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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., Competing Interests: Declaration of interests The authors declare no completing interests., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Considerable attention has recently been focused on the potential involvement of DNA methylation in regulating gene expression in cnidarians. Much of this work has been centered on corals, in the context of changes in methylation perhaps facilitating adaptation to higher seawater temperatures and other stressful conditions. Although first proposed more than 30 years ago, the possibility that DNA methylation systems function in protecting animal genomes against the harmful effects of transposon activity has largely been ignored since that time. Here, we show that transposons are specifically targeted by the DNA methylation system in cnidarians, and that the youngest transposons (i.e., those most likely to be active) are most highly methylated. Transposons in longer and highly active genes were preferentially methylated and, as transposons aged, methylation levels declined, reducing the potentially harmful side effects of CpG methylation. In Cnidaria and a range of other invertebrates, correlation between the overall extent of methylation and transposon content was strongly supported. Present transposon burden is the dominant factor in determining overall level of genomic methylation in a range of animals that diverged in or before the early Cambrian, suggesting that genome defense represents the ancestral role of CpG methylation., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Despite the ecological significance of the mutualistic relationship between Symbiodiniaceae and reef-building corals, the molecular interactions during establishment of this relationship are not well understood. This is particularly true of the transcriptional changes that occur in the symbiont. In the current study, a dual RNA-sequencing approach was used to better understand transcriptional changes on both sides of the coral-symbiont interaction during the colonization of Acropora tenuis by a compatible Symbiodiniaceae strain (Cladocopium goreaui; ITS2 type C1). Comparison of transcript levels of the in hospite symbiont 3, 12, 48 and 72 hr after exposure to those of the same strain in culture revealed that extensive and generalized down-regulation of symbiont gene expression occurred during the infection process. Included in this "symbiosis-derived transcriptional repression" were a range of stress response and immune-related genes. In contrast, a suite of symbiont genes implicated in metabolism was upregulated in the symbiotic state. The coral data support the hypothesis that immune-suppression and arrest of phagosome maturation play important roles during the establishment of compatible symbioses, and additionally imply the involvement of some SCRiP family members in the colonization process. Consistent with previous ecological studies, the transcriptomic data suggest that active translocation of metabolites to the host may begin early in the colonization process, and thus that the mutualistic relationship can be established at the larval stage. This dual RNA-sequencing study provides insights into the transcriptomic remodelling that occurs in C. goreaui during transition to a symbiotic lifestyle and the novel coral genes implicated in symbiosis., (© 2020 John Wiley & Sons Ltd.)

Reef-building corals live in a mutualistic relationship with photosynthetic algae (family Symbiodiniaceae) that usually provide most of the energy required by the coral host. This relationship is sensitive to temperature stress; as little as a 1°C increase often leads to the collapse of the association. This sensitivity has led to an interest in the potential of more stress-tolerant algae to supplement or substitute for the normal Symbiodiniaceae mutualists. In this respect, the apicomplexan-like microalga Chromera is of particular interest due to its greater temperature tolerance. We generated a de novo transcriptome for a Chromera strain isolated from a GBR coral ('GBR Chromera') and compared with those of the reference strain of Chromera ('Sydney Chromera'), and to those of Symbiodiniaceae (Fugacium kawagutii, Cladocopium goreaui and Breviolum minutum), as well as the apicomplexan parasite, Plasmodium falciparum. In contrast to the high sequence divergence amongst representatives of different genera within the family Symbiodiniaceae, the two Chromera strains featured low sequence divergence at orthologous genes, implying that they are likely to be conspecifics. Although KEGG categories provide few criteria by which true coral mutualists might be identified, they do supply a molecular rationalization that explains the ecological dominance of Cladocopium spp. amongst Indo-Pacific reef corals. The presence of HSP20 genes may contribute to the high thermal tolerance of Chromera., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Neuropeptides play critical roles in cnidarian development. However, although they are known to play key roles in settlement and metamorphosis, their temporal and spatial developmental expression has not previously been characterized in any coral. We here describe Acropora millepora LWamide and RFamide and their developmental expression from the time of their first appearance, using in situ hybridization and FMRFamide immunohistochemistry. AmRFamide transcripts first appear in the ectoderm toward the oral end of the planula larva following blastopore closure. This oral bias becomes less apparent as the planula develops. The cell bodies of AmRFamide-expressing cells are centrally located in the ectoderm, with narrow projections to the mesoglea and to the cell surface. As the planula approaches settlement, AmRFamide expression disappears and is undetectable in the newly settled polyp. Expressing cells then gradually reappear as the polyp develops, becoming particularly abundant on the tentacles. AmLWamide transcripts first appear in ectodermal cells of the developing planula, with minimal expression at its two ends. The cell bodies of expressing cells lie just above the mesoglea, in a position distinct from those of AmRFamide-expressing cells, and have a narrow projection extending across the ectoderm to its surface. AmLWamide-expressing cells persist for most of the planula stage, disappearing shortly before settlement, but later than AmRFamide-expressing cells. As is the case with AmRFamide, expressing cells are absent from the polyp immediately after settlement, reappearing later on its oral side. AmLWamide expression lags that of AmRFamide in both its disappearance and reappearance. Antibodies to FMRFamide stain cells in a pattern similar to that of the transcripts, but also cells in areas where there is no expression revealed by in situ hybridization, most notably at the aboral end of the planula and in the adult polyp. Adult polyps have numerous staining cells on the tentacles and oral discs, as well as an immunoreactive nerve ring around the mouth. There are scattered staining cells in the coenosarc between polyps and staining cells are abundant in the mesenterial filaments. The above results are discussed in the context of our knowledge of the behavior of coral planulae at the time of their settlement and metamorphosis. Corals are facing multiple environmental threats, and these results both highlight the need for, and bring us a step closer to, a mechanistic understanding of a process that is critical to their survival., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Despite the biological and economic significance of scleractinian reef-building corals, the lack of large molecular datasets for a representative range of species limits understanding of many aspects of their biology. Within the Scleractinia, based on molecular evidence, it is generally recognised that there are two major clades, Complexa and Robusta, but the genomic bases of significant differences between them remain unclear., Results: Draft genome assemblies and annotations were generated for three coral species: Galaxea fascicularis (Complexa), Fungia sp., and Goniastrea aspera (Robusta). Whilst phylogenetic analyses strongly support a deep split between Complexa and Robusta, synteny analyses reveal a high level of gene order conservation between all corals, but not between corals and sea anemones or between sea anemones. HOX-related gene clusters are, however, well preserved across all of these combinations. Differences between species are apparent in the distribution and numbers of protein domains and an apparent correlation between number of HSP20 proteins and stress tolerance. Uniquely amongst animals, a complete histidine biosynthesis pathway is present in robust corals but not in complex corals or sea anemones. This pathway appears to be ancestral, and its retention in the robust coral lineage has important implications for coral nutrition and symbiosis., Conclusions: The availability of three new coral genomes enabled recognition of a de novo histidine biosynthesis pathway in robust corals which is only the second identified biosynthetic difference between corals. These datasets provide a platform for understanding many aspects of coral biology, particularly the interactions of corals with their endosymbionts.

