The dynamic genome of Hydra.

The freshwater cnidarian Hydra was first described in 1702 and has been the object of study for 300 years. Experimental studies of Hydra between 1736 and 1744 culminated in the discovery of asexual reproduction of an animal by budding, the first description of regeneration in an animal, and successful transplantation of tissue between animals. Today, Hydra is an important model for studies of axial patterning, stem cell biology and regeneration. Here we report the genome of Hydra magnipapillata and compare it to the genomes of the anthozoan Nematostella vectensis and other animals. The Hydra genome has been shaped by bursts of transposable element expansion, horizontal gene transfer, trans-splicing, and simplification of gene structure and gene content that parallel simplification of the Hydra life cycle. We also report the sequence of the genome of a novel bacterium stably associated with H. magnipapillata. Comparisons of the Hydra genome to the genomes of other animals shed light on the evolution of epithelia, contractile tissues, developmentally regulated transcription factors, the Spemann-Mangold organizer, pluripotency genes and the neuromuscular junction.

How do environmental factors influence life cycles and development? An experimental framework for early-diverging metazoans.

Ecological developmental biology (eco-devo) explores the mechanistic relationships between the processes of individual development and environmental factors. Recent studies imply that some of these relationships have deep evolutionary origins, and may even pre-date the divergences of the simplest extant animals, including cnidarians and sponges. Development of these early diverging metazoans is often sensitive to environmental factors, and these interactions occur in the context of conserved signaling pathways and mechanisms of tissue homeostasis whose detailed molecular logic remain elusive. Efficient methods for transgenesis in cnidarians together with the ease of experimental manipulation in cnidarians and sponges make them ideal models for understanding causal relationships between environmental factors and developmental mechanisms. Here, we identify major questions at the interface between animal evolution and development and outline a road map for research aimed at identifying the mechanisms that link environmental factors to developmental mechanisms in early diverging metazoans. Also watch the Video Abstract.

Shaping the microenvironment: evidence for the influence of a host galaxin on symbiont acquisition and maintenance in the squid-Vibrio symbiosis.

Most bacterial species make transitions between habitats, such as switching from free living to symbiotic niches. We provide evidence that a galaxin protein, EsGal1, of the squid Euprymna scolopes participates in both: (i) selection of the specific partner Vibrio fischeri from the bacterioplankton during symbiosis onset and, (ii) modulation of V. fischeri growth in symbiotic maintenance. We identified two galaxins in transcriptomic databases and showed by quantitative reverse-transcriptase polymerase chain reaction that one (esgal1) was dominant in the light organ. Further, esgal1 expression was upregulated by symbiosis, a response that was partially achieved with exposure to symbiont cell-envelope molecules. Confocal immunocytochemistry of juvenile animals localized EsGal1 to the apical surfaces of light-organ epithelia and surrounding mucus, the environment in which V. fischeri cells aggregate before migration into the organ. Growth assays revealed that one repeat of EsGal1 arrested growth of Gram-positive bacterial cells, which represent the cell type first 'winnowed' during initial selection of the symbiont. The EsGal1-derived peptide also significantly decreased the growth rate of V. fischeri in culture. Further, when animals were exposed to an anti-EsGal1 antibody, symbiont population growth was significantly increased. These data provide a window into how hosts select symbionts from a rich environment and govern their growth in symbiosis.

The dual nature of haemocyanin in the establishment and persistence of the squid-vibrio symbiosis.

We identified and sequenced from the squid Euprymna scolopes two isoforms of haemocyanin that share the common structural/physiological characteristics of haemocyanin from a closely related cephalopod, Sepia officinalis, including a pronounced Bohr effect. We examined the potential roles for haemocyanin in the animal's symbiosis with the luminous bacterium Vibrio fischeri. Our data demonstrate that, as in other cephalopods, the haemocyanin is primarily synthesized in the gills. It transits through the general circulation into other tissues and is exported into crypt spaces that support the bacterial partner, which requires oxygen for its bioluminescence. We showed that the gradient of pH between the circulating haemolymph and the matrix of the crypt spaces in adult squid favours offloading of oxygen from the haemocyanin to the symbionts. Haemocyanin is also localized to the apical surfaces and associated mucus of a juvenile-specific epithelium on which the symbionts gather, and where their specificity is determined during the recruitment into the association. The haemocyanin has an antimicrobial activity, which may be involved in this enrichment of V. fischeri during symbiont initiation. Taken together, these data provide evidence that the haemocyanin plays a role in shaping two stages of the squid-vibrio partnership.

How Hydra senses and destroys microbes.

Molecular genetic evidence has revealed that the basic templates of innate immune sensors were laid down in ancient animals such as the cnidarian Hydra. Important functions of Hydra's innate immune sensors and effectors include not only protection against pathogens but also controlling tissue-microbiota homeostasis. The deep evolutionary connections imply that invertebrate and mammalian immune pathways have evolved from a reduced number of common ancestral building blocks to their present configurations.

