Gastrodermal Cells Research Articles

Pathology of tissue loss in three key gorgonian species in the Mediterranean Sea

The Mediterranean is known for its marine biodiversity, especially gorgonian forests. Unfortunately, these are experiencing rapid declines due to climate change, manifested by repeated marine heat waves resulting in mass mortality events since the early 1990 s. To better understand why gorgonians are declining, more systematic approaches to investigate the exact causes are needed, and pathology may aid in this goal.We described gross and microscopic pathology of tissue loss in three key gorgonian species in the Mediterranean region, Paramuricea clavata, Eunicella cavolini, and Leptogorgia sarmentosa, that were all experiencing various degrees of acute to subacute tissue loss characterized by exposed axial skeleton sometimes partly colonized by epibionts and thinning of adjacent tissues. The most significant variety of lesions was seen in P. clavata followed by L. sarmentosa and E. cavolini. For all species, dissociation of gastrodermal cells was the dominant microscopic lesion followed by necrosis of the gastrodermis. Ciliates invading gastrodermis and associated with necrosis of polyps were seen only in E. cavolini. Epidermal tissue loss was seen only in L. sarmentosa, while P. clavata was distinguished by a prominent inflammatory response and unidentified dark round structures within the tentacle epidermis and gastrodermis with no host response. Further work to understand the cause of death in gorgonians is needed, particularly to elucidate the role of ciliates and environmental co-factors or infectious agents not visible on light microscopy, as well as applications of additional tools such as cytology.

Photosynthesis and other factors affecting the establishment and maintenance of cnidarian-dinoflagellate symbiosis.

Coral growth depends on the partnership between the animal hosts and their intracellular, photosynthetic dinoflagellate symbionts. In this study, we used the sea anemone Aiptasia, a laboratory model for coral biology, to investigate the poorly understood mechanisms that mediate symbiosis establishment and maintenance. We found that initial colonization of both adult polyps and larvae by a compatible algal strain was more effective when the algae were able to photosynthesize and that the long-term maintenance of the symbiosis also depended on photosynthesis. In the dark, algal cells were taken up into host gastrodermal cells and not rapidly expelled, but they seemed unable to reproduce and thus were gradually lost. When we used confocal microscopy to examine the interaction of larvae with two algal strains that cannot establish stable symbioses with Aiptasia, it appeared that both pre- and post-phagocytosis mechanisms were involved. With one strain, algae entered the gastric cavity but appeared to be completely excluded from the gastrodermal cells. With the other strain, small numbers of algae entered the gastrodermal cells but appeared unable to proliferate there and were slowly lost upon further incubation. We also asked if the exclusion of either incompatible strain could result simply from their cells' being too large for the host cells to accommodate. However, the size distributions of the compatible and incompatible strains overlapped extensively. Moreover, examination of macerates confirmed earlier reports that individual gastrodermal cells could expand to accommodate multiple algal cells. This article is part of the theme issue 'Sculpting the microbiome: how host factors determine and respond to microbial colonization'.

Coral holobiont research needs spatial analyses at the microbial scale.

Coral holobiont research needs spatial analyses at the microbial scale.

Fasciola hepatica Gastrodermal Cells Selectively Release Extracellular Vesicles via a Novel Atypical Secretory Mechanism.

The liver fluke, Fasciola hepatica, is an obligate blood-feeder, and the gastrodermal cells of the parasite form the interface with the host’s blood. Despite their importance in the host–parasite interaction, in-depth proteomic analysis of the gastrodermal cells is lacking. Here, we used laser microdissection of F. hepatica tissue sections to generate unique and biologically exclusive tissue fractions of the gastrodermal cells and tegument for analysis by mass spectrometry. A total of 226 gastrodermal cell proteins were identified, with proteases that degrade haemoglobin being the most abundant. Other detected proteins included those such as proton pumps and anticoagulants which maintain a microenvironment that facilitates digestion. By comparing the gastrodermal cell proteome and the 102 proteins identified in the laser microdissected tegument with previously published tegument proteomic datasets, we showed that one-quarter of proteins (removed by freeze–thaw extraction) or one-third of proteins (removed by detergent extraction) previously identified as tegumental were instead derived from the gastrodermal cells. Comparative analysis of the laser microdissected gastrodermal cells, tegument, and F. hepatica secretome revealed that the gastrodermal cells are the principal source of secreted proteins, as well as showed that both the gastrodermal cells and the tegument are likely to release subpopulations of extracellular vesicles (EVs). Microscopical examination of the gut caeca from flukes fixed immediately after their removal from the host bile ducts showed that selected gastrodermal cells underwent a progressive thinning of the apical plasma membrane which ruptured to release secretory vesicles en masse into the gut lumen. Our findings suggest that gut-derived EVs are released via a novel atypical secretory route and highlight the importance of the gastrodermal cells in nutrient acquisition and possible immunomodulation by the parasite.

