Journal of Structural Biology 191 (2015) 165–174 Contents lists available at ScienceDirect Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi Electron microscope analyses of the bio-silica basal spicule from the Monorhaphis chuni sponge ⇑ Peter Werner a, , Horst Blumtritt a, Igor Zlotnikov b, Andreas Graff c, Yannicke Dauphin d, Peter Fratzl b a MPI of Microstructure Physics, Weinberg 2, D-06120 Halle (Saale), Germany b MPI of Colloids and Interfaces, Am Mühlenberg 1, D-14476 Potsdam, Germany c Fraunhofer Institute for Mechanics of Materials, 06120 Halle, Germany d Micropaléontologie, UFR TEB, Université P. & M. Curie, 75252 Paris Cedex 05, France article info abstract Article history: We report on a structural analysis of several basal spicules of the deep-sea silica sponge Monorhaphis Received 3 March 2015 chuni by electron microscope techniques supported by a precise focused ion beam (FIB) target prepara- Received in revised form 16 June 2015 tion. To get a deeper understanding of the spicules length growth, we concentrated our investigation onto Accepted 18 June 2015 the apical segments of two selected spicules with apparently different growth states and studied in detail Available online 19 June 2015 permanent and temporary growth structures in the central compact silica axial cylinder (AC) as well as the structure of the organic axial filament (AF) in its center. The new findings concern the following mor- Keywords: phology features: (i) at the tip we could identify thin silica layers, which overgrow as a tongue-like fea- Biosilica ture the front face of the AC and completely fuse during the subsequent growth state. This basically Biomineralization Silica sponge spicule differs from the radial growth of the surrounding lamellar zone of the spicules made of alternating silica SEM lamellae and organic interlayers. (ii) A newly detected disturbed cylindrical zone in the central region of TEM the AC (diameter about 30 lm) contains vertical and horizontal cavities, channels and agglomerates, STEM which can be interpreted as permanent leftover of a formerly open axial channel, later filled by silica. FIB (iii) The AF consists of a three-dimensional crystal-like arrangement of organic molecules and amorphous Monorhaphis chuni silica surrounding these molecules. Similar to an inorganic crystal, this encased protein crystal is typified by crystallographic directions, lattice planes and surface steps. The h001i growth direction is especially favored, thereby scaffolding the axial cylinders growth and consequently the spicules’ morphology. Ó 2015 Elsevier Inc. All rights reserved. 1. Introduction 2008), toughening and a reduced stiffness (Woesz et al., 2006; Zlotnikov et al., 2013). For both classes a thin organic axial filament Bio-minerals may have a complex mineral matrix structure (AF) with a diameter in the lm range is a typical feature of the spi- with organic interlayers or inclusions. Such composite structures cules’ center. It contains proteins, so-called silicatein (Shimizu are hierarchically organized with building units ranging from the et al., 1998; Müller et al., 2009), which are involved in the enzy- macroscopic to the nanometer scale. As an example, the two matic synthesis of the silica. The contribution of collagen for the classes of siliceous sponges – demosponges and hexactinellids – synthesis of silica is also under discussion (Ehrlich et al., 2008; build their skeletons of quite differently sized spicules. In the case Ehrlich, 2010). of demosponges, the mature spicules ultimately consist only of a The corresponding basic growth phenomena, including compact bio-silica cylinder with a central axial channel (Weaver bio-molecular processes as well as the morphologenesis, have been et al., 2010). In contrast, the spicules of hexactinellids are charac- intensively studied and widely understood in recent years (see, terized by an inner axial cylinder of more compact silica and a e.g., Wang et al., 2008, 2012). Nevertheless, several of detailed major outer lamellar structure of silica layers around it, which questions concerning structures and growth processes remain are separated by thin organic interlayers in the range of approxi- open. mately 50 nm. This laminar architecture generates improved The sponge Monorhaphis chuni is a well-known example of the mechanical properties, e.g., a fracture resistance (Miserez et al., hexactinellids. It represents one of the largest bio-silica structures (first described by Schulze, 1904). Its skeleton is built around a ⇑ giant basal spicule (GBS) of silica, which can reach a length up Corresponding author. to 2–3 m and a diameter up to approximately 1 cm. As an E-mail address: [email protected] (P. Werner). http://dx.doi.org/10.1016/j.jsb.2015.06.018 1047-8477/Ó 2015 Elsevier Inc. All rights reserved. 