Microbial and transcriptional response of Acropora valida and Turbinaria peltata to Vibrio coralliilyticus challenge: insights into corals disease resistance.

BACKGROUND: Coral diseases are significant drivers of global coral reef degradation, with pathogens dominated by Vibrio coralliilyticus playing a prominent role in the development of coral diseases. Coral phenotype, symbiotic microbial communities, and host transcriptional regulation have been well-established as factors involved in determining coral disease resistance, but the underlying mechanisms remain incompletely understood. METHODS: This study employs high-throughput sequencing to analyse the symbiotic microbial and transcriptional response of the hosts in order to evaluate the disease resistance of Acropora valida and Turbinaria peltata exposed to Vibrio coralliilyticus. RESULTS: A. valida exhibited pronounced bleaching and tissue loss within 7 h of pathogen infection, whereas T. peltata showed no signs of disease throughout the experiment. Microbial diversity analyses revealed that T. peltata had a more flexible microbial community and a higher relative abundance of potential beneficial bacteria compared to A. valida. Although Vibrio inoculation resulted in a more significant decrease in the Symbiodiniaceae density of A. valida compared to that of T. peltata, it did not lead to recombination of the coral host and Symbiodiniaceae in either coral species. RNA-seq analysis revealed that the interspecific differences in the transcriptional regulation of hosts after Vibrio inoculation. Differentially expressed genes in A. valida were mainly enriched in the pathways associated with energy supply and immune response, such as G protein-coupled receptor signaling, toll-like receptor signaling, regulation of TOR signaling, while these genes in T. peltata were mainly involved in the pathway related to immune homeostasis and ion transport, such as JAK-STAT signaling pathway and regulation of ion transport. CONCLUSIONS: Pathogenic challenges elicit different microbial and transcriptional shifts across coral species. This study offers novel insights into molecular mechanisms of coral resistance to disease.

Microbiome manipulation by corals and other Cnidaria via quorum quenching.

A dynamic mucous layer containing numerous micro-organisms covers the surface of corals and has multiple functions including both removal of sediment and "food gathering."1 It is likely to also act as the primary barrier to infection; various proteins and compounds with antimicrobial activity have been identified in coral mucus, though these are thought to be largely or exclusively of microbial origin. As in Hydra,2 anti-microbial peptides (AMPs) are likely to play major roles in regulating the microbiomes of corals.3,4 Some eukaryotes employ a complementary but less obvious approach to manipulate their associated microbiome by interfering with quorum signaling, effectively preventing bacteria from coordinating gene expression across a population. Our investigation of immunity in the reef-building coral Acropora millepora,5 however, led to the discovery of a coral gene referred to here as AmNtNH1 that can inactivate a range of acyl homoserine lactones (AHLs), common bacterial quorum signaling molecules, and is induced on immune challenge of adult corals and expressed during the larval settlement process. Closely related proteins are widely distributed within the Scleractinia (hard corals) and some other cnidarians, with multiple paralogs in Acropora, but their closest relatives are bacterial, implying that these are products of one or more lateral gene transfer events post-dating the cnidarian-bilaterian divergence. The deployment by corals of genes used by bacteria to compete with other bacteria reflects a mechanism of microbiome manipulation previously unknown in Metazoa but that may apply more generally.

Genetics of coral resilience.

Genome-wide study in staghorn coral identifies markers of disease resistance.

Functional Characterization of Hexacorallia Phagocytic Cells.

Phagocytosis is the cellular defense mechanism used to eliminate antigens derived from dysregulated or damaged cells, and microbial pathogens. Phagocytosis is therefore a pillar of innate immunity, whereby foreign particles are engulfed and degraded in lysolitic vesicles. In hexacorallians, phagocytic mechanisms are poorly understood, though putative anthozoan phagocytic cells (amoebocytes) have been identified histologically. We identify and characterize phagocytes from the coral Pocillopora damicornis and the sea anemone Nematostella vectensis. Using fluorescence-activated cell sorting and microscopy, we show that distinct populations of phagocytic cells engulf bacteria, fungal antigens, and beads. In addition to pathogenic antigens, we show that phagocytic cells engulf self, damaged cells. We show that target antigens localize to low pH phagolysosomes, and that degradation is occurring within them. Inhibiting actin filament rearrangement interferes with efficient particle phagocytosis but does not affect small molecule pinocytosis. We also demonstrate that cellular markers for lysolitic vesicles and reactive oxygen species (ROS) correlate with hexacorallian phagocytes. These results establish a foundation for improving our understanding of hexacorallian immune cell biology.