The characteristics of host lipid body biogenesis during coral-dinoflagellate endosymbiosis.

Intracellular lipid body (LB) biogenesis depends on the symbiosis between coral hosts and their Symbiodinaceae. Therefore, understanding the mechanism(s) behind LB biosynthesis in corals can portentially elucide the drivers of cellular regulation during endosymbiosis. This study assessed LB formation in the gastrodermal tissue layer of the hermatypic coral Euphyllia glabrescens. Diel rhythmicity in LB size and distribution was observed; solar irradiation onset at sunrise initiated an increase in LB formation, which continued throughout the day and peaked after sunset at 18:00. The LBs migrated from the area near the mesoglea to the gastrodermal cell border near the coelenteron. Micro-LB biogenesis occurred in the endoplasmic reticulum (ER) of the host gastrodermal cells. A transcriptomic analysis of genes related to lipogenesis indicated that binding immunoglobulin protein (BiP) plays a key role in metabolic signaling pathways. The diel rhythmicity of LB biogenesis was correlated with ER-localized BiP expression. BiP expression peaked during the period with the largest increase in LB formation, thereby indicating that the chaperoning reaction of abnormal protein folding inside the host ER is likely involved in LB biosynthesis. These findings suggest that the host ER, central to LB formation, potentially facilitates the regulation of endosymbiosis between coral hosts and Symbiodiniaceae.

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.

Regulation of the Fasciola hepatica newly excysted juvenile cathepsin L3 (FhCL3) by its propeptide: a proposed \u2018clamp-like\u2019 mechanism of binding and inhibition

BackgroundThe zoonotic worm parasite Fasciola hepatica secretes an abundance of cathepsin L peptidases that are associated with virulence, invasiveness, feeding and migration. The peptidases are produced as inactive zymogens that activate at low pH by autocatalytic removal of their N-terminal pro-domain or propeptide. Propeptides bind to their cognate enzyme with high specificity. Little is known, however, about the mechanism by which the propeptide of FhCL3, a cathepsin L peptidase secreted by the infective newly excysted juveniles (NEJs), regulates the inhibition and activation of the mature enzyme before it is secreted into host tissues.ResultsImmunolocalisation/immunoblotting studies show that the FhCL3 zymogen is produced and secreted by gastrodermal cells of the NEJs gut. A recombinant propeptide of FhCL3 (ppFhCL3) was shown to be a highly potent and selective inhibitor of native and recombinant F. hepatica FhCL3 peptidase, and other members of the cathepsin L family; inhibition constant (Ki) values obtained for FhCL1, FhCL2 and FhCL3 were 0.04 nM, 0.004 nM and < 0.002 nM, respectively. These values are at least 1000-fold lower than those Ki obtained for human cathepsin L (HsCL) and human cathepsin K (HsCK) demonstrating the selectivity of the ppFhCL3 for parasite cathepsins L. By exploiting 3-D structural data we identified key molecular interactions in the specific binding between the ppFhCL3 and FhCL3 mature domain. Using recombinant variants of ppFhCL3 we demonstrated the critical importance of a pair of propeptide residues (Tyr46Lys47) for the interaction with the propeptide binding loop (PBL) of the mature enzyme and other residues (Leu66 and Glu68) that allow the propeptide to block the active site.ConclusionsThe FhCL3 peptidase involved in host invasion by F. hepatica is produced as a zymogen in the NEJs gut. Regulation of its activation involves specific binding sites within the propeptide that are interdependent and act as a “clamp-like” mechanism of inhibition. These interactions are disrupted by the low pH of the NEJs gut to initiate autocatalytic activation. Our enzyme kinetics data demonstrates high potency and selectivity of the ppFhCL3 for its cognate FhCL3 enzyme, information that could be utilised to design inhibitors of parasite cathepsin L peptidases.

