ROCK and the actomyosin network control biomineral growth and morphology during sea urchin skeletogenesis.

Biomineralization had apparently evolved independently in different phyla, using distinct minerals, organic scaffolds, and gene regulatory networks (GRNs). However, diverse eukaryotes from unicellular organisms, through echinoderms to vertebrates, use the actomyosin network during biomineralization. Specifically, the actomyosin remodeling protein, Rho-associated coiled-coil kinase (ROCK) regulates cell differentiation and gene expression in vertebrates' biomineralizing cells, yet, little is known on ROCK's role in invertebrates' biomineralization. Here, we reveal that ROCK controls the formation, growth, and morphology of the calcite spicules in the sea urchin larva. ROCK expression is elevated in the sea urchin skeletogenic cells downstream of the Vascular Endothelial Growth Factor (VEGF) signaling. ROCK inhibition leads to skeletal loss and disrupts skeletogenic gene expression. ROCK inhibition after spicule formation reduces the spicule elongation rate and induces ectopic spicule branching. Similar skeletogenic phenotypes are observed when ROCK is inhibited in a skeletogenic cell culture, indicating that these phenotypes are due to ROCK activity specifically in the skeletogenic cells. Reduced skeletal growth and enhanced branching are also observed under direct perturbations of the actomyosin network. We propose that ROCK and the actomyosin machinery were employed independently, downstream of distinct GRNs, to regulate biomineral growth and morphology in Eukaryotes.

Myriad Mapping of nanoscale minerals reveals calcium carbonate hemihydrate in forming nacre and coral biominerals.

Calcium carbonate (CaCO3) is abundant on Earth, is a major component of marine biominerals and thus of sedimentary and metamorphic rocks and it plays a major role in the global carbon cycle by storing atmospheric CO2 into solid biominerals. Six crystalline polymorphs of CaCO3 are known-3 anhydrous: calcite, aragonite, vaterite, and 3 hydrated: ikaite (CaCO3·6H2O), monohydrocalcite (CaCO3·1H2O, MHC), and calcium carbonate hemihydrate (CaCO3·½H2O, CCHH). CCHH was recently discovered and characterized, but exclusively as a synthetic material, not as a naturally occurring mineral. Here, analyzing 200 million spectra with Myriad Mapping (MM) of nanoscale mineral phases, we find CCHH and MHC, along with amorphous precursors, on freshly deposited coral skeleton and nacre surfaces, but not on sea urchin spines. Thus, biomineralization pathways are more complex and diverse than previously understood, opening new questions on isotopes and climate. Crystalline precursors are more accessible than amorphous ones to other spectroscopies and diffraction, in natural and bio-inspired materials.

Gradients of Orientation, Composition, and Hydration of Proteins for Efficient Light Collection by the Cornea of the Horseshoe Crab.

The lateral eyes of the horseshoe crab, Limulus polyphemus, are the largest compound eyes within recent Arthropoda. The cornea of these eyes contains hundreds of inward projecting elongated cuticular cones and concentrate light onto proximal photoreceptor cells. Although this visual system has been extensively studied before, the precise mechanism allowing vision has remained controversial. Correlating high-resolution quantitative refractive index (RI) mapping and structural analysis, it is demonstrated how gradients of RI in the cornea stem from structural and compositional gradients in the cornea. In particular, these RI variations result from the chitin-protein fibers architecture, heterogeneity in protein composition, and bromine doping, as well as spatial variation in water content resulting from matrix cross-linking on the one hand and cuticle porosity on the other hand. Combining the realistic cornea structure and measured RI gradients with full-wave optical modeling and ray tracing, it is revealed that the light collection mechanism switches from refraction-based graded index (GRIN) optics at normal light incidence to combined GRIN and total internal reflection mechanism at high incident angles. The optical properties of the cornea are governed by different mechanisms at different hierarchical levels, demonstrating the remarkable versatility of arthropod cuticle.

Principles of elastic bridging in biological materials.

