Bioinspired synthesis of SnO crosses as backbone in artificial sponges.

The distinct electronic properties, including p-type semiconducting and a wide optical band gap, renders SnO suitable for applications such as microelectronic devices, gas sensors and electrodes. However, the synthesis of SnO is rather challenging due to the instability of the oxide, which is usually obtained as a by-product of SnO2 fabrication. In this work, we developed a bioinspired synthesis, based on a hydrothermal approach, for the direct production of SnO nanoparticles. The amount of mineralizer, inducing the precipitation, was identified, which supports a template-free formation of the nanosized SnO particles at low temperature and mild chemical conditions. Moreover, the SnO nanoparticles exhibit a shape of unique three-dimensional crosses similar to the calcite crosses present in the calcareous sponges. We demonstrated that SnO crosses are evenly distributed and embedded in an organic scaffold by an ice-templating approach, in this way closely mimicking the structure of calcareous sponges. Such scaffolds, reinforced by an active material, here SnO, could be used as filters, sensors or electrodes, where a high surface area and good accessibility are essential. This article is part of the theme issue 'Bioinspired materials and surfaces for green science and technology (part 2)'.

Incorporation of zinc into the coccoliths of the microalga Emiliania huxleyi.

The coccolithophore Emiliania huxleyi is covered with elaborated calcite plates, the so-called coccoliths, which are produced inside the cells. We investigated the incorporation of zinc into the coccoliths of E. huxleyi by applying different zinc and calcium amounts via the culture media and subsequently analyzing the zinc content in the cells and the Zn/Ca ratio of the coccoliths. To investigate the Zn/Ca ratio of coccoliths built in the manipulated media, the algae have first to be decalcified, i.e. coccolith free. We used a newly developed decalcification method to obtain 'naked' cells for cultivation. E. huxleyi proliferated and produced new coccoliths in all media with manipulated Zn/Ca ratios. The cells and the newly built coccoliths were investigated regarding their zinc content and their Zn/Ca ratio, respectively. High zinc amounts were taken up by the algae. The Zn/Ca ratio of the coccoliths was positively correlated to the Zn/Ca ratio of the applied media. The unique feature of the coccoliths was maintained also at high Zn/Ca ratios. We suggest the following pathway of the zinc ions into the coccoliths: first, the zinc ions are bound to the cell surface, followed by their transportation into the cytoplasm. Obviously, the zinc ions are removed afterwards into the coccolith vesicle, where the zinc is incorporated into the calcite coccoliths which are then extruded. The incorporation of toxic zinc ions into the coccoliths possibly due to a new function of the coccoliths as detoxification sites is discussed.

Behaviour of the plathelminth Symsagittifera roscoffensis under different light conditions and the consequences for the symbiotic algae Tetraselmis convolutae.

Symsagittifera roscoffensis is a plathelminth living in symbiosis with the green algae Tetraselmis convolutae. Host and symbiont are a model system for the study of endosymbiosis, which has so far mainly focused on their biochemical interactions. Symsagittifera roscoffensis is well known for its positive phototaxis that is hypothesized to optimize the symbiont's light perception for photosynthesis. In this study, we conducted a detailed analysis of phototaxis using light sources of different wavelength and brightness by videotracking. Furthermore, we compared the behavioural data with the electron transfer rate of the photosystem from cultured symbiotic cells. The symbiotic algae is adapted to low light conditions, showing a positive electron transfer rate at a photosynthetically active radiation of 0.112 µmol photons m(-2) s(-1), and S. roscoffensis showed a positive phototactic behaviour for light intensities up to 459.17 µmol photons m(-2) s(-1), which is not optimal regarding the needs of the symbiotic cells and may even harm host and symbiont. Red light cannot be detected by the animals and therefore their eyes seem not to be suitable for measuring the exact photosynthetically active radiation to the benefit of the photosymbionts.

Genesis of amorphous calcium carbonate containing alveolar plates in the ciliate Coleps hirtus (Ciliophora, Prostomatea).