Since the discovery of Chromera velia as a novel coral-associated microalga, this organism has attracted interest because of its unique evolutionary position between the photosynthetic dinoflagellates and the parasitic apicomplexans. The nature of the relationship between Chromera and its coral host is controversial. Is it a mutualism, from which both participants benefit, a parasitic relationship, or a chance association? To better understand the interaction, larvae of the common Indo-Pacific reef-building coral Acropora digitifera were experimentally infected with Chromera, and the impact on the host transcriptome was assessed at 4, 12, and 48 h post-infection using Illumina RNA-Seq technology. The transcriptomic response of the coral to Chromera was complex and implies that host immunity is strongly suppressed, and both phagosome maturation and the apoptotic machinery is modified. These responses differ markedly from those described for infection with a competent strain of the coral mutualist Symbiodinium, instead resembling those of vertebrate hosts to parasites and/or pathogens such as Mycobacterium tuberculosis. Consistent with ecological studies suggesting that the association may be accidental, the transcriptional response of A. digitifera larvae leads us to conclude that Chromera could be a coral parasite, commensal, or accidental bystander, but certainly not a beneficial mutualist.

Corallimorpharians (coral-like anemones) have a close phylogenetic relationship with scleractinians (hard corals) and can potentially provide novel perspectives on the evolution of biomineralization within the anthozoan subclass Hexacorallia. A survey of the transcriptomes of three representative corallimorpharians led to the identification of homologs of some skeletal organic matrix proteins (SOMPs) previously considered to be restricted to corals.Carbonic anhydrases (CAs), which are ubiquitous proteins involved in CO2 trafficking, are involved in both coral calcification and photosynthesis by endosymbiotic Symbiodinium (zooxanthellae). These multiple roles are assumed to place increased demands on the CA repertoire and have presumably driven the elaboration of the complex CA repertoires typical of corals (note that "corals" are defined here as reef-building Scleractinia). Comparison of the CA inventories of corallimorpharians with those of corals reveals that corals have specifically expanded the secreted and membrane-associated type CAs, whereas similar complexity is observed in the two groups with respect to other CA types.Comparison of the CA complement of the nonsymbiotic corallimorph Corynactis australis with that of Ricordea yuma, a corallimorph which normally hosts Symbiodinium, reveals similar numbers and distribution of CA types and suggests that an expansion of the CA repertoire has been necessary to enable calcification but may not be a requirement to enable symbiosis. Consistent with this idea, preliminary analysis suggests that the CA complexity of zooxanthellate and nonzooxanthellate sea anemones is similar.The comparisons above suggest that although there are relatively few new genes in the skeletal organic matrix of corals (which controls the skeleton deposition process), the evolution of calcification required an expanded repertoire of secreted and membrane-associated CAs., (© The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: Research into various aspects of coral biology has greatly increased in recent years due to anthropogenic threats to coral health including pollution, ocean warming and acidification. However, knowledge of coral early development has lagged. The present paper describes the embryonic development of two previously uncharacterized robust corals, Favia lizardensis (a massive brain coral) and Ctenactis echinata (a solitary coral) and compares it to that of the previously characterized complex coral, Acropora millepora, both morphologically and in terms of the expression of a set of key developmental genes., Results: Illumina sequencing of mixed age embryos was carried out, resulting in embryonic transcriptomes consisting of 40605 contigs for C.echinata (N50 = 1080 bp) and 48536 contigs for F.lizardensis (N50 = 1496 bp). The transcriptomes have been annotated against Swiss-Prot and were sufficiently complete to enable the identification of orthologs of many key genes controlling development in bilaterians. Developmental series of images of whole mounts and sections reveal that the early stages of both species contain a blastocoel, consistent with their membership of the robust clade. In situ hybridization was used to examine the expression of the developmentally important genes brachyury, chordin and forkhead. The expression of brachyury and forkhead was consistent with that previously reported for Acropora and allowed us to confirm that the pseudo-blastopore sometimes seen in robust corals such as Favia spp. is not directly associated with gastrulation. C.echinata chordin expression, however, differed from that seen in the other two corals., Conclusions: Embryonic transcriptomes were assembled for the brain coral Favia lizardensis and the solitary coral Ctenactis echinata. Both species have a blastocoel in their early developmental stages, consistent with their phylogenetic position as members of the robust clade. Expression of the key developmental genes brachyury, chordin and forkhead was investigated, allowing comparison to that of their orthologs in Acropora, Nematostella and bilaterians and demonstrating that even within the Anthozoa there are significant differences in expression patterns.