The cellular and molecular origins of extracellular vesicles released by the helminth pathogen, Fasciola hepatica

Parasitic helminths secrete extracellular vesicles (EVs) which have potent immunomodulatory effects. Whilst the cargo of EVs has been characterised for many species, we know little about the mechanisms that govern their biogenesis and release. Using antibodies raised against a panel of Fasciola hepatica EV (FhEV) marker proteins, we have identified multiple sites of EV production in the parasite. Discrete immunofluorescence patterns were observed within the gastrodermal cells and tegumental syncytium for different marker proteins whilst the protonephridial (excretory) system and parenchymal-type 2 cells were identified as additional sites of production (or transit) of FhEVs. Ligation was used to mechanically block the oral sucker, excretory pore, or both, to determine the effect on FhEV release from live adult flukes in vitro. This revealed that FhEVs are predominately derived from the gut, whilst the tegument releases EVs to a lesser extent. The data also suggest that the protonephridial system contributes to the small (120 K) EV sub-population. Sphingomyelinase (SMase) activity is a key driver of EV biogenesis in mammalian cells and we have previously identified SMases in FhEVs by mass spectrometry. SMase activity associated with isolated FhEVs was susceptible to the chemical inhibitor GW4869 and treatment of adult flukes with GW4869 led to a significant reduction in 120 K EV release in vitro, suggesting that a ceramide-dependent mechanism could drive 120 K EV formation. In contrast, the release of the larger 15 K EVs was only moderately impacted, indicating that they form independently of SMase activity. Ultrastructural observation of GW4869-treated F. hepatica tissue showed severe disruption to the parenchyma and vacuolation of the tegument, gastrodermal cells and epithelial lining of the excretory ducts. This work establishes that targeted disruption of EV biogenesis and release in helminths is possible, and provides proof-of-concept for future studies investigating EV secretion as a target for parasite control.

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.

A New Method for Collecting Large Amounts of Symbiotic Gastrodermal Cells from Octocorals.

The study of cnidarian-dinoflagellate endosymbiosis in octocorals is becoming increasingly important. As symbiotic gastrodermal cells (SGCs) are the key cells in a symbiotic relationship, obtaining SGCs and studying their functions represent an urgent need. The majority of the cells dissociated from octocoral tissues consist of host cells and algal cells, and very few intact SGCs can be observed. To solve this problem, we developed a new method to collect large amounts of SGCs from octocorals. We incubated the tissue of Sinularia flexibilis in high-salinity (60‰) filtered seawater for 6 h and were able to collect more than 18 times the number of SGCs from the control group. To test the quality of the dissociated cells, we performed three assays to evaluate their cell viability. All three assays demonstrated that cell viability was good after incubating in a high-salinity solution. We also used two other octocorals, Paralemnalia thyrsoides and Sinularia compressa, to perform the same experiment, and the results were similar to those for Sinularia flexibilis. Therefore, a high-salinity-induced increase in the SGC ratio is a common phenomenon among octocorals. This method allows researchers to collect large amounts of SGCs from octocorals and helps us to better understand the complex molecular interactions in cnidarian-dinoflagellate endosymbiosis.

A widespread necrotizing disease affecting Palythoa caribaeorum (Duchassaing and Michelotti, 1860) on coastal reefs in northeastern Brazil

Abstract A highly aggressive necrotizing disease affecting the zooantharian Palythoa caribaeorum was studied on coastal reefs in northeastern Brazil during March and April of 2008. Approximately 87% of the colonies at each locality showed wounds ranging from 27 cm to 1200 cm long and from 12 to 320 cm wide. This disease initiates with the darkening of the polyps, followed by the appearance of a rapidly enlarging wound that decomposes colony tissues and eventually exposes the substrate. The exposed areas are subsequently colonized by macroalgae, Zoanthus sociatus, and other benthic organisms. The necrotized areas were dark-colored and expelled fetid odors. Although extremely aggressive, complete mortality of the colonies was not observed. Surviving fragments recomposed the colonies within 6 to 9 months. Recurrences of the disease were in the summer of 2008 and from February/2013 to March/2014. During the study period, we found various colonies having black-spots (early stage of necrotizing disease), suggesting stress conditions. Zooxanthellae densities in those black-spots were always lower than in apparently healthy colonies, with reductions of up to 74%. More than one environmental variable seems to influence the zooxanthellae densities significantly, but there is no evidence of any association with abnormally high water temperature, as thermal variations in the study area were within their normal range. Histological analysis showed losses of tissue organization in colonies with black-spots and necrotizing components with hypertrophy and cell apoptosis of the gastrodermal cells; apoptosis of zooxanthellae and damage in completes mesenteries. All these features showed an inflammatory response to necrosis disease.

