation protocol was carried out but before the final RNALater fixation step, the experiment was then stopped at 12 h, and the live animals were removed from the fluid processing cassettes and aquarium bags to assess the effectiveness of colonization within the hardware. Individual animals in the SYM and APO conditions were assessed for luminescence, colony forming units, and bacteria-induced apoptosis (Supplemental Fig. S3 ). EVT results at 12 h indicated that the SYM animals exhibited significantly higher luminescence readings (2000–22,000 relative light units (RLU); p < 0.0001) compared to APO controls (< 500 RLU) suggesting effective colonization by the bioluminescent symbiont using the automated ADSEP hardware (Supplemental Fig. S3 a). Additionally, a subsample of SYM light organs in the EVT exhibiting high luminescence were subsequently homogenized and plated on seawater tryptone media resulting in a minimum of 2 x 10 5 cells per light organ, thereby confirming colonization by V. fischeri (Supplemental Fig. S3 b). APO light organs exhibited no colony forming units when plated. SYM light organs also exhibited the hallmarks of apoptosis with distinct patterns of pycnotic nuclei within the superficial ciliated epithelium of the light organ compared to APO controls (Supplemental Fig. S3 c). Together, these results suggest that the UMAMI experimental design, ADSEP hardware, and automated procedures were effective in inoculating the animals with V. fischeri during EVT testing. Transcriptome analyses revealed spaceflight was the primary driver of differential gene expression in the host tissues regardless of symbiotic state To investigate the effects of spaceflight and bacterial colonization on host gene expression, RNA sequencing was used to generate a total of 23,064,220,236 raw reads across 32 samples with a mean of 720,756,882 reads per sample. The reads were then mapped to the E. scolopes genome 53 , 54 with a success rate of 65–77% reads per sample. A PERMANOVA test was conducted on the transcriptome to statistically compare experimental groups. Strong statistical significance was found between the spaceflight and ground datasets (p-value < 0.0001), whereas the aposymbiotic and symbiotic datasets displayed little to no significance (p-value = 0.064). Additionally, a k-means analysis was conducted on the entire transcriptome and suggested the data separates according to the spaceflight condition, with a single ground cluster and three spaceflight clusters (Supplemental Fig. S4 ). Analysis of the transcriptomes using principal component analysis (PCA) revealed two distinct clusters, one each for flight and ground samples (Fig. 2 a). Collectively, these results suggest there is more variability in the spaceflight exposed animals compared to the ground controls and that spaceflight may be imprinting a stress that affects both the magnitude and variability in gene expression within the host animal. Although the specific rate in which V. fischeri colonized the light organ was not able to be directly measured due to spaceflight hardware limitations, gene expression markers typically associated with the initiation of symbiosis were examined and compared at all time points and treatments. Transcripts of target genes that encode for peptidoglycan receptor proteins (PGRPs), galaxins, cathepsin-L, and lipopolysaccharide binding proteins (LBPs), which all typically exhibit trends of increased expression during the normal colonization under unit gravity conditions 55 – 58 , were examined. Both flight and ground controls exhibited a non-significant, but overall trend of increased gene expression of several genes typically associated with colonization with low, but consistent upregulation in SYM animals relative to APO across most time points (Supplemental Fig. S5 ). The exception was transcripts of genes encoding cathepsin-Ls, which were increased under APO conditions beginning at 2 h only during spaceflight conditions (Supplemental Fig. S5 ). Cathepsins are known to be activated by caspases 59 , which can be increased under the stress of simulated microgravity conditions 60 , suggesting cathepsins are not a useful marker of symbiosis during spaceflight. However, the increased expression of other genes that are typically associated with the onset of symbiosis in the spaceflight and ground experiments in SYM animals compared to APO controls suggest that the host tissues were colonized by V. fischeri during the flight and ground conditions. Due to the large effect of spaceflight on overall gene expression in the host tissues gene expression analysis was performed on the flight and ground samples separately. To examine the effect of bacterial colonization on host gene expression under spaceflight and ground conditions, the SYM and APO transcriptomes were compared at each time point with differentially expressed genes (DEGs) defined as those under a false discovery rate (FDR) of 0.1 (Supplemental Fig. S6 ; (Supplemental Dataset S1). At the beginning of the experiment during spaceflight (t = 0 h), the ratio of DEGs up or downregulated under SYM and APO conditions was approximately even (i.e., the same number of DEGs increased in SYM and APO) (Supplemental Fig. S6 a). However, as the experiment progressed there were fewer significant DEGs increased under SYM conditions relative to APO. This apparent gene downregulation in SYM began at 2 h post-inoculation and continued at 6 h and 12 h. Similar results were observed in ground-based simulated microgravity conditions where APO animals exhibited much higher transcriptional activity than SYM animals 38 . Overall, the increased transcriptional activity during the spaceflight within aposymbiotic animals suggests that in the absence of the microbe the animal is activating a range of unique host gene expression responses to the novel environment of spaceflight that was not observed under ground controls. The presence of the symbiotic microbe in the spaceflight environment might “override” or help mitigate this increase of transcriptional activity within the animals. Gene set enrichment analyses revealed that several key animal developmental pathways were altered during spaceflight and dependent on the presence or absence of symbionts To identify common gene functions beyond just DEGs affected by colonization under spaceflight conditions, gene-set enrichment analysis (GSEA) was performed at all time points with significant gene sets (SGSs) defined with the default value of FDR < 0.25 (Fig. 2 b, c; Supplemental Fig. S7; Supplemental Dataset S2 - S3). GSEA uses a ranked list of genes that incorporates both the p-value and fold change in the expression to find gene functions that are statistically over- or underrepresented within the dataset 61 , 62 . A comparison of the SGSs at the start of the experiment (t = 0 h) revealed that the spaceflight and ground animal transcriptomes shared 30.8% of the SGS despite the flight animals being launched into space (Fig. 2 b). By 2 h post inoculation of the animals, however, there were few common SGS between the spaceflight and ground controls (Fig. 2 b; Supplemental Dataset S3). Under normal gravity conditions, there was an initial drop in overall SGSs at 2 h (Fig. 2 b), which matches gene expression patterns previously published in studies on the host animal 52 , 63 . By 6 h in ground controls, many of the SGSs that were enriched in SYM animals (Fig. 2 c; Supplemental Dataset S3) had functions associated with previously characterized bacteria-induced morphogenesis of the ciliated epithelium 47 , 49 , 50 . In spaceflight-exposed animals, however, at 2 h there were 102 SGSs enriched compared with only 7 SGSs in ground controls, with 81 SGSs increased in SYM and 21 increased in APO (Fig. 2 b; Supplemental Fig. S7). Furthermore, gene sets associated with bacteria-induced morphogenesis were enriched earlier in spaceflight conditions relative to ground controls (i.e., 2 h in flight and 6 h in ground controls), suggesting the host responded transcriptionally to the bacteria more rapidly in spaceflight than under unit gravity conditions (Fig. 2 c; Supplemental Fig. S7; Supplemental Dataset S3). Symbiotic animals exhibited lower oxidative stress gene expression in spaceflight compared to aposymbiotic controls One pronounced difference in the spaceflight animals was the enrichment of SGSs associated with oxidative stress in the light organs of APO animals, which did not receive their symbiotic microbes. Analysis of the gene expression of core and noncore genes associated with oxidative stress pathways revealed dynamic changes under spaceflight conditions (Fig. 3 a, d; Supplemental Dataset S2-S3). At the start of the experiment (t = 0), there was a slight increase in the expression of oxidative stress genes in spaceflight animals relative to their ground controls, but by 2 h post-inoculation, there was a reduction in the levels of gene expression in the SYM animals that persisted for the rest of the experimental timeline. During spaceflight, genes encoding several enzymatic antioxidants, such as superoxide dismutase and a wide range of peroxidases (e.g. glutathione peroxidases, glutathione reductase, and peroxiredoxins) exhibited increased expression in APO animals relative to SYM animals beginning at 2 h (Fig. 3 a) and continued through 6 h (Supplemental Dataset S2 – S3). These results suggest that aposymbiotic animals exhibited a higher and sustained oxidative stress response in their light organs during spaceflight compared to symbiotic animals. Symbiotic animals exhibited higher expression of genes associated with neurogenesis and cell morphogenesis under spaceflight conditions Numerous gene sets associated with the formation and stabilization of synaptic connections as well as the regulation of microtubule assembly and neuronal differentiation were significantly enriched in the SYM light organs compared to APO during spaceflight (Fig. 2 c). Several recent studies have shown the important role that symbiotic microbes can have in regulating the blood-brain barrier, homeostatic regulation of neuronal cells and in neurogenesis 64 , 65 . Beginning at 2 h post-inoculation, there were increases in genes encoding for several kalirin-like and Trio proteins (Fig. 3 b, e; Supplemental Dataset S2 – S3), which play important roles in nerve growth, remodeling of synapses, and axonal development 66 . The increased expression of these genes in SYM animals during spaceflight was also observed at 6 h post-inoculation, however, by 12 h post-inoculation expression levels of most of these genes were at background levels suggesting transient, but increased, gene transcription during spaceflight. Additionally, there was also a significant increase (P < 0.05) in expression of death-associated protein kinase (DAPK) early (i.e., 2 h) in the initiation of the symbiosis during spaceflight that was not significant under ground conditions (Fig. 3 b). DAPK is a molecular switch that has many different roles within animals including mediating ceramide and caspase-induced apoptosis 67 , 68 . DAPK also has important roles in regulating neuronal death, regulating synaptic plasticity, and is upregulated during recovery from injury 69 , 70 . There was also increased expression of titin isoforms during spaceflight in SYM animals, which encode proteins found in numerous muscles and neuronal cell types 71 , 72 and has been shown to regulate the structure of microvilli and trafficking of animal immune cells 73 . These results suggest that during spaceflight microbes can regulate aspects of neurogenesis during the bacteria-induced morphogenesis of the host light organ tissue. Aposymbiotic animals exhibited higher transcription of genes associated with lipid production, transport, and localization during spaceflight In animals that never received their beneficial symbiont, there were pronounced increases relative to SYM in the expression of genes associated with lipid transport and localization genes during spaceflight. Although many of the genes that were differentially expressed during spaceflight encode hypothetical or uncharacterized proteins, there were increases in the expression of genes associated with lipid transport proteins, such the oxysterol-binding proteins-related proteins (ORPs), apolipophorins, phospholipid scramblases, sterol-regulated proteins, and IgGFc-binding proteins (Fig. 3 c, f). Many of these lipid-transport proteins, such as apolipophorin and phospholipid scramblases, have dual functions and can bind microbial-associated molecular patterns, such as LPS and phosphatidylserines, as well as serve as mediators of apoptosis, innate immunity, oxidative stress responses, and other signaling processes in host cells 74 – 76 . Additionally, IgGFc-binding proteins, also known as Fc gamma binding proteins, are mucin-like glycoproteins that play roles in mucosal defense and are often secreted into the mucus to increase the structural integrity of the mucus layer 77 , 78 . The significant increase of these diverse lipid transport proteins in the APO animals during spaceflight may reflect a heightened stress response from animals that did not receive the normal microbial signals or cues for development. Spaceflight altered the host lipidome with increases in ceramide abundance in symbiotic animals In parallel with the transcriptomic analysis, the lipidome of the host animals was also profiled for each treatment and time point. Liquid chromatography/high-resolution mass spectrometry (LC/HRSM) with electrospray ionization (ESI) detected 3612 total peaks in positive ESI mode and 1812 total peaks in negative ESI, with 938 (~ 26%) and 373 (~ 21%), respectively, able to be characterized (Fig. 4 ; Supplemental Fig. S8; Supplemental Dataset S4). Principal component analysis showed that, as with the transcriptome, spaceflight strongly affected the lipidome at all time points. To characterize differential lipid abundances, a combination of multivariate orthogonal partial least squares discriminant analysis (OPLS-DA) and univariate (Student’s t-test) statistical tests were used with significant lipids being defined as having both a VIP > 1.5 for OPLS-DA and FDR < 0.1 for the t-test (Fig. 4 a; Supplemental Fig. S8). In normal gravity conditions, there was an initial enrichment of significant lipids (both positive and negative ESI) in SYM animals relative to APO at 0 h and 2 h, followed by a reversal in the pattern (i.e., APO > SYM) at 6 h and 12 h (Fig. 4 b, c; Supplemental Fig. S8). Beginning at 2 h in spaceflight, the majority of significant lipids exhibited a trend of being increased in SYM which continued at 6 h. Interestingly, significantly different lipids during spaceflight were only found at 12 h in SYM in the -ESI mode (Supplemental Fig. S8). Of those lipids that could be identified with tandem mass spectrometry (MS/MS), ceramides were the most abundant group, representing 36.6% and 41.7% of characterized lipids in flight and ground, respectively (Fig. 4 c; Supplemental Dataset S4). During spaceflight, ceramides were generally increased in SYM animals relative to APO with the highest levels at 2 and 6 h post-inoculation (Supplemental Fig. S9). In ground conditions at 2 h, however, the abundance of ceramides was evenly distributed between APO and SYM animals but by 6 and 12 h ceramides exhibited greater abundance in the APO animals compared to symbiotic animals (Supplemental Fig. S9). Ceramides are lipid modulators that regulate a wide range of functions in the cell, including cell differentiation, cell arrest, inflammation, and apoptosis 79 . Accumulation of ceramides within cells can affect membrane fluidity, alter mitochondrial function, and induce both extrinsic and intrinsic apoptosis pathways 80 primarily through the activation of caspases 81 . The elevated levels of ceramides in SYM light organs during spaceflight coupled with the observed decreases of transcription in genes associated with lipid transport (Fig. 3 c) suggest ceramides, as well as other lipids, may be accumulating in the light organs of symbiotic animals during spaceflight. The accumulation of this potent signaling molecule during spaceflight may be causing, and accelerating, several downstream effects, such as increases in initiator and executioner caspase gene expression and early onset of bacteria-induced apoptosis previously observed in ground-based simulated microgravity