Impact of modeled microgravity stress on innate immunity in a beneficial animal-microbe symbiosis.

The innate immune response is the first line of defense for all animals to not only detect invading microbes and toxins but also sense and interface with the environment. One such environment that can significantly affect innate immunity is spaceflight. In this study, we explored the impact of microgravity stress on key elements of the NFκB innate immune pathway. The symbiosis between the bobtail squid Euprymna scolopes and its beneficial symbiont Vibrio fischeri was used as a model system under a simulated microgravity environment. The expression of genes associated with the NFκB pathway was monitored over time as the symbiosis progressed. Results revealed that although the onset of the symbiosis was the major driver in the differential expression of NFκB signaling, the stress of simulated low-shear microgravity also caused a dysregulation of expression. Several genes were expressed at earlier time points suggesting that elements of the E. scolopes NFκB pathway are stress-inducible, whereas expression of other pathway components was delayed. The results provide new insights into the role of NFκB signaling in the squid-vibrio symbiosis, and how the stress of microgravity negatively impacts the host immune response. Together, these results provide a foundation to develop mitigation strategies to maintain host-microbe homeostasis during spaceflight.

Modeled microgravity alters apoptotic gene expression and caspase activity in the squid-vibrio symbiosis.

BACKGROUND: Spaceflight is a novel and profoundly stressful environment for life. One aspect of spaceflight, microgravity, has been shown to perturb animal physiology thereby posing numerous health risks, including dysregulation of normal developmental pathways. Microgravity can also negatively impact the interactions between animals and their microbiomes. However, the effects of microgravity on developmental processes influenced by beneficial microbes, such as apoptosis, remains poorly understood. Here, the binary mutualism between the bobtail squid, Euprymna scolopes, and the gram-negative bacterium, Vibrio fischeri, was studied under modeled microgravity conditions to elucidate how this unique stressor alters apoptotic cell death induced by beneficial microbes. RESULTS: Analysis of the host genome and transcriptome revealed a complex network of apoptosis genes affiliated with extrinsic/receptor-mediated and intrinsic/stress-induced apoptosis. Expression of apoptosis genes under modeled microgravity conditions occurred earlier and at high levels compared to gravity controls, in particular the expression of genes encoding initiator and executioner caspases. Functional assays of these apoptotic proteases revealed heightened activity under modeled microgravity; however, these increases could be mitigated using caspase inhibitors. CONCLUSIONS: The outcomes of this study indicated that modeled microgravity alters the expression of both extrinsic and intrinsic apoptosis gene expression and that this process is mediated in part by caspases. Modeled microgravity-associated increases of caspase activity can be pharmacologically inhibited suggesting that perturbations to the normal apoptosis signaling cascade can be mitigated, which may have broader implications for maintaining animal-microbial homeostasis in spaceflight.

Cellular Aquaculture: Prospects and Challenges.

Aquaculture plays an important role as one of the fastest-growing food-producing sectors in global food and nutritional security. Demand for animal protein in the form of fish has been increasing tremendously. Aquaculture faces many challenges to produce quality fish for the burgeoning world population. Cellular aquaculture can provide an alternative, climate-resilient food production system to produce quality fish. Potential applications of fish muscle cell lines in cellular aquaculture have raised the importance of developing and characterizing these cell lines. In vitro models, such as the mouse C2C12 cell line, have been extremely useful for expanding knowledge about molecular mechanisms of muscle growth and differentiation in mammals. Such studies are in an infancy stage in teleost due to the unavailability of equivalent permanent muscle cell lines, except a few fish muscle cell lines that have not yet been used for cellular aquaculture. The Prospect of cell-based aquaculture relies on the development of appropriate muscle cells, optimization of cell conditions, and mass production of cells in bioreactors. Hence, it is required to develop and characterize fish muscle cell lines along with their cryopreservation in cell line repositories and production of ideal mass cells in suitably designed bioreactors to overcome current cellular aquaculture challenges.

Publisher Correction: Gene knockdown via electroporation of short hairpin RNAs in embryos of the marine hydroid Hydractinia symbiolongicarpus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Gene knockdown via electroporation of short hairpin RNAs in embryos of the marine hydroid Hydractinia symbiolongicarpus.

Analyzing gene function in a broad range of research organisms is crucial for understanding the biological functions of genes and their evolution. Recent studies have shown that short hairpin RNAs (shRNAs) can induce gene-specific knockdowns in two cnidarian species. We have developed a detailed, straightforward, and scalable method to deliver shRNAs into fertilized eggs of the hydrozoan cnidarian Hydractinia symbiolongicarpus via electroporation, yielding effective gene-targeted knockdowns that can last throughout embryogenesis. Our electroporation protocol allows for the transfection of shRNAs into hundreds of fertilized H. symbiolongicarpus eggs simultaneously with minimal embryo death and no long-term harmful consequences on the developing animals. We show RT-qPCR and detailed phenotypic evidence of our method successfully inducing effective knockdowns of an exogenous gene (eGFP) and an endogenous gene (Nanos2), as well as knockdown confirmation by RT-qPCR of two other endogenous genes. We also provide visual confirmation of successful shRNA transfection inside embryos through electroporation. Our detailed protocol for electroporation of shRNAs in H. symbiolongicarpus embryos constitutes an important experimental resource for the hydrozoan community while also serving as a successful model for the development of similar methods for interrogating gene function in other marine invertebrates.