Cnidarian Pattern Recognition Receptor Repertoires Reflect Both Phylogeny and Life History Traits.

Pattern recognition receptors (PRRs) are evolutionarily ancient and crucial components of innate immunity, recognizing danger-associated molecular patterns (DAMPs) and activating host defenses. Basal non-bilaterian animals such as cnidarians must rely solely on innate immunity to defend themselves from pathogens. By investigating cnidarian PRR repertoires we can gain insight into the evolution of innate immunity in these basal animals. Here we utilize the increasing amount of available genomic resources within Cnidaria to survey the PRR repertoires and downstream immune pathway completeness within 15 cnidarian species spanning two major cnidarian clades, Anthozoa and Medusozoa. Overall, we find that anthozoans possess prototypical PRRs, while medusozoans appear to lack these immune proteins. Additionally, anthozoans consistently had higher numbers of PRRs across all four classes relative to medusozoans, a trend largely driven by expansions in NOD-like receptors and C-type lectins. Symbiotic, sessile, and colonial cnidarians also have expanded PRR repertoires relative to their non-symbiotic, mobile, and solitary counterparts. Interestingly, cnidarians seem to lack key components of mammalian innate immune pathways, though similar to PRR numbers, anthozoans possess more complete immune pathways than medusozoans. Together, our data indicate that anthozoans have greater immune specificity than medusozoans, which we hypothesize to be due to life history traits common within Anthozoa. Overall, this investigation reveals important insights into the evolution of innate immune proteins within these basal animals.

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.

Dinoflagellate symbionts escape vomocytosis by host cell immune suppression.

Alveolata comprises diverse taxa of single-celled eukaryotes, many of which are renowned for their ability to live inside animal cells. Notable examples are apicomplexan parasites and dinoflagellate symbionts, the latter of which power coral reef ecosystems. Although functionally distinct, they evolved from a common, free-living ancestor and must evade their host's immune response for persistence. Both the initial cellular events that gave rise to this intracellular lifestyle and the role of host immune modulation in coral-dinoflagellate endosymbiosis are poorly understood. Here, we use a comparative approach in the cnidarian endosymbiosis model Aiptasia, which re-establishes endosymbiosis with free-living dinoflagellates every generation. We find that uptake of microalgae is largely indiscriminate, but non-symbiotic microalgae are expelled by vomocytosis, while symbionts induce host cell innate immune suppression and form a lysosomal-associated membrane protein 1-positive niche. We demonstrate that exogenous immune stimulation results in symbiont expulsion and, conversely, inhibition of canonical Toll-like receptor signalling enhances infection of host animals. Our findings indicate that symbiosis establishment is dictated by local innate immune suppression, to circumvent expulsion and promote niche formation. This work provides insight into the evolution of the cellular immune response and key steps involved in mediating endosymbiotic interactions.

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.

Innate immune gene expression in Acropora palmata is consistent despite variance in yearly disease events.

Coral disease outbreaks are expected to increase in prevalence, frequency and severity due to climate change and other anthropogenic stressors. This is especially worrying for the Caribbean branching coral Acropora palmata which has already seen an 80% decrease in cover primarily due to disease. Despite the importance of this keystone species, there has yet to be a characterization of its transcriptomic response to disease exposure. In this study we provide the first transcriptomic analysis of 12 A. palmata genotypes and their symbiont Symbiodiniaceae exposed to disease in 2016 and 2017. Year was the primary driver of gene expression variance for A. palmata and the Symbiodiniaceae. We hypothesize that lower expression of ribosomal genes in the coral, and higher expression of transmembrane ion transport genes in the Symbiodiniaceae indicate that a compensation or dysbiosis may be occurring between host and symbiont. Disease response was the second driver of gene expression variance for A. palmata and included a core set of 422 genes that were significantly differentially expressed. Of these, 2 genes (a predicted cyclin-dependent kinase 11b and aspartate 1-decarboxylase) showed negative Log2 fold changes in corals showing transmission of disease, and positive Log2 fold changes in corals showing no transmission of disease, indicating that these may be important in disease resistance. Co-expression analysis identified two modules positively correlated to disease exposure, one enriched for lipid biosynthesis genes, and the other enriched in innate immune genes. The hub gene in the immune module was identified as D-amino acid oxidase, a gene implicated in phagocytosis and microbiome homeostasis. The role of D-amino acid oxidase in coral immunity has not been characterized but could be an important enzyme for responding to disease. Our results indicate that A. palmata mounts a core immune response to disease exposure despite differences in the disease type and virulence between 2016 and 2017. These identified genes may be important for future biomarker development in this Caribbean keystone species.