In the protist world, the ciliate Coleps hirtus (phylum Ciliophora, class Prostomatea) synthesizes a peculiar biomineralized test made of alveolar plates, structures located within alveolar vesicles at the cell cortex. Alveolar plates are arranged by overlapping like an armor and they are thought to protect and/or stiffen the cell. Although their morphology is species-specific and of complex architecture, so far almost nothing is known about their genesis, their structure and their elemental and mineral composition. We investigated the genesis of new alveolar plates after cell division and examined cells and isolated alveolar plates by electron microscopy, energy-dispersive X-ray spectroscopy, FTIR and X-ray diffraction. Our investigations revealed an organic mesh-like structure that guides the formation of new alveolar plates like a template and the role of vesicles transporting inorganic material. We further demonstrated that the inorganic part of the alveolar plates is composed out of amorphous calcium carbonate. For stabilization of the amorphous phase, the alveolar vesicles, the organic fraction and the element phosphorus may play a role.

Functional mimicry of sea urchin biomineralization proteins with CaCO3-binding peptides selected by phage display.

The intricate process of biomineralization, e.g. in sea urchins, involves the precise interplay of highly regulated mineralization proteins and the spatiotemporal coordination achieved through compartmentalization. However, the investigation of biomineralization effector molecules, e.g. proteins, is challenging, due to their very low abundance. Therefore, we investigate the functional mimicry in the bioinspired precipitation of calcium carbonate (CaCO3) with artificial peptides selected from a peptide library by phage display based on peptide-binding to calcite and aragonite, respectively. The structure-directing effects of the identified peptides were compared to those of natural protein mixes isolated from skeletal (test) structures of two sea urchin species (Arbacia lixula and Paracentrotus lividus). The calcium carbonate samples deposited in the absence or presence of peptides were analyzed with a set of complementary techniques with regard to morphology, polymorph, and nanostructural motifs. Remarkably, some of the CaCO3-binding peptides induced morphological features in calcite that appeared similar to those obtained in the presence of the natural protein mixes. Many of the peptides identified as most effective in exerting a structure-directing effect on calcium carbonate crystallization were rich in basic amino acid residues. Hence, our in vitro mineralization study further highlights the important, but often neglected, role of positively charged soluble organic matrices associated with biological and bioinspired CaCO3 deposition.

Targeting of pancreatic and prostate cancer stem cell characteristics by Crambe crambe marine sponge extract.

Cancer stem cells (CSCs) are suggested as reason for resistance of tumors toward conventional tumor therapy including pancreatic and advanced prostate cancer. New therapeutic agents are urgently needed for targeting of CSCs. Marine sponges harbor novel and undefined compounds with antineoplastic activity but their potential to eliminate CSC characteristics is not examined so far. We collected 10 marine sponges and one freshwater sponge by diving at the seaside and prepared crude methanolic extracts. The effect to established pancreatic and prostate CSC lines was evaluated by analysis of apoptosis, cell cycle, side population, colony and spheroid formation, migratory potential in vitro and tumorigenicity in vivo. While each sponge extract at a 1:10 dilution efficiently diminished viability, Crambe crambe marine sponge extract (CR) still strongly reduced viability of tumor cells at a dilution of 1:1,000 but was less toxic to normal fibroblasts and endothelial cells. CR inhibited self-renewal capacity, apoptosis resistance, and proliferation even in gemcitabine-selected pancreatic cancer cells with acquired therapy resistance and enhanced CSC characteristics. CR pretreatment of tumor cells diminished tumorigenicity of gemcitabine-resistant tumor cells in mice and totally abolished tumor take upon combination with gemcitabine. Our data suggest that CR contains substances, which render standard cancer therapy more effective by targeting of CSC characteristics. Isolation of bioactive metabolites from CR and evaluation in mice are required for development of new CSC-specific chemotherapeutic drugs from a marine sponge.

Interaction between honeybee mandibles and propolis.

In a biomimetic top-down process, challenging the problem of resin deposition on woodworking machine tools, an adequate biological model was sought, which hypothetically could have developed evolutionary anti-adhesive strategies. The honeybee (Apis mellifera) was identified as an analogue model since it collects and processes propolis, which largely consists of collected tree resin. Propolis is a sticky substance used by bees to seal their hive and protect the colony against pathogens. In spite of its stickiness, honeybees are able to handle and manipulate propolis with their mandibles. We wanted to know if beneficial anti-adhesive properties of bee mandibles reduce propolis adhesion. The anatomy of bee mandibles was studied in a (cryo-)scanning electron microscope. Adhesion experiments were performed with propolis on bee mandibles to find out if bee mandibles have anti-adhesive properties that enable bees to handle the sticky material. A scale-like pattern was found on the inside of the mandible. Fresh mandibles were covered with a seemingly fluid substance that was at least partially removed during the washing process. Propolis adhesion on bee mandibles was measured to be 1 J/m2 and was indeed significantly lower compared to five technical materials. Propolis adhesion was higher on mandibles that were washed compared to fresh, unwashed mandibles. Results indicate that the medial surface of the mandible is covered with a fluid substance that reduces propolis adhesion. First results suggested that the surface pattern does do not have a direct effect on propolis adhesion.

