Establishing Sustainable Cell Lines of a Coral, Acropora tenuis.

Planula larvae of the scleractinian coral, Acropora tenuis, consist of elongated ectodermal cells and developing inner endodermal cells. To establish in vitro cell lines for future studies of cellular and developmental potential of coral cells, larvae were successfully dissociated into single cells by treating them with a tissue dissociation solution consisting of trypsin, EDTA, and collagenase. Brown-colored cells, translucent cells, and pale blue cells were the major components of dissociated larvae. Brown-colored cells began to proliferate transiently in the culture medium that was devised for the coral, while translucent cells and pale blue cells decreased in number about 1 week after cell dissociation. In addition, when a modular protease, plasmin, was added to the cell culture medium, brown-colored cells extended pseudopodia and assumed amorphous shapes. They then continued to proliferate in clumps for more than 6 months with a doubling time of approximately 4-5 days. From 3 weeks of cell culture onward, brown-colored cells often aggregated and exhibited morphogenesis-like behavior to form flat sheets, and blastula-like clusters or gastrula-like spheres. Single cells or cell-clusters of the cell lines were analyzed by RNA-seq. This analysis showed that genes expressed in these cells in vitro were A. tenuis genes. Furthermore, each cell line expressed a specific set of genes, suggesting that their properties include gastroderm, secretory cells, undifferentiated cells, neuronal cells, and epidermis. All cell properties were maintained stably throughout successive cell cultures. These results confirm the successful establishment of a coral in vitro cell line.

Intracellular pH regulation: characterization and functional investigation of H+ transporters in Stylophora pistillata.

BACKGROUND: Reef-building corals regularly experience changes in intra- and extracellular H+ concentrations ([H+]) due to physiological and environmental processes. Stringent control of [H+] is required to maintain the homeostatic acid-base balance in coral cells and is achieved through the regulation of intracellular pH (pHi). This task is especially challenging for reef-building corals that share an endosymbiotic relationship with photosynthetic dinoflagellates (family Symbiodinaceae), which significantly affect the pHi of coral cells. Despite their importance, the pH regulatory proteins involved in the homeostatic acid-base balance have been scarcely investigated in corals. Here, we report in the coral Stylophora pistillata a full characterization of the genomic structure, domain topology and phylogeny of three major H+ transporter families that are known to play a role in the intracellular pH regulation of animal cells; we investigated their tissue-specific expression patterns and assessed the effect of seawater acidification on their expression levels. RESULTS: We identified members of the Na+/H+ exchanger (SLC9), vacuolar-type electrogenic H+-ATP hydrolase (V-ATPase) and voltage-gated proton channel (HvCN) families in the genome and transcriptome of S. pistillata. In addition, we identified a novel member of the HvCN gene family in the cnidarian subclass Hexacorallia that has not been previously described in any species. We also identified key residues that contribute to H+ transporter substrate specificity, protein function and regulation. Last, we demonstrated that some of these proteins have different tissue expression patterns, and most are unaffected by exposure to seawater acidification. CONCLUSIONS: In this study, we provide the first characterization of H+ transporters that might contribute to the homeostatic acid-base balance in coral cells. This work will enrich the knowledge of the basic aspects of coral biology and has important implications for our understanding of how corals regulate their intracellular environment.

Novel methods to establish whole-body primary cell cultures for the cnidarians Nematostella vectensis and Pocillopora damicornis.

Cnidarians are emerging model organisms for cell and molecular biology research. However, successful cell culture development has been challenging due to incomplete tissue dissociation and contamination. In this report, we developed and tested several different methodologies to culture primary cells from all tissues of two species of Cnidaria: Nematostella vectensis and Pocillopora damicornis. In over 170 replicated cell cultures, we demonstrate that physical dissociation was the most successful method for viable and diverse N. vectensis cells while antibiotic-assisted dissociation was most successful for viable and diverse P. damicornis cells. We also demonstrate that a rigorous antibiotic pretreatment results in less initial contamination in cell cultures. Primary cultures of both species averaged 12-13 days of viability, showed proliferation, and maintained high cell diversity including cnidocytes, nematosomes, putative gastrodermal, and epidermal cells. Overall, this work will contribute a needed tool for furthering functional cell biology experiments in Cnidaria.

