Physiological processes and lipidome dynamics in the soft coral Sinularia heterospiculata under experimental bleaching.

Coral polyps host intracellular symbiotic dinoflagellates (SD). The loss of SD (referred as bleaching) under stressful environmental conditions is the main reason of coral reef destruction, and therefore, intensively studied over the world. Lipids are the structural base of biomembranes and energy reserve of corals and are directly involved in the coral bleaching. In order to establish a relationship between coral tissue morphology, physiological processes and lipidome dynamics during bleaching, the soft coral Sinularia heterospiculata was exposed to experimental heat stress (33 °C) for 72 h. A chlorophyll content, structure of cells, the level of reactive oxygen species (ROS), and molecular species of storage and structural lipids were analyzed. After 24 h of heat exposure, the level of ROS-positive SD cells did not increase, but the host tissues lost a significant part of SD. The removal of SD cells by exocytosis were suggested. Exocytosis was presumed to prevail at earlier stages of the soft coral bleaching. Symbiophagosomes with degenerative SD were observed in the stressed coral host cells. After 24 h, the content of phosphatidylinositols, which involved in apoptosis and autophagy, was significantly decreased. The innate immune response was triggered, and SD were digested by the coral host. After 48 h, a degradation of SD chloroplasts and a decrease in the specific monogalactosyldiacylglycerol molecular species were detected that confirmed a disruption of lipid biosynthesis in chloroplasts. At the end of coral bleaching, the appearance of oxidized phosphatidylethanolamines, indicating damage to the host membranes, and the degradation of the coral tissues were simultaneously observed. Thus, a switch between dominant mechanisms of the SD loss during bleaching of S. heterospiculata was found and proved by certain variations of the lipidomic profile. Lipidomic parameters may become indicators of physiological processes occurring in the symbiotic coral organism and may be used for assessing anthropogenic or natural destructive effects on coral reefs.

AmAMP1 from Acropora millepora and damicornin define a family of coral-specific antimicrobial peptides related to the Shk toxins of sea anemones.

A candidate antimicrobial peptide (AmAMP1) was identified by searching the whole genome sequence of Acropora millepora for short (<125AA) cysteine-rich predicted proteins with an N-terminal signal peptide but lacking clear homologs in the SwissProt database. It resembled but was not closely related to damicornin, the only other known AMP from a coral, and was shown to be active against both Gram-negative and Gram-positive bacteria. These proteins define a family of AMPs present in corals and their close relatives, the Corallimorpharia, and are synthesised as preproproteins in which the C-terminal mature peptide contains a conserved arrangement of six cysteine residues. Consistent with the idea of a common origin for AMPs and toxins, this Cys motif is shared between the coral AMPs and the Shk neurotoxins of sea anemones. AmAMP1 is expressed at late stages of coral development, in ectodermal cells that resemble the "ganglion neurons" of Hydra, in which it has recently been demonstrated that a distinct AMP known as NDA-1 is expressed.

Transcriptome Analyses of Immune System Behaviors in Primary Polyp of Coral Acropora digitifera Exposed to the Bacterial Pathogen Vibrio coralliilyticus under Thermal Loading.

Elevated sea surface temperature associated with global warming is a serious threat to coral reefs. Elevated temperatures directly or indirectly alter the distribution of coral-pathogen interactions and thereby exacerbate infectious coral diseases. The pathogenic bacterium Vibrio coralliilyticus is well-known as a causative agent of infectious coral disease. Rising sea surface temperature promotes the infection of corals by this bacterium, which causes several coral pathologies, such as bacterial bleaching, tissue lysis, and white syndrome. However, the effects of thermal stress on coral immune responses to the pathogen are poorly understood. To delineate the effects of thermal stress on coral immunity, we performed transcriptome analysis of aposymbiotic primary polyps of the reef-building coral Acropora digitifera exposed to V. coralliilyticus under thermal stress conditions. V. coralliilyticus infection of coral that was under thermal stress had negative effects on various molecular processes, including suppression of gene expression related to the innate immune response. In response to the pathogen, the coral mounted various responses including changes in protein metabolism, exosome release delivering signal molecules, extracellular matrix remodeling, and mitochondrial metabolism changes. Based on these results, we provide new insights into innate immunity of A. digitifera against pathogen infection under thermal stress conditions.