Assessing the impacts of cryopreservation on the mitochondria of a thermotolerant Symbiodinium lineage: Implications for reef coral conservation

Earth's coral reefs are threatened by a barrage of anthropogenic insults, and cryopreservation-based conservation measures are warranted. Successfully cryopreserved corals could then thawed and out-planted on reefs when ocean temperatures stabilize. In such efforts, it will be necessary to also cryopreserve the photosynthetic dinoflagellates (genus Symbiodinium) that reside within the corals' gastrodermal cells. Given this need, Symbiodinium (clade D) cells were cryopreserved in 2 M propylene glycol by a two-step freezing protocol herein and then cultured for 42 days post-thaw. To gauge the effect of cryopreservation, mitochondrial DNA content and intracellular ATP concentration were assessed, and the former parameter was nearly 2-fold higher in freeze-thawed cells compared to controls after 14 days of post-thaw culture. In contrast, intracellular ATP concentration was relatively lower in freeze-thawed cells after seven days of post-thaw culture, though returned to control levels in samples cultured for 42 days post-thaw.

Immature Fasciola gigantica: Time-dependent ultrastructural changes following in vivo treatment with triclabendazole

An in vivo study was carried out to investigate the ultrastructural effects of triclabendazole (TCBZ) on immature Fasciola gigantica in a goat model. Five goats were infected with an oral gavage of 150 metacercarial cysts of F. gigantica and anthelmintic treatment occurred at 4 weeks post infection with an oral dose of 10 mg/kg. They were euthanized at 0 (untreated), 24, 48, 72 and 96 h post treatment (h pt). Juvenile flukes were recovered from each of the goat’s liver and processed for transmission electron microscopy (TEM). The untreated control flukes showed normal ultrastructure and no apparent changes were observed at 24 h pt. At 48 h pt, moderate levels of disruption were observed to the tegument and minor changes to the sub-tegument which included widespread blebbing and disruption of apical tegumental membrane, swollen mitochondria, reduced number of secretory bodies, swelling of basal infolds leading to severe vacuolation, and relatively mild disruption to the subtegumental muscle fibres, parenchyma and tegumental cells, whereas the gastrodermal cells appeared less affected. By 72 h pt, sloughing of the tegumental syncytium was evident leading to the exposure of the basal lamina and the disruption was severe in the subtegument too. At 96 h pt, the flukes were totally devoid of tegument and the disruption was extremely severe, distorting the ultrastructure of the entire fluke’s body. The results of the present study revealed that the flukes showed time-dependent progressive disruption to the internal tissues which became increasingly severe over time pt. This is the first study to detail the time-scale and impacts on ultrastructural morphology of the in vivo TCBZ treatment of the immature tropical liver fluke, Fasciola gigantica.

Surface molecules of extracellular vesicles secreted by the helminth pathogen Fasciola hepatica direct their internalisation by host cells

Helminth parasites secrete extracellular vesicles (EVs) that can be internalised by host immune cells resulting in modulation of host immunity. While the molecular cargo of EVs have been characterised in many parasites, little is known about the surface-exposed molecules that participate in ligand-receptor interactions with the host cell surface to initiate vesicle docking and subsequent internalisation. Using a membrane-impermeable biotin reagent to capture proteins displayed on the outer membrane surface of two EV sub-populations (termed 15k and 120k EVs) released by adult F. hepatica, we describe 380 surface proteins including an array of virulence factors, membrane transport proteins and molecules involved in EV biogenesis/trafficking. Proteomics and immunohistochemical analysis show that the 120k EVs have an endosomal origin and may be released from the parasite via the protonephridial (excretory) system whilst the larger 15k EVs are released from the gastrodermal epithelial cells that line the fluke gut. A parallel lectin microarray strategy was used to profile the topology of major surface oligosaccharides of intact fluorogenically-labelled EVs as they would be displayed to the host. Lectin profiles corresponding to glycoconjugates exposed on the surface of the 15 K and 120K EV sub-populations are practically identical but are distinct from those of the parasite surface tegument, although all are predominated by high mannose sugars. We found that while the F. hepatica EVs were resistant to exo- and endo-glycosidases, the glyco-amidase PNGase F drastically remodelled the surface oligosaccharides and blocked the uptake of EVs by host macrophages. In contrast, pre-treatment with antibodies obtained from infected hosts, or purified antibodies raised against the extracellular domains of specific EV surface proteins (DM9-containing protein, CD63 receptor and myoferlin), significantly enhanced their cellular internalisation. This work highlights the diversity of EV biogenesis and trafficking pathways used by F. hepatica and sheds light on the molecular interaction between parasite EVs and host cells.