Evidence that Indo-Pacific bottlenose dolphins self-medicate with invertebrates in coral reefs.

Indo-Pacific bottlenose dolphins (Tursiops aduncus) have been observed queueing up in natural environments to rub particular body parts against selected corals (Rumphella aggregata, Sarcophyton sp.) and sponges (Ircinia sp.) in the Egyptian Northern Red Sea. It was hypothesized that the presence of bioactive metabolites accounts for this selective rubbing behavior. The three invertebrates preferentially accessed by the dolphins, collected and analyzed by hyphenated high-performance thin-layer chromatography contained seventeen active metabolites, providing evidence of potential self-medication. Repeated rubbing allows these active metabolites to come into contact with the skin of the dolphins, which in turn could help them achieve skin homeostasis and be useful for prophylaxis or auxiliary treatment against microbial infections. This interdisciplinary research in behavior, separation science, and effect-directed analysis highlighted the importance of particular invertebrates in coral reefs, the urgent need to protect coral reefs for dolphins and other species, and calls for further vertebrate-invertebrate interaction studies.

In situ laboratory for plastic degradation in the Red Sea.

Degradation and fragmentation of plastics in the environment are still poorly understood. This is partly caused by the lack of long-term studies and methods that determine weathering duration. We here present a novel study object that preserves information on plastic age: microplastic (MP) resin pellets from the wreck of the SS Hamada, a ship that foundered twenty-nine years ago at the coast of Wadi el Gemal national park, Egypt. Its sinking date enabled us to precisely determine how long MP rested in the wreck and a nearby beach, on which part of the load was washed off. Pellets from both sampling sites were analyzed by microscopy, X-ray tomography, spectroscopy, calorimetry, gel permeation chromatography, and rheology. Most pellets were made of low-density polyethylene, but a minor proportion also consisted of high-density polyethylene. MP from inside the wreck showed no signs of degradation compared to pristine reference samples. Contrary, beached plastics exhibited changes on all structural levels, which sometimes caused fragmentation. These findings provide further evidence that plastic degradation under saltwater conditions is comparatively slow, whereas UV radiation and high temperatures on beaches are major drivers of that process. Future long-term studies should focus on underlying mechanisms and timescales of plastic degradation.

Mitochondrial genome evolution in Ophiuroidea, Echinoidea, and Holothuroidea: insights in phylogenetic relationships of Echinodermata.

The genome architecture and amino acid sequences of six new complete mitochondrial genomes were determined from representatives of Hemichordata (1), Ophiuroidea (3), Echinoidea (1) and Holothuroidea (1) and were analysed together with previously known sequences. Phylogenetic analyses recovered three lineages within echinoderms, Crinoidea, Ophiuroidea and a group comprising Holothuroidea, Echinoidea, and Asteroidea. In contrast to previous analyses of mitochondrial genomes the increased data set recovered the classical echinoderm phylogeny of Eleutherozoa and Echinozoa in Maximum Likelihood and Bayesian analyses using hemichordate out-group representatives. However, an inconsistent ramification appeared with vertebrate out-groups and in Maximum Parsimony and Neighbour Joining reconstructions. The basal (consensus) gene orders of all three lineages could be derived from a hypothetical ancestral crinoid gene order by one single rearrangement in each lineage. The genome architecture was highly conserved in Echinoidea, whereas the highest gene order differences and large amounts of unassigned sequences (UAS) were detected in Ophiuroidea, supporting a higher evolutionary rate than in any other echinoderm lineage. The variability in gene order and UAS regions in ophiuroid genomes suggest dominating rearrangement mechanisms by duplication events.

Cultivation-Independent Analysis of the Bacterial Community Associated With the Calcareous Sponge Clathrina clathrus and Isolation of Poriferisphaera corsica Gen. Nov., Sp. Nov., Belonging to the Barely Studied Class Phycisphaerae in the Phylum Planctomycetes.