Background: Apoptotic cell death is a defining and ubiquitous characteristic of metazoans, but its evolutionary origins are unclear. Although Caenorhabditis and Drosophila played key roles in establishing the molecular bases of apoptosis, it is now clear that cell death pathways of these animals do not reflect ancestral characteristics. Conversely, recent work suggests that the apoptotic networks of cnidarians may be complex and vertebrate-like, hence characterization of the apoptotic complement of representatives of the basal cnidarian class Anthozoa will help us to understand the evolution of the vertebrate apoptotic network., Results: We describe the Bcl-2 and caspase protein repertoires of the coral Acropora millepora, making use of the comprehensive transcriptomic data available for this species. Molecular phylogenetics indicates that some Acropora proteins are orthologs of specific mammalian pro-apoptotic Bcl-2 family members, but the relationships of other Bcl-2 and caspases are unclear. The pro- or anti-apoptotic activities of coral Bcl-2 proteins were investigated by expression in mammalian cells, and the results imply functional conservation of the effector/anti-apoptotic machinery despite limited sequence conservation in the anti-apoptotic Bcl-2 proteins. A novel caspase type ("Caspase-X"), containing both inactive and active caspase domains, was identified in Acropora and appears to be restricted to corals. When expressed in mammalian cells, full-length caspase-X caused loss of viability, and a truncated version containing only the active domain was more effective in inducing cell death, suggesting that the inactive domain might modulate activity in the full-length protein. Structure prediction suggests that the active and inactive caspase domains in caspase-X are likely to interact, resulting in a structure resembling that of the active domain in procaspase-8 and the inactive caspase domain in the mammalian c-FLIP anti-apoptotic factor., Conclusions: The data presented here confirm that many of the basic mechanisms involved in both the intrinsic and extrinsic apoptotic pathways were in place in the common ancestor of cnidarians and bilaterians. With the identification of most or all of the repertoires of coral Bcl-2 and caspases, our results not only provide new perspectives on the evolution of apoptotic pathways, but also a framework for future experimental studies towards a complete understanding of coral bleaching mechanisms, in which apoptotic cell death might be involved.

Organizer activity, once thought to be restricted to vertebrates, has ancient origins. However, among non-bilaterians, it has only been subjected to detailed investigation during embryonic development of the sea anemone, Nematostella vectensis. As a step toward establishing the extent to which findings in Nematostella can be generalized across the large and diverse phylum Cnidaria, we examined the expression of some key organizer and gastrulation genes during the embryonic development of the coral Acropora millepora. Although anemones and corals both belong to the cnidarian class Anthozoa, the two lineages diverged during the Cambrian and the morphological development of Acropora differs in several important respects from that of Nematostella. While the expression patterns of the key genes brachyury, bmp2/4, chordin, goosecoid and forkhead are broadly similar, developmental differences between the two species enable novel observations, and new interpretations of their significance. Specifically, brachyury expression during the flattened prawnchip stage before gastrulation, a developmental peculiarity of Acropora, leads us to suggest that it is the key gene demarcating ectoderm from endoderm in Acropora, and by implication in other cnidarians, whereas previous studies in Nematostella proposed that forkhead plays this role. Other novel observations include the transient expression of Acropora forkhead in scattered ectodermal cells shortly after gastrulation, and in the developing mesenterial filaments, with no corresponding expression reported in Nematostella. In addition, the expression patterns of goosecoid and bmp2/4 confirm the fundamental bilaterality of the Anthozoa., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A comprehensive understanding of coral reproduction and development is needed because corals are threatened in many ways by human activity. Major threats include the loss of their photosynthetic symbionts (Symbiodinium) caused by rising temperatures (bleaching), reduced ability to calcify caused by ocean acidification, increased storm severity associated with global climate change and an increase in predators caused by runoff from human agricultural activity. In spite of these threats, detailed descriptions of embryonic development are not available for many coral species. The current consensus is that there are two major groups of stony corals, the "complex" and the "robust". In this paper we describe the embryonic development of four "complex" species, Pseudosiderastrea tayamai, Galaxea fascicularis, Montipora hispida, and Pavona Decussata, and seven "robust" species, Oulastrea crispata, Platygyra contorta, Favites abdita, Echinophyllia aspera, Goniastrea favulus, Dipsastraea speciosa (previously Favia speciosa), and Phymastrea valenciennesi (previously Montastrea valenciennesi). Data from both histologically sectioned embryos and whole mounts are presented. One apparent difference between these two major groups is that before gastrulation the cells of the complex corals thus far described (mainly Acropora species) spread and flatten to produce the so-called prawn chip, which lacks a blastocoel. Our present broad survey of robust and complex corals reveals that prawn chip formation is not a synapomorphy of complex corals, as Pavona Decussata does not form a prawn chip and has a well-developed blastocoel. Although prawn chip formation cannot be used to separate the two clades, none of the robust corals which we surveyed has such a stage. Many robust coral embryos pass through two periods of invagination, separated by a return to a spherical shape. However, only the second of these periods is associated with endoderm formation. We have therefore termed the first invagination a pseudo-blastopore.