Load-bearing biological materials employ specialized elastic bridging regions to connect material parts with substantially different properties. While such bridging regions emerge in diverse systems of biological systems, their functional-mechanical origins are yet disclosed. Here, we hypothesize that these elastic bridging regions evolved primarily to minimize the near-interface stress effects in the biological material and, supported by experiments and simulations, we develop a simple theoretical model for such stress-minimizing bridging modulus. Our theoretical model describes well extensive experimental data of diverse biomechanical systems, suggesting that despite their compositionally distinct bridging regions, they share a similar mechanical adaptation strategy for stress minimization. The theoretical model developed in this study may directly serve as a design guideline for bio-inspired materials, biomedical applications, and advanced interfacial architectures with high resilience to mechanical failure. STATEMENT OF SIGNIFICANCE: Biological materials exhibit unconventional structural-mechanical strategies allowing them to attain extreme load-bearing capabilities. Here, we identify the strategy of biological materials to connect parts of distinct elastic properties in an optimal manner of stress minimization. Our findings are compatible with broad types of biological materials, including biopolymers, biominerals, and their bio-composite combinations, and may promote novel engineering designs of advanced biomedical and synthetic materials.

Multiscale X-ray study of Bacillus subtilis biofilms reveals interlinked structural hierarchy and elemental heterogeneity.

Biofilms are multicellular microbial communities that encase themselves in an extracellular matrix (ECM) of secreted biopolymers and attach to surfaces and interfaces. Bacterial biofilms are detrimental in hospital and industrial settings, but they can be beneficial, for example, in agricultural as well as in food technology contexts. An essential property of biofilms that grants them with increased survival relative to planktonic cells is phenotypic heterogeneity, the division of the biofilm population into functionally distinct subgroups of cells. Phenotypic heterogeneity in biofilms can be traced to the cellular level; however, the molecular structures and elemental distribution across whole biofilms, as well as possible linkages between them, remain unexplored. Mapping X-ray diffraction across intact biofilms in time and space, we revealed the dominant structural features in Bacillus subtilis biofilms, stemming from matrix components, spores, and water. By simultaneously following the X-ray fluorescence signal of biofilms and isolated matrix components, we discovered that the ECM preferentially binds calcium ions over other metal ions, specifically, zinc, manganese, and iron. These ions, remaining free to flow below macroscopic wrinkles that act as water channels, eventually accumulate and may possibly lead to sporulation. The possible link between ECM properties, regulation of metal ion distribution, and sporulation across whole, intact biofilms unravels the importance of molecular-level heterogeneity in shaping biofilm physiology and development.

The spider cuticle: a remarkable material toolbox for functional diversity.

Engineered systems are typically based on a large variety of materials differing in composition and processing to provide the desired functionality. Nature, however, has evolved materials that are used for a wide range of functional challenges with minimal compositional changes. The exoskeletal cuticle of spiders, as well as of other arthropods such as insects and crustaceans, is based on a combination of chitin, protein, water and small amounts of organic cross-linkers or minerals. Spiders use it to obtain mechanical support structures and lever systems for locomotion, protection from adverse environmental influences, tools for piercing, cutting and interlocking, auxiliary structures for the transmission and filtering of sensory information, structural colours, transparent lenses for light manipulation and more. This paper illustrates the 'design space' of a single type of composite with varying internal architecture and its remarkable capability to serve a diversity of functions. This article is part of the theme issue 'Bio-derived and bioinspired sustainable advanced materials for emerging technologies (part 1)'.

Epidermal Cell Surface Structure and Chitin-Protein Co-assembly Determine Fiber Architecture in the Locust Cuticle.

The geometrical similarity of helicoidal fiber arrangement in many biological fibrous extracellular matrices, such as bone, plant cell wall, or arthropod cuticle, to that of cholesteric liquid mesophases has led to the hypothesis that they may form passively through a mesophase precursor rather than by direct cellular control. In search of direct evidence to support or refute this hypothesis, here, we studied the process of cuticle formation in the tibia of the migratory locust, Locusta migratoria, where daily growth layers arise by the deposition of fiber arrangements alternating between unidirectional and helicoidal structures. Using focused ion beam/scanning electron microscopy (FIB/SEM) volume imaging and scanning X-ray scattering, we show that the epidermal cells determine an initial fiber orientation, from which the final architecture emerges by the self-organized co-assembly of chitin and proteins. Fiber orientation in the locust cuticle is therefore determined by both active and passive processes.

Mineral formation in the primary polyps of pocilloporoid corals.