Marine ecosystems serve as global carbon sinks and nutrient source or breeding ground for aquatic animals. Sponges are ancient parts of these important ecosystems and can be found in caves, the deep-sea, clear waters, or more turbid environments. Here, we studied the bacterial community composition of the calcareous sponge Clathrina clathrus sampled close to the island Corsica in the Mediterranean Sea with an emphasis on planctomycetes. We show that the phylum Planctomycetes accounts for 9% of the C. clathrus-associated bacterial community, a 5-fold enrichment compared to the surrounding seawater. Indeed, the use of C. clathrus as a yet untapped source of novel planctomycetal strains led to the isolation of strain KS4T. The strain represents a novel genus and species within the class Phycisphaerae in the phylum Planctomycetes and displays interesting cell biological features, such as formation of outer membrane vesicles and an unexpected mode of cell division.

Cultivation and functional characterization of 79 planctomycetes uncovers their unique biology.

When it comes to the discovery and analysis of yet uncharted bacterial traits, pure cultures are essential as only these allow detailed morphological and physiological characterization as well as genetic manipulation. However, microbiologists are struggling to isolate and maintain the majority of bacterial strains, as mimicking their native environmental niches adequately can be a challenging task. Here, we report the diversity-driven cultivation, characterization and genome sequencing of 79 bacterial strains from all major taxonomic clades of the conspicuous bacterial phylum Planctomycetes. The samples were derived from different aquatic environments but close relatives could be isolated from geographically distinct regions and structurally diverse habitats, implying that 'everything is everywhere'. With the discovery of lateral budding in 'Kolteria novifilia' and the capability of the members of the Saltatorellus clade to divide by binary fission as well as budding, we identified previously unknown modes of bacterial cell division. Alongside unobserved aspects of cell signalling and small-molecule production, our findings demonstrate that exploration beyond the well-established model organisms has the potential to increase our knowledge of bacterial diversity. We illustrate how 'microbial dark matter' can be accessed by cultivation techniques, expanding the organismic background for small-molecule research and drug-target detection.

Diversity and abundance of photosynthetic sponges in temperate Western Australia.

BACKGROUND: Photosynthetic sponges are important components of reef ecosystems around the world, but are poorly understood. It is often assumed that temperate regions have low diversity and abundance of photosynthetic sponges, but to date no studies have investigated this question. The aim of this study was to compare the percentages of photosynthetic sponges in temperate Western Australia (WA) with previously published data on tropical regions, and to determine the abundance and diversity of these associations in a range of temperate environments. RESULTS: We sampled sponges on 5 m belt transects to determine the percentage of photosynthetic sponges and identified at least one representative of each group of symbionts using 16S rDNA sequencing together with microscopy techniques. Our results demonstrate that photosynthetic sponges are abundant in temperate WA, with an average of 63% of sponge individuals hosting high levels of photosynthetic symbionts and 11% with low to medium levels. These percentages of photosynthetic sponges are comparable to those found on tropical reefs and may have important implications for ecosystem function on temperate reefs in other areas of the world. A diverse range of symbionts sometimes occurred within a small geographic area, including the three "big" cyanobacterial clades, Oscillatoria spongeliae, "Candidatus Synechococcus spongiarum" and Synechocystis species, and it appears that these clades all occur in a wide range of sponges. Additionally, spongin-permeating red algae occurred in at least 7 sponge species. This study provides the first investigation of the molecular phylogeny of rhodophyte symbionts in sponges. CONCLUSION: Photosynthetic sponges are abundant and diverse in temperate WA, with comparable percentages of photosynthetic to non-photosynthetic sponges to tropical zones. It appears that there are three common generalist clades of cyanobacterial symbionts of sponges which occur in a wide range of sponges in a wide range of environmental conditions.

DNA damage in storage cells of anhydrobiotic tardigrades.

In order to recover without any apparent damage, tardigrades have evolved effective adaptations to preserve the integrity of cells and tissues in the anhydrobiotic state. Despite those adaptations and the fact that the process of biological ageing comes to a stop during anhydrobiosis, the time animals can persist in this state is limited; after exceedingly long anhydrobiotic periods tardigrades fail to recover. Using the single cell gel electrophoresis (comet assay) technique to study the effect of anhydrobiosis on the integrity of deoxyribonucleic acid, we showed that the DNA in storage cells of the tardigrade Milnesium tardigradum was well protected during transition from the active into the anhydrobiotic state. Specimens of M. tardigradum that had been desiccated for two days had only accumulated minor DNA damage (2.09 +/- 1.98% DNA in tail, compared to 0.44 +/- 0.74% DNA in tail for the negative control with active, hydrated animals). Yet the longer the anhydrobiotic phase lasted, the more damage was inflicted on the DNA. After six weeks in anhydrobiosis, 13.63 +/- 6.41% of DNA was found in the comet tail. After ten months, 23.66 +/- 7.56% of DNA was detected in the comet tail. The cause for this deterioration is unknown, but oxidative processes mediated by reactive oxygen species are a possible explanation.