Background: As a step towards understanding coral immunity we present the first whole transcriptome analysis of the acute responses of Acropora millepora to challenge with the bacterial cell wall derivative MDP and the viral mimic poly I:C, defined immunogens provoking distinct but well characterised responses in higher animals., Results: These experiments reveal similarities with the responses both of arthropods and mammals, as well as coral-specific effects. The most surprising finding was that MDP specifically induced three members of the GiMAP gene family, which has been implicated in immunity in mammals but is absent from Drosophila and Caenorhabditis. Like their mammalian homologs, GiMAP genes are arranged in a tandem cluster in the coral genome., Conclusions: A phylogenomic survey of this gene family implies ancient origins, multiple independent losses and lineage-specific expansions during animal evolution. Whilst functional convergence cannot be ruled out, GiMAP expression in corals may reflect an ancestral role in immunity, perhaps in phagolysosomal processing.

Secreted peptides, produced by enzymatic processing of larger precursor molecules, are found throughout the animal kingdom and play important regulatory roles as neurotransmitters and hormones. Many require a carboxy-terminal modification, involving the conversion of a glycine residue into an α-amide, for their biological activity. Two sequential enzymatic activities catalyze this conversion: a monooxygenase (peptidylglycine α-hydroxylating monooxygenase or PHM) and an amidating lyase (peptidyl-α-hydroxyglycine α-amidating lyase or PAL). In vertebrates, these activities reside in a single polypeptide known as peptidylglycine α-amidating monooxygenase (PAM), which has been extensively studied in the context of neuropeptide modification. Bifunctional PAMs have been reported from some invertebrates, but the phylogenetic distribution of PAMs and their evolutionary relationship to PALs and PHMs is unclear. Here, we report sequence and expression data for two PAMs from the coral Acropora millepora (Anthozoa, Cnidaria), as well as providing a comprehensive survey of the available sequence data from other organisms. These analyses indicate that bifunctional PAMs predate the origins of the nervous and endocrine systems, consistent with the idea that within the Metazoa their ancestral function may have been to amidate epitheliopeptides. More surprisingly, the phylogenomic survey also revealed the presence of PAMs in green algae (but not in higher plants or fungi), implying that the bifunctional enzyme either predates the plant/animal divergence and has subsequently been lost in a number of lineages or perhaps that convergent evolution or lateral gene transfer has occurred. This finding is consistent with recent discoveries that other molecules once thought of as "neural" predate nervous systems.

Members of the universal stress protein (USP) family were originally identified in stressed bacteria on the basis of a shared domain, which has since been reported in a phylogenetically diverse range of prokaryotes, fungi, protists, and plants. Although not previously characterized in metazoans, here we report that USP genes are distributed in animal genomes in a unique pattern that reflects frequent independent losses and independent expansions. Multiple USP loci are present in urochordates as well as all Cnidaria and Lophotrochozoa examined, but none were detected in any of the available ecdysozoan or non-urochordate deuterostome genome data. The vast majority of the metazoan USPs are short, single-domain proteins and are phylogenetically distinct from the prokaryotic, plant, protist, and fungal members of the protein family. Whereas most of the metazoan USP genes contain introns, with few exceptions those in the cnidarian Hydra are intronless and cluster together in phylogenetic analyses. Expression patterns were determined for several cnidarian USPs, including two genes belonging to the intronless clade, and these imply diverse functions. The apparent paradox of implied diversity of roles despite high overall levels of sequence (and implied structural) similarity parallels the situation in bacteria. The absence of USP genes in ecdysozoans and most deuterostomes may be a consequence of functional redundancy or specialization in taxon-specific roles.