In an early branching metazoan, bacterial colonization of the embryo is controlled by maternal antimicrobial peptides.

Early embryos of many organisms develop outside the mother and are immediately confronted with myriads of potential colonizers. How these naive developmental stages control and shape the bacterial colonization is largely unknown. Here we show that early embryonic stages of the basal metazoan Hydra are able to control bacterial colonization by using maternal antimicrobial peptides. Antimicrobial peptides of the periculin family selecting for a specific bacterial colonization during embryogenesis are produced in the oocyte and in early embryos. If overexpressed in hydra ectodermal epithelial cells, periculin1a drastically reduces the bacterial load, indicating potent antimicrobial activity. Unexpectedly, transgenic polyps also revealed that periculin, in addition to bactericidal activity, changes the structure of the bacterial community. These findings delineate a role for antimicrobial peptides both in selecting particular bacterial partners during development and as important components of a "be prepared" strategy providing transgenerational protection.

Phylogenomics reveals an anomalous distribution of USP genes in metazoans.

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.

More than just orphans: are taxonomically-restricted genes important in evolution?

Comparative genome analyses indicate that every taxonomic group so far studied contains 10-20% of genes that lack recognizable homologs in other species. Do such 'orphan' or 'taxonomically-restricted' genes comprise spurious, non-functional ORFs, or does their presence reflect important evolutionary processes? Recent studies in basal metazoans such as Nematostella, Acropora and Hydra have shed light on the function of these genes, and now indicate that they are involved in important species-specific adaptive processes. Here we focus on evidence from Hydra suggesting that taxonomically-restricted genes play a role in the creation of phylum-specific novelties such as cnidocytes, in the generation of morphological diversity, and in the innate defence system. We propose that taxon-specific genes drive morphological specification, enabling organisms to adapt to changing conditions.

Where simplicity meets complexity: hydra, a model for host-microbe interactions.

For a long time, the main purpose of microbiology and immunology was to study pathogenic bacteria and infectious disease; the potential benefit of commensal bacteria remained unrecognised. Discovering that individuals from Hydra to man are not solitary, homogenous entities but consist of complex communities of many species that likely evolved during a billion years of coexistence (Fraune and Bosch 2010) led to the hologenome theory of evolution (Zilber-Rosenberg and Rosenberg 2008) which considers the holobiont with its hologenome as the unit of selection in evolution. Defining the individual microbe-host conversations in these consortia is a challenging but necessary step on the path to understanding the function of the associations as a whole. Untangling the complex interactions requires simple animal models with only a few specific bacterial species. Such models can function as living test tubes and may be key to dissecting the fundamental principles that underlie all host-microbe interactions. Here we introduce Hydra (Bosch et al. 2009) as such a model with one of the simplest epithelia in the animal kingdom (only two cell layers), with few cell types derived from only three distinct stem cell lineages, and with the availability of a fully sequenced genome and numerous genomic tools including transgenesis. Recognizing the entire system with its inputs, outputs and the interconnections (Fraune and Bosch 2010; Bosch et al. 2009; Fraune and Bosch 2007; Fraune et al. 2009a) we here present observations which may have profound impact on understanding a strictly microbe-dependent life style and its evolutionary consequences.

Cnidarian immunity: a tale of two barriers.

The phylum Cnidariais one of the earliest branches in the animal tree of life providing crucial insights into the early evolution of immunity. The diversity in cnidarian life histories and habitats raises several important issues relating to immunity. First, in the absence of specific immune cells, cnidarians must have effective mechanisms to defend against microbial pathogens. Second, to maintain tissue integrity, colonial forms have to rely on their capacity of self/nonself discrimination to rapidly detect approaching allogeneic cells as foreign and to eliminate them. And third, since cnidarians are colonized by complex bacterial communities and in many cases are home to algal symbionts, successful growth means for cnidarians to be able to distinguish between beneficial symbionts and pathogenic intruders. The aim of this chapter is to review the experimental evidence for innate immune reactions in Cnidaria. We show that in these diploblastic animals consisting of only two cell layers; the epithelial cells are able to mediate all innate immune responses. The endodermal epithelium appears as a chemical barrier employing antimicrobial peptides while the ectodermal epithelium is a physicochemical barrier supported by a glycocalix. Microbial recognition is mediated by pattern recognition receptors such as Toll- and Nod-like receptors. Together, the data support the hypothesis that the establishment of epithelial barriers represents an important step in evolution of host defense in eumetazoan animals more than 600 million years ago.

Activity of the novel peptide arminin against multiresistant human pathogens shows the considerable potential of phylogenetically ancient organisms as drug sources.

