Cultivation of primmorphs from the marine sponge Suberites domuncula: morphogenetic potential of silicon and iron

Marine demosponges (phylum Porifera) are rich sources for potent bioactive compounds. With the establishment of the primmorph system from sponges, especially from Suberites domuncula, the technology to cultivate sponge cells in vitro improved considerably. This progress was possible after the elucidation that sponges are provided with characteristic metazoan cell adhesion receptors and extracellular matrix molecules which allow their cells a positioning in a complex organization pattern. This review summarizes recent data on the cultivation of sponges in aquaria and--with main emphasis--of primmorphs in vitro. It is outlined that silicon and Fe(+++) contribute substantially to the formation of larger primmorphs (size of 10 mm) as well as of a canal system in primmorphs; canals are probably required for an improved oxygen and food supply. We conclude that the primmorph system will facilitate a sustainable use of sponges in the production of bioactive compounds; it may furthermore allow new and hitherto not feasible insights into basic questions on the origin of Metazoa.     

Contribution of sponge genes to unravel the genome of the hypothetical ancestor of Metazoa (Urmetazoa)

Recently the term Urmetazoa, as the hypothetical metazoan ancestor, was introduced to highlight the finding that all metazoan phyla including the Porifera (sponges) are derived from one common ancestor. Sponges as the evolutionarily oldest, still extant phylum, are provided with a complex network of structural and functional molecules. Analyses of sponge genomes from Demospongiae (Suberites domuncula and Geodia cydonium), Calcarea (Sycon raphanus) and Hexactinellida (Aphrocallistes vastus) have contributed also to the reconstruction of the evolutionary position of Metazoa with respect to Fungi. Furthermore, these analyses have provided evidence that the characteristic evolutionary novelties of Metazoa, such as the extracellular matrix molecules, the cell surface receptors, the nervous signal transduction molecules as well as the immune molecule existing in Porifera, share high sequence and in some aspects also functional similarities to related polypeptides found in other metazoan phyla. During the transition to Metazoa new domains occurred; as one example, the formation of the death domain from the ankyrin is outlined. In parallel, domanial proteins have been formed, such as the receptor tyrosine kinases. The metazoan essentials have been defined by analyzing and comparing the sponge sequences with the related sequences from the metazoans Homo sapiens, Caenorhabditis elegans and Drosophila melanogaster, the fungus Saccharomyces cerevisiae and the plant Arabidopsis thaliana. The data revealed that those sponge molecules grouped to cell adhesion cell recognition proteins are predominantly found in Protostomia and Deuterostomia while they are missing in Fungi and Viridiplantae. Moreover, evidence is presented allowing the conclusion that the sponge molecules are more closely related to the corresponding molecules from H. sapiens than to those of C. elegans or D. melanogaster. Especially surprising was the finding that the Demospongiae are provided with elements of adaptive immunity.     

Sponge paraphyly and the origin of Metazoa

In order to allow critical evaluation of the interrelationships between the three sponge classes, and to resolve the question of mono‐ or paraphyly of sponges (Porifera), we used the polymerase chain reaction (PCR) to amplify almost the entire nucleic acid sequence of the 18S rDNA from several hexactinellid, demosponge and calcareous sponge species. The amplification products were cloned, sequenced and then aligned with previously reported sequences from other sponges and nonsponge metazoans and variously distant outgroups, and trees were constructed using both neighbour‐joining and maximum parsimony methods. Our results suggest that sponges are paraphyletic, the Calcarea being more related to monophyletic Eumetazoa than to the siliceous sponges (Demospongiae, Hexactinellida). These results have important implications for our understanding of metazoan origins, because they suggest that the common ancestor of Metazoa was a sponge. They also have consequences for basal metazoan classification, implying that the phylum Porifera should be abandoned. Our results support the upgrading of the calcareous sponge class to the phylum level.     

