NOTE: End date changed to 02/29/2025 per F. Hernandez/ARC (Ed., 4/25/22)

The current project is centered around addressing two major knowledge gaps in space biology research. First, fundamental understanding is lacking as to how differences in microbiomes contribute to changes in gut colonization, organ-level physiology, and whole-organism function under microgravity. Second, even though it is recognized on Earth that individual genetic variation can have a large impact when organisms within a species are exposed to new environmental conditions, very little knowledge exists on how genetic diversity within individual species impacts the integrative physiology of organisms when exposed to microgravity since most flight studies to date have focused on genetically homogenous rodent models or cell cultures.

To address these knowledge gaps, we plan to use C. elegans, which is an established and low-cost invertebrate model for space biology, microbiome studies, and genetic diversity research. We plan to use recently established gut microbial communities to investigate the impact of the gut microbiome on host physiology. In a parallel advance, using genetically diverse wild isolates of C. elegans, we will study host-microbome interactions in spaceflight.

Our project plan involves testing the following hypotheses in International Space Station (ISS) flight studies with ground study components: (i) Microbial membership of C. elegans gut influences host transcriptional response, tissue-level physiology, and whole-organism function, (ii) Genetic diversity of host influences gut membership of individual microbes, tissue-level physiology, and whole-organism function, and (iii) Insulin signaling pathway play a central role in driving microbiome-induced host response in spaceflight.

The proposed studies are aligned with the stated strategic goals of NASA Space Biology, which defines over-arching guiding questions focused on integrated biological approaches to understand physiological and molecular mechanisms in living systems that respond to space exploration environments. Pre-biotic and probiotic therapies could be potentially realized from our investigations to improve crew health, along with the dissemination of new flight-tested protocols and molecular characterization tools for the spaceflight community.

• Optimization of temperature and collection timepoints for worm culture and bacterial colonization in polyethylene (PE) bags.

Our efforts have focused on optimizing conditions for analyzing bacterial colonization in C. elegans. We examined worm length and bacterial colonization at temperatures of 15°C, 18°C, and 20°C across various timepoints within PE bags. The optimal timeframe for visualizing bacterial colonization in the C. elegans gut, while minimizing offspring production, was identified to be between 7-9 days in PE bags. In parallel, a three-member microbial community was established to accurately replicate the BIGbiome community. [Ed. Note: The BIGbiome community is a model of diverse bacterial species designed to represent the complexity of the wild microbiome of C. elegans.] The combination of Ochrobactrum pituitosum BH3, Lelliottia amnigena JUb66, and Myroides odoratus BIGb0244 was confirmed to best replicate the BIGbiome gut profile at these temperatures. Additionally, optimal conditions for auxin-based sterility were determined. These advancements collectively enhance the study of bacterial interactions within C. elegans.

• Evaluation of NemaCapsules for C. elegans culture and assess imaging quality.

-- Significant improvements were made to the auxin exposure protocol prior to worm loading in assays, successfully preventing progeny production. -- Testing of the BIGbiome, alongside the three-biome community and OP50, was conducted through two trials using wild-type (WT) worms in the NemaCapsule lab hardware. These tests assessed worm survival and locomotion under different bacterial diets. In parallel, efforts have been made to develop low-resolution imaging stations and evaluate their imaging quality.

• Successfully completed science verification testing (SVT). Comprehensive analyses were completed to verify that bag culture conditions, including freezing, shipping, thawing, and fixing samples at various temperatures, consistently produced the required number and quality of animal samples suitable for planned imaging. Representative animals from six fluorescently labeled test strains were imaged to assess high resolution outcomes, successfully meeting the success criteria necessary to proceed to Environmental Verification Testing (EVT).

• Successful completion of experiment verification testing (EVT). In experiment verification testing, an end-to-end run with all flight samples in PE bags was successfully completed, meeting all required success criteria to advance to the Systems Readiness Flight Review (SRFR).