Transcription Profiling of Cultured Acropora digitifera Adult Cells Reveals the Existence of Ancestral Genome Regulatory Modules Underlying Pluripotency and Cell Differentiation in Cnidaria.

Due to their pluripotent nature and unlimited cell renewal, stem cells have been proposed as an ideal material for establishing long-term cnidarian cell cultures. However, the lack of unifying principles associated with "stemness" across the phylum complicates stem cells' identification and isolation. Here, we for the first time report gene expression profiles for cultured coral cells, focusing on regulatory gene networks underlying pluripotency and differentiation. Cultures were initiated from Acropora digitifera tip fragments, the fastest growing tissue in Acropora. Overall, in vitro transcription resembled early larvae, overexpressing orthologs of premetazoan and Hydra stem cell markers, and transcripts with roles in cell division, migration, and differentiation. Our results suggest the presence of pluripotent cell types in cultures and indicate the existence of ancestral genome regulatory modules underlying pluripotency and cell differentiation in cnidaria. Cultured cells appear to be synthesizing protein, differentiating, and proliferating.

Rapid counting and spectral sorting of live coral larvae using large-particle flow cytometry.

Research with coral embryos and larvae often requires laborious manual counting and sorting of individual specimens, usually via microscopy. Because many coral species spawn only once per year during a narrow temporal window, sample processing is a time-limiting step for research on the early life-history stages of corals. Flow cytometry, an automated technique for measuring and sorting particles, cells, and cell-clusters, is a potential solution to this bottleneck. Yet most flow cytometers do not accommodate live organisms of the size of most coral embryos (> 250 µm), and sample processing is often destructive. Here we tested the ability of a large-particle flow cytometer with a gentle pneumatic sorting mechanism to process and spectrally sort live and preserved Montipora capitata coral embryos and larvae. Average survival rates of mechanically-sorted larvae were over 90% and were comparable to those achieved by careful hand-sorting. Preserved eggs and embryos remained intact throughout the sorting process and were successfully sorted based on real-time size and fluorescence detection. In-line bright-field microscopy images were captured for each sample object as it passed through the flow-cell, enabling the identification of early-stage embryos (2-cell to morula stage). Samples were counted and sorted at an average rate of 4 s larva-1 and as high as 0.2 s larva-1 for high-density samples. Results presented here suggest that large-particle flow cytometry has the potential to significantly increase efficiency and accuracy of data collection and sample processing during time-limited coral spawning events, facilitating larger-scale and higher-replication studies with an expanded number of species.

Caspases from scleractinian coral show unique regulatory features.

Coral reefs are experiencing precipitous declines around the globe with coral diseases and temperature-induced bleaching being primary drivers of these declines. Regulation of apoptotic cell death is an important component in the coral stress response. Although cnidaria are known to contain complex apoptotic signaling pathways, similar to those in vertebrates, the mechanisms leading to cell death are largely unexplored. We identified and characterized two caspases each from Orbicella faveolata, a disease-sensitive reef-building coral, and Porites astreoides, a disease-resistant reef-building coral. The caspases are predicted homologs of the human executioner caspases-3 and -7, but OfCasp3a (Orbicella faveolata caspase-3a) and PaCasp7a (Porites astreoides caspase-7a), which we show to be DXXDases, contain an N-terminal caspase activation/recruitment domain (CARD) similar to human initiator/inflammatory caspases. OfCasp3b (Orbicella faveolata caspase-3b) and PaCasp3 (Porites astreoides caspase-3), which we show to be VXXDases, have short pro-domains, like human executioner caspases. Our biochemical analyses suggest a mechanism in coral which differs from that of humans, where the CARD-containing DXXDase is activated on death platforms but the protease does not directly activate the VXXDase. The first X-ray crystal structure of a coral caspase, of PaCasp7a determined at 1.57 Å resolution, reveals a conserved fold and an N-terminal peptide bound near the active site that may serve as a regulatory exosite. The binding pocket has been observed in initiator caspases of other species. These results suggest mechanisms for the evolution of substrate selection while maintaining common activation mechanisms of CARD-mediated dimerization.