The emergence of multidrug-resistant bacteria highlights the need for new antibacterial agents. Arminin 1a is a novel antimicrobial peptide discovered during investigations of the epithelial defense of the ancient metazoan Hydra. Following proteolytic processing, the 31-amino-acid-long positively charged C-terminal part of arminin 1a exhibits potent and broad-spectrum activity against bacteria, including multiresistant human pathogenic strains, such as methicillin-resistant Staphylococcus aureus (MRSA) strains (minimal bactericidal concentration, 0.4 microM to 0.8 microM). Ultrastructural observations indicate that bacteria are killed by disruption of the bacterial cell wall. Remarkably, the antibacterial activity of arminin 1a is not affected under the physiological salt conditions of human blood. In addition, arminin 1a is a selective antibacterial agent that does not affect human erythrocyte membranes. Arminin 1a shows no sequence homology to any known antimicrobial peptide. Because of its high level of activity against multiresistant bacterial strains pathogenic for humans, the peptide arminin 1a is a promising template for a new class of antibiotics. Our data suggest that ancient metazoan organisms such as Hydra hold promise for the detection of novel antimicrobial molecules and the treatment of infections caused by multiresistant bacteria.

Spontaneous body contractions are modulated by the microbiome of Hydra.

Spontaneous contractile activity, such as gut peristalsis, is ubiquitous in animals and is driven by pacemaker cells. In humans, disruption of the contraction pattern leads to gastrointestinal conditions, which are also associated with gut microbiota dysbiosis. Spontaneous contractile activity is also present in animals lacking gastrointestinal tract. Here we show that spontaneous body contractions in Hydra are modulated by symbiotic bacteria. Germ-free animals display strongly reduced and less regular contraction frequencies. These effects are partially restored by reconstituting the natural microbiota. Moreover, soluble molecule(s) produced by symbiotic bacteria may be involved in contraction frequency modulation. As the absence of bacteria does not impair the contractile ability itself, a microbial effect on the pacemakers seems plausible. Our findings indicate that the influence of bacteria on spontaneous contractile activity is present in the early-branching cnidarian hydra as well as in Bilateria, and thus suggest an evolutionary ancient origin of interaction between bacteria and metazoans, opening a window into investigating the roots of human motility disorders.

A secreted antibacterial neuropeptide shapes the microbiome of Hydra.

Colonization of body epithelial surfaces with a highly specific microbial community is a fundamental feature of all animals, yet the underlying mechanisms by which these communities are selected and maintained are not well understood. Here, we show that sensory and ganglion neurons in the ectodermal epithelium of the model organism hydra (a member of the animal phylum Cnidaria) secrete neuropeptides with antibacterial activity that may shape the microbiome on the body surface. In particular, a specific neuropeptide, which we call NDA-1, contributes to the reduction of Gram-positive bacteria during early development and thus to a spatial distribution of the main colonizer, the Gram-negative Curvibacter sp., along the body axis. Our findings warrant further research to test whether neuropeptides secreted by nerve cells contribute to the spatial structure of microbial communities in other organisms.Certain neuropeptides, in addition to their neuromodulatory functions, display antibacterial activities of unclear significance. Here, the authors show that a secreted neuropeptide modulates the distribution of bacterial communities on the body surface during development of the model organism Hydra.

Bacteria-bacteria interactions within the microbiota of the ancestral metazoan Hydra contribute to fungal resistance.

Epithelial surfaces of most animals are colonized by diverse microbial communities. Although it is generally agreed that commensal bacteria can serve beneficial functions, the processes involved are poorly understood. Here we report that in the basal metazoan Hydra, ectodermal epithelial cells are covered with a multilayered glycocalyx that provides a habitat for a distinctive microbial community. Removing this epithelial microbiota results in lethal infection by the filamentous fungus Fusarium sp. Restoring the complex microbiota in gnotobiotic polyps prevents pathogen infection. Although mono-associations with distinct members of the microbiota fail to provide full protection, additive and synergistic interactions of commensal bacteria are contributing to full fungal resistance. Our results highlight the importance of resident microbiota diversity as a protective factor against pathogen infections. Besides revealing insights into the in vivo function of commensal microbes in Hydra, our findings indicate that interactions among commensal bacteria are essential to inhibit pathogen infection.

Initial symbiont contact orchestrates host-organ-wide transcriptional changes that prime tissue colonization.

Upon transit to colonization sites, bacteria often experience critical priming that prepares them for subsequent, specific interactions with the host; however, the underlying mechanisms are poorly described. During initiation of the symbiosis between the bacterium Vibrio fischeri and its squid host, which can be observed directly and in real time, approximately five V. fischeri cells aggregate along the mucociliary membranes of a superficial epithelium prior to entering host tissues. Here, we show that these few early host-associated symbionts specifically induce robust changes in host gene expression that are critical to subsequent colonization steps. This exquisitely sensitive response to the host's specific symbiotic partner includes the upregulation of a host endochitinase, whose activity hydrolyzes polymeric chitin in the mucus into chitobiose, thereby priming the symbiont and also producing a chemoattractant gradient that promotes V. fischeri migration into host tissues. Thus, the host responds transcriptionally upon initial symbiont contact, which facilitates subsequent colonization.