Background: A successful metamorphosis from a planktonic larva to a settled polyp, which under favorable conditions will establish a future colony, is critical for the survival of corals. However, in contrast to the situation in other animals, e.g., frogs and insects, little is known about the molecular basis of coral metamorphosis. We have begun to redress this situation with previous microarray studies, but there is still a great deal to learn. In the present paper we have utilized a different technology, subtractive hybridization, to characterize genes differentially expressed across this developmental transition and to compare the success of this method to microarray., Methodology/principal Findings: Suppressive subtractive hybridization (SSH) was used to identify two pools of transcripts from the coral, Acropora millepora. One is enriched for transcripts expressed at higher levels at the pre-settlement stage, and the other for transcripts expressed at higher levels at the post-settlement stage. Virtual northern blots were used to demonstrate the efficacy of the subtractive hybridization technique. Both pools contain transcripts coding for proteins in various functional classes but transcriptional regulatory proteins were represented more frequently in the post-settlement pool. Approximately 18% of the transcripts showed no significant similarity to any other sequence on the public databases. Transcripts of particular interest were further characterized by in situ hybridization, which showed that many are regulated spatially as well as temporally. Notably, many transcripts exhibit axially restricted expression patterns that correlate with the pool from which they were isolated. Several transcripts are expressed in patterns consistent with a role in calcification., Conclusions: We have characterized over 200 transcripts that are differentially expressed between the planula larva and post-settlement polyp of the coral, Acropora millepora. Sequence, putative function, and in some cases temporal and spatial expression are reported.

Coral bleaching is a major threat to coral reefs worldwide and is predicted to intensify with increasing global temperature. This study represents the first investigation of gene expression in an Indo-Pacific coral species undergoing natural bleaching which involved the loss of algal symbionts. Quantitative real-time polymerase chain reaction experiments were conducted to select and evaluate coral internal control genes (ICGs), and to investigate selected coral genes of interest (GOIs) for changes in gene expression in nine colonies of the scleractinian coral Acropora millepora undergoing bleaching at Magnetic Island, Great Barrier Reef, Australia. Among the six ICGs tested, glyceraldehyde 3-phosphate dehydrogenase and the ribosomal protein genes S7 and L9 exhibited the most constant expression levels between samples from healthy-looking colonies and samples from the same colonies when severely bleached a year later. These ICGs were therefore utilised for normalisation of expression data for seven selected GOIs. Of the seven GOIs, homologues of catalase, C-type lectin and chromoprotein genes were significantly up-regulated as a result of bleaching by factors of 1.81, 1.46 and 1.61 (linear mixed models analysis of variance, P < 0.05), respectively. We present these genes as potential coral bleaching response genes. In contrast, three genes, including one putative ICG, showed highly variable levels of expression between coral colonies. Potential variation in microhabitat, gene function unrelated to the stress response and individualised stress responses may influence such differences between colonies and need to be better understood when designing and interpreting future studies of gene expression in natural coral populations.

Recent thought on genome evolution has focused on the creation of new genes and changes in regulatory mechanisms while ignoring the role of selective gene loss in shaping genomes. Using data from two cnidarians, the jellyfish Clytia and the coral Acropora, we examined the relative significance of new 'taxonomically restricted' genes and selectively retained ancestral genes in enabling the evolution of novel traits. Consistent with its more complex life-cycle, the proportion of novel genes identified in Clytia was higher than that in the 'polyp only' cnidarians Nematostella and Hydra, but each of these cnidarians has retained a proportion of ancestral genes not present in the other two. The ubiquity and near-stochastic nature of gene loss can explain the discord between patterns of gene distribution and taxonomy., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Background: The coral skeleton consists of CaCO3 deposited upon an organic matrix primarily as aragonite. Currently galaxin, from Galaxea fascicularis, is the only soluble protein component of the organic matrix that has been characterized from a coral. Three genes related to galaxin were identified in the coral Acropora millepora., Results: One of the Acropora genes (Amgalaxin) encodes a clear galaxin ortholog, while the others (Amgalaxin-like 1 and Amgalaxin-like 2) encode larger and more divergent proteins. All three proteins are predicted to be extracellular and share common structural features, most notably the presence of repetitive motifs containing dicysteine residues. In situ hybridization reveals distinct, but partially overlapping, spatial expression of the genes in patterns consistent with distinct roles in calcification. Both of the Amgalaxin-like genes are expressed exclusively in the early stages of calcification, while Amgalaxin continues to be expressed in the adult, consistent with the situation in the coral Galaxea., Conclusion: Comparisons with molluscs suggest functional convergence in the two groups; lustrin A/pearlin proteins may be the mollusc counterparts of galaxin, whereas the galaxin-like proteins combine characteristics of two distinct proteins involved in mollusc calcification. Database searches indicate that, although sequences with high similarity to the galaxins are restricted to the Scleractinia, more divergent members of this protein family are present in other cnidarians and some other metazoans. We suggest that ancestral galaxins may have been secondarily recruited to roles in calcification in the Triassic, when the Scleractinia first appeared. Understanding the evolution of the broader galaxin family will require wider sampling and expression analysis in a range of cnidarians and other animals.

Expressed sequence tag analyses of the annelid Pomatoceros lamarckii, recently published in BMC Evolutionary Biology, are consistent with less extensive gene loss in the Lophotrochozoa than in the Ecdysozoa, but it would be premature to generalize about patterns of gene loss on the basis of the limited data available.