Egg size and fecundity of biannually spawning corals at Scott Reef.

Egg size and fecundity are often used as proxies for coral reproductive success and health. The amount of energy a coral invests in reproduction reflects its environmental conditions during gametogenesis. Additionally, assuming resources for reproduction are limited, it is thought that an increase in egg size should result in a decrease in the number of eggs produced i.e. investing in many small eggs or fewer larger eggs. The biannually spawning populations of Scott Reef offer a unique opportunity to compare the egg size and polyp fecundity of corals exposed to different environmental conditions during gametogenesis, prior to spawning in autumn (March) and spring (October). In this study, we investigated the relationship between egg size and polyp fecundity within and between seven Acropora species from 2008 to 2010. We also quantified the fecundity and egg size of four Acropora species that spawn during both autumn and spring (2008-2010). We found no seasonal variability in egg size and fecundity in the species studied here, possibly as a result of a summer light regime being impacted by high cloud cover in cyclone season. There was high natural variability and no apparent trade-off between egg size and fecundity, both within and between each species. These findings challenge the assumption that egg size and fecundity are negatively correlated, or that a simple, energetically constrained trade-off exists between the two.

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 mechanisms are not well understood. To investigate coral response to stressors, we need tools for analyzing cellular responses. In particular, we need tools that facilitate the application of functional assays to better understand how cell populations are reacting to stress. In the current study, we use fluorescence-activated cell sorting (FACS) to isolate and separate different cell populations in stony corals. This protocol includes: (1) the separation of coral tissues from the skeleton, (2) creation of a single cell suspension, (3) labeling the coral cells using various markers for flow cytometry, and (4) gating and cell sorting strategies. This method will enable researchers to work on corals at the cellular level for analysis, functional assays, and gene expression studies of different cell populations.

Lineage dynamics of the endosymbiotic cell type in the soft coral Xenia.

Many corals harbour symbiotic dinoflagellate algae. The algae live inside coral cells in a specialized membrane compartment known as the symbiosome, which shares the photosynthetically fixed carbon with coral host cells while host cells provide inorganic carbon to the algae for photosynthesis1. This endosymbiosis-which is critical for the maintenance of coral reef ecosystems-is increasingly threatened by environmental stressors that lead to coral bleaching (that is, the disruption of endosymbiosis), which in turn leads to coral death and the degradation of marine ecosystems2. The molecular pathways that orchestrate the recognition, uptake and maintenance of algae in coral cells remain poorly understood. Here we report the chromosome-level genome assembly of a Xenia species of fast-growing soft coral3, and use this species as a model to investigate coral-alga endosymbiosis. Single-cell RNA sequencing identified 16 cell clusters, including gastrodermal cells and cnidocytes, in Xenia sp. We identified the endosymbiotic cell type, which expresses a distinct set of genes that are implicated in the recognition, phagocytosis and/or endocytosis, and maintenance of algae, as well as in the immune modulation of host coral cells. By coupling Xenia sp. regeneration and single-cell RNA sequencing, we observed a dynamic lineage progression of the endosymbiotic cells. The conserved genes associated with endosymbiosis that are reported here may help to reveal common principles by which different corals take up or lose their endosymbionts.

Survivorship and growth in staghorn coral (Acropora cervicornis) outplanting projects in the Florida Keys National Marine Sanctuary.

Significant population declines in Acropora cervicornis and A. palmata began in the 1970s and now exceed over 90%. The losses were caused by a combination of coral disease and bleaching, with possible contributions from other stressors, including pollution and predation. Reproduction in the wild by fragment regeneration and sexual recruitment is inadequate to offset population declines. Starting in 2007, the Coral Restoration Foundation™ evaluated the feasibility of outplanting A. cervicornis colonies to reefs in the Florida Keys to restore populations at sites where the species was previously abundant. Reported here are the results of 20 coral outplanting projects with each project defined as a cohort of colonies outplanted at the same time and location. Photogrammetric analysis and in situ monitoring (2007 to 2015) measured survivorship, growth, and condition of 2419 colonies. Survivorship was initially high but generally decreased after two years. Survivorship among projects based on colony counts ranged from 4% to 89% for seven cohorts monitored at least five years. Weibull survival models were used to estimate survivorship beyond the duration of the projects and ranged from approximately 0% to over 35% after five years and 0% to 10% after seven years. Growth rate averaged 10 cm/year during the first two years then plateaued in subsequent years. After four years, approximately one-third of surviving colonies were ≥ 50 cm in maximum diameter. Projects used three to sixteen different genotypes and significant differences did not occur in survivorship, condition, or growth. Restoration times for three reefs were calculated based on NOAA Recovery Plan (NRP) metrics (colony abundance and size) and the findings from projects reported here. Results support NRP conclusions that reducing stressors is required before significant population growth and recovery will occur. Until then, outplanting protects against local extinction and helps to maintain genetic diversity in the wild.