experiments 60 , 82 . Symbiotic state altered metabolites associated with the regulation of the host immune system during spaceflight To assess how spaceflight impacted the overall pool of metabolites within the host light organ, untargeted metabolomics using LC/HRMS was used to generate profiles of the different treatments. Analysis revealed a total of 3500 peaks in positive ESI (+ ESI) and 818 peaks in negative ESI (-ESI) mode (Supplementary Dataset S5). Of the recovered metabolites, 129 (~ 4%) for + ESI and 55 (~ 7%) for -ESI could be annotated at the highest confidence identification (level 1). Principal component analysis showed that as in the transcriptome and lipidome the metabolome was strongly influenced by spaceflight (Fig. 5 a). Statistical analysis of metabolites was performed using OPLS-DA and a Student’s t-test, with significant metabolites being defined as having both a VIP > 1.5 and FDR < 0.1, respectively (Supplemental Dataset S4; Supplemental Figures S10-S11). Analysis of the + ESI data revealed that at the start of the UMAMI experiment (t = 0), the metabolome profiles of the animals in spaceflight and ground conditions were comparable (Supplemental Fig. S10). By 2 h post-inoculation, however, there was a pronounced disparity in both the + ESI and -ESI data in SYM animals between spaceflight and ground samples with a reduction in the significant metabolites observed in spaceflight samples compared to ground controls (Fig. 5 b; Supplemental Fig. S10 – S11). Although most of the significant metabolites could not be fully identified to the highest level of confidence, the molecules that could be characterized with high confidence possessed similar bioactivities related to the modulation of the host immune system. For example, by 2 h in spaceflight conditions, metabolites known to act as immunostimulants were increased in APO animals relative to SYM animals, including mevalonolactone and azelaic acid (Supplemental Dataset S5) 83 – 85 . Additionally, in APO animals there were significant increases in formylkynurenine, formyl-5-hydroxykynurenamine, and 5-hydroxy-N-formylkynurenine, which are metabolites involved in the kynurenine pathway (Fig. 5 c; Supplemental Dataset S5). The kynurenine pathway is critical for tryptophan catabolism and is highly regulated by the immune system 86 . Under high oxidative stress and inflammation conditions, the kynurenine pathway can act as a negative feedback system to help scavenge reactive oxygen species and suppress the inflammatory response through tryptophan depletion 87 . Metabolites associated with the kynurenine pathway were increased in APO in both conditions relative to SYM, however, the differences due to symbiotic state were lower under flight conditions compared to ground controls (Fig. 5 c; Supplementary Dataset S5). These results were corroborated by increases in the expression of genes associated with tryptophan metabolism (e.g. tryptophanase isoforms, tryptophan 2,3-diooxygenase; and kynurenine 3-monooxygenase) in the APO animals during spaceflight beginning 2 h after the start of the experiment (Supplemental Fig. S6 ). These data may reflect the host attempting to compensate for the lack of symbiotic microbes by increasing expression of host-derived tryptophanases. Interestingly, tryptophan abundance was maintained approximately equal between APO and SYM animals under both spaceflight and ground conditions (Fig. 5 c; Supplementary Dataset S5). There was, however, a pronounced reduction in the accumulation of kynurenine pathway metabolites in APO animals under spaceflight compared to ground controls (Fig. 5 c; Supplemental Dataset S5). The reduction of the immunosuppressive kynurenine pathway in the APO flight animals may increase the stress environment in the APO light organs during spaceflight. In contrast, the SYM animals exhibited elevated levels of immunosuppressant metabolites, such as urocanate and xanthine, which were increased 6 h post-inoculation in SYM animals relative to APO controls (Fig. 5 b; Supplemental Dataset S5). Additionally, the decrease in kynurenine-pathway metabolites in SYM animals relative to APO suggests that V. fischeri played a major role in regulating tryptophan metabolism, perhaps through the metabolization of tryptophan 88 along the kynurenine pathway during spaceflight, thereby modulating the oxidative stress and immune responses in the host during spaceflight. Overall, these results suggest that colonization of the light organ by the symbiont may act as a “calming” mechanism for host stress and the innate immune response during spaceflight. Discussion Microbes play a major role in maintaining human health and strategies to ensure effective microbiome homeostasis and resilience during long-duration spaceflight are critical. The outcomes of our multi-omics approach, as summarized in Fig. 6 , suggest that microbes can effectively initiate the bacterial colonization process of the nascent host epithelial cells during spaceflight, thereby triggering the activation of key bacteria-induced pathways needed for the natural maturation of host tissues. However, the results also show that the onset of some aspects of normal developmental timeline were altered and accelerated under the stress of spaceflight. Additionally, the results show that the microbes may confer stress resistance to the host to help potentially mitigate perturbations caused by spaceflight. Together, these results suggest there is alteration of the molecular dialog occurring between the host and symbiont, which is an important finding for the field of space biology. The spaceflight environment has widespread effects on the physiology of animals that can negatively impact host-microbe interactions, including changes in body fluid dynamics, shortening of cilia, reductions in mucin production, and reorganization of the cellular cytoskeleton 89 – 93 . Despite these potential challenges, our results suggest that the host was able to effectively recruit V. fischeri from the environment and initiate symbiosis under the stress of spaceflight. Much like the mammalian digestive tract where microbes must travel through numerous chemical and physically distinct environments to associate with the surfaces of mucosal epithelial cells 94 , V. fischeri physically interface with mucociliary epithelial surfaces of the squid light organ (Fig. 1 ) where they aggregate within a mucus matrix containing antimicrobial molecules that serves to enrich V. fischeri from surrounding environmental microbes 95 – 98 . Analysis of the host gene expression responses suggests that V. fischeri was able induce the key host molecular and biochemical responses in the squid within the 12 h UMAMI experimental timeline, suggesting that normal colonization of host epithelial tissues by beneficial microbes will not be impeded during space travel. Additional experimentation, however, will be required to more fully assess whether certain aspects of the colonization processes, such as mucin production, surface-attachment strategies, aggregate formation, and bacterial strain population dynamics are altered during spaceflight, as simulated microgravity has been shown to impact these colonization phenotypes in animal-microbe symbioses 36 , 37 , 82 . For example, it is possible that the rate of bacterial colonization within the host light organ during spaceflight was altered, potentially accounting for many of the observed changes in the host transcriptome, metabolome and lipidome. In addition to the initiation of colonization, the UMAMI experiment showed that there were several changes to the host developmental timeline during spaceflight, including the rapid onset of an oxidative stress gene expression response. Although no organism on Earth has specifically evolved to cope with spaceflight, the innate immune system of eukaryotes has evolved to sense and respond rapidly to environmental perturbations, such as increases in oxidative stress and colonization by microbes 99 . There is a long history of spaceflight studies demonstrating that the elevated radiation and microgravity environment can induce oxidative stress responses throughout all organ systems in the body 100 . As redox homeostasis is fundamental to all metabolic processes, life has evolved rapid system-wide oxidative stress responses 101 . The increase in oxidative stress responses in both the symbiotic and aposymbiotic animals after the onset of the UMAMI experiment likely reflects the immediate stress animals experienced in the spaceflight environment. However, within 2 h of colonization by V. fischeri , the presence of the microbes appeared to attenuate the magnitude of the stress response to the perturbation of spaceflight. The attenuation of stress responses in symbiotic animals may be a by-product of the bacteria using the host stress response as a cue for normal colonization, thereby stabilizing the symbiosis during spaceflight. The use of stress to cue and shape host-microbe interactions has a long evolutionary history and is conserved in both plants and animals 99 . In plants, one well-studied example is in leguminous plants, such as the clover Medicago truncatula , whose symbiotic interactions with the nitrogen-fixing Sinorhizobium meliloti results in the formation of nodules in the host plant to improve the nutritional environment 102 . The host plant can initiate a generalized oxidative stress response that can trigger transcriptional changes in the symbiont to increase extracellular polymeric substances, which coupled with the production of Nod factors, contributes to the formation of an infection thread and the onset of bacteria-induce nodule formation within the plants 103 , 104 . The results of the UMAMI experiment suggest that the spaceflight environment may increase physical and chemical stimuli in the host, such as reactive oxygen species and antimicrobial peptides, acting as cues for the microbial response. Bacteria can then regulate the transcriptional responses of the host subsequently reducing stress and accelerating normal bacteria-induced development. The impact of the bacterial symbiont on host physiology during spaceflight was also reflected in the lipidome and metabolome analyses. The pronounced increase in lipids in symbiotic animals, in particular ceramides, during spaceflight suggests that bacteria-induced lipid accumulation may be triggering important signaling functions in the host. For example, ceramides can induce a range of physiological responses including, neuronal differentiation, the acceleration of apoptosis signaling, and increased exosome production to mediate communication between different cell types 105 – 107 that are highly dependent on the intracellular location 108 . Additionally, changes in the metabolome also revealed the critical role that the bacteria were playing in mediating the host stress response during spaceflight. The changes in metabolites associated with tryptophan metabolism may play a critical role in maintaining, or restoring, the homeostasis of the host light organ during spaceflight. In mammals, regulation of tryptophan metabolite abundance is coordinated between the gut microbiome and intestinal epithelial cells, and the kynurenine pathway and its metabolic derivatives are being targeted as potential therapeutic agents for inflammatory and autoimmune diseases 87 . Together, analyses of the transcriptional and small molecule changes in the host tissues revealed the pronounced impact that spaceflight can have on symbiotic interactions. Evidence suggested there was extensive crosstalk between V. fischeri and the host tissues during spaceflight at the initiation of the colonization process, and that the bacteria appeared to help the host squid mitigate the stress responses that the aposymbiotic animals exhibited during spaceflight. These results serve as an important foundation for building future strategies to maintain host-symbiont health in closed ecosystems, such as spacecraft. Predicting microbiome homeostasis and resiliency to the perturbations of spaceflight will require an improved understanding of the mechanisms regarding the initiation, establishment, maintenance, and loss of symbiotic interactions with their eukaryotic hosts. Additionally, to support sustainable long-term habitation beyond Earth, more complex holobiomic analyses of how collections of multicellular hosts and their associated microbiomes interconnect will be required to more fully understand the direct and indirect effects of spaceflight on beneficial interactions with microbes. Methods Ethics statement All experimental procedures using live cephalopods were approved by the University of Florida (201910899), NASA Flight (SQD01), and the Kennedy Space Center (FLT-20-129) Institutional Animal Care and Use Committees (IACUC). Animal husbandry A breeding colony of adult field-caught E. scolopes was maintained at the Space Life Sciences Laboratory in Merritt Island, FL in a recirculating seawater system with a water temperature of ~ 23°C and a 12h:12h day:night light cycle. Egg clutches were maintained separately in 2.5-gallon aquariums under the same temperature and lighting conditions for the duration of their development. One day before hatching, eggs were placed in individual bowls with aeration, and upon hatching, squid were removed within 1 h and placed into filtered seawater (FSW). Bacteria cultures Approximately 48 h before launch, cultures of V. fischeri strain ES114 were grown overnight in Luria Broth Saline to stationary phase as previously described 109 . Bacteria were rinsed once by centrifuging at 15,000 x g for 1 minute, the supernatant removed, and the pellet resuspended in FSW. The resuspended V. fischeri were then diluted 1:1000 in FSW for an approximate concentration of 5 x 10 5 cells per ml of FSW. UMAMI spaceflight experimental design and gravity controls The UMAMI spaceflight experiment was performed within the ADvanced Space Experiment Processor (ADSEP) using two Fluid Processing Cassettes (Fig. 1 d; Supplemental Fig. S1 ) designed and constructed by Redwire Space (Formerly Techshot, Greenville, IN, USA). Each cassette housed eight experimental loops, consisting of three fluorinated ethylene propylene (FEP) 25-mL cell culture bags (Saint-Gobain, Gaithersburg, MD), which are permeable to O 2 and CO 2 but impermeable to water. For each bag, there were two ports connected by Masterflex™ 1/16” ID tubing made of platinum-cured silicone and two peristaltic pump motors, one on each end of the bag. The three bags within each of the loops were: 1) Inoculum, 2) Aquarium, and 3) Fixative (Supplemental Fig. S1 ). Approximately 32 h before launch, the Inoculum bags were readied by loading either 5 mL of V. fischeri inoculum for the SYM colonization condition or 5 mL FSW for the APO colonization condition. Each of the Aquarium bags was prepared by loading eight squid hatchlings into the FEP bags containing 25 mL of FSW. The Fixative bags contained 25 mL of RNAlater™ (ThermoFisher Scientific, Waltham, MA, USA). There were two sets of FEP bags per treatment enabling 16 biological replicates and two technical replicates. Once the bags were aseptically assembled, they were connected to the appropriate pump and tested to ensure no leakage. All APO loops were contained separately in FPC module A, whereas all SYM loops were contained in FPC module B to ensure no potential in-flight contamination by V. fischeri . ADSEP hardware was turned over to SpaceX 24 h prior to launch. The temperature was maintained at 23°C +/- 0.5 for the duration of the UMAMI experiment. Approximately 7.5 h after the successful launch of SpaceX CRS-22, but before the Dragon capsule docked to the International Space Station (ISS), the UMAMI experiment was successfully run for a duration of 12 h. The UMAMI experiment was automatically controlled by Redwire Space from their headquarters in Greenville, IN. The experimental sequence in the ADSEP FPC cassettes initiated with a backflow of 5 mL of FSW from the Aquarium bags into the Inoculum bags to enable the mixing of the culture and provide space in the Aquarium bags for the inoculum (SYM) or seawater control (APO). Following the backflow, the colonization step was initiated by pumping 5 mL of inoculum into the Aquarium bags resulting in a final colonization of 1 x 10 5 cells per mL of FSW. In the APO cassette, 5 mL of FSW was added to the Aquarium