Formation of giant spicules in the deep-sea hexactinellid Monorhaphis chuni (Schulze 1904): electron-microscopic and biochemical studies

Werner E. G. Mller 0 1 2 Carsten Eckert 0 1 2 Klaus Kropf 0 1 2 Xiaohong Wang 0 1 2 Ute Schlomacher 0 1 2 Christopf Seckert 0 1 2 Stephan E. Wolf 0 1 2 Wolfgang Tremel 0 1 2 Heinz C. Schrder 0 1 2 0 This work was supported by grants from the European Commission, the Deutsche Forschungsgemeinschaft, the Bundesministerium fr 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 1 Carsten Eckert was previously with the Museum fr Naturkunde, Invalidenstrasse 43, 10115 Berlin, Germany 2 X. Wang National Research Center for Geoanalysis , 26 Baiwanzhuang Dajie, 100037 Beijing, China 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 m). Between 400 and 600 lamellae have been counted in one giant basal spicule. An axial canal (diameter: ~2 m) is located in the center of the spicules; it harbors the axial filament and is surrounded by an axial cylinder (100-150 m) 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. - The collagen sequence from Aphrocallistes vastus reported here, viz., [COL_APHRO] APHVACOL (accession number AM411124), has been deposited in the EMBL/GenBank data base. The phylum Porifera (sponges) has been subdivided into three classes, viz., the Hexactinellida, Demospongiae, and Calcarea, whereby the first two taxa comprise individuals with a siliceous skeleton composed of spicules, and members of the last-mentioned taxon have calcareous (calcium carbonate) skeletal elements. Based on molecular biological (Kruse et al. 1997) and geological (Reitner and Wrheide 2002) studies, the Hexactinellida taxon has been established as the oldest, having evolved between Neoproterozoic glaciations (720585 Ma), sometimes referred to as snowball Earth (for a review, see Corsetti et al. 2006). During this period, a cycle of silicate weathering and carbonate precipitation, prior to or simultaneously with the glaciations, has been proposed to have resulted in the dissolution of surface rocks composed of insoluble silicates (CaSiO3). Subsequently, soluble calcium carbonate (CaCO3) and soluble silica (SiO2) was formed by reaction with atmospheric CO2 (Walker et al. 1981). In consequence, the level of dissolved silicic acid in seawater increased providing the inorganic starting material for the formation of siliceous spicules. Physical and chemical analyses have revealed that the siliceous skeletons of the two siliceous sponge classes are composed of amorphous silica with a water content of around 10% (Sandford 2003). Analysis of infrared spectra have shown a distinct difference between the two taxa of between 1,080 and 1,100 cm1 suggesting a different molecular configuration involving the Si-O-Si linkage (Sandford 2003). Other than silicic acid, only small amounts of trace elements (<3%) are found in the spicules. During the past few years, some aspects of the mechanism of spicule formation in Demospongiae have been clarified. Work mainly with Tethya aurantium (Shimizu et al. 1998; Cha et al. 1999) and Suberites domuncula (Krasko et al. 2000) has established that, in contrast to silica deposition in other organisms (e.g., in diatoms), biosilica formation in sponges is mediated enzymatically via the enzyme silicatein. Silicatein forms the axial filament of the spicules from which the growth of the spicules starts intracellularly (Shimizu et al. 1998; Mller et al. 2003, 2005; Wang and Wang 2006). Although marine sponges contain two isoforms of silicatein at least, freshwater sponges contain up to five silicatein isoforms, indicating the more complex formation and organization of the spicules in these animals (Mller et al. 2006). In S. domuncula, and probably also in other Demospongiae, spicule formation starts intracellularly. After reaching a size of approximately 68 m, the macroscleres (spicules of >10 m) are extruded from the cells (sclerocytes) and completed extracellularly, where they may reach lengths of over 100 m (Mller et al. 2005). In the extracellular space, both the final longitudinal size and the final thickness of the spicules are reached by appositional growth (Mller et al. 2006). These processes are mediated by silicatein and controlled by galectin, which acts as an organic matrix for the attachment of silicatein molecules (Schrder et al. 2006). Collagen probably provides the structural groove along which the galectin-silicatein strings are guided, thus allowing the formation of the intricately architectured spicules (Eckert et al. 2006). The monophyletic group of Hexactinellida is, according to the form and organization of its spicules, divided into two subclasses: the Hexasterophora and Amphidiscophora (for a review, see Reiswig 2006). In the latter subclass, amphidiscs are the (main) microscleres and never fuse. One member of this subclass is the family Monorhaphididae, which includes three described species: Monorhaphis chuni, M. dives, and M. intermedia. Since the discovery of these sponges during the first German deep-sea expedition (RV Valdivia) and the first descriptions of these animals by Chun (1900) and Schulze (1904), only little additional information has been added regarding the form and construction of their skeletal systems. Nothing has been published concerning the synthesis of their giant spicules. Worldwide, M. chuni has been documented only at a few sampling sites (Tabachnick 2002), suggesting a distribution in the deep sea in the Indian Ocean and in the West Pacific. One outstanding feature of Monorhaphis is its anchoring spicule, which can reach lengths of up to 3 m with a maximum diameter of 8.5 mm (Schulze 1904). M. chuni thus produces the largest biosilica structure known on Earth. Polished cross sections confirm the existence of up to 500 highly regular concentric rings that are (...truncated)