Silicatein Genes in Spicule-Forming and Nonspicule-forming Pacific Demosponges

Silicatein genes are known to be involved in siliceous spicule formation in marine sponges. Proteins encoded by these genes, silicateins, were recently proposed for nanobiotechnological applications. We studied silicatein genes of marine sponges Latrunculia oparinae collected in the west Pacific region, shelf of Kuril Islands. Five silicatein genes, LoSilA1, LoSilA1a, LoSilA2, and LoSilA3 (silicatein-α group), LoSilB (silicatein-β group), and one cathepsin gene, LoCath, were isolated from the sponge L. oparinae for the first time. The deduced amino acid sequence of L. oparinae silicateins showed high-sequence identity with silicateins described previously. LoCath contains the catalytic triad of amino acid residues Cys-His-Asn characteristic for cathepsins as well as motifs typical for silicateins. A phylogenetic analysis places LoCath between sponge silicateins-β and L-cathepsins suggesting that the LoCath gene represents an intermediate form between silicatein and cathepsin genes. Additionally, we identified, for the first time, silicatein genes (AcSilA and AcSilB) in nonspicule-forming marine sponge, Acаnthodendrilla sp. The results suggest that silicateins could participate also in the function(s) unrelated to spiculogenesis.     

Silicateins, the major biosilica forming enzymes present in demosponges: protein analysis and phylogenetic relationship

Silicateins are enzymes, which are restricted to sponges (phylum Porifera), that mediate the catalytic formation of biosilica from monomeric silicon compounds. The silicatein protein is compartmented in the sponges in the axial filaments which reside in the axial canals of the siliceous spicules. In the present study silicatein has been isolated from the freshwater sponge Lubomirskia baicalensis where it occurs in isoforms with sizes of 23 kDa, 24 kDa and 26 kDa. Since the larger protein is glycosylated we posit that it is a processed form of one of the smaller size forms. The silicatein isoforms are post-translationally modified by phosphorylation; at least four isoforms exist with pI's of 5.4, of 5.2, of 4.9 and of 4.7. Surprisingly silicatein not only mediates polymerization of silicate, but also displays proteolytic activity which is specific for cathepsin L enzymes, thus underscoring the high relationship of the silicateins to cathepsin L. The cDNAs from L. baicalensis for silicatein and cathepsin L, as well as the respective genes, were cloned. It was found that the five introns present in the sponge genes are highly conserved up to human cathepsin L. This analysis has been completed by sequencing of two silicatein genes (both for silicatein-alpha and -beta) and of cathepsin L from another demosponge, Suberites domuncula. A comprehensive phylogenetic analysis with these new sequences shed new light upon the evolution of cathepsin L and silicatein families which occurred at the base of the metazoan phyla. It is concluded, that in parallel with the emergence of these enzymes at first the number of introns increased, especially in the coding region of the mature enzyme. Later in evolution the number of introns decreased again. We postulate that modification of the catalytic triad, especially of its first amino acid, is a suitable target for a chemical modulation of enzyme function of the silicateins/cathepsin L.     

Analysis of the axial filament in spicules of the demosponge Geodia cydonium: different silicatein composition in microscleres (asters) and megascleres (oxeas and triaenes)

The skeleton of the siliceous sponges (Porifera: Hexactinellida and Demospongiae) is supported by spicules composed of bio-silica. In the axial canals of megascleres, harboring the axial filaments, three isoforms of the enzyme silicatein (-alpha, -beta and -gamma) have been identified until now, using the demosponges Tethya aurantium and Suberites domuncula. Here we describe the composition of the proteinaceous components of the axial filament from small spicules, the microscleres, in the demosponge Geodia cydonium that possesses megascleres and microscleres. The morphology of the different spicule types is described. Also in G. cydonium the synthesis of the spicules starts intracellularly and they are subsequently extruded to the extracellular space. In contrast to the composition of the silicateins in the megascleres (isoforms: -alpha, -beta and -gamma), the axial filaments of the microscleres contain only one form of silicatein, termed silicatein-alpha/beta, with a size of 25kDa. Silicatein-alpha/beta undergoes three phosphorylation steps. The gene encoding silicatein-alpha/beta was identified and found to comprise the same characteristic sites, described previously for silicateins-alpha or -beta. It is hypothesized, that the different composition of the axial filaments, with respect to silicateins, contributes to the morphology of the different types of spicules.     