bags. The colonization step was allowed to incubate for 0, 2, 6, or 12 h. After colonization, approximately 24 mL of FSW was pumped out of the Aquarium bag back into the Inoculation bags, which were then renamed the Waste bags after which 24 mL of RNALater was added to the Aquarium bags to arrest transcription in the host squid and prevent tissue degradation. This approach was used to increase the ratio of RNALater to water ratio and improve tissue fixation. The fixation process took approximately 2 min. Following the conclusion of the UMAMI experimental procedure, the cassettes were cooled to 10°C for 48 h until the bags could be manually removed by a crewmember and frozen at -80°C on the ISS. The Aquarium bags containing RNALater were stored on the ISS for 30 days at -80°C until their return to Earth. As a control, the entire procedure, including the 39.5-hour hold (32 h pre-launch and 7.5 h transit time), was repeated at the Space Life Science Laboratory under unit gravity conditions at 23°C. Upon completion of the control experiment, the sample bags were removed from the cassette and placed at -80°C for storage for 30 days. During postflight processing of the spaceflight and gravity controls, the frozen squid were thawed, photographed, placed into fresh RNAlater, and stored at -80°C until processing. Extraction of RNA and sequencing Light organs were dissected from 16 squid per treatment and pooled in groups of four to provide enough material for RNA extraction. Total RNA was extracted as previously described from each group enabling three RNA extractions per treatment 27 . Briefly, RNA was isolated using the RNeasy Kit (Qiagen, Hilden, Germany) and treated with the TURBO DNA-free KitTM (ThermoFisher Scientific, Waltham, MA, USA). The concentration of RNA for each biological replicate was determined using a Qubit 2.0 fluorometer (ThermoFisher Scientific, Waltham, MA, USA). RNA quality was determined with an Agilent 2100 Bioanalyzer using an RNA 6000 Nano Kit (Agilent Technologies, Palo Alto, CA, USA). Normalized RNA samples (5 ng per sample) then underwent library preparation using the Illumina low input RNA-Seq library prep (Illumina, San Diego, CA) and sequencing using 2 x 150 bp paired-end read sequencing on the Illumina NovaSeq6000 Sequencing system using an S4 flowcell. Both the library preparation and sequencing were performed at the University of Florida ICBR NextGen DNA Sequencing Core Facility (RRID:SCR_019152). Gene expression analysis All gene expression analyses was performed using default parameters or as otherwise noted. The resulting sequencing reads first underwent quality verification using FastQC (version 0.11.7) 110 . The reads were then mapped to the E. scolopes genome 53 , 54 using the splice-aware aligner STAR (version 2.7.9a) 111 with the minimum mapped length adjusted to 0.5 (i.e., --outFilterMatchNminOverLread and --outFilterScoreMinOverLread set to 0.5). After mapping, gene expression was quantified using RSEM (version 1.3.3) 112 . The gene expression counts were normalized by Trimmed Mean of M-values (TMM) and samples were compared using an exact test and p-values adjusted for multiple comparisons with the Benjamini-Hochberg method using edgeR (version 3.14.0) 113 . Significant differential gene expression was defined as a false discovery rate (FDR) < 0.1. Genes were first characterized using BLASTX 114 against the National Center for Biotechnology Information (NCBI) nonredundant protein sequence database and InterProScan (version 5) against the InterPro 100.0 database 115 . Characterized genes then underwent gene ontology (GO) mapping and functional annotation using Blast2GO Pro 116 in Omicsbox (version 2.2.4). For functional analysis of the gene expression, gene-set enrichment analysis (GSEA) was performed 61 to show GO functions that were significantly over or underrepresented for the condition of interest (e.g., SYM versus APO). The GSEA was performed using a ranked list factoring both fold change and p-value for each gene with the calculation sin(logFC) * -log10(p-value) and 1000 permutations. Extraction and analysis of small molecules Light organs were dissected from each squid, placed into individual 1.5 ml Eppendorf tubes, frozen at -80°C, and later analyzed at the University of Florida’s Southeast Center for Integrated Metabolomics (SECIM). Frozen samples were thawed on ice, after which one pre-chilled 3 mm stainless steel bead was added to the tube. Next, 50 µL of 5 mM ammonium acetate was added and the samples were vortexed using five cycles that included 10 sec vortexing with 5 min incubation on ice. The samples then underwent two additional cycles of vortexing for 10 sec and cooling on ice for 3.5 min. The sides of the tubes were washed by adding 50 µL of 5 mM ammonium acetate and then the tubes were centrifuged at 3260 x g for 2 min at 4℃. The supernatants (100 µL) were transferred to 5 mL glass conical centrifuge tubes. Next, 5 µL of the Splash Lipidomix internal standard mix (Avanti Polar Lipids, Alabaster, AL), diluted in half, was added. A blank tube was included as an extraction blank. A biphasic extraction was then conducted following the Folch method where 1200 µL of a 2:1 mixture of ice cold chloroform:methanol was added to each tube. The tubes were allowed to cool on ice for 20 min with vortexing at 10 min. Next, 100 µL of water was added to each tube and they were allowed to cool again on ice for 10 min with vortexing at 5 min, after which they were centrifuged at 3260 x g for 10 min at 4 ℃. The bottom layer (800 µL) was removed and transferred to a new glass tube with a screw cap. The aqueous portion was re-extracted by adding 400 µL of ice cold 2:1 chloroform:methanol, vortexed, cooled for 10 min at 4℃, and again centrifuged at 3260 x 6 for 10 min, 4℃. At this point, 200 µL of the bottom layer was removed and added to the original organic layer, whereas remaining aqueous portion was saved for later metabolomic processing. The organic layer was dried under a gentle stream of nitrogen at 30℃. The dried residue was reconstituted in 50 µL of 2-propanol containing the lipid injection standards, vortexed and centrifuged at 3260 x 6 for 10 min, 4℃. The reconstituted samples were then transferred to an LC vial with fused insert for LC-HRMS analysis. The aqueous portions were transferred to microcentrifuge tubes and centrifuged at 20,000 x g for 10 min, 4℃ to pellet any remaining protein. Only 250 µL of the supernatant was transferred and 5 µL of the metabolomics internal standard mix was added and vortex mixed 118 . The samples were then centrifuged again at 20,000 x g for 10 min, 4℃. The supernatants were transferred to new microcentrifuge tubes and dried under a gentle stream of nitrogen at 30 ℃. The dried residue was then reconstituted with 50 µL of water containing injection standards, vortexed and centrifuged at 20,000 x g for 10 min, 4℃. The samples were then transferred to plastic LC vials with inserts for analysis. Metabolomics and Lipidomics using LC-HRMS All analyses were conducted using liquid chromatography high-resolution mass spectrometry (LC-HRMS) on a Thermo Q Exactive using heated electrospray ionization with both positive and negative ion modes collected using separate injections. The source conditions for lipid analysis were 3.5 kV spray voltage, 5 (+ mode) and 15 (-mode) arb units for auxiliary gas, 30 (+ mode) and 25 (-mode) arb units for sheath gas, 1 (+ mode and 0 (-mode) arb units for sweep gas, 300℃ capillary temperature, and S-lens set to 35. The conditions for metabolomics were 3.5 (+ mode) and 3.0 kV (-mode) spray voltage, 10 arb units for auxiliary gas, 50 arb units for sheath gas, 1.0 arb units for sweep gas and S-lens set to 30. Samples were maintained at 9℃ in an autosampler. Lipids were separated on a Waters Aquity BEH C18 column (1.7 µm, 2.1 mm x 50 mm) with temperature set to 50℃. Mobile phase A was 60:40 acetonitrile:water with 10 mM ammonium formate and 0.1% formic acid and mobile phase B was 90:8:2 2-propanol:acetonitrile:water also with 10 mM ammonium formate and 0.1% formic acid. The flow rate was 500 µL/min and a multistep gradient was used. Exact gradient conditions were followed according to previous work 119 . Data were collected with a mass resolution setting of 70,000 for full scan mode and a setting of 35,000 for top 5 data dependent MS/MS (ddMS2). Lipidmatch was used for data compilation and lipid identification 120 . The injection volume was 3 µL for positive ions and 5 µL for negative ions. Metabolites were separated using an ACE-Excel C18-pfp column (2.0 µm, 2.1 mm x 100 mm, Mac-Mod Chadds Ford, PA) with temperature set to 25℃ and flow rate of 350 µL/min. Mobile phase A was 0.1% formic acid in water and mobile phase B was acetonitrile. The gradient was held at 100% A from 0–3 min, then increased to 80% B from 3–13 min, held constant at 80% B for 3 min, returned to initial conditions in 0.5 min. The flow rate was increased to 600 µL/min from 16.5–20 min to re-equilibrate the column. The flow rate was returned to 350 µL/min at 20 min and then held until 22.5 min before the next injection. The injection volume was 2 µL (positive ions) and 4 µL (negative ions). Data processing was conducted by converting the files to mzXML using MSconvert from ProteoWizard 121 then loaded into MZmine 2.53 where a batch method was used for peak picking, alignment, and metabolite identification using an internal retention time library of 1200 metabolites (5 ppm for positive, 10 ppm for negative, ∓ 0.25 min retention time window) for level 1 identification. Statistics and Reproducibility Statistical analysis of principal components analysis PCA was conducted using a T2 hotelling test, which is a multivariate extension of the t-test and evaluated whether the two groups are significantly different in a multivariate space 117 . Statistical significance for the GSEA was defined as the default FDR q-value < 0.25. Significant gene sets were ordered by the normalized enrichment score (NES), which indicates how much a gene set is overrepresented at the top or the bottom of the ranked list and is normalized for multiple comparisons. PERMANOVA and k-means analyses for all datasets were performed on the normalized data that was log-transformed. All statistical comparisons of the small molecule analysis were performed using Metaboanalyst 5.0 122 . Prior to statistical analysis, the metabolite and lipid datasets were normalized by sum, log-transformed, and underwent Pareto scaling. The normalized data was then visualized PCA and outliers outside of the 95% confidence intervals were removed. Orthogonal partial least squares discriminant analysis (oPLS-DA) was used to initially identify potentially significant molecules (VIP > 1.5), which were then confirmed using a Student’s t-test adjusted for multiple comparisons (FDR < 0.1). Declarations ARRIVE Statement All animal experiments were performed in accordance with ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). All experimental procedures using live cephalopods were approved by the University of Florida (201910899), NASA Flight (SQD01), and the Kennedy Space Center (FLT-20-129) Institutional Animal Care and Use Committees (IACUC) and were performed in accordance with the approved protocols and guidelines. Competing interests The authors declare no competing interests. Author Contribution E.J.K.: Flight Preparation, Data Generation, and Analysis; Writing - original draft preparation, Review, and Editing. A.C.: RNASeq Data Analysis, Writing - Review and Editing, T.J.G.: Metabolomic and Lipidomics Data Generation and Analysis, Writing - Review and Editing, R. O., Flight Hardware Design, Flight Implementation Administration, Writing - Review and Editing. R.B.: Flight Hardware Engineer, Flight Preparation. D.R.: Flight Hardware Design, Writing - Review and Editing. J.S.F: Conceptualization, Flight Preparation, Data Analysis, Project Administration, Writing - original draft preparation, Review, and Editing. Acknowledgement The authors thank Dr. Megan MacArthur for her technical assistance aboard the International Space Station. This work was supported by a NASA Space Biology Award 80NSSC19K0138 awarded to J.S.F.. Data Availability The RNASeq datasets generated as part of this project are available in the NCBI Sequence Read Archive archive under project PRJNA1066592 with biosamples SAMN39496488 - SAMN3946519. Gene set enrichment, lipidomic, and metabolomic analyses are available as datasets in the supplemental materials. References Durante M, Cucinotta FA. Heavy ion carcinogenesis and human space exploration. Nat. Rev. Cancer 8, 465–472 (2008). Blaber E, Marcal H, Burns BP. Bioastronautics: the influence of microgravity on astronaut health. Astrobiology 10, 463–473 (2010). Afshinnekoo E, et al. Fundamental biological features of spaceflight: advancing the field to enable deep-space exploration. Cell 183, 1162–1184 (2020). Foster JS, Wheeler RM, Pamphile R. Host-microbe interactions in microgravity: assessment and implications. Life 4, 250–266 (2014). Tesei D, Jewczynko A, Lynch AM, Urbaniak C. 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Ceramides and mitochondrial homeostasis. Cell Signal 117, 111099 (2024). Kagan T, Stoyanova G, Lockshin RA, Zakeri Z. Ceramide from sphingomyelin hydrolysis induces neuronal differentiation, whereas de novo ceramide synthesis and sphingomyelin hydrolysis initiate apoptosis after NGF withdrawal in PC12 Cells. Cell Commun. Signal. 20, 15 (2022). Boettcher KJ, Ruby EG. Depressed light emission by symbiotic Vibrio fischeri of the sepiolid squid Euprymna scolopes . J. Bacteriol. 172, 3701–3706 (1990). Andrews S. FastQC a quality-control tool for high-throughput sequence data ac. uk/projects/fastqc , (2014). Dobin A, Gingeras TR. Mapping RNA-seq Reads with STAR. Curr. Protoc. Bioinformatics 51, 11.14.11-19 (2015). Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinform. 12, 323 (2011). Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. 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LipidMatch: an automated workflow for rule-based lipid identification using untargeted high-resolution tandem mass spectrometry data. BMC Bioinform. 18, 331 (2017). Chambers MC, et al. A cross-platform toolkit for mass spectrometry and proteomics. Nature Biotechnol. 30, 918–920 (2012). Xia J, Psychogios N, Young N, Wishart DS. MetaboAnalyst: a web server for metabolomic data analysis and interpretation. Nucleic Acids Res. 37, W652-660 (2009). Additional Declarations No competing interests reported. Supplementary Files SupplementalMaterialsMerged.pdf DatasetS1DEGsAll.xlsx DatasetS2GSEAgenelist.xlsx DatasetS3GSEAOverview.xlsx DatasetS4Lipidomics.xlsx DatasetS5MetabolomicsV2.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7103661","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":501316717,"identity":"24e55dec-f1db-4bcc-8a40-a29fd5884b25","order_by":0,"name":"Eric J. Koch","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Eric","middleName":"J.","lastName":"Koch","suffix":""},{"id":501316718,"identity":"67585067-8a3e-492f-b17a-74c87485ae37","order_by":1,"name":"Ana Conesa","email":"","orcid":"","institution":"Spanish National Research Council","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"","lastName":"Conesa","suffix":""},{"id":501316720,"identity":"fe841355-96c5-4bd3-9b2a-93a31a315be9","order_by":2,"name":"Timothy J. Garrett","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Timothy","middleName":"J.","lastName":"Garrett","suffix":""},{"id":501316722,"identity":"6a3fef49-2748-4fa7-8e80-ffe006814178","order_by":3,"name":"Rachel Ormsby","email":"","orcid":"","institution":"Redwire Space Technologies, Inc","correspondingAuthor":false,"prefix":"","firstName":"Rachel","middleName":"","lastName":"Ormsby","suffix":""},{"id":501316725,"identity":"05930769-6ee7-4a9b-847b-9ad88d11679f","order_by":4,"name":"Ryan Bohl","email":"","orcid":"","institution":"Redwire Space Technologies, Inc","correspondingAuthor":false,"prefix":"","firstName":"Ryan","middleName":"","lastName":"Bohl","suffix":""},{"id":501316727,"identity":"2e2d12b2-85bd-4522-89f4-80c5d9e87e83","order_by":5,"name":"David W. Reed","email":"","orcid":"","institution":"Redwire Space Technologies, Inc","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"W.","lastName":"Reed","suffix":""},{"id":501316729,"identity":"45955ca7-6667-40c9-a98f-43cc9bcb5117","order_by":6,"name":"Jamie S. Foster","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYBACxuYDYFqOgflwAwMDGzFa2hIYGIC6jBnYEonUwsAG0ZLYQLQW5jbmw58/VNxL33CMsYHhQ9lhYhzGliZx4ExxLkgL44xzxGiZ32PGcLAtIXfD/cYGZt42omzhMf5w8F9CugHQFua/RGoxkDjYkJAA1sJInBagX84cSzCcCdRysOdcOmEthsAQ+1BRkyDPd4z54IMfZdZEaGlA4hwgrB4I5IlSNQpGwSgYBSMbAAC6cz8tiNgYxwAAAABJRU5ErkJggg==","orcid":"","institution":"University of Florida","correspondingAuthor":true,"prefix":"","firstName":"Jamie","middleName":"S.","lastName":"Foster","suffix":""}],"badges":[],"createdAt":"2025-07-11 17:23:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7103661/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7103661/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89398713,"identity":"3078b0e6-258a-4ea2-a252-4aeeff3dea39","added_by":"auto","created_at":"2025-08-19 13:53:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1021307,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of the squid-vibrio system and UMAMI experimental design. