The cyclic dinucleotide 2'3'-cGAMP induces a broad antibacterial and antiviral response in the sea anemone Nematostella vectensis.

In mammals, cyclic dinucleotides (CDNs) bind and activate STING to initiate an antiviral type I interferon response. CDNs and STING originated in bacteria and are present in most animals. By contrast, interferons are believed to have emerged in vertebrates; thus, the function of CDN signaling in invertebrates is unclear. Here, we use a CDN, 2'3' cyclic guanosine monophosphate-adenosine monophosphate (2'3'-cGAMP), to activate immune responses in a model cnidarian invertebrate, the starlet sea anemone Nematostella vectensis Using RNA sequencing, we found that 2'3'-cGAMP induces robust transcription of both antiviral and antibacterial genes in N. vectensis Many of the antiviral genes induced by 2'3'-cGAMP are homologs of vertebrate interferon-stimulated genes, implying that the interferon response predates the evolution of interferons. Knockdown experiments identified a role for NF-κB in specifically inducing antibacterial genes downstream of 2'3'-cGAMP. Some of these putative antibacterial genes were also found to be induced during Pseudomonas aeruginosa infection. We characterized the protein product of one of the putative antibacterial genes, the N. vectensis homolog of Dae4, and found that it has conserved antibacterial activity. This work suggests that a broad antibacterial and antiviral transcriptional response is an evolutionarily ancestral output of 2'3'-cGAMP signaling in animals.

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.

The Rapid Regenerative Response of a Model Sea Anemone Species Exaiptasia pallida Is Characterised by Tissue Plasticity and Highly Coordinated Cell Communication.

Regeneration of a limb or tissue can be achieved through multiple different pathways and mechanisms. The sea anemone Exaiptasia pallida has been observed to have excellent regenerative proficiency, but this has not yet been described transcriptionally. In this study, we examined the genetic expression changes during a regenerative timecourse and reported key genes involved in regeneration and wound healing. We found that the major response was an early (within the first 8 h) upregulation of genes involved in cellular movement and cell communication, which likely contribute to a high level of tissue plasticity resulting in the rapid regeneration response observed in this species. We find the immune system was only transcriptionally active in the first 8 h post-amputation and conclude, in accordance with previous literature, that the immune system and regeneration have an inverse relationship. Fifty-nine genes (3.8% of total) differentially expressed during regeneration were identified as having no orthologues in other species, indicating that regeneration in E. pallida may rely on the activation of species-specific novel genes. Additionally, taxonomically restricted novel genes, including species-specific novels, and highly conserved genes were identified throughout the regenerative timecourse, showing that both may work in concert to achieve complete regeneration.

Characterization and expression of tyrosinase-like genes in the anemone Exaiptasia pallida as a function of health and symbiotic state.

Coral disease is a major threat to reef ecosystems and therefore, understanding the cellular pathways underlying disease progression and resistance is critical to mitigating future outbreaks. This study focused on tyrosinase-like proteins in cnidarians, which contribute to melanin synthesis, an invertebrate innate immune defense. Specifically, characterization and phylogenetic analysis of cnidarian tyrosinases were performed, and their role in symbiosis and a "mystery disease" in the anemone Exaiptasia pallida was investigated using qPCR. The results reveal a diversity of tyrosinase-like proteins in cnidarians that separate into two major clades on a phylogenetic tree, suggesting functional divergence. Two E. pallida sequences, Ep_Tyr1 and Ep_Tyr2, were further investigated, and qPCR results revealed no gene expression differences as a function of symbiotic state, but decreased expression in late disease stages. Overall this work provides evidence for the participation of tyrosinases in the cnidarian immune response.

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.

Immune function and the decision to deploy weapons during fights in the beadlet anemone, Actinia equina.

The ability to mitigate the costs of engaging in a fight will depend on an individual's physiological state. However, the experience of fighting itself may, in turn, affect an individual's state, especially if the fight results in injury. Previous studies have found a correlation between immune state and fighting success, but the causal direction of this relationship remains unclear. Does immune state determine fighting success? Or does fighting itself influence subsequent immune state? Using the beadlet anemone, Actinia equina, we disentangled the cause and effect of this relationship, measuring immune response once pre-fight and twice post-fight. Contrary to previous findings, pre-fight immune response did not predict fighting success, but rather predicted whether an individual used its weapons during the fight. Furthermore, weapon use and contest outcome significantly affected post-fight immune response. Individuals that used their weapons maintained a stable immune response following the fight, whereas those that fought non-injuriously did not. Furthermore, although winners suffered a reduction in immune response similar to that of losers immediately post-fight, winners began to recover pre-fight levels within 24 h. Our findings indicate that immune state can influence strategic fighting decisions and, moreover, that fight outcome and the agonistic behaviours expressed can significantly affect subsequent immunity.