Increased gene expression of a cytokine-related molecule and profilin after activation of Suberites domuncula cells with xenogeneic sponge molecule(s)

Porifera (sponges) constitute the lowest metazoan phylum. Experiments examined whether sponges can recognize self/nonself molecules. Cells from the marine sponge Suberites domuncula were incubated with membranes from either S. domuncula or another marine sponge, Geodia cydonium, as well as with recombinant alpha-integrin from G. cydonium. The cells responded immediately with a rise of intracellular Ca2+ ([Ca2+i]) if they were treated with membranes from G. cydonium but not after treatment by those from S. domuncula. This change of [Ca2+i] was also recorded with G. cydonium alpha-integrin. In parallel, the expression of two genes was strongly upregulated; one codes for a cytokine-related molecule, pre-B-cell colony-enhancing factor, and the other for profilin. These genes have previously been found to be highly expressed in human or echinoderm cells in the presence of xenogeneic proteins. Our data support the hypothesis that a primordial immune response system is present in sponges.     

Isolation and characterization of a cDNA encoding a potential morphogen from the marine sponge Geodia cydonium that is conserved in higher metazoans.

Species belonging to the lowest metazoan phylum, the sponges (Porifera), exhibit a surprisingly complex and multifaceted Bauplan (body plan). Recently, key molecules have been isolated from sponges which demonstrate that the cells of these animals are provided with characteristic metazoan adhesion and signal transduction molecules, allowing tissue formation. In order to understand which factors control the spatial organization of these cells in the sponge body plan, we screened for a cDNA encoding a soluble modulator of the behaviour of endothelial cells. A cDNA encoding a putative protein, which is highly similar to the human and mouse endothelial monocyte-activating polypeptide (EMAP) II has been isolated from a library of the marine sponge Geodia cydonium. The sponge EMAP-related polypeptide (EMAPR) has been termed EMAPR1_GC. The full-length cDNA clone, GCEMAPR1, has a size of 592 nucleotides (nt) and contains a 447 nt-long potential open reading frame; the molecular weight (MW) of the deduced amino acid sequence, 16,499 Da, is close to that of mature mammalian EMAP II (ca. 18 kDa). The sponge polypeptide is also closely related to a deduced polypeptide from the cosmid clone F58B3 isolated from Caenorhabditis elegans. A phylogenetic analysis revealed that the sponge and the nematode EMAPR molecules form a cluster which is significantly separated from the corresponding mammalian EMAP molecules. The function of the first cloned putative soluble modulator of endothelial cells in sponges remains to be determined.     

Caspase-mediated apoptosis in sponges: cloning and function of the phylogenetic oldest apoptotic proteases from Metazoa

Sponges (phylum Porifera) represent the phylogenetically oldest metazoan phylum. These animals have complex cell adhesion and powerful immune systems which allow the formation of a distinct body plan. Consequently, an apoptotic machinery has to be predicted that allows sponges to eliminate unwanted cells accumulating during development. With the marine sponge Geodia cydonium, it is shown that allografts of these animals undergo apoptosis as demonstrated by apoptotic DNA fragmentation. Extracts from allografts contain an enzymic activity characteristic for caspases; as substrate to determine the cleavage activity, Ac-DEVD-AMC was applied. cDNAs encoding predicted caspase-3-related proteins were isolated; they comprise the characteristic structure known from caspases of other metazoan phyla. The two cDNAs are assumed to originate from one gene by alternative splicing; the longer form comprises a caspase recruitment domain (CARD), whereas the shorter one is missing CARD. The expression of sponge caspase genes is up-regulated during allograft rejection. In vivo incubation experiments with Ac-DEVD-CHO (a caspase-3 inhibitor) showed a reduction of apoptotic DNA fragmentation, whereas Ac-LEHD-CHO (an inhibitor of caspase-9) caused no effect. It is concluded, that for the establishment of the metazoan body plan, both the adhesion molecules and the apoptotic molecules (described here) were essential prerequisites.     

