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.     

Formation of siliceous spicules in the marine demosponge <Emphasis Type="Italic">Suberites domuncula</Emphasis>

The siliceous skeleton of demosponges is constructed of spicules. We have studied the formation of spicules in primmorphs from Suberites domuncula. Scanning electron microscopy and transmission electron-microscopical (TEM) analyses have revealed, in the center of the spicules, an axial canal that is 0.3–1.6 m wide and filled with an axial filament. This filament is composed of the enzyme silicatein, which synthesizes the spicules. TEM analysis has shown that spicule formation starts intracellularly and ends extracellularly in the mesohyl. At the initial stage, the axial canal is composed only of silicatein, whereas membranous structures and fibrils (10–15 nm in width) can later also be identified, suggesting that intracellular components protrude into the axial canal. Antibodies against silicatein have been applied for Western blotting; intracellularly, silicatein is processed to the mature form (24 kDa), whereas the pro-enzyme with the propeptide (33 kDa) is detected extracellularly. Silicatein undergoes phosphorylation at five sites. Immunohistological analysis has shown that silicatein exists in the axial canal (axial filament) and on the surface of the spicules, suggesting that they grow by apposition. Finally, we have demonstrated that the enzymic reaction of silicatein is inhibited by anti-silicatein antibodies. These data provide, for the first time, a comprehensive outline of spicule formation.This work was supported by grants from the European Commission (SILIBIOTEC), the Deutsche Forschungsgemeinschaft, the Bundesministerium für Bildung und Forschung Germany (project: Center of Excellence BIOTECmarin) and the International Human Frontier Science Program.     

Formation of giant spicules in the deep-sea hexactinellid <Emphasis Type="Italic">Monorhaphis chuni</Emphasis> (Schulze 1904): electron-microscopic and biochemical studies

The siliceous sponge Monorhaphis chuni (Hexactinellida) synthesizes the largest biosilica structures on earth (3 m). Scanning electron microscopy has shown that these spicules are regularly composed of concentrically arranged lamellae (width: 3–10 μm). Between 400 and 600 lamellae have been counted in one giant basal spicule. An axial canal (diameter: ~2 μm) is located in the center of the spicules; it harbors the axial filament and is surrounded by an axial cylinder (100–150 μm) of electron-dense homogeneous silica. During dissolution of the spicules with hydrofluoric acid, the axial filament is first released followed by the release of a proteinaceous tubule. Two major proteins (150 kDa and 35 kDa) have been visualized, together with a 24-kDa protein that cross-reacts with antibodies against silicatein. The spicules are surrounded by a collagen net, and the existence of a hexactinellidan collagen gene has been demonstrated by cloning it from Aphrocallistes vastus. During the axial growth of the spicules, silicatein or the silicatein-related protein is proposed to become associated with the surface of the spicules and to be finally internalized through the apical opening to associate with the axial filament. Based on the data gathered here, we suggest that, in the Hexactinellida, the growth of the spicules is mediated by silicatein or by a silicatein-related protein, with the orientation of biosilica deposition being controlled by lectin and collagen. Carsten Eckert was previously with the Museum für Naturkunde, Invalidenstrasse 43, 10115 Berlin, Germany. The collagen sequence from Aphrocallistes vastus reported here, viz., [COL_APHRO] APHVACOL (accession number AM411124), has been deposited in the EMBL/GenBank data base. This work was supported by grants from the European Commission, the Deutsche Forschungsgemeinschaft, the Bundesministerium für Bildung und Forschung Germany (project: Center of Excellence BIOTECmarin), the National Natural Science Foundation of China (grant no. 50402023), and the International Human Frontier Science Program.     

Bio-sintering processes in hexactinellid sponges: Fusion of bio-silica in giant basal spicules from Monorhaphis chuni

The two sponge classes, Hexactinellida and Demospongiae, comprise a skeleton that is composed of siliceous skeletal elements (spicules). Spicule growth proceeds by appositional layering of lamellae that consist of silica nanoparticles, which are synthesized via the sponge-specific enzyme silicatein. While in demosponges during maturation the lamellae consolidate to a solid rod, the lamellar organization of hexactinellid spicules largely persists. However, the innermost lamellae, near the spicule core, can also fuse to a solid axial cylinder. Similar to the fusion of siliceous nanoparticles and lamella, in several hexactinellid species individual spicules unify during sintering-like processes. Here, we study the different stages of a process that we termed bio-sintering, within the giant basal spicule (GBS) of Monorhaphis chuni. During this study, a major GBS protein component (27 kDa) was isolated and analyzed by MALDI-TOF-MS. The sequences were used to isolate and clone the encoding cDNA via degenerate primer PCR. Bioinformatic analyses revealed a significant sequence homology to silicatein. In addition, the native GBS protein was able to mediate bio-silica synthesis in vitro. We conclude that the syntheses of bio-silica in M. chuni, and the subsequent fusion of nanoparticles to lamellae, and finally to spicules, are enzymatically-driven by a silicatein-like protein. In addition, evidence is now presented that in hexactinellids those fusions involve sintering-like processes.     