Molecular Cloning of Silicatein Gene from Marine Sponge <Emphasis Type="Italic">Petrosia ficiformis</Emphasis> (Porifera,Demospongiae) and Development of Primmorphs as a Model for Biosilicification Studies

In some sponges peculiar proteins called silicateins catalyze silica polymerization in ordered structures, and their study is of high interest for possible biotechnological applications in the nanostructure industry. In this work we describe the isolation and the molecular characterization of silicatein from spicules of Petrosia ficiformis, a common Mediterranean sponge, and the development of a cellular model (primmorphs) suitable for in vitro studies of silicatein gene regulation. The spicule of P. ficiformis contains an axial filament composed of 2 insoluble proteins, of 30 and 23 kDa. The 23-kDa protein was characterized, and the full-length cDNA was cloned. The putative amino acid sequence has high homology with previously described silicateins from other sponge species and also is very similar to cathepsins, a cystein protease family. Finally, P. ficiformis primmorphs express the silicatein gene, suggesting that they should be a good model for biosilicification studies.     

Axial growth of hexactinellid spicules: formation of cone-like structural units in the giant basal spicules of the hexactinellid Monorhaphis

The glass sponge Monorhaphis chuni (Porifera: Hexactinellida) forms the largest bio-silica structures on Earth; their giant basal spicules reach sizes of up to 3 m and diameters of 8.5 mm. Previously, it had been shown that the thickness growth proceeds by appositional layering of individual lamellae; however, the mechanism for the longitudinal growth remained unstudied. Now we show, that the surface of the spicules have towards the tip serrated relief structures that are consistent in size and form with the protrusions on the surface of the spicules. These protrusions fit into the collagen net that surrounds the spicules. The widths of the individual lamellae do not show a pronounced size tendency. The apical elongation of the spicule proceeds by piling up cone-like structural units formed from silica. As a support of the assumption that in the extracellular space silicatein(-like) molecules exist that associate with the external surface of the respective spicule immunogold electron microscopic analyses were performed. With the primmorph system from Suberites domuncula we show that silicatein(-like) molecules assemble as string- and net-like arrangements around the spicules. At their tips the silicatein(-like) molecules are initially stacked and at a later stay also organized into net-like structures. Silicatein(-like) molecules have been extracted from the giant basal spicule of Monorhaphis. Applying the SDS–PAGE technique it could be shown that silicatein molecules associate to dimers and trimers. Higher complexes (filaments) are formed from silicatein(-like) molecules, as can be visualized by electron microscopy (SEM). In the presence of ortho-silicate these filaments become covered with 30–60 nm long small rod-like/cuboid particles of silica. From these data we conclude that the apical elongation of the spicules of Monorhaphis proceeds by piling up cone-like silica structural units, whose synthesis is mediated by silicatein(-like) molecules.     

Silicatein expression in the hexactinellid Crateromorpha meyeri: the lead marker gene restricted to siliceous sponges

The siliceous spicules of sponges (Porifera) are synthesized by the enzyme silicatein. This protein and its gene have been identified so far in the Demospongiae, e.g., Tethya aurantium and Suberites domuncula. In the Hexactinellida, the second class of siliceous sponges, the mechanism of synthesis of the largest bio-silica structures on Earth remains obscure. Here, we describe the morphology of the spicules (diactines and stauractines) of the hexactinellid Crateromorpha meyeri. These spicules are composed of silica lamellae concentrically arranged around a central axial canal and contain proteinaceous sheaths (within the siliceous mantel) and proteinaceous axial filaments (within the axial canal). The major protein in the spicules is a 24-kDa protein that strongly reacts with anti-silicatein antibodies in Western blots. Its cDNA has been successfully cloned; the deduced hexactinellid silicatein comprises, in addition to the characteristic catalytic triad amino acids Ser-His-Asn and the "conventional" serine cluster, a "hexactinellid C. meyeri-specific" Ser cluster. We show that anti-silicatein antibodies react specifically with the proteinaceous matrix of the C. meyeri spicules. The characterization of silicatein at the genetic level should contribute to an understanding of the molecular/biochemical mechanism of spiculogenesis in Hexactinellida. These data also indicate that silicatein is an autapomorphic molecule common to both classes of siliceous sponges.     