Transcriptional profiling of the mutualistic bacterium Vibrio fischeri and an hfq mutant under modeled microgravity.

For long-duration space missions, it is critical to maintain health-associated homeostasis between astronauts and their microbiome. To achieve this goal it is important to more fully understand the host-symbiont relationship under the physiological stress conditions of spaceflight. To address this issue we examined the impact of a spaceflight analog, low-shear-modeled microgravity (LSMMG), on the transcriptome of the mutualistic bacterium Vibrio fischeri. Cultures of V. fischeri and a mutant defective in the global regulator Hfq (∆hfq) were exposed to either LSMMG or gravity conditions for 12 h (exponential growth) and 24 h (stationary phase growth). Comparative transcriptomic analysis revealed few to no significant differentially expressed genes between gravity and the LSMMG conditions in the wild type or mutant V. fischeri at exponential or stationary phase. There was, however, a pronounced change in transcriptomic profiles during the transition between exponential and stationary phase growth in both V. fischeri cultures including an overall decrease in gene expression associated with translational activity and an increase in stress response. There were also several upregulated stress genes specific to the LSMMG condition during the transition to stationary phase growth. The ∆hfq mutants exhibited a distinctive transcriptome profile with a significant increase in transcripts associated with flagellar synthesis and transcriptional regulators under LSMMG conditions compared to gravity controls. These results indicate the loss of Hfq significantly influences gene expression under LSMMG conditions in a bacterial symbiont. Together, these results improve our understanding of the mechanisms by which microgravity alters the physiology of beneficial host-associated microbes.

A year in the life of a thrombolite: comparative metatranscriptomics reveals dynamic metabolic changes over diel and seasonal cycles.

Microbialites are one of the oldest known ecosystems on Earth and the coordinated metabolisms and activities of these mineral-depositing communities have had a profound impact on the habitability of the planet. Despite efforts to understand the diversity and metabolic potential of these systems, there has not been a systematic molecular analysis of the transcriptional changes that occur within a living microbialite over time. In this study, we generated metatranscriptomic libraries from actively growing thrombolites, a type of microbialite, throughout diel and seasonal cycles and observed dynamic shifts in the population and metabolic transcriptional activity. The most transcribed genes in all seasons were associated with photosynthesis, but only transcripts associated with photosystem II exhibited diel cycling. Photosystem I transcripts were constitutively expressed at all time points including midnight and sunrise. Transcripts associated with nitrogen fixation, methanogenesis and dissimilatory sulfate reduction exhibited diel cycling, and variability between seasons. Networking analysis of the metatranscriptomes showed correlated expression patterns helping to elucidate how metabolic interactions are coordinated within the thrombolite community. These findings have identified distinctive temporal patterns within the thrombolites and will serve an important foundation to understand the mechanisms by which these communities form and respond to changes in their environment.

Transcriptomic changes in an animal-bacterial symbiosis under modeled microgravity conditions.

Spaceflight imposes numerous adaptive challenges for terrestrial life. The reduction in gravity, or microgravity, represents a novel environment that can disrupt homeostasis of many physiological processes. Additionally, it is becoming increasingly clear that an organism's microbiome is critical for host health and examining its resiliency in microgravity represents a new frontier for space biology research. In this study, we examine the impact of microgravity on the interactions between the squid Euprymna scolopes and its beneficial symbiont Vibrio fischeri, which form a highly specific binary mutualism. First, animals inoculated with V. fischeri aboard the space shuttle showed effective colonization of the host light organ, the site of the symbiosis, during space flight. Second, RNA-Seq analysis of squid exposed to modeled microgravity conditions exhibited extensive differential gene expression in the presence and absence of the symbiotic partner. Transcriptomic analyses revealed in the absence of the symbiont during modeled microgravity there was an enrichment of genes and pathways associated with the innate immune and oxidative stress response. The results suggest that V. fischeri may help modulate the host stress responses under modeled microgravity. This study provides a window into the adaptive responses that the host animal and its symbiont use during modeled microgravity.

Characterization of the stromatolite microbiome from Little Darby Island, The Bahamas using predictive and whole shotgun metagenomic analysis.

Modern stromatolites represent ideal ecosystems to understand the biological processes required for the precipitation of carbonate due to their long evolutionary history and occurrence in a wide range of habitats. However, most of the prior molecular work on stromatolites has focused on understanding the taxonomic complexity and not fully elucidating the functional capabilities of these systems. Here, we begin to characterize the microbiome associated with stromatolites of Little Darby Island, The Bahamas using predictive metagenomics of the 16S rRNA gene coupled with direct whole shotgun sequencing. The metagenomic analysis of the Little Darby stromatolites revealed many shared taxa and core pathways associated with biologically induced carbonate precipitation, suggesting functional convergence within Bahamian stromatolites. A comparison of the Little Darby stromatolites with other lithifying microbial ecosystems also revealed that although factors, such as geographic location and salinity, do drive some differences within the population, there are extensive similarities within the microbial populations. These results suggest that for stromatolite formation, 'who' is in the community is not as critical as metabolic activities and environmental interactions. Together, these analyses help improve our understanding of the similarities among lithifying ecosystems and provide an important first step in characterizing the shared microbiome of modern stromatolites.