Apposition of silica lamellae during growth of spicules in the demosponge Suberites domuncula: biological/biochemical studies and chemical/biomimetical confirmation

Recently it has been discovered that the formation of the siliceous spicules of Demospongiae proceeds enzymatically (via silicatein) and occurs matrix guided (on galectin strings). In addition, it could be demonstrated that silicatein, if immobilized onto inorganic surfaces, provides the template for the synthesis of biosilica. In order to understand the formation of spicules in the intact organism, detailed studies with primmorphs from Suberites domuncula have been performed. The demosponge spicules are formed from several silica lamellae which are concentrically arranged around the axial canal, harboring the axial filament composed of silicatein. Now we show that the appositional growth of the spicules in radial and longitudinal direction proceeds in the extracellular space along hollow cylinders; their surfaces are formed by silicatein. The extracellularly located spicules are surrounded by sclerocytes which are filled with both electron-dense and electron-poor vesicles; energy dispersive X-ray analysis/scanning electron microscopical studies revealed that the electron-dense vesicles are filled of silicon/silica and therefore termed silicasomes. The release of the content of the silicasomes into the hollow cylinder suggests that the newly formed silica lamella originate there; in addition the data are compatible with the view that the silicatein molecules, attached at the centripetal and centrifugal surfaces, mediate biosilica formation. In a chemical/biomimetical approach silicatein is linked onto the organic material-free spicules after their functionalization with aminopropyltriethoxysilane [amino groups]-poly(acetoxime methacrylate) [reactive ester polymer]-N(epsilon)-benzyloxycarbonyl L-lysine tert-butyl ester-Ni(II); finally His-tagged silicatein is immobilized. The matrix-bound enzyme synthesized a new biosilica lamella. These bioinspired findings are considered as the basis for a technical use/application/utilization of hollow cylinders formed by matrix-guided silicatein molecules for the biocatalytic synthesis of nanostructured tubes.     

Biosilica formation in spicules of the sponge Suberites domuncula: synchronous expression of a gene cluster

The formation of spicules is a complicated morphogenetic process in sponges (phylum Porifera). The primmorph system was used to demonstrate that in the demosponge Suberites domuncula the synthesis of the siliceous spicules starts intracellularly and is dependent on the concentration of silicic acid. To understand spicule formation, a cluster of genes was isolated. In the center of this cluster is the silicatein gene, which codes for the enzyme that synthesizes spicules. This gene is flanked by an ankyrin repeat gene at one side and by a tumor necrosis factor receptor-associated factor and a protein kinase gene at the other side. All genes are strongly expressed in primmorphs and intact animals after exposure to silicic acid, and this expression is restricted to those areas where the spicule formation starts or where spicules are maintained in the animals. Our observations suggest that in S. domuncula a coordinated expression of physically linked genes is essential for the synthesis of the major skeletal elements.     

Expression of silicatein in spicules from the Baikalian sponge Lubomirskia baicalensis

Lake Baikal harbors the largest diversity of sponge species [phylum Porifera] among all freshwater biotopes. The abundantly occurring species Lubomirskia baicalensis was used to study the seasonal silicatein metabolism; the spicules of this species have an unusually thick axial filament, consisting of silicatein, which remains constant in diameter during their growth. In the course of maturation, the size of the silicic acid shell grows, until the final diameter of the spicules of about 8 microm is reached. The seasonal content of silicatein was assessed by use of antibodies raised against silicatein; they stained specifically the axial filaments. In addition we determined, by application of the enzyme-linked immunosorbent assay system, that the proteinaceous content of the spicules, the silicatein, increases from spring to late summer by 8-fold. As molecular markers to quantify the seasonal changes in expression levels of genes coding for proteins/enzymes, the genes for the calumenin-like protein and the kinesin-related protein, were selected. The expression of calumenin-like gene, involved in the intracellular signaling, is highest during September, whereas the expression of the kinesin-related protein does not change during the annual course. These results suggest that the highest metabolic activity of L. baicalensis occurs in late summer (September), in parallel with the highest accumulation of silicatein, a structural protein/enzyme of the spicules.     

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.     