Embryo protection in contemporary immunology: Why bacteria matter.

Early embryos of many vertebrates and invertebrates develop outside the mother and are exposed to a myriad of potential microbial colonizers. Here we discuss how these embryos are protected from microbial attacks and how they might control and shape their microbiota. In essence we delineate a new role for antimicrobial peptides both in selecting particular bacterial partners during early development and in being important components of a "be prepared" strategy providing transgenerational protection.

Identification of a kazal-type serine protease inhibitor with potent anti-staphylococcal activity as part of Hydra's innate immune system.

In the absence of migratory phagocytic cells the basal metazoan Hydra has developed a very effective immune system. Previous work has shown that epithelial cells, both in the ectoderm and endoderm, recognize PAMPs by TLR and produce a number of antimicrobial peptides. In this study we demonstrate that not only epithelial cells but also gland cells are critically involved in Hydra's innate host defense by producing a kazal-type serine protease inhibitor, kazal2, that has potent in vitro bactericidal activity against Staphylococcus aureus. The discovery of an antimicrobial serine protease inhibitor in Hydra may shed new light on the mechanisms of host defense early in metazoan evolution, and promises to open new avenues for the development of potent anti-staphylococcal compounds.

Is activated hemocyanin instead of phenoloxidase involved in immune response in woodlice?

In the Common woodlouse Porcellio scaber (Crustacea: Isopoda: Oniscidea), experimental immune challenge did not induce the expression of pro-phenoloxidase that, in most other invertebrates studied thus far, can be activated into phenoloxidase via an activation cascade upon immune challenge. Instead, Porcellio hemocyanin proved to exhibit catecholoxidase activity upon activation. However, none of the activating factors known from other invertebrates other than SDS-treatment resulted in activation of hemocyanin into a functional phenoloxidase in vitro. The distinct characteristics of isopod hemocyanin are reflected by the quaternary structure of the hemocyanin dodecamers that differs from that of other crustacean hemocyanins in that the two hexamers share a common 3-fold rotation axis and have an angular offset of 60 degrees against each other. Accordingly, the sequence of Porcellio hemocyanin can be distinguished clearly from other crustacean hemocyanins and in a phylogenetic analysis forms a cluster with other isopod and amphipod hemocyanins. We propose a peracarid-type hemocyanin that may have evolved in response to its required multiple functions in respiration and immune response, while phenoloxidase sensu strictu is lacking.

Hydramacin-1, structure and antibacterial activity of a protein from the basal metazoan Hydra.

Hydramacin-1 is a novel antimicrobial protein recently discovered during investigations of the epithelial defense of the ancient metazoan Hydra. The amino acid sequence of hydramacin-1 shows no sequence homology to any known antimicrobial proteins. Determination of the solution structure revealed that hydramacin-1 possesses a disulfide bridge-stabilized alphabeta motif. This motif is the common scaffold of the knottin protein fold. The structurally closest relatives are the scorpion oxin-like superfamily. Within this superfamily hydramacin-1 establishes a new family of proteins that all share antimicrobial activity. Hydramacin-1 is potently active against Gram-positive and Gram-negative bacteria including multi-resistant human pathogenic strains. It leads to aggregation of bacteria as an initial step of its bactericidal mechanism. Aggregated cells are connected via electron-dense contacts and adopt a thorn apple-like morphology. Analysis of the hydramacin-1 structure revealed an unusual distribution of amino acid side chains on the surface. A belt of positively charged residues is sandwiched by two hydrophobic areas. Based on this characteristic surface feature and on biophysical analysis of protein-membrane interactions, we propose a model that describes the aggregation effect exhibited by hydramacin-1.

Uncovering the evolutionary history of innate immunity: the simple metazoan Hydra uses epithelial cells for host defence.

Although many properties of the innate immune system are shared among multicellular animals, the evolutionary origin remains poorly understood. Here we characterize the innate immune system in Hydra, one of the simplest multicellular animals known. In the complete absence of both protective mechanical barriers and mobile phagocytes, Hydra's epithelium is remarkably well equipped with potent antimicrobial peptides to prevent pathogen infection. Induction of antimicrobial peptide production is mediated by the interaction of a leucine-rich repeats (LRRs) domain containing protein with a TIR-domain containing protein lacking LRRs. Conventional Toll-like receptors (TLRs) are absent in the Hydra genome. Our findings support the hypothesis that the epithelium represents the ancient system of host defence.