in-situ-hybridization-techniques-for-paraffin-embedded-adult-coral

<em>In Situ</em> Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ...

fluorescence-activated-cell-sorting-for-isolation-scleractinian-cell

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the ...

University of Miami, Rosenstiel School of Marine and Atmospheric Sciences

University of Miami

Wound healing is a critical physiological function needed for survival in all marine organisms. However, it is particularly critical in organisms like corals, which cannot escape predators. In this study, I characterized the gross morphology of wound healing in  and  found in two pools with distinct previously documented temperature profiles in Ofu, American Samoa. I observed differences between healing rates of  versus  but no significant difference in healing rates between  colonies found in different environmental regimes. Both coral species exhibit very distinct healing phenotypes, where  develops a pink pigmentation and  forms an algal/sand plug. The algal/sand plug appeared in corals found in the highly variable pool more quickly than in the corals from the moderately variable pool. Lastly,  appeared to never fully heal during this two-week study, indicating that it is a slower healer despite predation pressure.

Distinctive wound-healing characteristics in the corals  and  found in two different temperature regimes.

Previous transcriptional studies in heat-stressed corals have shown that many genes are responsive to generalized heat stress whereas the expression patterns of specific gene networks after heat stress show strong correlations with variation in bleaching outcomes. However, where these specific genes are expressed is unknown. In this study, we employed  hybridization to identify patterns of spatial gene expression of genes previously predicted to be involved in general stress response and bleaching. We found that tumor necrosis factor receptors (TNFRs), known to be strong responders to heat stress, were not expressed in gastrodermal symbiont-containing cells but were widely expressed in specific cells of the epidermal layer. The transcription factors AP-1 and FosB, implicated as early signals of heat stress, were widely expressed throughout the oral gastrodermis and epidermis. By contrast, a G protein-coupled receptor gene (GPCR) and a fructose bisphosphate aldolase C gene (aldolase), previously implicated in bleaching, were expressed in symbiont-containing gastrodermal cells and in the epidermal tissue. Finally, chordin-like/kielin (chordin-like), a gene highly correlated to bleaching, was expressed solely in the oral gastrodermis. From this study, we confirm that heat-responsive genes occur widely in coral tissues outside of symbiont-containing cells. Joint information about expression patterns in response to heat and cell specificity will allow greater dissection of the regulatory pathways and specific cell reactions that lead to coral bleaching.

The cell specificity of gene expression in the response to heat stress in corals.

Generalized methods for understanding the cell biology of non-model species are quite rare, yet very much needed. In order to address this issue, we have modified a technique traditionally used in the biomedical field for ecological and evolutionary research. Fluorescent activated cell sorting (FACS) is often used for sorting and identifying cell populations. In this study, we developed a method to identify and isolate different cell populations in corals and other cnidarians.

Coral cell separation and isolation by fluorescence-activated cell sorting (FACS).

Comparative analysis of the expanding genomic resources for scleractinian corals may provide insights into the evolution of these organisms, with implications for their continued persistence under global climate change. Here, we sequenced and annotated the genome of Pocillopora damicornis, one of the most abundant and widespread corals in the world. We compared this genome, based on protein-coding gene orthology, with other publicly available coral genomes (Cnidaria, Anthozoa, Scleractinia), as well as genomes from other anthozoan groups (Actiniaria, Corallimorpharia), and two basal metazoan outgroup phlya (Porifera, Ctenophora). We found that 46.6% of P. damicornis genes had orthologs in all other scleractinians, defining a coral 'core' genome enriched in basic housekeeping functions. Of these core genes, 3.7% were unique to scleractinians and were enriched in immune functionality, suggesting an important role of the immune system in coral evolution. Genes occurring only in P. damicornis were enriched in cellular signaling and stress response pathways, and we found similar immune-related gene family expansions in each coral species, indicating that immune system diversification may be a prominent feature of scleractinian coral evolution at multiple taxonomic levels. Diversification of the immune gene repertoire may underlie scleractinian adaptations to symbiosis, pathogen interactions, and environmental stress.

Comparative analysis of the Pocillopora damicornis genome highlights role of immune system in coral evolution.