Phylogenetic Position of the Hexactinellida Within the Phylum Porifera Based on the Amino Acid Sequence of the Protein Kinase C from Rhabdocalyptus dawsoni

Recent analyses of genes encoding proteins typical for multicellularity, especially adhesion molecules and receptors, favor the conclusion that all metazoan phyla, including the phylum Porifera (sponges), are of monophyletic origin. However, none of these data includes cDNA encoding a protein from the sponge class Hexactinellida. We have now isolated and characterized the cDNA encoding a protein kinase C, belonging to the C subfamily (cPKC), from the hexactinellid sponge Rhabdocalyptus dawsoni. The two conserved regions, the regulatory part with the pseudosubstrate site, the two zinc fingers, and the C2 domain, as well as the catalytic domain were used for phylogenetic analyses. Sequence alignment and construction of a phylogenetic tree from the catalytic domains revealed that the yeast Saccharomyces cerevisiae and the protozoan Trypanosoma brucei are at the base of the tree, while the hexactinellid R. dawsoni branches off first among the metazoan sequences; the other two classes of the Porifera, the Calcarea (the sequence from Sycon raphanus was used) and the Demospongiae (sequences from Geodia cydonium and Suberites domuncula were used), branch off later. The statistically robust tree also shows that the two cPKC sequences from the higher invertebrates Drosophila melanogaster and Lytechinus pictus are most closely related to the calcareous sponge. This finding was also confirmed by comparing the regulatory part of the kinase gene. We suggest, that (i) within the phylum Porifera, the class Hexactinellida diverged first from a common ancestor to the Calcarea and the Demospongiae, which both appeared later, and (ii) the higher invertebrates are more closely related to the calcareous sponges. Received: 6 August 1997 / Accepted: 24 October 1997     

A (1-->3)-beta-D-glucan recognition protein from the sponge Suberites domuncula. Mediated activation of fibrinogen-like protein and epidermal growth factor gene expression.

Sponges (phylum Porifera) live in a symbiotic relationship with microorganisms, primarily bacteria. Until now, molecular proof for the capacity of sponges to recognize fungi in the surrounding aqueous milieu has not been available. Here we demonstrate, for the demosponge Suberites domuncula (Porifera, Demospongiae, Hadromerida), a cell surface receptor that recognizes (1-->3)-beta-D-glucans, e.g. curdlan or laminarin. This receptor, the (1-->3)-beta-D-glucan-binding protein, was identified and its cDNA analysed. The gene coding for the 45 kDa protein was found to be upregulated in tissue after incubation with carbohydrate. Simultaneously with the increased expression of this gene, two further genes showed an elevated steady state level of expression; one codes for a fibrinogen-like protein and the other for the epidermal growth factor precursor. Expression of the (1-->3)-beta-D-glucan-binding protein and the fibrinogen-like protein occurred in cells on the sponge surface, in the pinacoderm. By Western blotting, the product of the fibrinogen-like protein gene was identified, the recombinant protein isolated, and antibodies raised to this protein. Their application revealed that a 5 kDa factor is produced, which is apparently processed from the 77 kDa epidermal growth factor precursor. Finally, we provided evidence that a tyrosine kinase pathway is initiated in response to exposure to D-glucan; its phosphorylation activity could be blocked by aeroplysinin. In turn, the increased expression of the downstream genes was suppressed. We conclude that sponges possess a molecular mechanism for recognizing fungi via the d-glucan carbohydrates on their surfaces.     

Genomic insights into the marine sponge microbiome

Marine sponges (phylum Porifera) often contain dense and diverse microbial communities, which can constitute up to 35% of the sponge biomass. The genome of one sponge, Amphimedon queenslandica, was recently sequenced, and this has provided new insights into the origins of animal evolution. Complementary efforts to sequence the genomes of uncultivated sponge symbionts have yielded the first glimpse of how these intimate partnerships are formed. The remarkable microbial and chemical diversity of the sponge-microorganism association, coupled with its postulated antiquity, makes sponges important model systems for the study of metazoan host-microorganism interactions, and their evolution, as well as for enabling access to biotechnologically important symbiont-derived natural products. In this Review, we discuss our current understanding of the interactions between marine sponges and their microbial symbiotic consortia, and highlight recent insights into these relationships from genomic studies.     