In reef-building corals, larval settlement and its rapid calcification provides a unique opportunity to study the bio-calcium carbonate formation mechanism involving skeleton morphological changes. Here we investigate the mineral formation of primary polyps, just after settlement, in two species of the pocilloporoid corals: Stylophora pistillata (Esper, 1797) and Pocillopora acuta (Lamarck, 1816). We show that the initial mineral phase is nascent Mg-Calcite, with rod-like morphology in P. acuta, and dumbbell morphology in S. pistillata. These structures constitute the first layer of the basal plate which is comparable to Rapid Accretion Deposits (Centers of Calcification, CoC) in adult coral skeleton. We found also that the rod-like/dumbbell Mg-Calcite structures in subsequent growth step will merge into larger aggregates by deposition of aragonite needles. Our results suggest that a biologically controlled mineralization of initial skeletal deposits occurs in three steps: first, vesicles filled with divalent ions are formed intracellularly. These vesicles are then transferred to the calcification site, forming nascent Mg-Calcite rod/pristine dumbbell structures. During the third step, aragonite crystals develop between these structures forming spherulite-like aggregates. STATEMENT OF SIGNIFICANCE: Coral settlement and recruitment periods are highly sensitive to environmental conditions. Successful mineralization during these periods is vital and influences the coral's chances of survival. Therefore, understanding the exact mechanism underlying carbonate precipitation is highly important. Here, we used in vivo microscopy, spectroscopy and molecular methods to provide new insights into mineral development. We show that the primary polyp's mineral arsenal consists of two types of minerals: Mg-Calcite and aragonite. In addition, we provide new insights into the ion pathway by showing that divalent ions are concentrated in intracellular vesicles and are eventually deposited at the calcification site.

A hydrated crystalline calcium carbonate phase: Calcium carbonate hemihydrate.

As one of the most abundant materials in the world, calcium carbonate, CaCO3, is the main constituent of the skeletons and shells of various marine organisms. It is used in the cement industry and plays a crucial role in the global carbon cycle and formation of sedimentary rocks. For more than a century, only three polymorphs of pure CaCO3-calcite, aragonite, and vaterite-were known to exist at ambient conditions, as well as two hydrated crystal phases, monohydrocalcite (CaCO3·1H2O) and ikaite (CaCO3·6H2O). While investigating the role of magnesium ions in crystallization pathways of amorphous calcium carbonate, we unexpectedly discovered an unknown crystalline phase, hemihydrate CaCO3·½H2O, with monoclinic structure. This discovery may have important implications in biomineralization, geology, and industrial processes based on hydration of CaCO3.

Growth and regrowth of adult sea urchin spines involve hydrated and anhydrous amorphous calcium carbonate precursors.

In various mineralizing marine organisms, calcite or aragonite crystals form through the initial deposition of amorphous calcium carbonate (ACC) phases with different hydration levels. Using X-ray PhotoEmission Electron spectroMicroscopy (X-PEEM), ACCs with varied spectroscopic signatures were previously identified. In particular, ACC type I and II were recognized in embryonic sea urchin spicules. ACC type I was assigned to hydrated ACC based on spectral similarity with synthetic hydrated ACC. However, the identity of ACC type II has never been unequivocally determined experimentally. In the present study we show that synthetic anhydrous ACC and ACC type II identified here in sea urchin spines, have similar Ca L 2,3-edge spectra. Moreover, using X-PEEM chemical mapping, we revealed the presence of ACC-H2O and anhydrous ACC in growing stereom and septa regions of sea urchin spines, supporting their role as precursor phases in both structures. However, the distribution and the abundance of the two ACC phases differ substantially between the two growing structures, suggesting a variation in the crystal growth mechanism; in particular, ACC dehydration, in the two-step reaction ACC-H2O → ACC → calcite, presents different kinetics, which are proposed to be controlled biologically.