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.

Dual recognition activity of a rhamnose-binding lectin to pathogenic bacteria and zooxanthellae in stony coral Pocillopora damicornis.

Rhamnose-binding lectin (RBL) is a type of Ca2+-independent lectin with tandem repeat carbohydrate-recognition domain, and is crucial for the innate immunity in many invertebrates. In this study, the cDNA sequence encoding RBL in coral Pocillopora damicornis (PdRBL-1) was cloned. The PdRBL-1 protein shared highest amino acid sequence similarity (55%) with the polyp of Hydra vulgaris, and contained a signal peptide and two tandem carbohydrate-recognition domains in which all cysteine residues were conserved. Surface plasmon resonance method revealed that the recombinant PdRBL-1 protein bound to LPS and Lipid A, but not to LTA, ß-glucan, mannose and Poly (I:C). Results also showed that it bonded with zooxanthellae using western blotting method, and that the bound protein was detectable only at concentrations higher than 102 zooxanthellae cell mL-1. When recombinant PdRBL-1 protein was preincubated with LPS, lower amounts of protein bound to zooxanthellae compared to cells not preincubated with LPS. Furthermore, PdRBL-1 mRNA expression increased significantly at 12 h, and declined to the baseline at 24 h after heat stress at 31 °C. These results collectively suggest that PdRBL-1 could recognize not only pathogenic bacteria but also symbiotic zooxanthellae, and that the recognition of zooxanthellae by PdRBL-1 could be repressed by pathogenic bacteria through competitive binding. This information allows us to gain new insights in the mechanisms influencing the establishment and maintenance of coral-zooxanthella symbiosis in coral P. damicornis.

Functional characterization of Vitellogenin_N domain, domain of unknown function 1943, and von Willebrand factor type D domain in vitellogenin of the non-bilaterian coral Euphyllia ancora: Implications for emergence of immune activity of vitellogenin in basal metazoan.

Our understanding of the function of vitellogenin (Vg) in reproduction has undergone a transformation over the past decade in parallel with new insights into the role of Vg in immunity. However, the time when Vg was endowed with immunological activities during animal evolution remains elusive. Here we demonstrate for the first time that the recombinant proteins rVitellogenin_N, rDUF1943, and rVWD from Vg of the basal metazoan coral Euphyllia ancora not only interact with Gram-positive and negative bacteria as well as their conserved surface components LTA and LPS but also enhance phagocytosis of bacteria by macrophages. Moreover, challenge with LPS results in a marked up-regulation of vg in the coral E. ancora. These data suggest that E. ancora Vg, like that described in the bilaterian oviparous animals fish and amphioxus, is a molecule related to antibacterial defense, indicating that the timing of the emergence of immune role of Vg predates the divergence of the cnidarian (non-bilaterian) and bilaterian lineages.

Histology and ultrastructure of the coenenchyme of the octocoral Swiftia exserta, a model organism for innate immunity/graft rejection.

The octocoral Swiftia exserta has been utilized extensively in our laboratory to study innate immune reactions in Cnidaria such as wound healing, auto- and allo-graft reactions, and for some classical "foreign body" phagocytosis experiments. All of these reactions occur in the coenenchyme of the animal, the colonial tissue surrounding the axial skeleton in which the polyps are embedded, and do not rely on nematocysts or directly involve the polyps. In order to better understand some of the cellular reactions occurring in the coenenchyme, the present study employed several cytochemical methods (periodic acid-Schiff reaction, Mallory's aniline blue collagen stain, and Gomori's trichrome stain) and correlated the observed structures with electron microscopy (both scanning and transmission). Eight types of cells were apparent in the coenenchyme of S. exserta, exclusive of gastrodermal tissue: (i) epithelial ectoderm cells, (ii) oblong granular cells, (iii) granular amoebocytes, (iv) morula-like cells, (v) mesogleal cells, (vi) sclerocytes, (vii) axial epithelial cells, and (viii) cnidocytes with mostly atrichous isorhiza nematocysts. Several novel organizational features are now apparent from transmission electron micrographs: the ectoderm consists of a single layer of flat epithelial cells, the cell types of the mesoglea extend from beneath the thin ectoderm throughout the mesogleal cell cords, the organization of the solenia gastroderm consists of a single layer of cells, and two nematocyst types have been found. A new interpretation of the cellular architecture of S. exserta, and more broadly, octocoral biology is now possible.