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Representative juvenile squid imaged after the completion of the UMAMI experiment. Blue box represents the location of the host light organ. Bar = 0.5 cm. (\u003cstrong\u003eb\u003c/strong\u003e) Representative epifluorescence image of light organ at the time of hatching depicting the pronounced ciliated epithelium appendage (CEA) structures on either side of the light organs that are used to recruit bacteria into the vicinity of pores on the surface of the light organ. Bar = 50 µm. (\u003cstrong\u003ec\u003c/strong\u003e) Cartoon of one half of the light organ depicting the path symbiosis competent \u003cem\u003eVibrio fischeri\u003c/em\u003e travel to colonize individual crypts lined with mucosal epithelial cells. (\u003cstrong\u003ed\u003c/strong\u003e) One of two Fluid Processing Cassettes (FPC) that housed either the symbiotic or aposymbiotic animals for the duration of the experiment. (e) individual Aquarium bags that each housed eight replicate juvenile squid connected to pumps in the FPC that would bring fluids in and out of the bag. Bar = 1 cm. (f) Diagram of the experimental flow of the spaceflight samples with 16 replicate Aquarium bags at four time points. At the end of the experiment, the FPCs were opened by a crew member, and Aquarium bags were removed and placed at -80°C until the return to Earth. A replicate experimental flow using the same flight hardware and timeline was completed on the ground as a control.\u003c/p\u003e","description":"","filename":"UMAMIFigure1V4.png","url":"https://assets-eu.researchsquare.com/files/rs-7103661/v1/0c8a54428b367cdcc603121b.png"},{"id":89398746,"identity":"ea74132c-7d92-4099-ae51-5761e247b1e2","added_by":"auto","created_at":"2025-08-19 13:53:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":73880,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of transcriptome data from the UMAMI flight experiment.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Principal component (PC) analysis of 32 transcriptomes derived from the aposymbiotic (Apo, orange) and symbiotic (Sym, blue) exposed to either spaceflight (triangle) or ground (square) conditions. The transcriptomes were clustered into two distinct groups including ground samples (red circle) and flight samples (green circle). Ellipses reflect 95% confidence that the data falls within the boundaries. (\u003cstrong\u003eb\u003c/strong\u003e) Gene set enrichment (GSEA) comparisons of transcriptomes derived from ground controls and flight samples at each time point of the experiment. Total number of significant gene sets are in black. Numbers of enriched gene sets in symbiotic animals relative to aposymbiotic animals are marked in blue, whereas genes that are decreased in symbiotic animals relative to aposymbiotic animals are listed in orange. (\u003cstrong\u003ec\u003c/strong\u003e) Histogram of GSEA analysis comparisons at 6 h post-inoculation depicting the distinct categories that are either enriched (blue) or decreased (orange) according to the normalized enrichment score (NES) in symbiotic animals compared to aposymbiotic animals.\u003c/p\u003e","description":"","filename":"UMAMIFigure26HV5.png","url":"https://assets-eu.researchsquare.com/files/rs-7103661/v1/19660842c0f533485cd7f23a.png"},{"id":89398744,"identity":"1849c1ce-1af2-4f8e-9a21-5412863eaafb","added_by":"auto","created_at":"2025-08-19 13:53:59","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":109536,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeted comparison of gene set enrichment analysis for oxidative stress, neurogenesis, and lipid transport genes in host transcriptomes\u003c/strong\u003e. Heat maps depicting the expression of transcripts for genes associated with \u003cstrong\u003e(a) \u003c/strong\u003eoxidative stress, \u003cstrong\u003e(b)\u003c/strong\u003e neurogenesis, and \u003cstrong\u003e(c)\u003c/strong\u003e lipid transport that were either enriched in symbiotic (sym, blue) or aposymbiotic (apo, orange) animals under both spaceflight (flight) or ground conditions. Numbers represent the hours post-inoculation with either \u003cem\u003eV. fischeri\u003c/em\u003e(sym) or filtered seawater (apo). Asterisks represent core genes within the oxidative stress gene set. Scatter plots of the fold change in expression of core genes associated with \u003cstrong\u003e(d)\u003c/strong\u003eoxidative stress, \u003cstrong\u003e(e)\u003c/strong\u003e neurogenesis, and \u003cstrong\u003e(f)\u003c/strong\u003e lipid transport over time that either increased under symbiotic (sym) or aposymbiotic (apo) conditions.\u003c/p\u003e","description":"","filename":"UMAMIFigure3heatmapsV3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7103661/v1/29d40d51d7a01863427078c7.jpg"},{"id":89398750,"identity":"689f92c2-f102-4d4e-896f-64ef19593829","added_by":"auto","created_at":"2025-08-19 13:54:00","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":38918,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of host lipidome during spaceflight in the presence and absence of symbiotic microbes\u003c/strong\u003e. \u003cstrong\u003e(a)\u003c/strong\u003e Principal component (PC) analysis of the positive electrospray ionization mass spectroscopy (ESI-MS) lipidome data depicting the differences between the flight (triangle) and ground (square) conditions of symbiotic (blue) and aposymbiotic (orange) animals. Ellipses reflect 95% confidence that the data falls within the boundaries. \u003cstrong\u003e(b)\u003c/strong\u003e\u0026nbsp; Timeline of differentially abundant lipids under spaceflight and ground conditions. For each time point, the top number reflects the total amount of significant lipids (VIP\u0026gt; 1.5, FDR \u0026lt; 0.1), the lower numbers indicate the amount of lipids that are either significantly increased (blue) or decreased (orange) in symbiotic (Sym) relative to aposymbiotic (Apo) animals. \u003cstrong\u003e(c)\u003c/strong\u003e Volcano plots of lipids detected at 2 h comparing Sym and Apo under flight (left) and ground (right) conditions. All recovered lipids from the positive-ESI data were plotted and each symbol represents one unique lipid.\u0026nbsp; The log fold change (FC) is represented on the x-axis, whereas the -log\u003csub\u003e10\u003c/sub\u003e of the p-value (P) is on the y-axis. Significant lipids (VIP\u0026gt; 1.5, FDR \u0026lt; 0.1) are indicated with blue (up in Sym\u0026gt;Apo) or orange (Apo\u0026gt;Sym), whereas non-significant lipids are in gray. The pie charts (inset) indicate the biochemical characterization of the significant lipids separated into three groupings: unknowns (dark gray), ceramides (green), and miscellaneous (purple).\u003c/p\u003e","description":"","filename":"UMAMIFigure4LipidomeV6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7103661/v1/f30f7c9c4f9ea1cf227b4298.jpg"},{"id":89399282,"identity":"914aee3d-fac1-49c9-a9f4-cc71499f43df","added_by":"auto","created_at":"2025-08-19 14:02:01","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":44798,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of host metabolome during spaceflight in the presence and absence of symbiotic microbes\u003c/strong\u003e. \u003cstrong\u003e(a)\u003c/strong\u003e Principal component (PC) analysis of the positive electrospray ionization mass spectroscopy (ESI-MS) metabolome data depicting the differences between the flight (triangle) and ground (square) conditions of symbiotic (blue) and aposymbiotic (orange) animals. Ellipses reflect 95% confidence that the data falls within the boundaries\u003cstrong\u003e. (b)\u003c/strong\u003e Volcano plots of metabolites detected at 2 h, comparing symbiotic (Sym) and aposymbiotic (Apo) animals under spaceflight (left) and ground (right) conditions. All recovered features from the positive-ESI data were plotted and each symbol represents one metabolite. The log\u003csub\u003e2\u003c/sub\u003e-fold change (FC) calculated as Sym/Apo is represented on the x-axis, whereas the -log\u003csub\u003e10\u003c/sub\u003e of the p-value (P) is on the y-axis. Statistically significant metabolites (VIP \u0026gt; 1.5, FDR \u0026lt; 0.1) are indicated in blue (Sym\u0026gt;Apo) or orange (Apo\u0026gt;Sym), whereas non-significant metabolites are in gray. \u003cstrong\u003e(c)\u003c/strong\u003e Heat map (left) of metabolites associated with the kynurenine pathway under flight and ground conditions. The log\u003csub\u003e2\u003c/sub\u003e-fold change calculated as Sym/Apo is displayed to show enrichment under symbiotic (blue) or aposymbiotic conditions (orange). A scatter plot of the same metabolites over time depicting metabolite enrichment in Sym (blue) relative to Apo (orange) animals under flight (triangle) or ground (square) conditions. The log\u003csub\u003e2\u003c/sub\u003e-fold change (FC) calculated as Sym/Apo is represented on the y-axis and the time of sampling post-inoculation is on the x-axis. Pink symbols reflect L-tryptophan abundance across all time points and conditions.\u003c/p\u003e","description":"","filename":"UMAMIFigure5MetabolomeV7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7103661/v1/5226739949df4aa32f129b30.jpg"},{"id":89398722,"identity":"18c5f5d8-9250-456d-a0e4-1e229fd21d57","added_by":"auto","created_at":"2025-08-19 13:53:57","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":118109,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSummary figure of the overarching trends within the host light organ in the presence and absence of symbiotic microbes during spaceflight. \u003c/strong\u003eOverall trends suggest increases and persistence of stress responses of host animals in the absence of symbiosis-competent microbes suggesting microbes may confer resistance to stress during spaceflight. Additionally, symbiotic animals exhibited increases in genes associated with neuronal differentiation and axonogenesis at earlier time points than ground controls suggesting the presence of the bacteria during spaceflight might transcriptionally activate developmental pathways faster than under normal gravity conditions.\u003c/p\u003e","description":"","filename":"UMAMIFigure6SummaryV5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7103661/v1/dbfea9ecc45efc16ce8cc60b.jpg"},{"id":91783649,"identity":"cf8f4d2a-1d6d-42f5-89e7-fde9862465f0","added_by":"auto","created_at":"2025-09-21 06:31:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2972626,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7103661/v1/98df50a5-a7ed-4a2d-bfec-8b09363464f5.pdf"},{"id":89398757,"identity":"aa2c4ed0-c9bb-4e4b-b487-95b65623e9f7","added_by":"auto","created_at":"2025-08-19 13:54:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1751283,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterialsMerged.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7103661/v1/f0580738abcdcf63b5f24a14.pdf"},{"id":89398761,"identity":"51c9a91f-f928-4067-8aa8-df14b49475bc","added_by":"auto","created_at":"2025-08-19 13:54:01","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":31206,"visible":true,"origin":"","legend":"","description":"","filename":"DatasetS1DEGsAll.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7103661/v1/6e233144ed0b3b630f3d8dd5.xlsx"},{"id":89398764,"identity":"0d97ff99-cbad-48ce-a494-b91a60724c73","added_by":"auto","created_at":"2025-08-19 13:54:01","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":27333,"visible":true,"origin":"","legend":"","description":"","filename":"DatasetS2GSEAgenelist.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7103661/v1/7a597d9a3fc703d86183d657.xlsx"},{"id":89398748,"identity":"c40ead2c-47b0-4991-8fd7-7c74c57f46e9","added_by":"auto","created_at":"2025-08-19 13:53:59","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":160342,"visible":true,"origin":"","legend":"","description":"","filename":"DatasetS3GSEAOverview.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7103661/v1/a67fb73668f4dfbc38130a9a.xlsx"},{"id":89398683,"identity":"1f842328-ba5d-4aa1-96a5-c5cfc4e1967e","added_by":"auto","created_at":"2025-08-19 13:53:54","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":4657204,"visible":true,"origin":"","legend":"","description":"","filename":"DatasetS4Lipidomics.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7103661/v1/d2a36fa9fc35043a04d89b56.xlsx"},{"id":89398677,"identity":"2dc198d9-35ab-4826-ba37-b60155576c56","added_by":"auto","created_at":"2025-08-19 13:53:52","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":3118307,"visible":true,"origin":"","legend":"","description":"","filename":"DatasetS5MetabolomicsV2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7103661/v1/fa3001d4f2d1d3dec6a0c638.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Beneficial microbes mitigate molecular stress responses and accelerate developmental pathways in host animals during spaceflight","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSpaceflight exerts numerous environmental and physiological challenges on life. Whether it be the reduction of gravity, increased exposure to radiation, or the mental health challenges of being in a confining and isolating environment \u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, working and living in space presents unique challenges to not only plant and animal hosts but also their associated microbiomes \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Eukaryotes live in a microbial world, and space travel is no exception. Microbes are transported and exchanged with the crew, food supplies, experimental cargo, flight hardware, and the spacecraft itself; however, the processes by which interdomain communication is happening in the spaceflight ecosystem are not fully understood and represent an important area of research for on-going human spaceflight activities.\u003c/p\u003e\u003cp\u003eRecently, there have been numerous studies on how the dynamics and diversity of the microbiome change within and amongst astronauts as well as the exchange between the crew and the built environment of the International Space Station (ISS) \u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10 CR11 CR12 CR13 CR14 CR15\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The results of these studies have been varied and appear to be highly dependent on the location within the body, with increased taxonomic diversity even after short exposures to space in the gut and saliva microbiomes, whereas other areas maintain homeostasis over time, such as the skin microbiome \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The changes in microbiome diversity and associated metabolism can be heavily influenced by additional modifiers, such as diet, age, medications, and flight durations \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Although efforts are underway to regularly track and monitor the microbiomes of crew and spacecraft to improve the overall understanding of what is a healthy microbiome during spaceflight \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, there are numerous unresolved questions including what drives the variation of the host microbiome in space and how resilient the microbiome is to the many hazards of spaceflight. Additionally, due to the overall complexity of the microbiome, fundamental questions persist regarding whether spaceflight alters the normal communication between host animal tissues and their associated microbes and how beneficial microbes facilitate and mediate the normal development of animals in environmentally stressful conditions.