Evagination of cells controls bio-silica formation and maturation during spicule formation in sponges

The enzymatic-silicatein mediated formation of the skeletal elements, the spicules of siliceous sponges starts intracellularly and is completed extracellularly. With Suberites domuncula we show that the axial growth of the spicules proceeds in three phases: (I) formation of an axial canal; (II) evagination of a cell process into the axial canal, and (III) assembly of the axial filament composed of silicatein. During these phases the core part of the spicule is synthesized. Silicatein and its substrate silicate are stored in silicasomes, found both inside and outside of the cellular extension within the axial canal, as well as all around the spicule. The membranes of the silicasomes are interspersed by pores of ≈ 2 nm that are likely associated with aquaporin channels which are implicated in the hardening of the initial bio-silica products formed by silicatein. We can summarize the sequence of events that govern spicule formation as follows: differential GENETIC READOUT (of silicatein) → FRACTAL ASSOCIATION of the silicateins → EVAGINATION of cells by hydro-mechanical forces into the axial canal → and finally PROCESSIVE BIO-SILICA POLYCONDENSATION around the axial canal. We termed this process, occurring sequentially or in parallel, BIO-INORGANIC SELF-ORGANIZATION.     

The dorid nudibranchs Peltodoris lentiginosa and Archidoris odhneri as predators of glass sponges

The dorid nudibranchs Peltodoris lentiginosa and Archidoris odhneri were found on glass sponges (Porifera, Hexactinellida) during remotely operated vehicle surveys of three reefs in the Strait of Georgia, British Columbia, Canada. Eight nudibranchs were sampled from 2009 to 2011. Identification of sponge spicules found in their gut and fecal contents confirmed the nudibranchs to be predators of the reef‐forming hexactinellids Aphrocallistes vastus and Heterochone calyx, as well as of the demosponge Desmacella austini, which encrusts skeletons of the glass sponges. Four of five nudibranchs dissected for gut content analysis had stomachs containing sponge spicules. Counts from high‐definition video footage taken during systematic surveys done in 2009 showed that nudibranchs were found in only two of the three glass sponge reefs. These data provide the first quantitative evidence of a molluscan predator on glass sponges found outside of Antarctica, and establish the first trophic link between glass sponges and their associated community of animals in a sponge reef ecosystem on the western Canadian continental shelf.     

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     

Glass sponges and bilaterian animals share derived mitochondrial genomic features: a common ancestry or parallel evolution?

Glass sponges (Hexactinellida) are a group of deep-water benthicanimals that have a unique syncytial organization and possessa characteristic siliceous skeleton. Although hexactinellidsare traditionally grouped with calcareous and demosponges inthe phylum Porifera, the monophyly of sponges and the phylogeneticposition of the Hexactinellida remain contentious. We determinedand analyzed the nearly complete mitochondrial genome sequencesof the hexactinellid sponges Iphiteon panicea and Sympagellanux. Unexpectedly, our analysis revealed several mitochondrialgenomic features shared between glass sponges and bilateriananimals, including an Arg Ser change in the genetic code, acharacteristic secondary structure of one of the serine tRNAs,highly derived tRNA and rRNA genes, and the presence of a singlelarge noncoding region. At the same time, glass sponge mtDNAcontains atp9, a gene previously found only in the mtDNA ofdemosponges (among animals), and encodes a with an atypical A11–U24 pair that is alsofound in demosponges and placozoans. Most of our sequence-basedphylogenetic analyses place Hexactinellida as the sister groupto the Bilateria; however, these results are suspect given acceleratedrates of mitochondrial sequence evolution in these groups. Thus,it remains an open question whether shared mitochondrial genomicfeatures in glass sponges and bilaterian animals reflect theirclose phylogenetic affinity or provide a remarkable exampleof parallel evolution.     