Review: How was metazoan threshold crossed? The hypothetical Urmetazoa.

The origin of Metazoa remained--until recently--the most enigmatic of all phylogenetic problems. Sponges [Porifera] as "living fossils", positioned at the base of multicellular animals, have been used to answer basic questions in metazoan evolution by molecular biological techniques. During the last few years, cDNAs/genes coding for informative proteins have been isolated and characterized from sponges, especially from the marine demosponges Suberites domuncula and Geodia cydonium. The analyses of their deduced amino acid sequences allowed a molecular biological resolution of the monophyly of Metazoa. Molecules of the extracellular matrix/basal lamina, with the integrin receptor, fibronectin and galectin as prominent examples, cell-surface receptors (tyrosine kinase receptors), elements of nerve system/sensory cells (metabotropic glutamate receptor), homologs/modules of an immune system [immunoglobulin-like molecules, SRCR- and SCR-repeats, cytokines, (2-5)A synthetase], as well as morphogens (myotrophin) classify the Porifera as true Metazoa. As "living fossils", provided with simple, primordial molecules allowing cell-cell and cell-matrix adhesion, as well as processes of signal transduction as known in a more complex manner from higher Metazoa, sponges also show peculiarities. Tissues of sponges are rich in telomerase activity, suggesting a high plasticity in the determination of cell lineages. It is concluded that molecular biological studies with sponges as models will not only help to understand the evolution to the Metazoa but also the complex, hierarchical regulatory network of cells in higher Metazoa [reviewed in Progress in Molecular Subcellular Biology, vols. 19, 21 (1998) Springer Verlag]. The hypothetical ancestral animal, the Urmetazoa, from which the metazoan lineages diverged (more than 600 MYA), may have had the following characteristics: cell adhesion molecules with intracellular signal transduction pathways, morphogens/growth factors forming gradients, a functional immune system, and a primordial nerve cell/receptor system.     

The Occurrence of Type S1A Serine Proteases in Sponge and Jellyfish

Although serine proteases are found in all kinds of cellular organisms and many viruses, the classic "chymotrypsin family" (Group S1A by the 1998 Barrett nomenclature) has an unusual phylogenetic distribution, being especially common in animals, entirely absent from plants and protists, and rare among fungi. The distribution in Bacteria is largely restricted to the genus Streptomyces, although a few isolated occurrences in other bacteria have been reported. The family may be entirely absent from Archaea. Although more than a thousand sequences have been reported for enzymes of this type from animals, none of them have been from early diverging phyla like Porifera or Cnidaria. We now report the existence of Group S1A serine proteases in a sponge (phylum Porifera) and a jellyfish (phylum Cnidaria), making it safe to conclude that all animal groups possess these enzymes.     

The occurrence of type S1A serine proteases in sponge and jellyfish

Although serine proteases are found in all kinds of cellular organisms and many viruses, the classic "chymotrypsin family" (Group S1A by the 1998 Barrett nomenclature) has an unusual phylogenetic distribution, being especially common in animals, entirely absent from plants and protists, and rare among fungi. The distribution in Bacteria is largely restricted to the genus Streptomyces, although a few isolated occurrences in other bacteria have been reported. The family may be entirely absent from Archaea. Although more than a thousand sequences have been reported for enzymes of this type from animals, none of them have been from early diverging phyla like Porifera or Cnidaria. We now report the existence of Group S1A serine proteases in a sponge (phylum Porifera) and a jellyfish (phylum Cnidaria), making it safe to conclude that all animal groups possess these enzymes.     

HBMMD: an enhanced database of the microorganisms associated with deeper water marine invertebrates

The Harbor Branch Marine Microbial Database (HBMMD) provides preliminary taxonomic identifications and features of microorganisms maintained in the Harbor Branch Oceanographic Institution Marine Microbial Culture Collection. The microbes are primarily derived from marine invertebrates such as sponges (phylum Porifera) and soft corals (phylum Cnidaria) found in deep water environments [>120 feet (>35 m) seawater]. The microbes isolated from within marine invertebrates represent some unique taxa and phylogenetic signatures. The database provides a user-friendly method to systemically search or sort a desired input. The site allows a powerful search for multiple parameters of any entry. Images of the microbes are contained within the database and can be accessed from the website. The HBMMD homepage is located at .     