RNA-seq profiles of immune related genes in the staghorn coral Acropora cervicornis infected with white band disease.

Coral diseases are among the most serious threats to coral reefs worldwide, yet most coral diseases remain poorly understood. How the coral host responds to pathogen infection is an area where very little is known. Here we used next-generation RNA-sequencing (RNA-seq) to produce a transcriptome-wide profile of the immune response of the Staghorn coral Acropora cervicornis to White Band Disease (WBD) by comparing infected versus healthy (asymptomatic) coral tissues. The transcriptome of A. cervicornis was assembled de novo from A-tail selected Illumina mRNA-seq data from whole coral tissues, and parsed bioinformatically into coral and non-coral transcripts using existing Acropora genomes in order to identify putative coral transcripts. Differentially expressed transcripts were identified in the coral and non-coral datasets to identify genes that were up- and down-regulated due to disease infection. RNA-seq analyses indicate that infected corals exhibited significant changes in gene expression across 4% (1,805 out of 47,748 transcripts) of the coral transcriptome. The primary response to infection included transcripts involved in macrophage-mediated pathogen recognition and ROS production, two hallmarks of phagocytosis, as well as key mediators of apoptosis and calcium homeostasis. The strong up-regulation of the enzyme allene oxide synthase-lipoxygenase suggests a key role of the allene oxide pathway in coral immunity. Interestingly, none of the three primary innate immune pathways--Toll-like receptors (TLR), Complement, and prophenoloxydase pathways, were strongly associated with the response of A. cervicornis to infection. Five-hundred and fifty differentially expressed non-coral transcripts were classified as metazoan (n = 84), algal or plant (n = 52), fungi (n = 24) and protozoans (n = 13). None of the 52 putative Symbiodinium or algal transcript had any clear immune functions indicating that the immune response is driven by the coral host, and not its algal symbionts.

Immune response to a pathogen in corals.

The sea fan coral (Gorgonia ventalina), one of the most abundant gorgonians in the tropical and subtropical Atlantic waters, have suffered several diseases that have diminished its abundance throughout their range. In this study, we present a model that analyzes the capacity of G. ventalina to eradicate a micro-pathogen under three immune responses: strong, moderate, and very weak. The model assumes that: (1) polyps are the main unit of the coral; (2) the population of polyps is homogeneously distributed; and (3) the immune system is activated by a signal. When an endosymbiont exceeds a density threshold, it becomes pathogenic, increasing polyp mortality. As a consequence, the colony emits a signal to its stem cells to differentiate into phagocytic and humoral cells, both of which combat the pathogen. Given a strong immune response, the pathogen is rapidly eradicated by the immune cells, and the coral polyp population returns to an equilibrium state. With a moderate immune response, polyps and pathogen coexist, but the maximum capacity of polyp density is never reached. An immunologically compromised colony offering a weak immune response is unable to stop pathogen growth, and the colony dies. This analysis suggests an alternative explanation for the spatial and temporal variability in disease incidence and mortality, which is based on the strength of the immune system of hosts rather than the virulence of the pathogen.

Patterns of coral ecological immunology: variation in the responses of Caribbean corals to elevated temperature and a pathogen elicitor.