The effects of in-vitro pH decrease on the gametogenesis of the red tree coral, Primnoa pacifica.

Primnoa pacifica is the most ecologically important coral species in the North Pacific Ocean and provides important habitat for commercially important fish and invertebrates. Ocean acidification (OA) is more rapidly increasing in high-latitude seas because anthropogenic CO2 uptake is greater in these regions. This is due to the solubility of CO2 in cold water and the reduced buffering capacity and low alkalinity of colder waters. Primnoa pacifica colonies were cultured for six to nine months in either pH 7.55 (predicted Year 2100 pH levels) or pH 7.75 (Control). Oocyte development and fecundity in females, and spermatocyst stages in males were measured to assess the effects of pH on gametogenesis. Oocyte diameters were 13.6% smaller and fecundities were 30.9% lower in the Year 2100 samples. A higher proportion of vitellogenic oocytes (65%) were also reabsorbed (oosorption) in the Year 2100 treatment. Lower pH appeared to advance the process of spermatogenesis with a higher percentage of later stage sperm compared to Control. There was a laboratory effect observed in all measurement types, however this only significantly affected the analyses of spermatogenesis. Based on the negative effect of acidification on oogenesis and increased rate of oosorption, successful spawning could be unlikely in an acidified ocean. If female gametes were spawned, they are likely to be insufficiently equipped to develop normally, based on the decreased overall size and therefore subsequent limited amount of lipids necessary for successful larval development.

Metabolomics Reveal That Octocrylene Accumulates in Pocillopora damicornis Tissues as Fatty Acid Conjugates and Triggers Coral Cell Mitochondrial Dysfunction.

Octocrylene (OC) is an ingredient used in many sunscreens and cosmetics worldwide. Our group evaluated the toxicity of OC in corals. Adult Pocillopora damicornis coral was treated with OC at concentrations of 5, 50, 300, and 1000 µg/L. Most polyps were closed at concentrations of 300 µg/L and higher. Further, metabolomic profiling provided crucial information regarding OC accumulation in coral tissues and OC toxicity. First, we demonstrated that OC was transformed into fatty acid conjugates via oxidation of the ethylhexyl chain, yielding very lipophilic OC analogues that accumulate in coral tissues. Second, the differential analysis of coral profiles revealed higher levels of 15 acylcarnitines, suggesting abnormal fatty acid metabolism related to mitochondrial dysfunction. The formation of OC analogues suggests that OC concentrations measured in the environment, and organisms may have been largely underestimated. Overall, these results call for an in-depth evaluation of OC toxicity and the reevaluation of the actual OC accumulation rate in the ocean's food chain, including OC-fatty acid conjugates.

In Situ 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 ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers' ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress. The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.

Molecular, Enzymatic, and Cellular Characterization of Soluble Adenylyl Cyclase From Aquatic Animals.

The enzyme soluble adenylyl cyclase (sAC) is the most recently identified source of the messenger molecule cyclic adenosine monophosphate. sAC is evolutionarily conserved from cyanobacteria to human, is directly stimulated by [Formula: see text] ions, and can act as a sensor of environmental and metabolic CO2, pH, and [Formula: see text] levels. sAC genes tend to have multiple alternative promoters, undergo extensive alternative splicing, be translated into low mRNA levels, and the numerous sAC protein isoforms may be present in various subcellular localizations. In aquatic organisms, sAC has been shown to mediate various functions including intracellular pH regulation in coral, blood acid/base regulation in shark, heart beat rate in hagfish, and NaCl absorption in fish intestine. Furthermore, sAC is present in multiple other species and tissues, and sAC protein and enzymatic activity have been reported in the cytoplasm, the nucleus, and other subcellular compartments, suggesting even more diverse physiological roles. Although the methods and experimental tools used to study sAC are conventional, the complexity of sAC genes and proteins requires special considerations that are discussed in this chapter.