Nanostructural features of demosponge biosilica

Recent interest in the optical and mechanical properties of silica structures made by living sponges, and the possibility of harnessing these mechanisms for the synthesis of advanced materials and devices, motivate our investigation of the nanoscale structure of these remarkable biomaterials. Scanning electron and atomic force microscopic (SEM and AFM) analyses of the annular substructure of demosponge biosilica spicules reveals that the deposited material is nanoparticulate, with a mean particle diameter of 74+/-13 nm. The nanoparticles are deposited in alternating layers with characteristic etchant reactivities. Further analyses of longitudinally fractured spicules indicate that each deposited layer is approximately monoparticulate in thickness and exhibits extensive long range ordering, revealing an unanticipated level of nanoscale structural complexity. NMR data obtained from differentially heated spicule samples suggest that the etch sensitivity exhibited by these annular domains may be related to variation in the degree of silica condensation, rather than variability in the inclusion of organics. In addition, AFM phase imaging in conjunction with results obtained from HF and alkaline etching experiments suggest that at various stages in spicule biosynthesis, regions of unusually low silica condensation are deposited, indicating a possible interruption in normal spicule formation. While this discovery of nanoparticulate silica aggregation in demosponge skeletal elements is likely to reflect the intrinsic kinetic tendency of silica to form such particles during polycondensation, the heirarchical organization of these nanoparticles is biologically unique.     

Investigation of the budding process in Tethya citrina and Tethya aurantium (Porifera, Demospongiae)

The budding process has been studied in two congeneric Mediterranean species belonging to Tethya from different sampling sites: Marsala and Venice Lagoons (Tethya citrina); Marsala Lagoon and Porto Cesareo Basin (Tethya aurantium). Buds, connected to the adult by a spiculated stalk, differ between the two species in morphology and size, since those of T. citrina are small with elongated bodies, showing only a few spicules protruding from the apical region, whereas those of T. aurantium are round, larger, and show spicules radiating from the peripheral border. In T. citrina, cells with inclusions, varying in electron density and size, represent the main cell types of the buds. In T. aurantium, the cell component shows a major diversification, resulting from spherulous cells, grey cells, vacuolar cells and peculiar micro-vesicle cells. Neither canals nor choanocyte chambers were observed in the buds of the two species. In T. citrina, bud production is similar in both sampling sites. In T. aurantium, budding occurs more rarely in Porto Cesareo Basin, probably in relation with environmental factors, such as the covering of the cortex by sediment and micro-algae. Finally, in the buds of both species, the spicule size does not differ from that of the cortex of the adult sponges, further supporting the main involvement of the cortex in organizing the skeletal architecture of the buds.     

A modern approach to demineralization of spicules in glass sponges (Porifera: Hexactinellida) for the purpose of extraction and examination of the protein matrix

Glass sponges of the class Hexactinellida are a group of the most ancient multicellular animals, whose fossil remnants from the early Proterozoic have been registered. In order to demineralize the skeletal structures of the glass sponge Hyalonema sieboldi, we have used for the first time a strategy of slow leaching of the silicon-bearing component, based on the usage of alkaline solutions of sodium hydroxide, sodium dodecyl sulfate, and an anionic biosurfactant of a rhamnolipid nature. The obtained data unequivocally corroborate the presence of a fibrillar protein matrix functioning as a basis for silicon biomineralization in the basal spicules of H. sieboldi. Also, it has been found for the first time that the protein matrix is constructed of a collagenous protein. The technical approach proposed here might appear important for the study of the structural organization of skeletons in other silicon-bearing animals and, in an applied aspect, to work out new biomaterials for implantology and biocomposites, in order to use the latter as bioactive additives.     

Intra-epithelial spicules in a homosclerophorid sponge

Attempts to understand the intricacies of biosilicification in sponges are hampered by difficulties in isolating and culturing their sclerocytes, which are specialized cells that wander at low density within the sponge body, and which are considered as being solely responsible for the secretion of siliceous skeletal structures (spicules). By investigating the homosclerophorid Corticium candelabrum, traditionally included in the class Demospongiae, we show that two abundant cell types of the epithelia (pinacocytes), in addition to sclerocytes, contain spicules intracellularly. The small size of these intracellular spicules, together with the ultrastructure of their silica layers, indicates that their silicification is unfinished and supports the idea that they are produced "in situ" by the epithelial cells rather than being incorporated from the intercellular mesohyl. The origin of small spicules that also occur (though rarely) within the nucleus of sclerocytes and the cytoplasm of choanocytes is more uncertain. Not only the location, but also the structure of spicules are unconventional in this sponge. Cross-sectioned spicules show a subcircular axial filament externally enveloped by a silica layer, followed by two concentric extra-axial organic layers, each being in turn surrounded by a silica ring. We interpret this structural pattern as the result of a distinctive three-step process, consisting of an initial (axial) silicification wave around the axial filament and two subsequent (extra-axial) silicification waves. These findings indicate that the cellular mechanisms of spicule production vary across sponges and reveal the need for a careful re-examination of the hitherto monophyletic state attributed to biosilicification within the phylum Porifera.     