Disease epizootics are increasing with climatic shifts, yet within each system only a subset of species are identified as the most vulnerable. Understanding ecological immunology patterns as well as environmental influences on immune defenses will provide insight into the persistence of a functional system through adverse conditions. Amongst the most threatened ecosystems are coral reefs, with coral disease epizootics and thermal stress jeopardizing their survival. Immune defenses were investigated within three Caribbean corals, Montastraea faveolata, Stephanocoenia intersepta and Porites astreoides, which represent a range of disease and bleaching susceptibilities. Levels of several immune parameters were measured in response to elevated water temperature and the presence of a commercial pathogen-associated molecular pattern (PAMP) - lipopolysaccharide (LPS) - as an elicitor of the innate immune response. Immune parameters included prophenoloxidase (PPO) activity, melanin concentration, bactericidal activity, the antioxidants peroxidase and catalase, and fluorescent protein (FP) concentration. LPS induced an immune response in all three corals, although each species responded differently to the experimental treatments. For example, M. faveolata, a disease-susceptible species, experienced significant decreases in bactericidal activity and melanin concentration after exposure to LPS and elevated temperature alone. Porites astreoides, a disease-resistant species, showed increased levels of enzymatic antioxidants upon exposure to LPS independently and increased PPO activity in response to the combination of LPS and elevated water temperature. This study demonstrates the ability of reef-building corals to induce immune responses in the presence of PAMPs, indicating activation of PAMP receptors and the transduction of appropriate signals leading to immune effector responses. Furthermore, these data address the emerging field of ecological immunology by highlighting interspecific differences in immunity and immunocompetences among Caribbean corals, which are reflected in their life-history characteristics, disease susceptibilities and bleaching-induced mortality.

Using the Acropora digitifera genome to understand coral responses to environmental change.

Despite the enormous ecological and economic importance of coral reefs, the keystone organisms in their establishment, the scleractinian corals, increasingly face a range of anthropogenic challenges including ocean acidification and seawater temperature rise. To understand better the molecular mechanisms underlying coral biology, here we decoded the approximately 420-megabase genome of Acropora digitifera using next-generation sequencing technology. This genome contains approximately 23,700 gene models. Molecular phylogenetics indicate that the coral and the sea anemone Nematostella vectensis diverged approximately 500 million years ago, considerably earlier than the time over which modern corals are represented in the fossil record (∼240 million years ago). Despite the long evolutionary history of the endosymbiosis, no evidence was found for horizontal transfer of genes from symbiont to host. However, unlike several other corals, Acropora seems to lack an enzyme essential for cysteine biosynthesis, implying dependency of this coral on its symbionts for this amino acid. Corals inhabit environments where they are frequently exposed to high levels of solar radiation, and analysis of the Acropora genome data indicates that the coral host can independently carry out de novo synthesis of mycosporine-like amino acids, which are potent ultraviolet-protective compounds. In addition, the coral innate immunity repertoire is notably more complex than that of the sea anemone, indicating that some of these genes may have roles in symbiosis or coloniality. A number of genes with putative roles in calcification were identified, and several of these are restricted to corals. The coral genome provides a platform for understanding the molecular basis of symbiosis and responses to environmental changes.

Innate immune responses of a scleractinian coral to vibriosis.

Scleractinian corals are the most basal eumetazoan taxon and provide the biological and physical framework for coral reefs, which are among the most diverse of all ecosystems. Over the past three decades and coincident with climate change, these phototrophic symbiotic organisms have been subject to increasingly frequent and severe diseases, which are now geographically widespread and a major threat to these ecosystems. Although coral immunity has been the subject of increasing study, the available information remains fragmentary, especially with respect to coral antimicrobial responses. In this study, we characterized damicornin from Pocillopora damicornis, the first scleractinian antimicrobial peptide (AMP) to be reported. We found that its precursor has a segmented organization comprising a signal peptide, an acidic proregion, and the C-terminal AMP. The 40-residue AMP is cationic, C-terminally amidated, and characterized by the presence of six cysteine molecules joined by three intramolecular disulfide bridges. Its cysteine array is common to another AMP and toxins from cnidarians; this suggests a common ancestor, as has been proposed for AMPs and toxins from arthropods. Damicornin was active in vitro against Gram-positive bacteria and the fungus Fusarium oxysporum. Damicornin expression was studied using a combination of immunohistochemistry, reverse phase HPLC, and quantitative RT-PCR. Our data show that damicornin is constitutively transcribed in ectodermal granular cells, where it is stored, and further released in response to nonpathogenic immune challenge. Damicornin gene expression was repressed by the coral pathogen Vibrio coralliilyticus. This is the first evidence of AMP gene repression in a host-Vibrio interaction.