First evidence of chitin as a component of the skeletal fibers of marine sponges. Part I. Verongidae (demospongia: Porifera)

The Porifera (sponges) are often regarded as the oldest, extant metazoan phylum, also bearing the ancestral stage for most features occurring in higher animals. The absence of chitin in sponges, except for the wall of peculiar resistance bodies produced by a highly derived fresh-water group, is puzzling, since it points out chitin to be an autapomorphy for a particular sponge family rather than the ancestral condition within the metazoan lineage. By investigating the internal proteinaceous (spongin) skeleton of two demosponges (Aplysina sp. and Verongula gigantea) using a wide array of techniques (Fourier transform infrared (FTIR), Raman, X-ray, Calcofluor White Staining, Immunolabeling, and chitinase test), we show that chitin is a component of the outermost layer (cuticle) of the skeletal fibers of these demosponges. FTIR and Raman spectra, as well as X-ray difractograms consistently revealed that sponge chitin is much closer to the alpha-chitin known from other animals than to beta-chitin. These findings support the view that the occurrence of a chitin-producing system is the ancestral condition in Metazoa, and that the alpha-chitin is the primitive form in animals.     

Polarity factor 'Frizzled' in the demosponge Suberites domuncula: identification, expression and localization of the receptor in the epithelium/pinacoderm(1)

Until recently, it was assumed that polarity and axis formation have evolved only in metazoan phyla higher than Cnidaria. One key molecule involved in the signal transduction causing tissue polarity is Frizzled, a seven-transmembrane receptor that is activated by the Wnt family of secreted proteins. We report the isolation and characterization of a Frizzled gene from the demosponge Suberites domuncula (Sd-Fz). The deduced polypeptide comprises all characteristic domains known from Frizzled receptors of higher metazoans. In situ hybridization studies show that Sd-Fz is expressed in cells close to the surface of the sponges and in the pinacocytes of some canals. Northern blot analysis demonstrates its upregulation during the formation of three-dimensional sponge cell aggregates in culture. These data provide for the first time experimental evidence that already in the lowest metazoan phylum (Porifera) genes are present which are very likely involved in tissue polarity.     

Sustainable Production of Bioactive Compounds by Sponges—Cell Culture and Gene Cluster Approach: A Review

Sponges (phylum Porifera) are sessile marine filter feeders that have developed efficient defense mechanisms against foreign attackers such as viruses, bacteria, or eukaryotic organisms. Protected by a highly complex immune system, as well as by the capacity to produce efficient antiviral compounds (e.g., nucleoside analogues), antimicrobial compounds (e.g., polyketides), and cytostatic compounds (e.g., avarol), they have not become extinct during the last 600 million years. It can be assumed that during this long period of time, bacteria and microorganisms coevolved with sponges, and thus acquired a complex common metabolism. It is suggested that (at least) some of the bioactive secondary metabolites isolated from sponges are produced by functional enzyme clusters, which originated from the sponges and their associated microorganisms. As a consequence, both the host cells and the microorganisms lost the ability to grow independently from each other. Therefore, it was—until recently—impossible to culture sponge cells in vitro. Also the predominant number of symbiotic bacteria proved to be nonculturable. In order to exploit the bioactive potential of both the sponge and the symbionts, a 3D-aggregate primmorph culture system was established; also it was proved that one bioactive compound, avarol/avarone, is produced by the sponge Dysidea avara. Another promising way to utilize the bioactive potential of the microorganisms is the cloning and heterologous expression of enzymes involved in secondary metabolism, such as the polyketide synthases. From the consortium German Center of Excellence [BiotecMarin]. Dedicated to Dr. Paul J. Scheuer (University of Hawaii) who created the basis for the progress in the biomedical application of the bioactive potential of the marine environment.