\u003c/p\u003e\u003cp\u003eOne approach to address these fundamental questions of how microbes, particularly beneficial microbes, impact the normal health and development of animals during spaceflight is to use simplified model systems. As in Earth-based research, it is necessary to use multiple model systems in space biology research to generate a robust understanding of the impact that spaceflight has on animal-microbe communication and physiology. No single model system can address all questions regarding the impact of spaceflight on animal and human health \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Although there has been significant progress regarding the use of tissue and organoid chips for spaceflight experiments \u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, there are still significant limitations for examining complex organismal-level functions throughout the body as well as temporal and spatial variations between microbes and their hosts \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, therefore using experimentally tractable animal-bacterial models that have short time frames of initiation and development are pivotal for space life sciences research.\u003c/p\u003e\u003cp\u003eIn this study, we examined how microbes colonize nascent animal epithelial tissues under the physiological stress of spaceflight using the binary symbiosis between the Hawaiian bobtail squid \u003cem\u003eEuprymna scolopes\u003c/em\u003e and its bioluminescent bacterial partner \u003cem\u003eVibrio fischeri\u003c/em\u003e. The squid-vibrio symbiosis has served as a valuable model to help untangle the dynamic interplay between environmentally acquired bacteria, the host epithelial barrier, and the innate immune system for more than 30 years \u003csup\u003e\u003cspan additionalcitationids=\"CR26 CR27 CR28 CR29 CR30\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The squid harbors a specialized organ that houses the symbiosis and is embryologically derived from the squid hindgut \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The light organ exhibits both ciliated and microvillous epithelia, which are the two most common types of polarized mucosal epithelial within the animal kingdom that interface with bacterial symbionts \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Upon colonization, the light organ exhibits a mucosal innate immune response that shares extensive similarity to mammalian systems \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Additionally, previous research has shown that under simulated microgravity conditions wild-type strains of \u003cem\u003eV. fischeri\u003c/em\u003e do not exhibit noticeable changes to their growth rate compared to gravity-based controls \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Together, these features coupled with the small size of the hatchling squid (~\u0026thinsp;3 mm, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), short embryogenesis (~\u0026thinsp;21 days), and rapid colonization and developmental timelines make this system ideal for spaceflight experimentation \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWithin minutes of the squid hatching, microbes are recruited from the environment to the light organ surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Ciliated epithelial cells on the exterior of the light organ aggregate the bacteria into the vicinity of pores on the light organ surface that connect to six independent crypt spaces lined with mucosal epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec) \u003csup\u003e\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Evidence suggests that regardless of inoculum size, typically only one \u003cem\u003eV. fischeri\u003c/em\u003e cell is required to enter the pore and migrate to each of the six individual crypt spaces to initiate colonization \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Once symbiosis-competent \u003cem\u003eV. fischeri\u003c/em\u003e enter the crypt spaces they begin to replicate clonally and interact with the host epithelium \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. During these interactions, the bacteria release diffusible molecules into the crypt spaces that can influence and modulate the host responses as well as foster communication between \u003cem\u003eV. fischeri\u003c/em\u003e symbionts in the other crypt spaces \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, thereby rapidly inducing host light organ morphogenesis \u003csup\u003e\u003cspan additionalcitationids=\"CR48 CR49 CR50 CR51\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTo explore how spaceflight impacts the onset of beneficial animal-microbe interactions, newly hatched squid were sent to the ISS aboard the SpaceX Commercial Resupply Services-22 mission (SpX-22) where, once in the microgravity environment, they were inoculated with mutualistic \u003cem\u003eV. fischeri\u003c/em\u003e. The transcriptomes, metabolomes, and lipidomes of the juvenile squid, in the presence and absence of the beneficial microbes, were sampled over time to provide a broad overview of how spaceflight impacted the initiation of the symbiosis as well as the onset of bacteria-induced developmental pathways. The results of this study provide new insights into how the space environment alters the normal timeline of bacteria-induced developmental processes in host tissues and how microbes can potentially mitigate stress responses and promote the stability of symbiotic interactions following environmental perturbations, such as spaceflight.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eSpaceflight experimental overview and hardware performance validation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe experiment entitled Understanding of Microgravity on Animal-Microbe Interactions (UMAMI) was designed to monitor the initiation and early developmental pathways of the host animal in the presence and absence of beneficial microbes in the spaceflight environment. The UMAMI experiment was completed in two fluid processing cassettes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed), one with \u003cem\u003eV. fischeri\u003c/em\u003e (i.e., symbiotic, SYM) and one without \u003cem\u003eV. fischeri\u003c/em\u003e (i.e., aposymbiotic, APO), and integrated into the ADvanced Space Experiment Processor (ADSEP) where it was launched aboard SpX-22 from the Kennedy Space Center. A detailed overview of the experimental design is provided in the methods section and visualized in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplemental Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Briefly, the automated experiment used a loop design where animals were housed within cell culture bags (i.e., aquariums; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee) and upon reaching microgravity motors pumped in either filtered-sterilized seawater containing \u003cem\u003eV. fischeri\u003c/em\u003e inoculant as SYM or only filtered seawater as APO controls. Animals were then incubated as SYM or APO for 0, 2, 6, or 12 h and then were infiltrated with RNALater to terminate the experiment and the bags were later moved to -80\u0026deg;C aboard the ISS until their return to Earth 30 days later (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). This entire procedure was repeated under normal gravity conditions as a ground control. A total of 16 replicate animals were recovered for each colonization treatment with two technical replicates for both the spaceflight experiment (n\u0026thinsp;=\u0026thinsp;128) and ground controls (n\u0026thinsp;=\u0026thinsp;128).\u003c/p\u003e\u003cp\u003eElectrical current draws and temperature readings from the ADSEP hardware suggested motor and pumping operations for both the spaceflight and ground controls occurred as planned (Supplemental Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The temperature for the spaceflight and ground incubations ranged from 22.78\u0026deg;C \u0026minus;\u0026thinsp;23.32\u0026deg;C for the APO cassettes and 22.80\u0026deg;C \u0026minus;\u0026thinsp;23.67\u0026deg;C for the SYM cassettes, indicating less than 1\u0026deg;C fluctuation for the duration of the UMAMI experiment.\u003c/p\u003e\u003cp\u003eTo confirm that animals were effectively inoculated using the ADSEP hardware, replicate experiments, including the 39-h hold simulating the launch pad experience, were conducted on the ground during the Experiment Verification Test (EVT), a NASA requirement to ensure spaceflight readiness. The entire inoculation and incubation protocol was carried out but before the final RNALater fixation step, the experiment was then stopped at 12 h, and the live animals were removed from the fluid processing cassettes and aquarium bags to assess the effectiveness of colonization within the hardware. Individual animals in the SYM and APO conditions were assessed for luminescence, colony forming units, and bacteria-induced apoptosis (Supplemental Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). EVT results at 12 h indicated that the SYM animals exhibited significantly higher luminescence readings (2000\u0026ndash;22,000 relative light units (RLU); p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) compared to APO controls (\u0026lt;\u0026thinsp;500 RLU) suggesting effective colonization by the bioluminescent symbiont using the automated ADSEP hardware (Supplemental Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003ea). Additionally, a subsample of SYM light organs in the EVT exhibiting high luminescence were subsequently homogenized and plated on seawater tryptone media resulting in a minimum of 2 x 10\u003csup\u003e5\u003c/sup\u003e cells per light organ, thereby confirming colonization by \u003cem\u003eV. fischeri\u003c/em\u003e (Supplemental Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eb). APO light organs exhibited no colony forming units when plated. SYM light organs also exhibited the hallmarks of apoptosis with distinct patterns of pycnotic nuclei within the superficial ciliated epithelium of the light organ compared to APO controls (Supplemental Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003ec). Together, these results suggest that the UMAMI experimental design, ADSEP hardware, and automated procedures were effective in inoculating the animals with \u003cem\u003eV. fischeri\u003c/em\u003e during EVT testing.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTranscriptome analyses revealed spaceflight was the primary driver of differential gene expression in the host tissues regardless of symbiotic state\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the effects of spaceflight and bacterial colonization on host gene expression, RNA sequencing was used to generate a total of 23,064,220,236 raw reads across 32 samples with a mean of 720,756,882 reads per sample. The reads were then mapped to the \u003cem\u003eE. scolopes\u003c/em\u003e genome \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e with a success rate of 65\u0026ndash;77% reads per sample. A PERMANOVA test was conducted on the transcriptome to statistically compare experimental groups. Strong statistical significance was found between the spaceflight and ground datasets (p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), whereas the aposymbiotic and symbiotic datasets displayed little to no significance (p-value\u0026thinsp;=\u0026thinsp;0.064). Additionally, a k-means analysis was conducted on the entire transcriptome and suggested the data separates according to the spaceflight condition, with a single ground cluster and three spaceflight clusters (Supplemental Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Analysis of the transcriptomes using principal component analysis (PCA) revealed two distinct clusters, one each for flight and ground samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Collectively, these results suggest there is more variability in the spaceflight exposed animals compared to the ground controls and that spaceflight may be imprinting a stress that affects both the magnitude and variability in gene expression within the host animal.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAlthough the specific rate in which \u003cem\u003eV. fischeri\u003c/em\u003e colonized the light organ was not able to be directly measured due to spaceflight hardware limitations, gene expression markers typically associated with the initiation of symbiosis were examined and compared at all time points and treatments. Transcripts of target genes that encode for peptidoglycan receptor proteins (PGRPs), galaxins, cathepsin-L, and lipopolysaccharide binding proteins (LBPs), which all typically exhibit trends of increased expression during the normal colonization under unit gravity conditions \u003csup\u003e\u003cspan additionalcitationids=\"CR56 CR57\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, were examined. Both flight and ground controls exhibited a non-significant, but overall trend of increased gene expression of several genes typically associated with colonization with low, but consistent upregulation in SYM animals relative to APO across most time points (Supplemental Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). The exception was transcripts of genes encoding cathepsin-Ls, which were increased under APO conditions beginning at 2 h only during spaceflight conditions (Supplemental Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). Cathepsins are known to be activated by caspases \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, which can be increased under the stress of simulated microgravity conditions \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, suggesting cathepsins are not a useful marker of symbiosis during spaceflight. However, the increased expression of other genes that are typically associated with the onset of symbiosis in the spaceflight and ground experiments in SYM animals compared to APO controls suggest that the host tissues were colonized by \u003cem\u003eV. fischeri\u003c/em\u003e during the flight and ground conditions.\u003c/p\u003e\u003cp\u003eDue to the large effect of spaceflight on overall gene expression in the host tissues gene expression analysis was performed on the flight and ground samples separately. To examine the effect of bacterial colonization on host gene expression under spaceflight and ground conditions, the SYM and APO transcriptomes were compared at each time point with differentially expressed genes (DEGs) defined as those under a false discovery rate (FDR) of 0.1 (Supplemental Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e; (Supplemental Dataset S1). At the beginning of the experiment during spaceflight (t\u0026thinsp;=\u0026thinsp;0 h), the ratio of DEGs up or downregulated under SYM and APO conditions was approximately even (i.e., the same number of DEGs increased in SYM and APO) (Supplemental Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003ea). However, as the experiment progressed there were fewer significant DEGs increased under SYM conditions relative to APO. This apparent gene downregulation in SYM began at 2 h post-inoculation and continued at 6 h and 12 h. Similar results were observed in ground-based simulated microgravity conditions where APO animals exhibited much higher transcriptional activity than SYM animals \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Overall, the increased transcriptional activity during the spaceflight within aposymbiotic animals suggests that in the absence of the microbe the animal is activating a range of unique host gene expression responses to the novel environment of spaceflight that was not observed under ground controls. The presence of the symbiotic microbe in the spaceflight environment might \u0026ldquo;override\u0026rdquo; or help mitigate this increase of transcriptional activity within the animals.