Silicateins identification of the freshwater sponge L. baicalensis

Siliceous spicules of the freshwater Baikal sponge Lubomirskia baicalensis contain several proteins including silicateins. Existences of four different genes of silicatein alpha (alpha1, alpha2, alpha3, alpha4) which are related to silicatein alpha from the sea sponges were found when cDNA library analysis was made. The intron-exon structure of the full-size silicatein alpha1 gene was determined. This gene has total length of 1988 bp and includes 6 introns (1007 bp) and 7 exons (981 bp). With use of mass-spectrometric analysis of the spicule proteins tryptic digest, two silicateins alpha were authentically found.     

Phylogenetic diversity of Gram-positive bacteria cultured from Antarctic deep-sea sponges

Gram-positive bacteria, specifically actinobacteria and members of the order Bacillales, are well-known producers of important secondary metabolites. Little is known about the diversity of Gram-positive bacteria associated with Antarctic deep-sea sponges. In this study, cultivation-based approaches were applied to investigate the Gram-positive bacteria associated with the Antarctic sponges Rossella nuda, Rossella racovitzae (Porifera: Hexactinellida), and Myxilla mollis, Homaxinella balfourensis, Radiella antarctica (Porifera: Demospongiae). In total, 46 Gram-positive strains were cultured. Phylogenetic analysis revealed that 24 strains were affiliated with the Actinobacteria, including six genera Streptomyces, Nocardiopsis, Pseudonocardia, Dietzia, Brachybacterium, and Brevibacterium. The other 22 strains were affiliated with the Firmicutes, and among them two (V17-1 and V179-1) only shared 92–95% 16S rRNA gene sequence identity with the nearest type strain. To our knowledge, this is the first report on the isolation of strains belonging to genera Dietzia and Brevibacterium from Antarctic sponges. All of the 46 strains were PCR screened for genes encoding polyketide synthases (PKS), and a selection of 36 isolates were used in subsequent bioassay analyses. Eighty-eight percentage of the isolates that possess a PKS gene were active against at least one test organism. The study confirms the existence of diverse bacteria in Antarctic sponges and their potential for producing active compounds.     

Eight new mtDNA sequences of glass sponges reveal an extensive usage of + 1 frameshifting in mitochondrial translation

Three previously studied mitochondrial genomes of glass sponges (phylum Porifera, class Hexactinellida) contained single nucleotide insertions in protein coding genes inferred as sites of + 1 translational frameshifting. To investigate the distribution and evolution of these sites and to help elucidate the mechanism of frameshifting, we determined eight new complete or nearly complete mtDNA sequences from glass sponges and examined individual mitochondrial genes from three others. We found nine new instances of single nucleotide insertions in these sequences and analyzed them both comparatively and phylogenetically. The base insertions appear to have been gained and lost repeatedly in hexactinellid mt protein genes, suggesting no functional significance for the frameshifting sites. A high degree of sequence conservation, the presence of unusual tRNAs, and a distinct pattern of codon usage suggest the “out-of-frame pairing” model of translational frameshifting. Additionally, we provide evidence that relaxed selection pressure on glass sponge mtDNA – possibly a result of their low growth rates and deep-water lifestyle – has allowed frameshift insertions to be tolerated for hundreds of millions of years. Our study provides the first example of a phylogenetically diverse and extensive usage of translational frameshifting in animal mitochondrial coding sequences.     

Molecular phylogeny of glass sponges (Porifera, Hexactinellida): increased taxon sampling and inclusion of the mitochondrial protein-coding gene, cytochrome oxidase subunit I

Marine sponges of the class Hexactinellida (glass sponges) are among the most understudied groups of Porifera, and molecular approaches to investigating their evolution have only recently emerged. Although these first results appeared reliable as they largely corroborated morphology-based hypotheses, they were almost exclusively based on ribosomal RNA genes (rDNA) and should, therefore, be further tested with independent types of genetic data, such as protein-coding genes. To this end, we established the mitochondrial-encoded cytochrome oxidase subunit I gene (COI) as an additional marker, and conducted phylogenetic analyses on DNA- and amino-acid level, as well as a supermatrix analysis based on combined COI DNA and rDNA alignments. Furthermore, we increased taxon sampling compared to previous studies by adding seven additional species. The COI-based phylogenies were largely congruent with the rDNA-based phylogeny but suffered from poor bootstrap support for many nodes. However, addition of the COI sequences to the rDNA data set increased resolution of the overall molecular phylogeny. Thus, although obtaining COI sequences from glass sponges turned out to be quite challenging, this gene appears to be a valuable supplement to rDNA data for molecular evolutionary studies of this group. Some implications of our extended phylogeny for the evolution and systematics of Hexactinellida are discussed.     