Background: Anthozoan cnidarians are amongst the simplest animals at the tissue level of organization, but are surprisingly complex and vertebrate-like in terms of gene repertoire. As major components of tropical reef ecosystems, the stony corals are anthozoans of particular ecological significance. To better understand the molecular bases of both cnidarian development in general and coral-specific processes such as skeletogenesis and symbiont acquisition, microarray analysis was carried out through the period of early development - when skeletogenesis is initiated, and symbionts are first acquired., Results: Of 5081 unique peptide coding genes, 1084 were differentially expressed (P

Background: Sox genes encode transcription factors that function in a wide range of developmental processes across the animal kingdom. To better understand both the evolution of the Sox family and the roles of these genes in cnidarians, we are studying the Sox gene complement of the coral, Acropora millepora (Class Anthozoa)., Results: Based on overall domain structures and HMG box sequences, the Acropora Sox genes considered here clearly fall into four of the five major Sox classes. AmSoxC is expressed in the ectoderm during development, in cells whose morphology is consistent with their assignment as sensory neurons. The expression pattern of the Nematostella ortholog of this gene is broadly similar to that of AmSoxC, but there are subtle differences--for example, expression begins significantly earlier in Acropora than in Nematostella. During gastrulation, AmSoxBb and AmSoxB1 transcripts are detected only in the presumptive ectoderm while AmSoxE1 transcription is restricted to the presumptive endoderm, suggesting that these Sox genes might play roles in germ layer specification. A third type B Sox gene, AmSoxBa, and a Sox F gene AmSoxF also have complex and specific expression patterns during early development. Each of these genes has a clear Nematostella ortholog, but in several cases the expression pattern observed in Acropora differs significantly from that reported in Nematostella., Conclusion: These differences in expression patterns between Acropora and Nematostella largely reflect fundamental differences in developmental processes, underscoring the diversity of mechanisms within the anthozoan Sub-Class Hexacorallia (Zoantharia).

The genome sequence of Trichoplax adhaerens, the founding member of the enigmatic animal phylum Placozoa, has revealed that a surprising level of genetic complexity underlies its extremely simple body plan, indicating either that placozoans are secondarily simple or that there is an undiscovered morphologically complex life stage.

Background: Adhesion mediated through the integrin family of cell surface receptors is central to early development throughout the Metazoa, playing key roles in cell-extra cellular matrix adhesion and modulation of cadherin activity during the convergence and extension movements of gastrulation. It has been suggested that Caenorhabditis elegans, which has a single beta and two alpha integrins, might reflect the ancestral integrin complement. Investigation of the integrin repertoire of anthozoan cnidarians such as the coral Acropora millepora is required to test this hypothesis and may provide insights into the original roles of these molecules., Results: Two novel integrins were identified in Acropora. AmItgalpha1 shows features characteristic of alpha integrins lacking an I-domain, but phylogenetic analysis gives no clear indication of its likely binding specificity. AmItgbeta2 lacks consensus cysteine residues at positions 8 and 9, but is otherwise a typical beta integrin. In situ hybridization revealed that AmItgalpha1, AmItgbeta1, and AmItgbeta2 are expressed in the presumptive endoderm during gastrulation. A second anthozoan, the sea anemone Nematostella vectensis, has at least four beta integrins, two resembling AmItgbeta1 and two like AmItgbeta2, and at least three alpha integrins, based on its genomic sequence., Conclusion: In two respects, the cnidarian data do not fit expectations. First, the cnidarian integrin repertoire is more complex than predicted: at least two betas in Acropora, and at least three alphas and four betas in Nematostella. Second, whereas the bilaterian alphas resolve into well-supported groups corresponding to those specific for RGD-containing or laminin-type ligands, the known cnidarian alphas are distinct from these. During early development in Acropora, the expression patterns of the three known integrins parallel those of amphibian and echinoderm integrins.

The full genomic sequence of the sea anemone Nematostella vectensis, which is the first full genomic sequence for a representative of the Phylum Cnidaria, has recently been published, providing some surprising findings and a unique perspective on the evolution of animal genomes. Major conclusions are that, in gene number, composition and intron/exon structure, the anemone is more similar to vertebrates than are flies and nematodes and that this shared complexity must therefore be very ancient.

The past two years have seen a dramatic increase in the available data on gene sequence and gene expression for cnidarians and other "lower" Metazoa, and a flurry of recent papers has drawn on these to address the origins of bilaterality. Cnidarian homologs of many genes that play key roles in the specification of both the A/P and D/V axes of bilaterians have been characterized, and their patterns of expression determined. Some of these expression patterns are consistent with the possibility of conservation of function between Cnidaria and Bilateria, but others clearly differ. Moreover, in some cases very different interpretations have been made on the basis of the same, or similar, data. In part, these differences reflect the inevitable uncertainties associated with the depth of the divergence between cnidarians and bilaterians. In this article, we briefly summarize the cnidarian data on gene expression and organization relevant to axis formation, the varying interpretations of these data, and where they conflict. Our conclusion is that the presently available data do not allow us to unequivocally homologize the single overt axis of cnidarians with either of the bilaterian axes.

A measure of the functional importance of G protein-coupled receptors (GPCRs) as signalling molecules is that over seven hundred have been cloned and identified in the human genome alone. Yet few have been characterized in the lower metazoan phyla, especially in the phylum Cnidaria which is well positioned phylogenetically for tracing the early evolution of GPCRs owing to their possession of the first-evolved nervous systems. We report here the cloning and characterization of four novel rhodopsin-like GPCR cDNAs from the staghorn coral Acropora millepora that share significant similarity with each other but not with the majority of other members of the rhodopsin alpha subfamily. The deduced proteins lack many of the conserved residues and motifs that form the signature of the different groups of alpha rhodopsin receptors. Maximum likelihood phylogenetic analysis likewise implies that the coral receptors do not have a simple or close relationship with any of the major groups within the alpha rhodopsin subfamily. In situ hybridization revealed transcripts in endodermal cells of planula larvae of all ages and in post-settlement polyps. These GPCRs appear to belong to a alpha rhodopsin-like group unique to corals. Comparisons with other cnidarian GPCRs suggest also that GPCRs diverged early in metazoan evolution.