Cross-shelf investigation of coral reef cryptic benthic organisms reveals diversity patterns of the hidden majority.

Coral reefs harbor diverse assemblages of organisms yet the majority of this diversity is hidden within the three dimensional structure of the reef and neglected using standard visual surveys. This study uses Autonomous Reef Monitoring Structures (ARMS) and amplicon sequencing methodologies, targeting mitochondrial cytochrome oxidase I and 18S rRNA genes, to investigate changes in the cryptic reef biodiversity. ARMS, deployed at 11 sites across a near- to off-shore gradient in the Red Sea were dominated by Porifera (sessile fraction), Arthropoda and Annelida (mobile fractions). The two primer sets detected different taxa lists, but patterns in community composition and structure were similar. While the microhabitat of the ARMS deployment affected the community structure, a clear cross-shelf gradient was observed for all fractions investigated. The partitioning of beta-diversity revealed that replacement (i.e. the substitution of species) made the highest contribution with richness playing a smaller role. Hence, different reef habitats across the shelf are relevant to regional diversity, as they harbor different communities, a result with clear implications for the design of Marine Protected Areas. ARMS can be vital tools to assess biodiversity patterns in the generally neglected but species-rich cryptic benthos, providing invaluable information for the management and conservation of hard-bottomed habitats over local and global scales.

Using NanoSIMS coupled with microfluidics to visualize the early stages of coral infection by Vibrio coralliilyticus.

BACKGROUND: Global warming has triggered an increase in the prevalence and severity of coral disease, yet little is known about coral/pathogen interactions in the early stages of infection. The point of entry of the pathogen and the route that they take once inside the polyp is currently unknown, as is the coral's capacity to respond to infection. To address these questions, we developed a novel method that combines stable isotope labelling and microfluidics with transmission electron microscopy (TEM) and nanoscale secondary ion mass spectrometry (NanoSIMS), to monitor the infection process between Pocillopora damicornis and Vibrio coralliilyticus under elevated temperature. RESULTS: Three coral fragments were inoculated with 15N-labeled V. coralliilyticus and then fixed at 2.5, 6 and 22 h post-inoculation (hpi) according to the virulence of the infection. Correlative TEM/NanoSIMS imaging was subsequently used to visualize the penetration and dispersal of V. coralliilyticus and their degradation or secretion products. Most of the V. coralliilyticus cells we observed were located in the oral epidermis of the fragment that experienced the most virulent infection (2.5 hpi). In some cases, these bacteria were enclosed within electron dense host-derived intracellular vesicles. 15N-enriched pathogen-derived breakdown products were visible in all tissue layers of the coral polyp (oral epidermis, oral gastrodermis, aboral gastrodermis), at all time points, although the relative 15N-enrichment depended on the time at which the corals were fixed. Tissues in the mesentery filaments had the highest density of 15N-enriched hotspots, suggesting these tissues act as a "collection and digestion" site for pathogenic bacteria. Closer examination of the sub-cellular structures associated with these 15N-hotspots revealed these to be host phagosomal and secretory cells/vesicles. CONCLUSIONS: This study provides a novel method for tracking bacterial infection dynamics at the levels of the tissue and single cell and takes the first steps towards understanding the complexities of infection at the microscale, which is a crucial step towards understanding how corals will fare under global warming.

Intra-colony disease progression induces fragmentation of coral fluorescent pigments.