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.

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.

Lipopolysaccharide treatment stimulates Pocillopora coral genotype-specific immune responses but does not alter coral-associated bacteria communities.

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

Investigating the roles of transforming growth factor-beta in immune response of Orbicella faveolata, a scleractinian coral.

Symbiotic relationships range from parasitic to mutualistic, yet all endosymbionts face similar challenges, including evasion of host immunity. Many symbiotic organisms have evolved similar mechanisms to face these challenges, including manipulation of the host's transforming growth factor-beta (TGFß) pathway. Here we investigate the TGFß pathway in scelaractinian corals which are dependent on symbioses with dinoflagellates from the family Symbiodiniaceae. Using the Caribbean coral, Orbicella faveolata, we explore the effects of enhancement and inhibition of the TGFß pathway on host gene expression. Following transcriptomic analyses, we demonstrated limited effects of pathway manipulation in absence of immune stimulation. However, manipulation of the TGFß pathway significantly affects the subsequent ability of host corals to mount an immune response. Enhancement of the TGFß pathway eliminates transcriptomic signatures of host coral immune response, while inhibition of the pathway maintains the response. This is, to our knowledge, the first evidence of an immunomodulatory role for TGFß in a scelaractinian coral. These findings suggest variation in TGFß signaling may have implications in the face of increasing disease prevelance. Our results suggest that the TGFß pathway can modulate tradeoffs between symbiosis and immunity. Further study of links between symbiosis, TGFß, and immunity is needed to better understand the ecological implications of these findings.

Ecological Factors Mediate Immunity and Parasitic Co-Infection in Sea Fan Octocorals.

The interplay among environment, demography, and host-parasite interactions is a challenging frontier. In the ocean, fundamental changes are occurring due to anthropogenic pressures, including increased disease outbreaks on coral reefs. These outbreaks include multiple parasites, calling into question how host immunity functions in this complex milieu. Our work investigates the interplay of factors influencing co-infection in the Caribbean sea fan octocoral, Gorgonia ventalina, using metrics of the innate immune response: cellular immunity and expression of candidate immune genes. We used existing copepod infections and live pathogen inoculation with the Aspergillus sydowii fungus, detecting increased expression of the immune recognition gene Tachylectin 5A (T5A) in response to both parasites. Cellular immunity increased by 8.16% in copepod infections compared to controls and single Aspergillus infections. We also detected activation of cellular immunity in reef populations, with a 13.6% increase during copepod infections. Cellular immunity was similar in the field and in the lab, increasing with copepod infections and not the fungus. Amoebocyte density and the expression of T5A and a matrix metalloproteinase (MMP) gene were also positively correlated across all treatments and colonies, irrespective of parasitic infection. We then assessed the scaling of immune metrics to population-level disease patterns and found random co-occurrence of copepods and fungus across 15 reefs in Puerto Rico. The results suggest immune activation by parasites may not alter parasite co-occurrence if factors other than immunity prevail in structuring parasite infection. We assessed non-immune factors in the field and found that sea fan colony size predicted infection by the copepod parasite. Moreover, the effect of infection on immunity was small relative to that of site differences and live coral cover, and similar to the effect of reproductive status. While additional immune data would shed light on the extent of this pattern, ecological factors may play a larger role than immunity in controlling parasite patterns in the wild. Parsing the effects of immunity and ecological factors in octocoral co-infection shows how disease depends on more than one host and one parasite and explores the application of co-infection research to a colonial marine organism.