Sea anemone model has a single Toll-like receptor that can function in pathogen detection, NF-κB signal transduction, and development.

In organisms from insects to vertebrates, Toll-like receptors (TLRs) are primary pathogen detectors that activate downstream pathways, specifically those that direct expression of innate immune effector genes. TLRs also have roles in development in many species. The sea anemone Nematostella vectensis is a useful cnidarian model to study the origins of TLR signaling because its genome encodes a single TLR and homologs of many downstream signaling components, including the NF-κB pathway. We have characterized the single N. vectensis TLR (Nv-TLR) and demonstrated that it can activate canonical NF-κB signaling in human cells. Furthermore, we show that the intracellular Toll/IL-1 receptor (TIR) domain of Nv-TLR can interact with the human TLR adapter proteins MAL and MYD88. We demonstrate that the coral pathogen Vibrio coralliilyticus causes a rapidly lethal disease in N. vectensis and that heat-inactivated V. coralliilyticus and bacterial flagellin can activate a reconstituted Nv-TLR-to-NF-κB pathway in human cells. By immunostaining of anemones, we show that Nv-TLR is expressed in a subset of cnidocytes and that many of these Nv-TLR-expressing cells also express Nv-NF-κB. Additionally, the nematosome, which is a Nematostella-specific multicellular structure, expresses Nv-TLR and many innate immune pathway homologs and can engulf V. coralliilyticus Morpholino knockdown indicates that Nv-TLR also has an essential role during early embryonic development. Our characterization of this primitive TLR and identification of a bacterial pathogen for N. vectensis reveal ancient TLR functions and provide a model for studying the molecular basis of cnidarian disease and immunity.

Evolutionary conserved mechanisms pervade structure and transcriptional modulation of allograft inflammatory factor-1 from sea anemone Anemonia viridis.

Gene family encoding allograft inflammatory factor-1 (AIF-1) is well conserved among organisms; however, there is limited knowledge in lower organisms. In this study, the first AIF-1 homologue from cnidarians was identified and characterised in the sea anemone Anemonia viridis. The full-length cDNA of AvAIF-1 was of 913 bp with a 5' -untranslated region (UTR) of 148 bp, a 3'-UTR of 315 and an open reading frame (ORF) of 450 bp encoding a polypeptide with149 amino acid residues and predicted molecular weight of about 17 kDa. The predicted protein possesses evolutionary conserved EF hand Ca2+ binding motifs, post-transcriptional modification sites and a 3D structure which can be superimposed with human members of AIF-1 family. The AvAIF-1 transcript was constitutively expressed in all tested tissues of unchallenged sea anemone, suggesting that AvAIF-1 could serve as a general protective factor under normal physiological conditions. Moreover, we profiled the transcriptional activation of AvAIF-1 after challenges with different abiotic/biotic stresses showing induction by warming conditions, heavy metals exposure and immune stimulation. Thus, mechanisms associated to inflammation and immune challenges up-regulated AvAIF-1 mRNA levels. Our results suggest its involvement in the inflammatory processes and immune response of A. viridis.

Insights into the innate immunome of actiniarians using a comparative genomic approach.

BACKGROUND: Innate immune genes tend to be highly conserved in metazoans, even in early divergent lineages such as Cnidaria (jellyfish, corals, hydroids and sea anemones) and Porifera (sponges). However, constant and diverse selection pressures on the immune system have driven the expansion and diversification of different immune gene families in a lineage-specific manner. To investigate how the innate immune system has evolved in a subset of sea anemone species (Order: Actiniaria), we performed a comprehensive and comparative study using 10 newly sequenced transcriptomes, as well as three publically available transcriptomes, to identify the origins, expansions and contractions of candidate and novel immune gene families. RESULTS: We characterised five conserved genes and gene families, as well as multiple novel innate immune genes, including the newly recognised putative pattern recognition receptor CniFL. Single copies of TLR, MyD88 and NF-κB were found in most species, and several copies of IL-1R-like, NLR and CniFL were found in almost all species. Multiple novel immune genes were identified with domain architectures including the Toll/interleukin-1 receptor (TIR) homology domain, which is well documented as functioning in protein-protein interactions and signal transduction in immune pathways. We hypothesise that these genes may interact as novel proteins in immune pathways of cnidarian species. Novelty in the actiniarian immunome is not restricted to only TIR-domain-containing proteins, as we identify a subset of NLRs which have undergone neofunctionalisation and contain 3-5 N-terminal transmembrane domains, which have so far only been identified in two anthozoan species. CONCLUSIONS: This research has significance in understanding the evolution and origin of the core eumetazoan gene set, including how novel innate immune genes evolve. For example, the evolution of transmembrane domain containing NLRs indicates that these NLRs may be membrane-bound, while all other metazoan and plant NLRs are exclusively cytosolic receptors. This is one example of how species without an adaptive immune system may evolve innovative solutions to detect pathogens or interact with native microbiota. Overall, these results provide an insight into the evolution of the innate immune system, and show that early divergent lineages, such as actiniarians, have a diverse repertoire of conserved and novel innate immune genes.