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.     

Isolation and characterization of two T-box genes from sponges,the phylogenetically oldest metazoan taxon

It is now well established that all metazoan phyla derived from one common ancestor, the hypothetical Urmetazoa. Due to the basal position of Porifera (Demospongiae) in the phylogenetic tree of Metazoa, studies on the mechanisms controlling the development of these animals can provide clues on the understanding of the origin of multicellular animals and on how the first organization of the body plan evolved. In this report we describe the isolation and genomic characterization of two T-box genes from the siliceous sponge Suberites domuncula. The phylogenetic analysis classifies one into the subfamily of Brachyury, Sd-Bra, and the second into the Tbx2 subfamily, Sd-Tbx2. Analyses of the Sd-Bra and Sd-Tbx2 sequences and their intron-exon structures demonstrate their basal position in the phylogeny of the T-box family, and allows us to hypothesize a model of the phylogenetic evolution of all T-box genes. Furthermore, we report the presence of two different products of alternative splicing of Sd-Bra, and demonstrate that they exist in different phosphorylation and glycosylation states in the sponge tissue. Sd-Bra expression in tissue and 3D-cell aggregates (primmorphs) is analyzed, suggesting that Sd-Bra might also have a role in Porifera morphogenesis.Edited by N. SatohThe sequences from Suberites domuncula reported here are deposited in the EMBL/GenBank data base: the cDNA of Brachyury (Sd-Bra; accession number AJ544242) as well as the second T-box gene Sd-Tbx2 (AJ544241).     

Differential gene expression in response to brown planthopper feeding in rice

Plant responses to herbivores are complex. 108 cDNA clones representing genes relating to plant responses to chewing insect-feeding, pathogen infection, wounding and other stresses were collected. Northern blot and cDNA array analysis were employed to investigate gene expression regulated by piercing-sucking insect, brown planthopper (BPH), Nilaparvata lugens (Homoptera: Dephacidae) on both the resistant and susceptible rice genotypes. After BPH feeding in rice for 72 h, the expression of most tested genes was affected. 14 genes in resistant rice variety B5 and 44 genes in susceptible MH63 were significantly up- or down-regulated. Most of the well-regulated genes were grouped in the categories of signaling pathways, oxidative stress/apoptosis, wound-response, drought-inducible and pathogen-related proteins. Those related to the flavonoid pathway, aromatic metabolidsm and the octadecanoid pathway were mostly kept unchanged or down-regulated. Our results indicate that BPH feeding induces plant responses which would take part in a jasmonic acid-independent pathway and crosstalk with those related to abiotic stress, pathogen invasion and phytohormone signaling pathways.     

The pleiotropic effects induced by therolC gene in transgenic plants are caused by expression restricted to protophloem and companion cells

Expression of therolC gene fromAgrobacterium rhizogenes causes morphological and developmental alterations in transgenic plants. The histological alterations underlying the macroscopic changes and the cellular localization of the site of expression of therolC gene have shown that: (i) the expression of therolC gene is developmentally regulated, (ii) in vegetative transgenic plants, the expression of therolC gene under the control of its own promoter is restricted to companion and protophloem cells, (iii) the site of action of the product(s) of the activity of the rolC enzyme is distinct from its site of expression, (iv) precise localization of the rolC peptide has been achieved by immunocytochemistry but not by the histochemical GUS assay. These results imply that the sites of action and expression of therolC gene in trangenic plants are physically separated. Thus the product(s) of the activity of the rolC enzyme must be a factor capable of being transported. Current models forrolC gene action are discussed taking into account the reported results.     

Allocation of chemical and structural defenses in the sponge Melophlus sarasinorum

Sponges have evolved a variety of chemical and structural defense mechanisms to avoid predation. While chemical defense is well established in sponges, studies on structural defense are rare and with ambiguous results. We used field and laboratory experiments to investigate predation patterns and the anti-predatory defense mechanisms of the sponge Melophlus sarasinorum, a common inhabitant of Indo-pacific coral reefs. Specifically, we aimed to investigate whether M. sarasinorum is chemically or structurally defended against predation and if the defenses are expressed differently in the ectosomal and choanosomal tissue of the sponge. Chemical defense was measured as feeding deterrence, structural defense as feeding deterrence and toughness. Our results demonstrated that chemical defense is evenly distributed throughout the sponge and works in conjunction with a structurally defended ectosome to further reduce predation levels. The choanosome of the sponge contained higher protein levels, but revealed no structural defense. We conclude that the equal distribution of chemical defenses throughout M. sarasinorum is in accordance with Optimal Defense Theory (ODT) in regards to fish predation, while structural defense supports ODT by being restricted to the surface layer which experiences the highest predation risks from mesograzers.