A Combined Strategy to Improve the Development of a Coral Antivenom Against Micrurus spp.

Accidents involving Micrurus snakes are not the most common ones but are noteworthy due to their severity. Victims envenomed by Micrurus snakes are at high risk of death and therefore must be treated with coral antivenom. In Brazil, the immunization mixture used to fabricate coral antivenom contains Micrurus frontalis and Micrurus corallinus venoms, which are difficult to be obtained in adequate amounts. Different approaches to solve the venom limitation problem have been attempted, including the use of synthetic and recombinant antigens as substitutes. The present work proposes a combined immunization protocol, using priming doses of M. frontalis venom and booster doses of synthetic B-cell epitopes derived from M. corallinus toxins (four three-finger toxins-3FTX; and one phospholipase A2-PLA2) to obtain coral antivenom in a rabbit model. Immunized animals elicited a humoral response against both M. frontalis and M. corallinus venoms, as detected by sera reactivity in ELISA and Western Blot. Relevant cross-reactivity of the obtained sera with other Micrurus species (Micrurus altirostris, Micrurus lemniscatus, Micrurus spixii, Micrurus surinamensis) venoms was also observed. The elicited antibodies were able to neutralize PLA2 activity of both M. frontalis and M. corallinus venoms. In vivo, immunized rabbit sera completely protected mice from a challenge with 1.5 median lethal dose (LD50) of M. corallinus venom and 50% of mice challenged with 1.5 LD50 of M. frontalis venom. These results show that this combined protocol may be a suitable alternative to reduce the amount of venom used in coral antivenom production in Brazil.

Variable effects of local management on coral defenses against a thermally regulated bleaching pathogen.

Bleaching and disease are decimating coral reefs especially when warming promotes bleaching pathogens, such as Vibrio coralliilyticus. We demonstrate that sterilized washes from three common corals suppress V. coralliilyticus but that this defense is compromised when assays are run at higher temperatures. For a coral within the ecologically critical genus Acropora, inhibition was 75 to 154% greater among colonies from coral-dominated marine protected areas versus adjacent fished areas that were macroalgae-dominated. Acropora microbiomes were more variable within fished areas, suggesting that reef degradation may also perturb coral microbial communities. Defenses of a robust poritid coral and a weedy pocilloporid coral were not affected by reef degradation, and microbiomes were unaltered for these species. For some ecologically critical, but bleaching-susceptible, corals such as Acropora, local management to improve reef state may bolster coral resistance to global change, such as bacteria-induced coral bleaching during warming events.

Proteomic Investigation of a Diseased Gorgonian Coral Indicates Disruption of Essential Cell Function and Investment in Inflammatory and Other Immune Processes.

As scleractinian coral cover declines in the face of increased frequency in disease outbreaks, future reefs may become dominated by octocorals. Understanding octocoral disease responses and consequences is therefore necessary if we are to gain insight into the future of ecosystem services provided by coral reefs. In Florida, populations of the octocoral Eunicea calyculata infected with Eunicea black disease (EBD) were observed in the field in the fall of 2011. This disease was recognized by a stark, black pigmentation caused by heavy melanization. Histological preparations of E. calyculata infected with EBD demonstrated granular amoebocyte (GA) mobilization, melanin granules in much of the GA population, and the presence of fungal hyphae penetrating coral tissue. Previous transcriptomic analysis also identified immune trade-offs evidenced by increased immune investment at the expense of growth. Our investigation utilized proteogenomic techniques to reveal decreased investment in general cell signaling while increasing energy production for immune responses. Inflammation was also prominent in diseased E. calyculata and sheds light on factors driving the extreme phenotype observed with EBD. With disease outbreaks continuing to increase in frequency, our results highlight new targets within the cnidarian immune system and provide a framework for understanding transcriptomics in the context of an organismal disease phenotype and its protein expression.

Responding to Threats Both Foreign and Domestic: NOD-Like Receptors in Corals.