Depth-related alkaloid variation in Mediterranean Aplysina sponges.

Total amounts and patterns of bromoisoxazoline alkaloids of Aplysina sponges from Croatia (Mediterranean Sea) were analyzed along an underwater slope ranging from 1.8 to 38.5 m. Total amounts of alkaloids varied from sample to sample and showed no correlation with depth. In contrast, striking differences of alkaloid patterns were found between sponges from shallow sites (1.8-11.8 m) and those collected from deeper sites (11.8-38.5 m). Sponges from shallow depths consistently exhibited alkaloid patterns typical for Aplysina aerophoba with aerophobin-2 (2) and isofistularin-3 (3) as main constituents. Sponges from deeper sites (below 11.8 m) resembled Aplysina cavernicola with aerothionin (4) and aplysinamisin-1 (1) as major compounds. The typical A. cavernicola pigment 3,4-dihydroxyquinoline-2-carboxylic acid (6), however, could not be detected in A. aerophoba sponges but was replaced by the A. aerophoba pigment uranidine (5) which appeared to be present in all sponge samples analyzed. During transplantation experiments sponges from sites below 30 m featuring the A. cavernicola chemotype of bromoisoxazoline alkaloids were translocated to shallower habitats (10 m). The alkaloid patterns in transplanted sponges were found to be stable over a period of 12 months and unaffected by this change in depth. In a further experiment, clones of Aplysina sponges from shallow depths of 5-6 m resembling the A. aerophoba chemotype were either kept in situ under natural light conditions or artificially shaded by excluding photosynthetically active radiation (PAR). Neither 4 nor 1 were detected in artificially shaded sponges over an observation period of 12 months. In summary, two chemically distinct types of Aplysina sponges were discovered in this study that proved to be remarkably stable with regard to the bromoisoxazoline patterns and unaffected either by changing the light conditions or depth. It is not clear presently whether the Aplysina sponges collected from depths < 11.8 m represent a new chemotype of A. cavernicola lacking the pigment 6 or whether we have incidentally come across a so far undescribed species of the genus Aplysina.

Heat shock proteins (Hsp70) and water content in the estivating Mediterranean Grunt Snail (Cantareus apertus).

Pulmonate land snails often are able to estivate to survive dry hot seasons were water and food are scarce. The aperture of the shell is closed with an epiphragm, and metabolism is depressed to approximately one fourth of basal metabolism. We investigated a molecular aspect of estivation focussing on the heat shock protein 70 (Hsp70) stress response during estivation in the Mediterranean Grunt Snail Cantareus apertus. Sequences of a new inducible hsp70 and of actin are presented and expression of the hsp70 gene as well as Hsp70 protein content was measured in estivating animals. Both Hsp70 protein and mRNA do not show a significant change from the control, although there is a trend that hsp70 mRNA is less abundant in estivating specimens. After heat shock, the expression of hsp70 increased and a higher Hsp70 protein content was detected. Water relations were also investigated. After a period of 6 months in the dormant state, the snails contained 14% less water than active ones, implying a constricted protection against desiccation, compared to the desert snail Sphincterochila zonata, and a Mediterranean-type water economy.

Metallic nickel nanoparticles and their effect on the embryonic development of the sea urchin Paracentrotus lividus.

The presence of nanoparticles in many industrial applications and daily products is making it nowadays crucial to assess their impact when exposed to the environment. Metallic nickel nanoparticles (Ni NPs) are of high industrial interest due to their ability to catalyze the reversible hydration of CO2 to carbonic acid at ambient conditions. We characterized metallic Ni NPs by XRD, HRTEM and EDS and determined the solubility of free nickel ions from 3 mg/L metallic Ni NPs in seawater by ICP-MS over 96 h, which was below 3%. Further, embryonic development of the sea urchin Paracentrotus lividus was investigated for 48 h in the presence of metallic Ni NPs (0.03 mg/L to 3 mg/L), but no lethal effects were observed. However, 3 mg/L metallic Ni NPs caused a size reduction similar to 1.2 mg/L NiCl2*6 H2O. The obtained results contribute to current studies on metallic Ni NPs and point to their consequences for the marine ecosystem.