Defending against pathogens - immunological priming and its molecular basis in a sea anemone, cnidarian.

Cnidarians, in general, are long-lived organisms and hence may repeatedly encounter common pathogens during their lifespans. It remains unknown whether these early diverging animals possess some type of immunological reaction that strengthens the defense response upon repeated infections, such as that described in more evolutionary derived organisms. Here we show results that sea anemones that had previously encountered a pathogen under sub-lethal conditions had a higher survivorship during a subsequently lethal challenge than naïve anemones that encountered the pathogen for the first time. Anemones subjected to the lethal challenge two and four weeks after the sub-lethal exposure presented seven- and five-fold increases in survival, respectively, compared to the naïve anemones. However, anemones challenged six weeks after the sub-lethal exposure showed no increase in survivorship. We argue that this short-lasting priming of the defense response could be ecologically relevant if pathogen encounters are restricted to short seasons characterized by high stress. Furthermore, we discovered significant changes in proteomic profiles between naïve sea anemones and those primed after pathogen exposure suggesting a clear molecular signature associated with immunological priming in cnidarians. Our findings reveal that immunological priming may have evolved much earlier in the tree of life than previously thought.

Anaphylaxis.

The term anaphylaxis was coined by Charles Richet and Paul Portier when they tried to immunize dogs with actinia extracts, but after a repeated injection of a small amount of the toxin the dog died within 25 min. The new term rapidly spread all over the world. The discovery of the phenomenon of anaphylaxis showed that by immunization not only protection but also harmful events could be induced. For this discovery Richet received the Nobel Prize in 1913, but he still believed the condition of anaphylaxis was a lack of protection to the poisonous effect of the substance. Already earlier similar clinical phenomena had been observed but not well described. A major breakthrough in understanding the pathophysiology came through the experiments of Dale and Laidlaw who showed that the newly discovered histamine was able to induce quite similar symptoms to anaphylaxis. For decades reactions mimicking anaphylaxis but without involvement of the immune systems were called 'anaphylactoid', 'allergy-like' or 'pseudo-allergic'. Since the new definition of the World Allergy Organization (WAO) anaphylaxis is defined on the basis of clinical symptoms independent of pathomechanisms involved: one distinguishes between allergic and non-immune anaphylaxis. Epinephrine (Adrenalin) was soon recognized as treatment of choice of this dramatic condition.

Regulation of cnidarian-dinoflagellate mutualisms: Evidence that activation of a host TGFß innate immune pathway promotes tolerance of the symbiont.

Animals must manage interactions with beneficial as well as detrimental microbes. Immunity therefore includes strategies for both resistance to and tolerance of microbial invaders. Transforming growth factor beta (TGFß) cytokines have many functions in animals including a tolerance-promoting (tolerogenic) role in immunity in vertebrates. TGFß pathways are present in basal metazoans such as cnidarians but their potential role in immunity has never been explored. This study takes a two-part approach to examining an immune function for TGFß in cnidarians. First bioinformatic analyses of the model anemone Aiptasia pallida were used to identify TGFß pathway components and explore the hypothesis that an immune function for TGFßs existed prior to the evolution of vertebrates. A TGFß ligand from A. pallida was identified as one that groups closely with vertebrate TGFßs that have an immune function. Second, cellular analyses of A. pallida were used to examine a role for a TGFß pathway in the regulation of cnidarian-dinoflagellate mutualisms. These interactions are stable under ambient conditions but collapse under elevated temperature, a phenomenon called cnidarian bleaching. Addition of exogenous human TGFß suppressed an immune response measured as LPS-induced nitric oxide (NO) production by the host. Addition of anti-TGFß to block a putative TGFß pathway resulted in immune stimulation and a failure of the symbionts to successfully colonize the host. Finally, addition of exogenous TGFß suppressed immune stimulation in heat-stressed animals and partially abolished a bleaching response. These findings suggest that the dinoflagellate symbionts somehow promote host tolerance through activation of tolerogenic host immune pathways, a strategy employed by some intracellular protozoan parasites during their invasion of vertebrates. Insight into the ancient, conserved nature of host-microbe interactions gained from this cnidarian-dinoflagellate model is valuable to understanding the evolution of immunity and its role in the regulation of both beneficial and detrimental associations.