Historically mechanisms with which basal animals such as reef-building corals use to respond to changing and increasingly stressful environments have remained elusive. However, the increasing availability of genomic and transcriptomic data from these organisms has provided fundamental insights into the biology of these critically important ecosystem engineers. Notably, insights into cnidarians gained in the post-genomics age have revealed a surprisingly complex immune system which bears a surprising level of similarity with the vertebrate innate immune system. This system has been critically linked to how corals respond to the two most prominent threats on a global scale, emerging coral diseases and increasing water temperature, which are recognized cellularly as either foreign or domestic threats, respectively. These threats can arise from pathogenic microbes or internal cellular dysfunction, underscoring the need to further understand mechanisms corals use to sense and respond to threats to their cellular integrity. In this investigation and meta-analysis, we utilize resources only recently available in the post-genomic era to identify and characterize members of an underexplored class of molecules known as NOD-like receptors in the endangered Caribbean coral Orbicella faveolata. We then leverage these data to identify pathways possibly mediated by NLRs in both O. faveolata and the ecologically important branching coral Acropora digitifera. Overall, we find support that this class of proteins may provide a mechanistic link to how reef-building corals respond to threats both foreign and domestic.

Cell Biology of Coral Symbiosis: Foundational Study Can Inform Solutions to the Coral Reef Crisis.

Coral reefs are faced with almost complete destruction by the end of the century due to global warming unless humanity can cap global temperature rise. There is now a race to develop a diverse set of solutions to save coral reefs. In this perspective, a case is made for understanding the cell biology of coral-dinoflagellate symbiosis to help inform development of solutions for saving reefs. Laboratory model systems for the study of coral symbiosis, including the sea anemone Exaiptasia pallida, are featured as valuable tools in the fight to save corals. The roles of host innate immunity and inter-partner nutrient dynamics in the onset, ongoing maintenance, and dysregulation of symbiosis are reviewed and discussed. Key innate immune genes and pathways, such as glycan-lectin interactions, the sphingosine rheostat, and the cytokine transforming growth factor beta are shown to modulate a host immune response in the symbiotic state. An upset in the homeostatic inorganic nutrient balance during heat stress and high exogenous nutrient availability is credited with driving the partnership toward dysregulation and coral bleaching. Specific examples are given where knowledge of the cell biology of symbiosis is informing the development of solutions, including studies showing clear limitations in the value of partner switching and acclimatization protocols. Finally, emphasis is placed on rapid advancement of knowledge to try to meet the urgent need for solutions. This includes real-time open communication with colleagues on successes and failures, sharing of resources and information, and working together in the spirit of a collective mission to save coral reefs.

Temperature regulates the recognition activities of a galectin to pathogen and symbiont in the scleractinian coral Pocillopora damicornis.

Lectins serve as essential pattern recognition receptors, and play important roles in the recognition of non-self and mediation of innate immune response in metazoans. Scleractinian corals are vulnerable to pathogen infection and endosymbiosis disruption under heat stress that can finally lead to coral bleaching. In this study, a cDNA sequence encoding one galectin was cloned in scleractinian coral Pocillopora damicornis (PdGLT-1). The deduced PdGLT-1 protein shared highest amino acid sequence similarity (99%) with galectin from Stylophora pistillata (XP_022806650.1), and was composed of one signal peptide, one Collagen domain and one Gal-Lectin domain. PdGLT-1 recombinant protein (rPdGLT-1) was expressed and purified in vitro. Binding activities of rPdGLT-1 to bacteria and symbiont were determined using western blotting method. Results showed that rPdGLT-1 was able to bind to gram-positive bacterium Streptococcus mutans, gram-negative bacteria Vibrio coralliilyticus and Escherichia coli, with the highest activity for V. coralliilyticus, and further agglutinated them. The bound rPdGLT-1 to Symbiodinium (10-104 cells mL-1) was detectable, and its binding ability was concentration-dependent. Furthermore, dual binding activities were determined under different temperatures (20, 25, 30 and 35 °C), and the optimal temperatures were found to be 25 and 30 °C for V. coralliilyticus and Symbiodinium, respectively. Results suggested that PdGLT-1 could recognize pathogenic bacteria and symbiotic dinoflagellates Symbiodinium. However, their recognition activities were repressed under high temperature (>30 °C). This study provided insights into the underlying mechanism of lectin modulation to heat bleaching through its pathogen and Symbiodinium recognition in the scleractinian coral P. damicornis.