166 P. Werner et al. / Journal of Structural Biology 191 (2015) 165–174 introduction, Fig. 1 represents the basic morphological features of of a crystalline organization within the AFs was received by such a well-developed mature GBS of this species, observed by transmission electron microscopy (Garonne et al., 1981). The scanning electron microscopy (SEM) and by light optical micro- existence of crystalline phases in several sponge species was scopy. The present paper deals specifically with structural investi- later proven by X-ray diffraction, too (Croce et al., 2007). gations of such tip segments where the length growth takes place. Therefore, a similar phenomenon could be expected for the The SEM micrograph in Fig. 1a shows the typical silica lamellae, hexactinellids. Indeed, in a recent work the authors could which have a thickness in the range of 2–10 lm. Large, mature show unambiguously by high-resolution transmission GBSs can contain up to several hundreds of such lamellae. The electron microscopy (TEM), energy-dispersive X-ray lamellar outer zone imbeds a compact, non-lamellar, inner axial spectroscopy (EDX) and X-ray diffraction that the AF in cylinder of silica (AC, in the insert below Fig. 1a). Fig. 1b and c show the spicule of M. chuni is actually a hybrid material where corresponding light-microscopic images of the tip region from the silicatein molecules are arranged on a regular such a spicule. These – and most of the following optical micro- body-centered tetragonal lattice encased in mesoporous sil- graphs – were taken in dark-field mode since this technique is ica (Zlotnikov et al., 2014). In which way corresponding more sensitive to inner structural details within the silica rod. results would refer to the formation of the GBS? Thereby Fig. 1b and c clearly show a light-blue cylinder, which cor- (iii) Moreover, even by optical microscopy one easily can detect responds to the AC. In the present sample, it has a diameter of a heavily disturbed cylindrical zone (DCZ) with a diameter of approximately 150–200 lm along the whole investigated segment approximately 20–30 lm (see inside the white rectangle in of the spicule (see cylindrical scheme in Fig. 1c). Fig. 1d). This zone is magnified in Fig. 1e, where the distur- While the outer lamellar zone of the M. chuni GBS has been bances are visible as small bright dots. What is the morphol- studied in detail including its bio-mechanical properties (e.g., ogy of this DCZ and in which way it could be attributed to Miserez et al., 2008; Mayer, 2009), till now no deeper analysis of the GBS growth? the morphology of the AC, e.g., by electron microscopy techniques, was performed up to the knowledge of the authors. Open questions Related to these aspects, the present study is concentrated onto concern, e.g., the following aspects: the morphology and structure of the inner region of the AC, espe- cially at the tip segment of the spicules, where its length growth (i) What is the structure and morphology of the compact proceeds. We show that the disturbed zone in the AC around the AC? Is it formed in a subsequent sintering process from axial filament is likely to be associated with the initially open ‘‘ax- originally grown silica lamellae or is its growth mecha- ial channel’’ that is described in former studies of young spicule nism different from that of the lamellar zone from the tips (see, e.g., Müller et al., 2009). In this tip region, the length beginning? development proceeds and it can be regarded as the active and (ii) A second topic concerns the so-called axial filament in the youngest zone of the spicule. Here also temporary growth struc- AC axis. Inside this zone, along the axis of the spicule, a thin tures may be found, the analysis of which would lead to a better vertical stripe with a diameter of approximately 2 lmis insight in the length growth mechanism. Next, we demonstrate located (marked by an arrow in Fig. 1e) described already that the AF located in the center of the amorphous silica matrix, in the earliest reports of M. chuni (e.g., by Schulze, 1904) has a crystalline quality. Its building blocks consist of organic as an organic axial filament (see also Fig. 1a, insert). molecules imbedded in silica. Its crystallinity shows similarities Concerning its morphology recent studies discuss, on the to inorganic crystals. Furthermore, organic inclusions and precipi- one side, that the silicatein molecules within the AF are tates occur in the AC. arranged as bundles of fibers (Müller et al., 2008). On the For the structural and chemical analysis, we combined SEM, other side, in the case of demosponges, even a first evidence focused ion beam target milling (FIB), transmission electron Fig. 1. Morphology and cylindrical structure of the tip region of the basal spicule of Monorhaphis chuni observed by SEM (a) and by light microscopy (b–e). (a) The glass spicule consists of a major, lamellar silica shell and a non-lamellar silica core region, the axial cylinder (AC), schematically shown in the lower insert.