Endosymbiotic green algae in European Hydra strains show quantitative difference on morphological and isoenzyme level

The process of endosymbiosis is one of the promoters of speciation. The green hydra (Hydra viridissima Pallas, 1766) is a typical example of endosymbiosis. Its gastrodermal myoepithelial cells harbour the individuals of unicellular photoautotrophic algae. In this study we have developed healthy laboratory cultures of endosymbiotic green algae isolated from green hydra strains. The Hydra strains were collected from four different geographical localities, two of which were in Croatia (strains BV, T), one in Israel (strain M9) and one in Germany (strain HV). For the first time, endosymbiotic algae isolated from green hydra strains have been visualized and described by scanning and transmission electron microscopy. Isolated endosymbiotic algae were characterized by cytological morphometric parameters, enzyme activity and isoenzyme pattern analysis (catalase, peroxidase and esterase). In addition, cells of endosymbiotic algae were characterized by morphometric measurements of diameter, perimeter and area, and chloroplast area. Endosymbiotic algae HV (collected in Germany) and M9 (collected in Israel) were significantly different when compared with endosymbiotic algae T and BV collected in Croatia. The endosymbionts were more different with referent alga Parachlorella kessleri than with referent alga Chlorella vulgaris. The results obtained by isoenzyme pattern analysis also suggested that there was a difference between Croatian and European algal endosymbionts, i.e. the results indicate biological diversity among algal symbionts isolated from the green hydras from different geographical localities. Ultimately, five isoenzymes of catalase and five isoenzymes of peroxidase were resolved by PAGE electrophoresis. Catalase isoenzyme K1 appeared only in endosymbiotic alga M9 collected in Israel and catalase isoenzyme K3 only in endosymbiotic alga T collected in Croatia. Peroxidase isoenzyme P1 was observed only in endosymbiont HV while peroxidase isoenzyme P5 was observed in both endosymbionts HV and M9. Peroxidase isoenzymes P3 and P4 were visible in endosymbiotic algae BV and T collected in Croatia.

Infection by the Helminth Parasite Fasciola hepatica Requires Rapid Regulation of Metabolic, Virulence, and Invasive Factors to Adjust to Its Mammalian Host

The parasite Fasciola hepatica infects a broad range of mammals with impunity. Following ingestion of parasites (metacercariae) by the host, newly excysted juveniles (NEJ) emerge from their cysts, rapidly penetrate the duodenal wall and migrate to the liver. Successful infection takes just a few hours and involves negotiating hurdles presented by host macromolecules, tissues and micro-environments, as well as the immune system. Here, transcriptome and proteome analysis of ex vivo F. hepatica metacercariae and NEJ reveal the rapidity and multitude of metabolic and developmental alterations that take place in order for the parasite to establish infection. We found that metacercariae despite being encased in a cyst are metabolically active, and primed for infection. Following excystment, NEJ expend vital energy stores and rapidly adjust their metabolic pathways to cope with their new and increasingly anaerobic environment. Temperature increases induce neoblast proliferation and the remarkable up-regulation of genes associated with growth and development. Cysteine proteases synthesized by gastrodermal cells are secreted to facilitate invasion and tissue degradation, and tegumental transporters, such as aquaporins, are varied to deal with osmotic/salinity changes. Major proteins of the total NEJ secretome include proteases, protease inhibitors and anti-oxidants, and an array of immunomodulators that likely disarm host innate immune effector cells. Thus, the challenges of infection by F. hepatica parasites are met by rapid metabolic and physiological adjustments that expedite tissue invasion and immune evasion; these changes facilitate parasite growth, development and maturation. Our molecular analysis of the critical processes involved in host invasion has identified key targets for future drug and vaccine strategies directed at preventing parasite infection.

Astacin gene family of metalloproteinases in planarians: Structural organization and tissue distribution

Planarian flatworms possess extraordinary regenerative capability and body plasticity, which rely on a composite population of stem cells, the neoblasts. Despite impressive advances have been recently achieved in the knowledge of neoblast biology, few is still known about factors that are released by differentiated tissues and influence the neoblast fate. Extracellular matrix (ECM) is a fundamental component of the stem cell niche and its remodeling affects stem cell fate. Here we provide the characterization of the astacin gene family of metalloproteinases in planarians, good candidate enzymes for generating dynamicity in the ECM. Ten and eighteen astacin isoforms were identified in the planarian species Schmidtea mediterranea and Dugesia japonica, respectively. Besides the already characterized Smedolloid, in Schmidtea mediterranea are present eight astacins with a minimal structure (a signal peptide, an activation domain and a Zn-binding catalytic domain), that are colocalized in large cells organized in a peculiar, not yet morphologically characterized, two-ring-shaped structure located in the middle of the body. A single astacin, characterized by a ShK toxin domain in its C-terminal region, has been found to be produced in gastrodermal cells.