Silicateins, silicatein interactors and cellular interplay in sponge skeletogenesis: formation of glass fiber-like spicules

Biomineralization processes are characterized by controlled deposition of inorganic polymers/minerals mediated by functional groups linked to organic templates. One metazoan taxon, the siliceous sponges, has utilized these principles and even gained the ability to form these polymers/minerals by an enzymatic mechanism using silicateins. Silicateins are the dominant protein species present in the axial canal of the skeletal elements of the siliceous sponges, the spicules, where they form the axial filament. Silicateins also represent a major part of the organic components of the silica lamellae, which are cylindrically arranged around the axial canal. With the demosponge Suberites domuncula as a model, quantitative enzymatic studies revealed that both the native and the recombinant enzyme display in vitro the same biosilica-forming activity as the enzyme involved in spicule formation in vivo. Monomeric silicatein molecules assemble into filaments via fractal intermediates, which are stabilized by the silicatein-interacting protein silintaphin-1. Besides the silicateins, a silica-degrading enzyme silicase acting as a catabolic enzyme has been identified. Growth of spicules proceeds in vivo in two directions: first, by axial growth, a process that is controlled by evagination of cell protrusions and mediated by the axial filament-associated silicateins; and second, by appositional growth, which is driven by the extraspicular silicateins, a process that provides the spicules with their final size and morphology. This radial layer-by-layer accretion is directed by organic cylinders that are formed around the growing spicule and consist of galectin and silicatein. The cellular interplay that controls the morphogenetic processes during spiculogenesis is outlined.     

Phylogeny and evolution of glass sponges (porifera, hexactinellida)

Reconstructing the phylogeny of sponges (Porifera) is one of the remaining challenges to resolve the metazoan Tree of Life and is a prerequisite for understanding early animal evolution. Molecular phylogenetic analyses for two of the three extant classes of the phylum, Demospongiae and Calcarea, are largely incongruent with traditional classifications, most likely because of a paucity of informative morphological characters and high levels of homoplasy. For the third class, Hexactinellida (glass sponges)--predominantly deep-sea inhabitants with unusual morphology and biology--we present the first molecular phylogeny, along with a cladistic analysis of morphological characters. We collected 18S, 28S, and mitochondrial 16S ribosomal DNA sequences of 34 glass sponge species from 27 genera, 9 families, and 3 orders and conducted partitioned Bayesian analyses using RNA secondary structure-specific substitution models (paired-sites models) for stem regions. Bayes factor comparisons of different paired-sites models against each other and conventional (independent-sites) models revealed a significantly better fit of the former but, contrary to previous predictions, the least parameter-rich of the tested paired-sites models provided the best fit to our data. In contrast to Demospongiae and Calcarea, our rDNA phylogeny agrees well with the traditional classification and a previously proposed phylogenetic system, which we ascribe to a more informative morphology in Hexactinellida. We find high support for a close relationship of glass sponges and Demospongiae sensu stricto, though the latter may be paraphyletic with respect to Hexactinellida. Homoscleromorpha appears to be the sister group of Calcarea. Contrary to most previous findings from rDNA, we recover Porifera as monophyletic, although support for this clade is low under paired-sites models.     

Dynamics of spicule production in the marine sponge <Emphasis Type="Italic">Hymeniacidon perlevis</Emphasis> during in vitro cell culture and seasonal development in the field