Background: Characterization of the innate immune repertoire of extant cnidarians is of both fundamental and applied interest--it not only provides insights into the basic immunological 'tool kit' of the common ancestor of all animals, but is also likely to be important in understanding the global decline of coral reefs that is presently occurring. Recently, whole genome sequences became available for two cnidarians, Hydra magnipapillata and Nematostella vectensis, and large expressed sequence tag (EST) datasets are available for these and for the coral Acropora millepora., Results: To better understand the basis of innate immunity in cnidarians, we scanned the available EST and genomic resources for some of the key components of the vertebrate innate immune repertoire, focusing on the Toll/Toll-like receptor (TLR) and complement pathways. A canonical Toll/TLR pathway is present in representatives of the basal cnidarian class Anthozoa, but neither a classic Toll/TLR receptor nor a conventional nuclear factor (NF)-kappaB could be identified in the anthozoan Hydra. Moreover, the detection of complement C3 and several membrane attack complex/perforin domain (MAC/PF) proteins suggests that a prototypic complement effector pathway may exist in anthozoans, but not in hydrozoans. Together with data for several other gene families, this implies that Hydra may have undergone substantial secondary gene loss during evolution. Such losses are not confined to Hydra, however, and at least one MAC/PF gene appears to have been lost from Nematostella., Conclusion: Consideration of these patterns of gene distribution underscores the likely significance of gene loss during animal evolution whilst indicating ancient origins for many components of the vertebrate innate immune system.

Cnidarians are animals with a single (oral/aboral) overt body axis and with origins that nominally predate bilaterality. To better understand the evolution of axial patterning mechanisms, we characterized genes from the coral, Acropora millepora (Class Anthozoa) that are considered to be unambiguous markers of the bilaterian anterior/posterior and dorsal/ventral axes. Homologs of Otx/otd and Emx/ems, definitive anterior markers across the Bilateria, are expressed at opposite ends of the Acropora larva; otxA-Am initially around the blastopore and later preferentially toward the oral end in the ectoderm, and emx-Am predominantly in putative neurons in the aboral half of the planula larva, in a domain overlapping that of cnox-2Am, a Gsh/ind gene. The Acropora homologs of Pax-3/7, NKX2.1/vnd and Msx/msh are expressed in axially restricted and largely non-overlapping patterns in larval ectoderm. In Acropora, components of both the D/V and A/P patterning systems of bilateral animals are therefore expressed in regionally restricted patterns along the single overt body axis of the planula larva, and two 'anterior' markers are expressed at opposite ends of the axis. Thus, although some specific gene functions appear to be conserved between cnidarians and higher animals, no simple relationship exists between axial patterning systems in the two groups.

The phylogenetic conundrum posed by the Chaetognatha, a cryptic phylum consisting largely of planktonic predators, is the subject of two short papers in this issue of Current Biology. These analyses go some way towards defining the phylogenetic position of the chaetognaths, which possess features apparently spanning the protostome/deuterostome divide.

Cnidarians are among the simplest extant animals; however EST analyses reveal that they have a remarkably high level of genetic complexity. In this article, we show that the full diversity of metazoan signaling pathways is represented in this phylum, as are antagonists previously known only in chordates. Many of the cnidarian ESTs match genes previously known only in non-animal kingdoms. At least some of these represent ancient genes lost by all bilaterians examined so far, rather than genes gained by recent lateral gene transfer.

Eleven of the twelve recognized wingless (Wnt) subfamilies are represented in the sea anemone Nematostella vectensis, indicating that this developmentally important gene family was already fully diversified in the common ancestor of 'higher' animals. In deuterostomes, although duplications have occurred, no novel subfamilies of Wnts have evolved. By contrast, the protostomes Drosophila and Caenorhabditis have lost half of the ancestral Wnts. This pattern -- loss of genes from an ancestrally complex state -- might be more important in animal evolution than previously recognized.

A number of examples of independently duplicated regulatory genes have been identified in cnidarians, but the extent of this phenomenon and organization of these duplicated genes are unknown. Here we describe the identification of three pairs of independently duplicated homeobox genes in the anthozoan cnidarian, Acropora millepora. In each case, the pairs of paralogous genes are tightly linked, but the extent of sequence divergence implies that these do not reflect recent duplication events. The phenomenon is likely to be more general, as the examples reported here represent most of the limited number of Acropora homeobox genes for which genomic data are yet available.

A recent report of high levels of genetic variation between strains of Trichoplax adhaerens challenges the traditional view that the phylum Placozoa comprises only one species. At the morphological level, placozoans are amongst the simplest extant animals, but molecular evidence suggests that they may have more complex origins.