As disease spreads through living coral, it can induce changes in the distribution of coral's naturally fluorescent pigments, making fluorescence a potentially powerful non-invasive intrinsic marker of coral disease. Here, we show the usefulness of live-imaging laser scanning confocal microscopy to investigate coral health state. We demonstrate that the Hawaiian coral Montipora capitata consistently emits cyan and red fluorescence across a depth gradient in reef habitats, but the micro-scale spatial distribution of those pigments differ between healthy coral and coral affected by a tissue loss disease. Naturally diseased and laboratory infected coral systematically exhibited fragmented fluorescent pigments adjacent to the disease front as indicated by several measures of landscape structure (e.g., number of patches) relative to healthy coral. Histology results supported these findings. Pigment fragmentation indicates a disruption in coral tissue that likely impedes translocation of energy within a colony. The area of fragmented fluorescent pigments in diseased coral extended 3.03 mm ± 1.80 mm adjacent to the disease front, indicating pathogenesis was highly localized rather than systemic. Our study demonstrates that coral fluorescence can be used as a proxy for coral health state, and, such patterns may help refine hypotheses about modes of pathogenesis.

Coral individuality - confluence of change physical splitting and developmental ability of embryos.

Previous studies have suggested that blastomeres from the 2-, 4-, or 8-cell stage of corals have the ability to develop into normal primary polyps. However, it is still not known which developmental stage's blastomere produces which juvenile. In this study, we demonstrated that only the blastomeres with animal hemispheres have the capacity to develop into normal primary polyps. Individuality was evaluated using blastomeres isolated from the corals Acropora digitifera, A. intermedia, Dipsastraea lizardensis, and Favites chinensis. On commencement of embryo cleavage, the animal pole was marked using Neutral red staining, and at the 2-, 4-, and 8-cell stages, embryos were divided into individual blastomeres using glass needles. We found that the survival rate and percentage metamorphosis were higher in the larger-sized blastomeres with animal hemispheres. The vegetal hemisphere alone is incapable of developing into a normal primary polyp; however, a ball-shaped embryo with incomplete mesenteries and no pharynx developed in some cases. These results indicate that the animal hemisphere is needed for corals to develop into normal primary polyps, and that the individuality of corals is possibly determined by a combination of the chance physical splitting of embryos by waves and their innate developmental ability.

Immunodetection of acetylated alpha-tubulin in stony corals: Evidence for the existence of flagella in coral male germ cells.

The molecular and cellular characteristics of male germ cell development remain largely unknown in corals. This study focused on the expression pattern of acetylated α-tubulin (Ac-α-Tu), which is involved in male germ cell development in various animals across taxa, to gain a better understanding of male germ cell development in the stony coral Euphyllia ancora. Immunohistochemical analysis of the different stages of male germ cells showed the presence of filamentous Ac-α-Tu in the early to late stages of male germ cells-such as spermatogonia, spermatocytes, and spermatids-as well as in the flagella of mature sperm. Immunocytochemical and transmission electron microscope analyses demonstrated that early-stage male germ cells possess long flagella containing Ac-α-Tu. The presence of filamentous Ac-α-Tu was also immunohistochemically demonstrated in the male germ cells from 14 other coral species, implying that possession of flagella with Ac-α-Tu is a common characteristic of male germ cells in stony corals. Therefore, as a distinctive cellular characteristic of male germ cells, Ac-α-Tu could be used as a male germ cell marker in stony corals; indeed, immunolabeling for Ac-α-Tu may be a useful method to aid in the identification and morphological observation of male germ cells in various corals in basic and applied biology (e.g., aquaculture) as well as in ecological studies.

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

BACKGROUND: 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. METHODS: Using fluorescence-activated cell sorting (FACS), coral cell suspension were sorted into different cellular populations using fluorescent cell markers that are non-species specific. Over 30 different cell markers were tested. Additionally, cell suspension from Aiptasia pallida was also tested, and a phagocytosis test was done as a downstream functional assay. RESULTS: We found that 24 of the screened markers positively labeled coral cells and 16 differentiated cell sub-populations. We identified 12 different cellular sub-populations using three markers, and found that each sub-population is primarily homogeneous. Lastly, we verified this technique in a sea anemone, Aiptasia pallida, and found that with minor modifications, a similar gating strategy can be successfully applied. Additionally, within A. pallida, we show elevated phagocytosis of sorted cells based on an immune associated marker. CONCLUSIONS: In this study, we successfully adapted FACS for isolating coral cell populations and conclude that this technique is translatable for future use in other species. This technique has the potential to be used for different types of studies on the cellular stress response and other immunological studies.