NOTE: End date changed to 02/28/2025 per F. Hernandez/ARC (Ed., 4/25/22)

The current project is centered around addressing two major knowledge gaps in space biology research. First, fundamental understanding is lacking as to how differences in microbiomes contribute to changes in gut colonization, organ-level physiology, and whole-organism function under microgravity. Second, even though it is recognized on Earth that individual genetic variation can have a large impact when organisms within a species are exposed to new environmental conditions, very little knowledge exists on how genetic diversity within individual species impacts the integrative physiology of organisms when exposed to microgravity since most flight studies to date have focused on genetically homogenous rodent models or cell cultures.

To address these knowledge gaps, we plan to use C. elegans, which is an established and low-cost invertebrate model for space biology, microbiome studies, and genetic diversity research. We plan to use recently established gut microbial communities to investigate the impact of the gut microbiome on host physiology. In a parallel advance, using genetically diverse wild isolates of C. elegans, we will study host-microbome interactions in spaceflight.

Our project plan involves testing the following hypotheses in International Space Station (ISS) flight studies with ground study components: (i) Microbial membership of C. elegans gut influences host transcriptional response, tissue-level physiology, and whole-organism function, (ii) Genetic diversity of host influences gut membership of individual microbes, tissue-level physiology, and whole-organism function, and (iii) Insulin signaling pathway play a central role in driving microbiome-induced host response in spaceflight.

The proposed studies are aligned with the stated strategic goals of NASA Space Biology, which defines over-arching guiding questions focused on integrated biological approaches to understand physiological and molecular mechanisms in living systems that respond to space exploration environments. Pre-biotic and probiotic therapies could be potentially realized from our investigations to improve crew health, along with the dissemination of new flight-tested protocols and molecular characterization tools for the spaceflight community.

To achieve the spaceflight project aims, the science team pursued ground investigations and completed several tasks. We have successfully constructed transgenic C. elegans lines that will permit on-ground elimination of progeny for young adults in spaceflight. Culture experiments were conducted to identify microbial members of the microbiome community, based on their growth and gut colonization capabilities. We evaluated whether polyethylene culture bags can support growth of synchronized populations of C. elegans and the biomes of interest in this project. Finally, we evaluated microfluidic devices in terms of their ability to culture C. elegans in different bacterial diet conditions and found that the locomotory behavior depends on age and diet.

The current project is centered around addressing two major knowledge gaps in space biology research. First, fundamental understanding is lacking as to how differences in microbiomes contribute to changes in gut colonization, organ-level physiology, and whole-organism function under microgravity. Second, even though it is recognized on Earth that individual genetic variation can have a large impact when organisms within a species are exposed to new environmental conditions, very little knowledge exists on how genetic diversity within individual species impacts the integrative physiology of organisms when exposed to microgravity since most flight studies to date have focused on genetically homogenous rodent models or cell cultures.

To address these knowledge gaps, we plan to use C. elegans, which is an established and low-cost invertebrate model for space-biology, microbiome studies, and genetic diversity research. We plan to use recently established gut microbial communities to investigate the impact of the gut microbiome on host physiology. In a parallel advance, using genetically diverse wild isolates of C. elegans, we will study host-microbome interactions in spaceflight.

Our project plan involves testing the following hypotheses in International Space Station (ISS) flight studies with ground study components: (i) Microbial membership of C. elegans gut influences host transcriptional response, tissue-level physiology, and whole-organism function, (ii) Genetic diversity of host influences gut membership of individual microbes, tissue-level physiology, and whole-organism function, and (iii) Insulin signaling pathway play a central role in driving microbiome-induced host response in spaceflight.

The proposed studies are aligned with the stated strategic goals of NASA Space Biology, which defines over-arching guiding questions focused on integrated biological approaches to understand physiological and molecular mechanisms in living systems that respond to space exploration environments. Pre-biotic and probiotic therapies could be potentially realized from our investigations to improve crew health, along with the dissemination of new flight-tested protocols and molecular characterization tools for the spaceflight community.