Although corals are nominally diploblastic, the early development of Acropora millepora involves a process that clearly resembles gastrulation in higher metazoans. This similarity at the morphological level led us to search for the Acropora equivalents of genes whose key roles in gastrulation are conserved across the higher Metazoa. We here report the characterisation of one such gene, snail, which in both Drosophila and the mouse is expressed in cells undergoing an epithelial-mesenchyme transition and/or morphogenetic movements. In addition to an N-terminal SNAG domain, the Acropora snail protein contains four zinc fingers with sequences diagnostic for members of the snail protein subfamily. In situ hybridisation reveals expression in epithelial tissue in the central portion of one side of the flattened pre-gastrulation embryo, which continues to express snail as it is engulfed by its opposite layer. Comparison to snail expression during gastrulation in bilaterians such as Drosophila reveals striking similarities and suggests mechanistic, and possibly evolutionary, links between the processes of mesoderm formation in bilaterians and endoderm formation in the Cnidaria.

The ecdysone receptor(1), which is a heterodimer of EcR and the retinoic acid receptor (RXR) homolog, Ultraspiracle (USP), has been well studied in the evolutionarily advanced and derived insects, the flies and moths. It is less well characterized in more primitive insect orders such as the Orthoptera, which include the grasshoppers and locusts. Following our previous isolation from Locusta migratoria (Lm) of a shorter RXR isoform (now called LmRXR-S), the isolation of a second, longer isoform (LmRXR-L) that appears to have characteristics of a ligand-modulated nuclear receptor is reported here. Transcripts for both isoforms, as well as LmEcR, were detected in embryos and in females during oocyte maturation. After expression in E. coli, both LmRXR-S and LmRXR-L form heterodimers with recombinant LmEcR in vitro which bind the active ecdysteroid, ponasterone A. Binding was only weakly competed for by ecdysone agonists that are known to be toxic to more advanced insects, suggesting functionally significant divergence in EcR ligand binding domains. In contrast, the DNA binding domain of LmEcR is less divergent and a protein complex, presumably LmEcR/LmRXR, that bound the ecdysone response element, IR-1, was detected in locust nuclear extracts. Because of reports of juvenile hormone (JH III) binding to Drosophila USP and the observed in silico RXR-like ligand-binding site in LmRXR-L, the recombinant proteins were also tested for binding to JH III. Neither LmRXR isoform, alone or in combination with LmEcR, bound JH III at nanomolar concentrations.

The identification and functional studies of DM domain-containing proteins Doublesex, MAB-3, and DMRT1 indicated that flies, nematodes, and humans share at least some of the molecular mechanisms of sex determination. We identified a gene, AmDM1, from the coral Acropora millepora that encodes a homologous DM domain-containing protein. Molecular analyses show that the AmDM1 primary transcript is processed to generate four different messenger RNAs. Alternative use of two polyadenylation sites produces transcripts that vary only in the 3' untranslated regions, whereas alternative splicing generates transcripts with and without the region coding for the DM domain. All the transcripts include a second motif, the DMA domain, which is found in a number of other proteins containing a DM domain. Hermaphroditic A. millepora differentiates sexual cells seasonally before the spring spawn, and Northern blot analysis shows that the AmDM1 transcripts are present at higher levels during sexual differentiation. The non-DM domain-containing messages are also present at significant levels in late embryos, but DM domain transcripts are extremely rare at this stage. These data suggest that the association of DM domain proteins and sexual determination or differentiation predates the separation of the Cnidaria from the rest of the Metazoa.

As the closest outgroup to the Bilateria, the Phylum Cnidaria is likely to be critical to understanding the origins and evolution of body axes. Proteins of the decapentaplegic (DPP)/bone morphogenetic protein (BMP) 2/4 subfamily are central to the specification of the dorsoventral (D/V) axis in bilateral animals, albeit with an axis inversion between arthropods and chordates. We show that a dpp/BMP2/4 ortholog (bmp2/4-Am) is present in the reef-building scleractinian coral, Acropora millepora (Class Anthozoa) and that it is capable of causing phenotypic effects in Drosophila that mimic those of the endogenous dpp gene. We also show that, during coral embryonic development, bmp2/4-Am expression is localized in an ectodermal region adjacent to the blastopore. Thus, a representative of the DPP/BMP2/4 subfamily of ligands was present in the common ancestor of diploblastic and triploblastic animals where it was probably expressed in a localized fashion during development. A localized source of DPP/BMP2/4 may have already been used in axis formation in this ancestor, or it may have provided a means by which an axis could evolve in triploblastic animals.

The phylum Cnidaria is the closest outgroup to the triploblastic metazoans and as such offers unique insights into evolutionary questions at several levels. In the post-genomic era, a knowledge of the gene complement of representative cnidarians will be important for understanding the relationship between the expansion of gene families and the evolution of morphological complexity among more highly evolved metazoans. Studies of cnidarian development and its molecular control will provide information about the origins of the major bilaterian body axes, the origin of the third tissue layer, the mesoderm, and the evolution of nervous system patterning. We are studying the cnidarian Acropora millepora, a reef building scleractinian coral, and a member of the basal cnidarian class, the Anthozoa. We review ourwork on descriptive embryology and studies of selected transcription factor gene families, where our knowledge from Acropora is particularly advanced relative to other cnidarians. We also describe a recent preliminary whole genome initiative, a coral EST database.