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGene set enrichment analyses revealed that several key animal developmental pathways were altered during spaceflight and dependent on the presence or absence of symbionts\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo identify common gene functions beyond just DEGs affected by colonization under spaceflight conditions, gene-set enrichment analysis (GSEA) was performed at all time points with significant gene sets (SGSs) defined with the default value of FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.25 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c; Supplemental Fig. S7; Supplemental Dataset S2 - S3). GSEA uses a ranked list of genes that incorporates both the p-value and fold change in the expression to find gene functions that are statistically over- or underrepresented within the dataset \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. A comparison of the SGSs at the start of the experiment (t\u0026thinsp;=\u0026thinsp;0 h) revealed that the spaceflight and ground animal transcriptomes shared 30.8% of the SGS despite the flight animals being launched into space (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eBy 2 h post inoculation of the animals, however, there were few common SGS between the spaceflight and ground controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb; Supplemental Dataset S3). Under normal gravity conditions, there was an initial drop in overall SGSs at 2 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), which matches gene expression patterns previously published in studies on the host animal \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. By 6 h in ground controls, many of the SGSs that were enriched in SYM animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec; Supplemental Dataset S3) had functions associated with previously characterized bacteria-induced morphogenesis of the ciliated epithelium \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. In spaceflight-exposed animals, however, at 2 h there were 102 SGSs enriched compared with only 7 SGSs in ground controls, with 81 SGSs increased in SYM and 21 increased in APO (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb; Supplemental Fig. S7). Furthermore, gene sets associated with bacteria-induced morphogenesis were enriched earlier in spaceflight conditions relative to ground controls (i.e., 2 h in flight and 6 h in ground controls), suggesting the host responded transcriptionally to the bacteria more rapidly in spaceflight than under unit gravity conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec; Supplemental Fig. S7; Supplemental Dataset S3).\u003c/p\u003e\u003cp\u003e\u003cb\u003eSymbiotic animals exhibited lower oxidative stress gene expression in spaceflight compared to aposymbiotic controls\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOne pronounced difference in the spaceflight animals was the enrichment of SGSs associated with oxidative stress in the light organs of APO animals, which did not receive their symbiotic microbes. Analysis of the gene expression of core and noncore genes associated with oxidative stress pathways revealed dynamic changes under spaceflight conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, d; Supplemental Dataset S2-S3).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAt the start of the experiment (t\u0026thinsp;=\u0026thinsp;0), there was a slight increase in the expression of oxidative stress genes in spaceflight animals relative to their ground controls, but by 2 h post-inoculation, there was a reduction in the levels of gene expression in the SYM animals that persisted for the rest of the experimental timeline. During spaceflight, genes encoding several enzymatic antioxidants, such as superoxide dismutase and a wide range of peroxidases (e.g. glutathione peroxidases, glutathione reductase, and peroxiredoxins) exhibited increased expression in APO animals relative to SYM animals beginning at 2 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) and continued through 6 h (Supplemental Dataset S2 \u0026ndash; S3). These results suggest that aposymbiotic animals exhibited a higher and sustained oxidative stress response in their light organs during spaceflight compared to symbiotic animals.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSymbiotic animals exhibited higher expression of genes associated with neurogenesis and cell morphogenesis under spaceflight conditions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNumerous gene sets associated with the formation and stabilization of synaptic connections as well as the regulation of microtubule assembly and neuronal differentiation were significantly enriched in the SYM light organs compared to APO during spaceflight (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Several recent studies have shown the important role that symbiotic microbes can have in regulating the blood-brain barrier, homeostatic regulation of neuronal cells and in neurogenesis \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Beginning at 2 h post-inoculation, there were increases in genes encoding for several kalirin-like and Trio proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, e; Supplemental Dataset S2 \u0026ndash; S3), which play important roles in nerve growth, remodeling of synapses, and axonal development \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. The increased expression of these genes in SYM animals during spaceflight was also observed at 6 h post-inoculation, however, by 12 h post-inoculation expression levels of most of these genes were at background levels suggesting transient, but increased, gene transcription during spaceflight.\u003c/p\u003e\u003cp\u003eAdditionally, there was also a significant increase (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in expression of death-associated protein kinase (DAPK) early (i.e., 2 h) in the initiation of the symbiosis during spaceflight that was not significant under ground conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). DAPK is a molecular switch that has many different roles within animals including mediating ceramide and caspase-induced apoptosis \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. DAPK also has important roles in regulating neuronal death, regulating synaptic plasticity, and is upregulated during recovery from injury \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. There was also increased expression of titin isoforms during spaceflight in SYM animals, which encode proteins found in numerous muscles and neuronal cell types \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e and has been shown to regulate the structure of microvilli and trafficking of animal immune cells \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. These results suggest that during spaceflight microbes can regulate aspects of neurogenesis during the bacteria-induced morphogenesis of the host light organ tissue.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAposymbiotic animals exhibited higher transcription of genes associated with lipid production, transport, and localization during spaceflight\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn animals that never received their beneficial symbiont, there were pronounced increases relative to SYM in the expression of genes associated with lipid transport and localization genes during spaceflight. Although many of the genes that were differentially expressed during spaceflight encode hypothetical or uncharacterized proteins, there were increases in the expression of genes associated with lipid transport proteins, such the oxysterol-binding proteins-related proteins (ORPs), apolipophorins, phospholipid scramblases, sterol-regulated proteins, and IgGFc-binding proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, f). Many of these lipid-transport proteins, such as apolipophorin and phospholipid scramblases, have dual functions and can bind microbial-associated molecular patterns, such as LPS and phosphatidylserines, as well as serve as mediators of apoptosis, innate immunity, oxidative stress responses, and other signaling processes in host cells \u003csup\u003e\u003cspan additionalcitationids=\"CR75\" citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. Additionally, IgGFc-binding proteins, also known as Fc gamma binding proteins, are mucin-like glycoproteins that play roles in mucosal defense and are often secreted into the mucus to increase the structural integrity of the mucus layer \u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. The significant increase of these diverse lipid transport proteins in the APO animals during spaceflight may reflect a heightened stress response from animals that did not receive the normal microbial signals or cues for development.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSpaceflight altered the host lipidome with increases in ceramide abundance in symbiotic animals\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn parallel with the transcriptomic analysis, the lipidome of the host animals was also profiled for each treatment and time point. Liquid chromatography/high-resolution mass spectrometry (LC/HRSM) with electrospray ionization (ESI) detected 3612 total peaks in positive ESI mode and 1812 total peaks in negative ESI, with 938 (~\u0026thinsp;26%) and 373 (~\u0026thinsp;21%), respectively, able to be characterized (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Supplemental Fig. S8; Supplemental Dataset S4). Principal component analysis showed that, as with the transcriptome, spaceflight strongly affected the lipidome at all time points. To characterize differential lipid abundances, a combination of multivariate orthogonal partial least squares discriminant analysis (OPLS-DA) and univariate (Student\u0026rsquo;s t-test) statistical tests were used with significant lipids being defined as having both a VIP\u0026thinsp;\u0026gt;\u0026thinsp;1.5 for OPLS-DA and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.1 for the t-test (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea; Supplemental Fig. S8). In normal gravity conditions, there was an initial enrichment of significant lipids (both positive and negative ESI) in SYM animals relative to APO at 0 h and 2 h, followed by a reversal in the pattern (i.e., APO\u0026thinsp;\u0026gt;\u0026thinsp;SYM) at 6 h and 12 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, c; Supplemental Fig. S8). Beginning at 2 h in spaceflight, the majority of significant lipids exhibited a trend of being increased in SYM which continued at 6 h. Interestingly, significantly different lipids during spaceflight were only found at 12 h in SYM in the -ESI mode (Supplemental Fig. S8).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOf those lipids that could be identified with tandem mass spectrometry (MS/MS), ceramides were the most abundant group, representing 36.6% and 41.7% of characterized lipids in flight and ground, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec; Supplemental Dataset S4). During spaceflight, ceramides were generally increased in SYM animals relative to APO with the highest levels at 2 and 6 h post-inoculation (Supplemental Fig. S9). In ground conditions at 2 h, however, the abundance of ceramides was evenly distributed between APO and SYM animals but by 6 and 12 h ceramides exhibited greater abundance in the APO animals compared to symbiotic animals (Supplemental Fig. S9).\u003c/p\u003e\u003cp\u003eCeramides are lipid modulators that regulate a wide range of functions in the cell, including cell differentiation, cell arrest, inflammation, and apoptosis \u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e. Accumulation of ceramides within cells can affect membrane fluidity, alter mitochondrial function, and induce both extrinsic and intrinsic apoptosis pathways \u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e primarily through the activation of caspases \u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e. The elevated levels of ceramides in SYM light organs during spaceflight coupled with the observed decreases of transcription in genes associated with lipid transport (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec) suggest ceramides, as well as other lipids, may be accumulating in the light organs of symbiotic animals during spaceflight. The accumulation of this potent signaling molecule during spaceflight may be causing, and accelerating, several downstream effects, such as increases in initiator and executioner caspase gene expression and early onset of bacteria-induced apoptosis previously observed in ground-based simulated microgravity experiments \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSymbiotic state altered metabolites associated with the regulation of the host immune system during spaceflight\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess how spaceflight impacted the overall pool of metabolites within the host light organ, untargeted metabolomics using LC/HRMS was used to generate profiles of the different treatments. Analysis revealed a total of 3500 peaks in positive ESI (+\u0026thinsp;ESI) and 818 peaks in negative ESI (-ESI) mode (Supplementary Dataset S5). Of the recovered metabolites, 129 (~\u0026thinsp;4%) for +\u0026thinsp;ESI and 55 (~\u0026thinsp;7%) for -ESI could be annotated at the highest confidence identification (level 1). Principal component analysis showed that as in the transcriptome and lipidome the metabolome was strongly influenced by spaceflight (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eStatistical analysis of metabolites was performed using OPLS-DA and a Student\u0026rsquo;s t-test, with significant metabolites being defined as having both a VIP\u0026thinsp;\u0026gt;\u0026thinsp;1.5 and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.1, respectively (Supplemental Dataset S4; Supplemental Figures S10-S11). Analysis of the +\u0026thinsp;ESI data revealed that at the start of the UMAMI experiment (t\u0026thinsp;=\u0026thinsp;0), the metabolome profiles of the animals in spaceflight and ground conditions were comparable (Supplemental Fig. S10). By 2 h post-inoculation, however, there was a pronounced disparity in both the +\u0026thinsp;ESI and -ESI data in SYM animals between spaceflight and ground samples with a reduction in the significant metabolites observed in spaceflight samples compared to ground controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb; Supplemental Fig. S10 \u0026ndash; S11).\u003c/p\u003e\u003cp\u003eAlthough most of the significant metabolites could not be fully identified to the highest level of confidence, the molecules that could be characterized with high confidence possessed similar bioactivities related to the modulation of the host immune system. For example, by 2 h in spaceflight conditions, metabolites known to act as immunostimulants were increased in APO animals relative to SYM animals, including mevalonolactone and azelaic acid (Supplemental Dataset S5) \u003csup\u003e\u003cspan additionalcitationids=\"CR84\" citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e. Additionally, in APO animals there were significant increases in formylkynurenine, formyl-5-hydroxykynurenamine, and 5-hydroxy-N-formylkynurenine, which are metabolites involved in the kynurenine pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec; Supplemental Dataset S5).\u003c/p\u003e\u003cp\u003eThe kynurenine pathway is critical for tryptophan catabolism and is highly regulated by the immune system \u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u003c/sup\u003e. Under high oxidative stress and inflammation conditions, the kynurenine pathway can act as a negative feedback system to help scavenge reactive oxygen species and suppress the inflammatory response through tryptophan depletion \u003csup\u003e\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e. Metabolites associated with the kynurenine pathway were increased in APO in both conditions relative to SYM, however, the differences due to symbiotic state were lower under flight conditions compared to ground controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec; Supplementary Dataset S5). These results were corroborated by increases in the expression of genes associated with tryptophan metabolism (e.g. tryptophanase isoforms, tryptophan 2,3-diooxygenase; and kynurenine 3-monooxygenase) in the APO animals during spaceflight beginning 2 h after the start of the experiment (Supplemental Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e). These data may reflect the host attempting to compensate for the lack of symbiotic microbes by increasing expression of host-derived tryptophanases. Interestingly, tryptophan abundance was maintained approximately equal between APO and SYM animals under both spaceflight and ground conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec; Supplementary Dataset S5). There was, however, a pronounced reduction in the accumulation of kynurenine pathway metabolites in APO animals under spaceflight compared to ground controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec; Supplemental Dataset S5). The reduction of the immunosuppressive kynurenine pathway in the APO flight animals may increase the stress environment in the APO light organs during spaceflight.