It is well understood that heat stress causes bleaching in corals. Much work has focused on the way heat stress disrupts corals' symbiotic relationship with endosymbiotic algal dinoflagellate, Symbiodiniaceae, a process called bleaching. However, the damage to the coral tissue that occurs during the bleaching process and, importantly, the factors that contribute to subsequent recovery, are not well understood. I hypothesize that the host tissue damage created by heat stress initiates cascades of wound healing factors that maintain epithelial integrity. These factors may be found to contribute to the coral's potential capacity to recover. In this study, I present evidence that heat stress causes damage to the coral host tissue and that collagen is present in the gastrodermis of heat-stressed corals. I found that, during the early stages of bleaching, an important transcription factor for wound healing, Grainyhead, is expressed throughout the gastrodermis, where the cellular and tissue rearrangements occur. Lastly, using phylogenetics, I found that cnidarian Grainyhead proteins evolved three distinct groups and that evolution of this protein family likely happened within each taxonomic group. These findings have important implications for our study of coral resiliency in the face of climate change.

Heat stress compromises epithelial integrity in the coral, .

Innate immunity is an ancient physiological response critical for protecting metazoans from invading pathogens. It is the primary pathogen defense mechanism among invertebrates. While innate immunity has been studied extensively in diverse invertebrate taxa, including mollusks, crustaceans, and cnidarians, this system has not been well characterized in ctenophores. The ctenophores comprise an exclusively marine, non-bilaterian lineage that diverged early during metazoan diversification. The phylogenetic position of ctenophore lineage suggests that characterization of the ctenophore innate immune system will reveal important features associated with the early evolution of the metazoan innate immune system. Here, we review current understanding of the ctenophore immune repertoire and identify innate immunity genes recovered from three ctenophore species. We also isolate and characterize Mnemiopsis leidyi cells that display macrophage-like behavior when challenged with bacteria. Our results indicate that ctenophores possess cells capable of phagocytosing microbes and that two distantly related ctenophores, M. leidyi and Hormiphora californiensis, possess many candidate innate immunity proteins.

Still Enigmatic: Innate Immunity in the Ctenophore Mnemiopsis leidyi.

In recent decades coral gardening has become increasingly popular to restore degraded reef ecosystems. However, the growth and survivorship of nursery-reared outplanted corals are highly variable. Scientists are trying to identify genotypes that show signs of disease resistance and leverage these genotypes in restoring more resilient populations. In a previous study, a field disease grafting assay was conducted on nursery-reared Acropora cervicornis and Acropora palmata to quantify relative disease susceptibility. In this study, we further evaluate this field assay by investigating putative disease-causing agents and the microbiome of corals with disease-resistant phenotypes. We conducted 16S rRNA gene high-throughput sequencing on A. cervicornis and A. palmata that were grafted (inoculated) with a diseased A. cervicornis fragment. We found that independent of health state, A. cervicornis and A. palmata had distinct alpha and beta diversity patterns from one another and distinct dominant bacteria. In addition, despite different microbiome patterns between both inoculated coral species, the genus Sphingomonadaceae was significantly found in both diseased coral species. Additionally, a core bacteria member from the order Myxococcales was found at relatively higher abundances in corals with lower rates of disease development following grafting. In all, we identified Sphingomonadaceae as a putative coral pathogen and a bacterium from the order Myxococcales associated with corals that showed disease resistant phenotypes.

Microbiome differences in disease-resistant vs. susceptible Acropora corals subjected to disease challenge assays.

Corals are comprised of a coral host and associated microbes whose interactions are mediated by the coral innate immune system. The diversity of immune factors identified in the Pocillopora damicornis genome suggests that immunity is linked to maintaining microbial symbioses while also being able to detect pathogens. However, it is unclear which immune factors respond to specific microbe-associated molecular patterns and how these immune reactions simultaneously affect coral-associated bacteria. To investigate this, fragments of P. damicornis and P. acuta colonies from Taiwan were subjected to lipopolysaccharide (LPS) treatment to stimulate immune responses and measure bacteria community shifts. RNA-seq revealed genotype-specific immune responses to LPS involving the upregulation of immune receptors, transcription factors, and pore-forming toxins. Bacteria 16S sequencing revealed significantly different bacteria communities between coral genotypes but no differences in bacteria communities were caused by LPS. Our findings confirm that Pocillopora corals activate conserved immune factors in response to LPS and identify transcription factors coordinating Pocillopora corals' immune responses. Additionally, the strong effect of coral genotype on gene expression and bacteria communities highlights the importance of coral genotype in the investigation of coral host-microbe interactions.

Nikki Traylor-Knowles

Department of Marine Biology and Ecology

University of Miami, Rosenstiel School of Marine and Atmospheric Sciences

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

Nikki Traylor-Knowles

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Marine Biology and Ecology

University of Miami, Rosenstiel School of Marine and Atmospheric Sciences

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

Department of Marine Biology and Ecology