To characterize the formation of silica spicules, the dynamics of spiculogenesis of an intertidal marine sponge Hymeniacidon perlevis (Montagu 1818) (Porifera: Demospongiae) were investigated by measuring the gene expression of silicatein (the enzyme responsible for spicule silicification) and the dimensional changes of spicules during the developmental process of individual sponges and in cell cultures of primmorphs of archaeocyte-dominant cell populations. The different developmental stages of spicules were documented by time-lapse microscopy and observed by transmission electron microscopy during a 1-month culture period. During its annual life cycle, H. perlevis has four different developmental stages: dormancy, resuscitation, bloom, and decline. Field-grown individual sponge samples at different stages were collected over 7 months (March to September 2005). The dimensions of the silica spicules from these samples were microscopically measured and statistically analyzed. This analysis and the material properties of the spicules allowed them to be classified into four groups representing the different developmental stages of spiculogenesis. Silicatein expression in the bloom stage was more than 100 times higher than that in the other stages and was correlated with the spicule developmental stage. The trend of spicule formation in field-grown sponges was consistent with the trend in cell culture. A new parameter, the maturation degree (MD) of spicules (defined as the ratio of actual to theoretical silica deposition of mature spicules), was introduced to quantify spicule development. Silica spiculogenesis during H. perlevis development was delineated by comparing MD and silicatein expression.     

Systematics and spicule evolution in dictyonal sponges (Hexactinellida: Sceptrulophora) with description of two new species

In this paper we report on recently collected specimens of glass sponges belonging to Farreidae Gray, 1872, and Tretodictyidae Schulze, 1886 (Porifera: Hexactinellida: Hexactinosida). All specimens represent new geographical records for their genera: Coral Sea for Aspidoscopulia Reiswig, 2002 (Farreidae) and Psilocalyx Ijima, 1927 (Tretodictyidae); north‐west Atlantic for Sarostegia Topsent, 1904 (Farreidae). Two new species, Aspidoscopulia australia Dohrmann, Göcke & Janussen sp. nov. and Aspidoscopulia ospreya Dohrmann, Göcke & Janussen sp. nov. , are described. To investigate further the evolution of hexactinosidan sponges, we sequenced two nuclear (18S and 28S rDNA) and two mitochondrial [16S ribosomal rDNA, cytochrome oxidase subunit I (COI)] genes from these specimens, as well as from a recently described new species of Lonchiphora Ijima, 1927 (Farreidae). Besides corroborating the monophyly of Tretodictyidae, our molecular phylogenetic analyses support a clade of clavule‐bearing sponges with a farreoid dictyonal framework (i.e. Farreidae sensu stricto). In contrast, Sarostegia, which lacks these features, appears unrelated to this clade – instead our data are consistent with an earlier placement of this genus in Euretidae Zittel, 1877. We introduce formally the taxon Sceptrulophora Mehl 1992, and emend the classification of Hexactinosida to reflect this move and our new findings regarding the position of Sarostegia. Finally, we discuss implications of the molecular phylogeny for the evolution of sceptrules, the defining autapomorphy of Sceptrulophora. © 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011, 163 , 1003–1025.     

Isolation of Ef silicatein and Ef lectin as molecular markers for sclerocytes and cells involved in innate immunity in the freshwater sponge Ephydatia fluviatilis

Sponges (phylum Porifera) have remarkable regenerative and reconstitutive abilities and represent evolutionarily the oldest metazoans. To investigate sponge stem cell differentiation, we have focused on the asexual reproductive system in the freshwater sponge Ephydatia fluviatilis. During germination, thousands of stem cells proliferate and differentiate to form a fully functional sponge. As an initial step of our investigation of stem cell (archeocyte) differentiation, we isolated molecular markers for two differentiated cell types: spicule-making sclerocyte cells, and cells involved in innate immunity. Sclerocyte lineage-specific Ef silicatein shares 45% to 62% identity with other sponge silicateins. As in situ hybridization of Ef silicatein specifically detects archeocytes possibly committed to sclerocytes, as well as sclerocytes with an immature or mature spicule, therefore covering all the developmental stages, we conclude that Ef silicatein is a suitable sclerocyte lineage marker. Ef lectin, a marker for the cell type involved in innate immunity, shares 59% to 65% identity with the marine sponge Suberites domuncula galactose-binding protein (Sd GBP) and horseshoe crab Tachypleus tridentatus tachylectin1/lectinL6. Since Sd GBP and tachylectin1 are known to bind to bacterial lipopolysaccharides and inhibit the growth of bacteria, Ef lectin may have a similar function and be expressed in a specialized type of cell involved in defense against invading bacteria. Ef lectin mRNA and protein are not expressed in early stages of development, but are detected in late stages. Therefore, Ef lectin may be specifically expressed in differentiating and/or differentiated cells. We suggest Ef lectin as a marker for cells that assume innate immunity in freshwater sponges.