\u003c/p\u003e\u003cp\u003eIn contrast, the SYM animals exhibited elevated levels of immunosuppressant metabolites, such as urocanate and xanthine, which were increased 6 h post-inoculation in SYM animals relative to APO controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb; Supplemental Dataset S5). Additionally, the decrease in kynurenine-pathway metabolites in SYM animals relative to APO suggests that \u003cem\u003eV. fischeri\u003c/em\u003e played a major role in regulating tryptophan metabolism, perhaps through the metabolization of tryptophan \u003csup\u003e\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e along the kynurenine pathway during spaceflight, thereby modulating the oxidative stress and immune responses in the host during spaceflight. Overall, these results suggest that colonization of the light organ by the symbiont may act as a \u0026ldquo;calming\u0026rdquo; mechanism for host stress and the innate immune response during spaceflight.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMicrobes play a major role in maintaining human health and strategies to ensure effective microbiome homeostasis and resilience during long-duration spaceflight are critical. The outcomes of our multi-omics approach, as summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, suggest that microbes can effectively initiate the bacterial colonization process of the nascent host epithelial cells during spaceflight, thereby triggering the activation of key bacteria-induced pathways needed for the natural maturation of host tissues. However, the results also show that the onset of some aspects of normal developmental timeline were altered and accelerated under the stress of spaceflight. Additionally, the results show that the microbes may confer stress resistance to the host to help potentially mitigate perturbations caused by spaceflight. Together, these results suggest there is alteration of the molecular dialog occurring between the host and symbiont, which is an important finding for the field of space biology.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe spaceflight environment has widespread effects on the physiology of animals that can negatively impact host-microbe interactions, including changes in body fluid dynamics, shortening of cilia, reductions in mucin production, and reorganization of the cellular cytoskeleton \u003csup\u003e\u003cspan additionalcitationids=\"CR90 CR91 CR92\" citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e–\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e\u003c/sup\u003e. Despite these potential challenges, our results suggest that the host was able to effectively recruit \u003cem\u003eV. fischeri\u003c/em\u003e from the environment and initiate symbiosis under the stress of spaceflight. Much like the mammalian digestive tract where microbes must travel through numerous chemical and physically distinct environments to associate with the surfaces of mucosal epithelial cells \u003csup\u003e\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eV. fischeri\u003c/em\u003e physically interface with mucociliary epithelial surfaces of the squid light organ (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) where they aggregate within a mucus matrix containing antimicrobial molecules that serves to enrich \u003cem\u003eV. fischeri\u003c/em\u003e from surrounding environmental microbes \u003csup\u003e\u003cspan additionalcitationids=\"CR96 CR97\" citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e–\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e\u003c/sup\u003e. Analysis of the host gene expression responses suggests that \u003cem\u003eV. fischeri\u003c/em\u003e was able induce the key host molecular and biochemical responses in the squid within the 12 h UMAMI experimental timeline, suggesting that normal colonization of host epithelial tissues by beneficial microbes will not be impeded during space travel. Additional experimentation, however, will be required to more fully assess whether certain aspects of the colonization processes, such as mucin production, surface-attachment strategies, aggregate formation, and bacterial strain population dynamics are altered during spaceflight, as simulated microgravity has been shown to impact these colonization phenotypes in animal-microbe symbioses \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e. For example, it is possible that the rate of bacterial colonization within the host light organ during spaceflight was altered, potentially accounting for many of the observed changes in the host transcriptome, metabolome and lipidome.\u003c/p\u003e\u003cp\u003eIn addition to the initiation of colonization, the UMAMI experiment showed that there were several changes to the host developmental timeline during spaceflight, including the rapid onset of an oxidative stress gene expression response. Although no organism on Earth has specifically evolved to cope with spaceflight, the innate immune system of eukaryotes has evolved to sense and respond rapidly to environmental perturbations, such as increases in oxidative stress and colonization by microbes \u003csup\u003e\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e\u003c/sup\u003e. There is a long history of spaceflight studies demonstrating that the elevated radiation and microgravity environment can induce oxidative stress responses throughout all organ systems in the body \u003csup\u003e\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e\u003c/sup\u003e. As redox homeostasis is fundamental to all metabolic processes, life has evolved rapid system-wide oxidative stress responses \u003csup\u003e\u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e\u003c/sup\u003e. The increase in oxidative stress responses in both the symbiotic and aposymbiotic animals after the onset of the UMAMI experiment likely reflects the immediate stress animals experienced in the spaceflight environment. However, within 2 h of colonization by \u003cem\u003eV. fischeri\u003c/em\u003e, the presence of the microbes appeared to attenuate the magnitude of the stress response to the perturbation of spaceflight. The attenuation of stress responses in symbiotic animals may be a by-product of the bacteria using the host stress response as a cue for normal colonization, thereby stabilizing the symbiosis during spaceflight.\u003c/p\u003e\u003cp\u003eThe use of stress to cue and shape host-microbe interactions has a long evolutionary history and is conserved in both plants and animals \u003csup\u003e\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e\u003c/sup\u003e. In plants, one well-studied example is in leguminous plants, such as the clover \u003cem\u003eMedicago truncatula\u003c/em\u003e, whose symbiotic interactions with the nitrogen-fixing \u003cem\u003eSinorhizobium meliloti\u003c/em\u003e results in the formation of nodules in the host plant to improve the nutritional environment \u003csup\u003e\u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e\u003c/sup\u003e. The host plant can initiate a generalized oxidative stress response that can trigger transcriptional changes in the symbiont to increase extracellular polymeric substances, which coupled with the production of Nod factors, contributes to the formation of an infection thread and the onset of bacteria-induce nodule formation within the plants \u003csup\u003e\u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e, \u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e\u003c/sup\u003e. The results of the UMAMI experiment suggest that the spaceflight environment may increase physical and chemical stimuli in the host, such as reactive oxygen species and antimicrobial peptides, acting as cues for the microbial response. Bacteria can then regulate the transcriptional responses of the host subsequently reducing stress and accelerating normal bacteria-induced development.\u003c/p\u003e\u003cp\u003eThe impact of the bacterial symbiont on host physiology during spaceflight was also reflected in the lipidome and metabolome analyses. The pronounced increase in lipids in symbiotic animals, in particular ceramides, during spaceflight suggests that bacteria-induced lipid accumulation may be triggering important signaling functions in the host. For example, ceramides can induce a range of physiological responses including, neuronal differentiation, the acceleration of apoptosis signaling, and increased exosome production to mediate communication between different cell types \u003csup\u003e\u003cspan additionalcitationids=\"CR106\" citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e–\u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e\u003c/sup\u003e that are highly dependent on the intracellular location \u003csup\u003e\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e\u003c/sup\u003e. Additionally, changes in the metabolome also revealed the critical role that the bacteria were playing in mediating the host stress response during spaceflight. The changes in metabolites associated with tryptophan metabolism may play a critical role in maintaining, or restoring, the homeostasis of the host light organ during spaceflight. In mammals, regulation of tryptophan metabolite abundance is coordinated between the gut microbiome and intestinal epithelial cells, and the kynurenine pathway and its metabolic derivatives are being targeted as potential therapeutic agents for inflammatory and autoimmune diseases \u003csup\u003e\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTogether, analyses of the transcriptional and small molecule changes in the host tissues revealed the pronounced impact that spaceflight can have on symbiotic interactions. Evidence suggested there was extensive crosstalk between \u003cem\u003eV. fischeri\u003c/em\u003e and the host tissues during spaceflight at the initiation of the colonization process, and that the bacteria appeared to help the host squid mitigate the stress responses that the aposymbiotic animals exhibited during spaceflight. These results serve as an important foundation for building future strategies to maintain host-symbiont health in closed ecosystems, such as spacecraft. Predicting microbiome homeostasis and resiliency to the perturbations of spaceflight will require an improved understanding of the mechanisms regarding the initiation, establishment, maintenance, and loss of symbiotic interactions with their eukaryotic hosts. Additionally, to support sustainable long-term habitation beyond Earth, more complex holobiomic analyses of how collections of multicellular hosts and their associated microbiomes interconnect will be required to more fully understand the direct and indirect effects of spaceflight on beneficial interactions with microbes.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eEthics statement\u003c/b\u003e\u003c/p\u003e\u003cp\u003e All experimental procedures using live cephalopods were approved by the University of Florida (201910899), NASA Flight (SQD01), and the Kennedy Space Center (FLT-20-129) Institutional Animal Care and Use Committees (IACUC).\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnimal husbandry\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA breeding colony of adult field-caught \u003cem\u003eE. scolopes\u003c/em\u003e was maintained at the Space Life Sciences Laboratory in Merritt Island, FL in a recirculating seawater system with a water temperature of ~ 23°C and a 12h:12h day:night light cycle. Egg clutches were maintained separately in 2.5-gallon aquariums under the same temperature and lighting conditions for the duration of their development. One day before hatching, eggs were placed in individual bowls with aeration, and upon hatching, squid were removed within 1 h and placed into filtered seawater (FSW).\u003c/p\u003e\u003cp\u003e\u003cb\u003eBacteria cultures\u003c/b\u003e\u003c/p\u003e\u003cp\u003eApproximately 48 h before launch, cultures of \u003cem\u003eV. fischeri\u003c/em\u003e strain ES114 were grown overnight in Luria Broth Saline to stationary phase as previously described \u003csup\u003e\u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e\u003c/sup\u003e. Bacteria were rinsed once by centrifuging at 15,000 x g for 1 minute, the supernatant removed, and the pellet resuspended in FSW. The resuspended \u003cem\u003eV. fischeri\u003c/em\u003e were then diluted 1:1000 in FSW for an approximate concentration of 5 x 10\u003csup\u003e5\u003c/sup\u003e cells per ml of FSW.\u003c/p\u003e\u003cp\u003e\u003cb\u003eUMAMI spaceflight experimental design and gravity controls\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe UMAMI spaceflight experiment was performed within the ADvanced Space Experiment Processor (ADSEP) using two Fluid Processing Cassettes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed; Supplemental Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) designed and constructed by Redwire Space (Formerly Techshot, Greenville, IN, USA). Each cassette housed eight experimental loops, consisting of three fluorinated ethylene propylene (FEP) 25-mL cell culture bags (Saint-Gobain, Gaithersburg, MD), which are permeable to O\u003csub\u003e2\u003c/sub\u003e and CO\u003csub\u003e2\u003c/sub\u003e but impermeable to water. For each bag, there were two ports connected by Masterflex™ 1/16” ID tubing made of platinum-cured silicone and two peristaltic pump motors, one on each end of the bag. The three bags within each of the loops were: 1) Inoculum, 2) Aquarium, and 3) Fixative (Supplemental Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eApproximately 32 h before launch, the Inoculum bags were readied by loading either 5 mL of \u003cem\u003eV. fischeri\u003c/em\u003e inoculum for the SYM colonization condition or 5 mL FSW for the APO colonization condition. Each of the Aquarium bags was prepared by loading eight squid hatchlings into the FEP bags containing 25 mL of FSW. The Fixative bags contained 25 mL of RNAlater™ (ThermoFisher Scientific, Waltham, MA, USA). There were two sets of FEP bags per treatment enabling 16 biological replicates and two technical replicates. Once the bags were aseptically assembled, they were connected to the appropriate pump and tested to ensure no leakage. All APO loops were contained separately in FPC module A, whereas all SYM loops were contained in FPC module B to ensure no potential in-flight contamination by \u003cem\u003eV. fischeri\u003c/em\u003e. ADSEP hardware was turned over to SpaceX 24 h prior to launch. The temperature was maintained at 23°C +/- 0.5 for the duration of the UMAMI experiment.\u003c/p\u003e\u003cp\u003eApproximately 7.5 h after the successful launch of SpaceX CRS-22, but before the Dragon capsule docked to the International Space Station (ISS), the UMAMI experiment was successfully run for a duration of 12 h. The UMAMI experiment was automatically controlled by Redwire Space from their headquarters in Greenville, IN. The experimental sequence in the ADSEP FPC cassettes initiated with a backflow of 5 mL of FSW from the Aquarium bags into the Inoculum bags to enable the mixing of the culture and provide space in the Aquarium bags for the inoculum (SYM) or seawater control (APO). Following the backflow, the colonization step was initiated by pumping 5 mL of inoculum into the Aquarium bags resulting in a final colonization of 1 x 10\u003csup\u003e5\u003c/sup\u003e cells per mL of FSW. In the APO cassette, 5 mL of FSW was added to the Aquarium bags. The colonization step was allowed to incubate for 0, 2, 6, or 12 h. After colonization, approximately 24 mL of FSW was pumped out of the Aquarium bag back into the Inoculation bags, which were then renamed the Waste bags after which 24 mL of RNALater was added to the Aquarium bags to arrest transcription in the host squid and prevent tissue degradation. This approach was used to increase the ratio of RNALater to water ratio and improve tissue fixation. The fixation process took approximately 2 min. Following the conclusion of the UMAMI experimental procedure, the cassettes were cooled to 10°C for 48 h until the bags could be manually removed by a crewmember and frozen at -80°C on the ISS. The Aquarium bags containing RNALater were stored on the ISS for 30 days at -80°C until their return to Earth.