Cnidarian Primary Cell Culture as a Tool to Investigate the Effect of Thermal Stress at Cellular Level.

In the context of global change, symbiotic cnidarians are largely affected by seawater temperature elevation leading to symbiosis breakdown. This process, also called bleaching, is triggered by the dysfunction of the symbiont photosystems causing an oxidative stress and cell death to both symbiont and host cells. In our study, we wanted to elucidate the intrinsic capacity of isolated animal cells to deal with thermal stress in the absence of symbiont. In that aim, we have characterized an animal primary cell culture form regenerating tentacles of the temperate sea anemone Anemonia viridis. We first compared the potential of whole tissue tentacle or separated epidermal or gastrodermal monolayers as tissue sources to settle animal cell cultures. Interestingly, only isolated cells extracted from whole tentacles allowed establishing a viable and proliferative primary cell culture throughout 31days. The analysis of the expression of tissue-specific and pluripotency markers defined cultivated cells as differentiated cells with gastrodermal origin. The characterization of the animal primary cell culture allowed us to submit the obtained gastrodermal cells to hyperthermal stress (+ 5 and + 8°C) during 1 and 7days. Though cell viability was not affected at both hyperthermal stress conditions, cell growth drastically decreased. In addition, only a + 8°C hyperthermia induced a transient increase of antioxidant defences at 1day but no ubiquitin or carbonylation protein damages. These results demonstrated an intrinsic resistance of cnidarian gastrodermal cells to hyperthermal stress and then confirmed the role of symbionts in the hyperthermia sensitivity leading to bleaching.

Generation of clade- and symbiont-specific antibodies to characterize marker molecules during Cnidaria-Symbiodinium endosymbiosis

The endosymbiosis between cnidarians and dinoflagellates is responsible for the formation of coral reefs. Changes in molecules have been identified during the process of cnidaria-Symbiodinium endosymbiosis. However, the complexity of the molecular interaction has prevented the establishment of a mechanistic explanation of cellular regulation in this mutualistic symbiosis. To date, no marker molecules have been identified to specifically represent the symbiotic status. Because the endosymbiotic association occurs in the symbiotic gastrodermal cells (SGCs), whole cells of isolated SGCs were used as an antigen to generate monoclonal antibodies (mAb) to screen possible molecular candidates of symbiotic markers. The results showed that one of the generated monoclonal antibodies, 2–6F, specifically recognized clade C symbiotic Symbiodinium but not its free-living counterpart or other Symbiodinium clades. The expression levels of 2–6F mAb-recognized proteins are highly correlated with the symbiotic status, and these proteins were characterized as N-linked glycoproteins via treatment with peptide N-glycosidase F. Furthermore, their glycan moieties were markedly different from those of free-living Symbiodinium, potentially suggesting host regulation of post-translational modification. Consequently, the 2–6F mAb can be used to detect the symbiotic state of corals and investigate the complex molecular interactions in cnidaria-Symbiodinium endosymbiosis.

Coral lipid bodies as the relay center interconnecting diel-dependent lipidomic changes in different cellular compartments

Lipid bodies (LBs) in the coral gastrodermal tissues are key organelles in the regulation of endosymbiosis and exhibit a diel rhythmicity. Using the scleractinian Euphyllia glabrescens collected across the diel cycle, we observed temporally dynamic lipid profiles in three cellular compartments: host coral gastrodermal cells, LBs, and in hospite Symbiodinium. Particularly, the lipidome varied over time, demonstrating the temporally variable nature of the coral–Symbiodinium endosymbiosis. The lipidome-scale data highlight the dynamic, light-driven metabolism of such associations and reveal that LBs are not only lipid storage organelles but also act as a relay center in metabolic trafficking. Furthermore, lipogenesis in LBs is significantly regulated by coral hosts and the lipid metabolites within holobionts featured predominantly triacylglycerols, sterol esters, and free fatty acids. Given these findings through a time-varied lipidome status, the present study provided valuable insights likely to be crucial to understand the cellular biology of the coral–Symbiodinium endosymbiosis.