The biology of coral metamorphosis: molecular responses of larvae to inducers of settlement and metamorphosis.

Like many other cnidarians, corals undergo metamorphosis from a motile planula larva to a sedentary polyp. In some sea anemones such as Nematostella this process is a smooth transition requiring no extrinsic stimuli, but in many corals it is more complex and is cue-driven. To better understand the molecular events underlying coral metamorphosis, competent larvae were treated with either a natural inducer of settlement (crustose coralline algae chips/extract) or LWamide, which bypasses the settlement phase and drives larvae directly into metamorphosis. Microarrays featuring >8000 Acropora unigenes were used to follow gene expression changes during the 12h period after these treatments, and the expression patterns of specific genes, selected on the basis of the array experiments, were investigated by in situ hybridization. Three patterns of expression were common-an aboral pattern restricted to the searching/settlement phase, a second phase of aboral expression corresponding to the beginning of the development of the calicoblastic ectoderm and continuing after metamorphosis, and a later orally-restricted pattern.

Coral immunology and resistance to disease.

Scleractinian corals (phylum Cnidaria, class Anthozoa) have innate immunological responses against infections. Research has recently suggested that corals also possess an adaptive-like immunological repertoire that recognizes specific pathogens and allografts. While evolutionarily distinct, the corals' innate and adaptive-like immunity systems are not mutually exclusive because the phagocytic cells of the non-specific, innate immune system may activate specific adaptive immunological responses. Warming oceans may immunocompromise coral hosts, making them more susceptible to tropical marine diseases, independent of the virulence of the pathogen. The ability of corals to ward off both primary and opportunistic infections, through adaptive-like mechanisms, may play a critical role in the corals' ability to fight future disease infection. Here we show evidence that corals possess immunological repertoires that extend well beyond simple innate defenses. The extent to which corals have developed such an adaptive-like immune repertoire will determine whether corals will survive climate change and other anthropogenic disturbances.

High amino acid diversity and positive selection at a putative coral immunity gene (tachylectin-2).

BACKGROUND: Genes involved in immune functions, including pathogen recognition and the activation of innate defense pathways, are among the most genetically variable known, and the proteins that they encode are often characterized by high rates of amino acid substitutions, a hallmark of positive selection. The high levels of variation characteristic of immunity genes make them useful tools for conservation genetics. To date, highly variable immunity genes have yet to be found in corals, keystone organisms of the world's most diverse marine ecosystem, the coral reef. Here, we examine variation in and selection on a putative innate immunity gene from Oculina, a coral genus previously used as a model for studies of coral disease and bleaching. RESULTS: In a survey of 244 Oculina alleles, we find high nonsynonymous variation and a signature of positive selection, consistent with a putative role in immunity. Using computational protein structure prediction, we generate a structural model of the Oculina protein that closely matches the known structure of tachylectin-2 from the Japanese horseshoe crab (Tachypleus tridentatus), a protein with demonstrated function in microbial recognition and agglutination. We also demonstrate that at least three other genera of anthozoan cnidarians (Acropora, Montastrea and Nematostella) possess proteins structurally similar to tachylectin-2. CONCLUSIONS: Taken together, the evidence of high amino acid diversity, positive selection and structural correspondence to the horseshoe crab tachylectin-2 suggests that this protein is 1) part of Oculina's innate immunity repertoire, and 2) evolving adaptively, possibly under selective pressure from coral-associated microorganisms. Tachylectin-2 may serve as a candidate locus to screen coral populations for their capacity to respond adaptively to future environmental change.