\u003c/p\u003e\u003cp\u003eAs a control, the entire procedure, including the 39.5-hour hold (32 h pre-launch and 7.5 h transit time), was repeated at the Space Life Science Laboratory under unit gravity conditions at 23°C. Upon completion of the control experiment, the sample bags were removed from the cassette and placed at -80°C for storage for 30 days. During postflight processing of the spaceflight and gravity controls, the frozen squid were thawed, photographed, placed into fresh RNAlater, and stored at -80°C until processing.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExtraction of RNA and sequencing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLight organs were dissected from 16 squid per treatment and pooled in groups of four to provide enough material for RNA extraction. Total RNA was extracted as previously described from each group enabling three RNA extractions per treatment\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Briefly, RNA was isolated using the RNeasy Kit (Qiagen, Hilden, Germany) and treated with the TURBO DNA-free KitTM (ThermoFisher Scientific, Waltham, MA, USA). The concentration of RNA for each biological replicate was determined using a Qubit 2.0 fluorometer (ThermoFisher Scientific, Waltham, MA, USA). RNA quality was determined with an Agilent 2100 Bioanalyzer using an RNA 6000 Nano Kit (Agilent Technologies, Palo Alto, CA, USA). Normalized RNA samples (5 ng per sample) then underwent library preparation using the Illumina low input RNA-Seq library prep (Illumina, San Diego, CA) and sequencing using 2 x 150 bp paired-end read sequencing on the Illumina NovaSeq6000 Sequencing system using an S4 flowcell. Both the library preparation and sequencing were performed at the University of Florida ICBR NextGen DNA Sequencing Core Facility (RRID:SCR_019152).\u003c/p\u003e\u003cp\u003e\u003cb\u003eGene expression analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll gene expression analyses was performed using default parameters or as otherwise noted. The resulting sequencing reads first underwent quality verification using FastQC (version 0.11.7) \u003csup\u003e\u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e\u003c/sup\u003e. The reads were then mapped to the \u003cem\u003eE. scolopes\u003c/em\u003e genome \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e using the splice-aware aligner STAR (version 2.7.9a) \u003csup\u003e\u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e111\u003c/span\u003e\u003c/sup\u003e with the minimum mapped length adjusted to 0.5 (i.e., --outFilterMatchNminOverLread and --outFilterScoreMinOverLread set to 0.5).\u003c/p\u003e\u003cp\u003eAfter mapping, gene expression was quantified using RSEM (version 1.3.3) \u003csup\u003e\u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e112\u003c/span\u003e\u003c/sup\u003e. The gene expression counts were normalized by Trimmed Mean of M-values (TMM) and samples were compared using an exact test and p-values adjusted for multiple comparisons with the Benjamini-Hochberg method using edgeR (version 3.14.0) \u003csup\u003e\u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e113\u003c/span\u003e\u003c/sup\u003e. Significant differential gene expression was defined as a false discovery rate (FDR) \u0026lt; 0.1. Genes were first characterized using BLASTX \u003csup\u003e\u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e114\u003c/span\u003e\u003c/sup\u003e against the National Center for Biotechnology Information (NCBI) nonredundant protein sequence database and InterProScan (version 5) against the InterPro 100.0 database \u003csup\u003e\u003cspan citationid=\"CR115\" class=\"CitationRef\"\u003e115\u003c/span\u003e\u003c/sup\u003e. Characterized genes then underwent gene ontology (GO) mapping and functional annotation using Blast2GO Pro \u003csup\u003e\u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e116\u003c/span\u003e\u003c/sup\u003e in Omicsbox (version 2.2.4).\u003c/p\u003e\u003cp\u003eFor functional analysis of the gene expression, gene-set enrichment analysis (GSEA) was performed \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e to show GO functions that were significantly over or underrepresented for the condition of interest (e.g., SYM versus APO). The GSEA was performed using a ranked list factoring both fold change and p-value for each gene with the calculation sin(logFC) * -log10(p-value) and 1000 permutations.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExtraction and analysis of small molecules\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLight organs were dissected from each squid, placed into individual 1.5 ml Eppendorf tubes, frozen at -80°C, and later analyzed at the University of Florida’s Southeast Center for Integrated Metabolomics (SECIM). Frozen samples were thawed on ice, after which one pre-chilled 3 mm stainless steel bead was added to the tube. Next, 50 µL of 5 mM ammonium acetate was added and the samples were vortexed using five cycles that included 10 sec vortexing with 5 min incubation on ice. The samples then underwent two additional cycles of vortexing for 10 sec and cooling on ice for 3.5 min. The sides of the tubes were washed by adding 50 µL of 5 mM ammonium acetate and then the tubes were centrifuged at 3260 x g for 2 min at 4℃. The supernatants (100 µL) were transferred to 5 mL glass conical centrifuge tubes. Next, 5 µL of the Splash Lipidomix internal standard mix (Avanti Polar Lipids, Alabaster, AL), diluted in half, was added. A blank tube was included as an extraction blank.\u003c/p\u003e\u003cp\u003eA biphasic extraction was then conducted following the Folch method where 1200 µL of a 2:1 mixture of ice cold chloroform:methanol was added to each tube. The tubes were allowed to cool on ice for 20 min with vortexing at 10 min. Next, 100 µL of water was added to each tube and they were allowed to cool again on ice for 10 min with vortexing at 5 min, after which they were centrifuged at 3260 x g for 10 min at 4 ℃. The bottom layer (800 µL) was removed and transferred to a new glass tube with a screw cap. The aqueous portion was re-extracted by adding 400 µL of ice cold 2:1 chloroform:methanol, vortexed, cooled for 10 min at 4℃, and again centrifuged at 3260 x 6 for 10 min, 4℃. At this point, 200 µL of the bottom layer was removed and added to the original organic layer, whereas remaining aqueous portion was saved for later metabolomic processing. The organic layer was dried under a gentle stream of nitrogen at 30℃. The dried residue was reconstituted in 50 µL of 2-propanol containing the lipid injection standards, vortexed and centrifuged at 3260 x 6 for 10 min, 4℃. The reconstituted samples were then transferred to an LC vial with fused insert for LC-HRMS analysis.\u003c/p\u003e\u003cp\u003eThe aqueous portions were transferred to microcentrifuge tubes and centrifuged at 20,000 x g for 10 min, 4℃ to pellet any remaining protein. Only 250 µL of the supernatant was transferred and 5 µL of the metabolomics internal standard mix was added and vortex mixed \u003csup\u003e\u003cspan citationid=\"CR118\" class=\"CitationRef\"\u003e118\u003c/span\u003e\u003c/sup\u003e. The samples were then centrifuged again at 20,000 x g for 10 min, 4℃. The supernatants were transferred to new microcentrifuge tubes and dried under a gentle stream of nitrogen at 30 ℃. The dried residue was then reconstituted with 50 µL of water containing injection standards, vortexed and centrifuged at 20,000 x g for 10 min, 4℃. The samples were then transferred to plastic LC vials with inserts for analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMetabolomics and Lipidomics using LC-HRMS\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll analyses were conducted using liquid chromatography high-resolution mass spectrometry (LC-HRMS) on a Thermo Q Exactive using heated electrospray ionization with both positive and negative ion modes collected using separate injections. The source conditions for lipid analysis were 3.5 kV spray voltage, 5 (+ mode) and 15 (-mode) arb units for auxiliary gas, 30 (+ mode) and 25 (-mode) arb units for sheath gas, 1 (+ mode and 0 (-mode) arb units for sweep gas, 300℃ capillary temperature, and S-lens set to 35. The conditions for metabolomics were 3.5 (+ mode) and 3.0 kV (-mode) spray voltage, 10 arb units for auxiliary gas, 50 arb units for sheath gas, 1.0 arb units for sweep gas and S-lens set to 30. Samples were maintained at 9℃ in an autosampler.\u003c/p\u003e\u003cp\u003eLipids were separated on a Waters Aquity BEH C18 column (1.7 µm, 2.1 mm x 50 mm) with temperature set to 50℃. Mobile phase A was 60:40 acetonitrile:water with 10 mM ammonium formate and 0.1% formic acid and mobile phase B was 90:8:2 2-propanol:acetonitrile:water also with 10 mM ammonium formate and 0.1% formic acid. The flow rate was 500 µL/min and a multistep gradient was used. Exact gradient conditions were followed according to previous work \u003csup\u003e\u003cspan citationid=\"CR119\" class=\"CitationRef\"\u003e119\u003c/span\u003e\u003c/sup\u003e. Data were collected with a mass resolution setting of 70,000 for full scan mode and a setting of 35,000 for top 5 data dependent MS/MS (ddMS2). Lipidmatch was used for data compilation and lipid identification \u003csup\u003e\u003cspan citationid=\"CR120\" class=\"CitationRef\"\u003e120\u003c/span\u003e\u003c/sup\u003e. The injection volume was 3 µL for positive ions and 5 µL for negative ions.\u003c/p\u003e\u003cp\u003eMetabolites were separated using an ACE-Excel C18-pfp column (2.0 µm, 2.1 mm x 100 mm, Mac-Mod Chadds Ford, PA) with temperature set to 25℃ and flow rate of 350 µL/min. Mobile phase A was 0.1% formic acid in water and mobile phase B was acetonitrile. The gradient was held at 100% A from 0–3 min, then increased to 80% B from 3–13 min, held constant at 80% B for 3 min, returned to initial conditions in 0.5 min. The flow rate was increased to 600 µL/min from 16.5–20 min to re-equilibrate the column. The flow rate was returned to 350 µL/min at 20 min and then held until 22.5 min before the next injection. The injection volume was 2 µL (positive ions) and 4 µL (negative ions). Data processing was conducted by converting the files to mzXML using MSconvert from ProteoWizard \u003csup\u003e\u003cspan citationid=\"CR121\" class=\"CitationRef\"\u003e121\u003c/span\u003e\u003c/sup\u003e then loaded into MZmine 2.53 where a batch method was used for peak picking, alignment, and metabolite identification using an internal retention time library of 1200 metabolites (5 ppm for positive, 10 ppm for negative, ∓ 0.25 min retention time window) for level 1 identification.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistics and Reproducibility\u003c/b\u003e\u003c/p\u003e\u003cp\u003eStatistical analysis of principal components analysis PCA was conducted using a T2 hotelling test, which is a multivariate extension of the t-test and evaluated whether the two groups are significantly different in a multivariate space \u003csup\u003e\u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e117\u003c/span\u003e\u003c/sup\u003e. Statistical significance for the GSEA was defined as the default FDR q-value \u0026lt; 0.25. Significant gene sets were ordered by the normalized enrichment score (NES), which indicates how much a gene set is overrepresented at the top or the bottom of the ranked list and is normalized for multiple comparisons. PERMANOVA and k-means analyses for all datasets were performed on the normalized data that was log-transformed.\u003c/p\u003e\u003cp\u003eAll statistical comparisons of the small molecule analysis were performed using Metaboanalyst 5.0 \u003csup\u003e122\u003c/sup\u003e. Prior to statistical analysis, the metabolite and lipid datasets were normalized by sum, log-transformed, and underwent Pareto scaling. The normalized data was then visualized PCA and outliers outside of the 95% confidence intervals were removed. Orthogonal partial least squares discriminant analysis (oPLS-DA) was used to initially identify potentially significant molecules (VIP \u0026gt; 1.5), which were then confirmed using a Student’s t-test adjusted for multiple comparisons (FDR \u0026lt; 0.1).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eARRIVE Statement\u003c/h2\u003e\u003cp\u003e All animal experiments were performed in accordance with ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). All experimental procedures using live cephalopods were approved by the University of Florida (201910899), NASA Flight (SQD01), and the Kennedy Space Center (FLT-20-129) Institutional Animal Care and Use Committees (IACUC) and were performed in accordance with the approved protocols and guidelines.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eE.J.K.: Flight Preparation, Data Generation, and Analysis; Writing - original draft preparation, Review, and Editing. A.C.: RNASeq Data Analysis, Writing - Review and Editing, T.J.G.: Metabolomic and Lipidomics Data Generation and Analysis, Writing - Review and Editing, R. O., Flight Hardware Design, Flight Implementation Administration, Writing - Review and Editing. R.B.: Flight Hardware Engineer, Flight Preparation. D.R.: Flight Hardware Design, Writing - Review and Editing. J.S.F: Conceptualization, Flight Preparation, Data Analysis, Project Administration, Writing - original draft preparation, Review, and Editing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank Dr. Megan MacArthur for her technical assistance aboard the International Space Station. This work was supported by a NASA Space Biology Award 80NSSC19K0138 awarded to J.S.F..\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe RNASeq datasets generated as part of this project are available in the NCBI Sequence Read Archive archive under project PRJNA1066592 with biosamples SAMN39496488 - SAMN3946519. Gene set enrichment, lipidomic, and metabolomic analyses are available as datasets in the supplemental materials.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDurante M, Cucinotta FA. 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Here, we explore whether the onset of symbiotic associations between microbes and animals are impacted during spaceflight. We used the association between the bobtail squid \u003cem\u003eEuprymna scolopes\u003c/em\u003e and its beneficial bacterium \u003cem\u003eVibrio fischeri\u003c/em\u003e as an animal model system to examine how spaceflight affects symbiotic interactions at the transcriptomic, metabolomic, and lipidomic levels over time. Our results suggest that in the spaceflight environment, symbiotic microbes can mitigate molecular stress responses of the host animal and accelerate normal developmental pathways, such as neurogenesis and tissue morphogenesis. Overall, this work provides evidence that beneficial microbes can effectively colonize nascent host epithelial tissues in microgravity and play a critical role in shaping the host tissue environment to promote stability of symbiosis during spaceflight.\u003c/p\u003e","manuscriptTitle":"Beneficial microbes mitigate molecular stress responses and accelerate developmental pathways in host animals during spaceflight","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-19 13:53:45","doi":"10.21203/rs.3.rs-7103661/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"813490d8-2ce3-496c-9284-52c9486ec262","owner":[],"postedDate":"August 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":53250191,"name":"Biological sciences/Cell biology"},{"id":53250192,"name":"Biological sciences/Microbiology"},{"id":53250193,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-09-21T06:23:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-19 13:53:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7103661","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7103661","identity":"rs-7103661","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}