Characterization of an Alpha Type Carbonic Anhydrase from Paracentrotus lividus Sea Urchin Embryos.

Carbonic anhydrases (CA) are zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate. In the sea urchin, CA has a role in the formation of the calcitic skeleton during embryo development. Here, we report a newly identified mRNA sequence from embryos of the sea urchin Paracentrotus lividus, referred to as Pl-can. The complete coding sequence was identified with the aid of both EST databases and experimental procedures. Pl-CAN is a 447 aa-long protein, with an estimated molecular mass of 48.5 kDa and an isoelectric point of 6.83. The in silico study of functional domains showed, in addition to the alpha type CA-specific domain, the presence of an unexpected glycine-rich region at the N-terminal of the molecule. This is not found in any other species described so far, but probably it is restricted to the sea urchins. The phylogenetic analysis indicated that Pl-CAN is evolutionarily closer to human among chordates than to other species. The putative role(s) of the identified domains is discussed. The Pl-can temporal and spatial expression profiles, analyzed throughout embryo development by comparative qPCR and whole-mount in situ hybridization (WMISH), showed that Pl-can mRNA is specifically expressed in the primary mesenchyme cells (PMC) of the embryo and levels increase along with the growth of the embryonic skeleton, reaching a peak at the pluteus stage. A recombinant fusion protein was produced in E. coli and used to raise specific antibodies in mice recognized the endogenous Pl-CAN by Western blot in embryo extracts from gastrula and pluteus.

The test skeletal matrix of the black sea urchin Arbacia lixula.

In the field of biomineralization, the past decade has been marked by the increasing use of high throughput techniques, i.e. proteomics, for identifying in one shot the protein content of complex macromolecular mixtures extracted from mineralized tissues. Although crowned with success, this approach has been restricted so far to a limited set of key-organisms, such as the purple sea urchin Strongylocentrotus purpuratus, the pearl oyster or the abalone, leaving in the shadow non-model organisms. As a consequence, it is still unknown to what extent the calcifying repertoire varies, from group to group, at high (phylum, class), median (order, family) or low (genus, species) taxonomic rank. The present paper shows the first biochemical and proteomic characterization of the test matrix of the Mediterranean black sea urchin Arbacia lixula (Arbacioida). Our work suggests that the skeletal repertoire of A. lixula exhibits some similarities but also several differences with that of the few sea urchin species (S. purpuratus, Paracentrotus lividus), for which molecular data are already available. The differences may be attributable to the taxonomic position of the species considered: A. lixula belongs to an order - Arbacioida - that diverged more than one hundred million years ago from the Camarodonta, which includes the two species S. purpuratus and P. lividus. For the echinoid class, we suggest that large-scale proteomic screening should be performed in order to understand which molecular functions related to calcification are conserved and which ones have been co-opted for biomineralization in particular lineages.

Spine and test skeletal matrices of the Mediterranean sea urchin Arbacia lixula--a comparative characterization of their sugar signature.

Calcified structures of sea urchins are biocomposite materials that comprise a minor fraction of organic macromolecules, such as proteins, glycoproteins and polysaccharides. These macromolecules are thought to collectively regulate mineral deposition during the process of calcification. When occluded, they modify the properties of the mineral. In the present study, the organic matrices (both soluble and insoluble in acetic acid) of spines and tests from the Mediterranean black sea urchin Arbacia lixula were extracted and characterized, in order to determine whether they exhibit similar biochemical signatures. Bulk characterizations were performed by mono-dimensional SDS/PAGE, FT-IR spectroscopy, and an in vitro crystallization assay. We concentrated our efforts on characterization of the sugar moieties. To this end, we determined the monosaccharide content of the soluble and insoluble organic matrices of A. lixula spines and tests by HPAE-PAD, together with their respective lectin-binding profiles via enzyme-linked lectin assay. Finally, we performed in situ localization of N-acetyl glucosamine-containing saccharides on spines and tests using gold-conjugated wheatgerm agglutinin. Our data show that the test and spine matrices exhibit different biochemical signatures with regard to their saccharidic fraction, suggesting that future studies should analyse the regulation of mineral deposition by the matrix in these two mineralized structures in detail. This study re-emphasizes the importance of non-protein moieties, i.e. sugars, in calcium carbonate systems, and highlights the need to clearly identify their function in the biomineralization process.