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.

Shared antigenicity between the polar filaments of myxosporeans and other Cnidaria.

Nematocysts containing coiled polar filaments are a distinguishing feature of members of the phylum Cnidaria. As a first step to characterizing the molecular structure of polar filaments, a polyclonal antiserum was raised in rabbits against a cyanogen bromide-resistant protein extract of mature cysts containing spores of Myxobolus pendula. The antiserum reacted only with proteins associated with extruded polar filaments. Western blot and whole-mount immunohistochemical analyses indicated a conservation of polar filament epitopes between M. pendula and 2 related cnidarians, i.e., the anthozoan, Nematostella vectensis, and the hydrozoan, Hydra vulgaris. This conservation of polar filament epitopes lends further support to a shared affinity between Myxozoa and cnidarians.

Evolution of thioester-containing proteins revealed by cloning and characterization of their genes from a cnidarian sea anemone, Haliplanella lineate.

To elucidate the evolutionary origin of genes encoding thioester-containing proteins (TEPs), TEP genes were isolated from a cnidarian, a sea anemone, Haliplanella lineate. Phylogenetic tree analysis of the four identified cnidarian TEP genes and various TEP genes of many metazoa, indicated that they could be classified into two subfamilies: the alpha-2-macroglobulin (A2M) subfamily encodining A2M, CD109 and insect TEPs, and the C3 subfamily encoding complement C3, C4 and C5. Two of the four cnidarian TEP genes belonged to the A2M subfamily, showing a close similarity to human A2M and CD109, respectively and thus were termed HaliA2M and HaliCD109. The other two genes belonged to the C3 subfamily, and were termed HaliC3-1 and HaliC3-2. Cnidarian TEPs retained the basic domain structure and functionally important residues for each molecule, and their mRNA were detected at different parts of the sea anemone body. These results suggest that gene duplication and subsequent functional differentiation among C3, A2M and CD109 were very ancient events predating the divergence of the cnidaria and bilateria.

Multi-component complement system of Cnidaria: C3, Bf, and MASP genes expressed in the endodermal tissues of a sea anemone, Nematostella vectensis.

The origin of the complement system, one of the major systems of mammalian innate immunity, is more ancient than that of the adaptive immune system, as shown by the identification of the gene for the complement component 3 (C3) in a basic metazoa, cnidarian coral. Only a few reports on the other complement genes of non-chordates have been published, and the composition of the ancient complement system has not been clarified. We performed comprehensive cloning of the complement genes with characteristic domain structures using a Cnidarian, the sea anemone, Nematostella vectensis. Partial sequences of the two C3, two factor B (Bf), and one mannan-binding protein-associated serine protease (MASP) genes were identified in the draft genome data, and the complete coding sequences of these genes were elucidated by RT-PCR and 5'- and 3'-RACE. In contrast, no C6 and factor I family genes were identified. These cnidarian components shared the unique domain structures and most of the functionally critical amino acid residues with their mammalian counterparts, suggesting the conservation of their basic biochemical functions throughout metazoan evolution. In situ hybridization analysis indicated that all five genes are expressed in the tentacles, pharynx, and mesentery in an endoderm-specific manner. These results suggest that the multi-component complement system comprising at least C3, Bf, and MASP was established in a common ancestor of Cnidaria and Bilateria more than 600 million years ago to protect the coelenteron, the primitive gut cavity with putative circulatory functions.

Anaphylactic shock caused by exposure to sea anemones.

BACKGROUND: Since the first report of a dog that developed severe systemic symptoms in response to a second injection of sea anemone toxin by Richet and Portier in 1902, no clear human cases of anaphylaxis related to exposure to sea anemones has been reported in the literature. METHODS: A 24-year-old man with an episode of local urticaria on his first contact with a sea anemone (Stichodactyla haddoni), developed dyspnea, severe urticaria and hypotension on exposure to water containing the dead bodies of the organism. To study whether this reaction was mediated by antigen-specific IgE, we performed a histamine release test with blood, Western blotting with serum and lymphocyte proliferating test with peripheral blood mononuclear cells of the patient, for the homogenate of sea anemones. RESULTS: The homogenate of sea anemones induced histamine release from the blood of the patient, but it also induced histamine release from the blood of control subjects. Moreover, it also caused hemolysis of blood of all donors. However, Western-blotting demonstrated the presence of an 86 kd protein-specific IgE in the serum of the patient. CONCLUSIONS: Protein antigen(s) in sea anemones may cause anaphylactic shock under the influence of the cytolytic effects and/or lymphocyte-stimulating activity elicited by the toxin of sea anemones.