Integrated Cooling-Vacuum-Assisted Non-Fractional 1540-nm Erbium:Glass Laser: A New Modality for the Simultaneous Effective Treatment of Acne Lesions and Scars

Introduction: Acne vulgaris is a common skin disorder with a significant impact on patients' quality of life. There is currently no treatment designated to treat acne lesions and scars concurrently. However, mid-infrared lasers may promote neocollagenesis within atrophic scars, while exerting a beneficial effect on acne lesions. Objectives: To determine the safety and efficacy of an integrated cooling-vacuum-assisted non-fractional 1540-nm Erbium:Glass laser for the treatment of acne lesions and scars. Patients and Methods: Twenty-two patients (8 male, 14 female) with mild-to-moderate acne and moderate-to-severe acne scars were included. Patients were treated using a non-fractional 1540-nm Er:Glass laser (Harmony XL™, Alma Lasers Ltd.). Acne lesions and scars were exposed to 3-4 stacked pulses emitted at a rate of 3Hz for up to two passes per treatment session (spot size, 4 mm; fluence, 400-600 mJ/pulse), receiving overall 3-7 treatments with 2-3-week intervals. Patients were followed-up one and three months following their last treatment. Clinical evaluation including (i) overall aesthetic appearance, (ii) acne lesions, and (iii) acne scars, assessed independently by two dermatologists and graded on a scale of 0 (exacerbation) to 4 (76-100 percent improvement); and (iv) pain perception, adverse effects and patients' satisfaction. Results: All but one patient completed treatment and follow-up and had moderate-to-significant improvement in all outcomes (overall aesthetic appearance, mean 3.9 [1 month] and 3.75 [3 month] improvement; acne lesions, 3.5 [1 month] and 2.3 [3 month] improvement; scarring 4 [1 month] and 4.2 [3 month] improvement). Pain and adverse effects were mild and transient. Patients' mean satisfaction was 4.2. Conclusion: Cooling-vacuum-assisted 1540 nm laser is a safe and effective modality for the simultaneous treatment of acne lesions and scars.

The Crystallization of Amorphous Calcium Carbonate is Kinetically Governed by Ion Impurities and Water.

Many organisms use amorphous calcium carbonate (ACC) and control its stability by various additives and water; however, the underlying mechanisms are yet unclear. Here, the effect of water and inorganic additives commonly found in biology on the dynamics of the structure of ACC during crystallization and on the energetics of this process is studied. Total X-ray scattering and pair distribution function analysis show that the short- and medium-range order of all studied ACC samples are similar; however, the use of in situ methodologies allow the observation of small structural modifications that are otherwise easily overlooked. Isothermal calorimetric coupled with microgravimetric measurements show that the presence of Mg2+ and of PO43- in ACC retards the crystallization whereas increased water content accelerates the transformation. The enthalpy of ACC with respect to calcite appears, however, independent of the additive concentration but decreases with water content. Surprisingly, the enthalpic contribution of water is compensated for by an equal and opposite entropic term leading to a net independence of ACC thermodynamic stability on its hydration level. Together, these results point toward a kinetic stabilization effect of inorganic additives and water, and may contribute to the understanding of the biological control of mineral stability.

Additives influence the phase behavior of calcium carbonate solution by a cooperative ion-association process.

Amorphous calcium carbonate (ACC) has been widely found in biomineralization, both as a transient precursor and a stable phase, but how organisms accurately control its formation and crystallization pathway remains unclear. Here, we aim to illuminate the role of biologically relevant additives on the phase behaviour of calcium carbonate solution by investigating their effects on the formation of ACC. Results show that divalent cations like magnesium (Mg2+) ions and negatively charged small organic molecules like aspartic acid (Asp) have little/no effect on ACC formation. However, the particle size of ACC is significantly reduced by poly(aspartic acid) (pAsp) with long chain-length, but no effect on the position of the phase boundary for ACC formation was observed. Phosphate (PO4 3-) ions are even more effective in reducing ACC particle size, and shift the phase boundary for ACC formation to lower concentrations. These phenomena can be explained by a cooperative ion-association process where the formation of ACC is only influenced by additives that are able to attract either Ca2+ ions or CO3 2- ions and, more importantly, introduce an additional long range interaction between the CaCO complexes and promote the phase separation process. The findings corroborate with our proposed model of ACC formation via spinodal decomposition and provide a more realistic representation of how biology can direct mineralization processes.

Control of Polymorph Selection in Amorphous Calcium Carbonate Crystallization by Poly(Aspartic Acid): Two Different Mechanisms.

Poly(aspartic acid) (pAsp) is known to stabilize amorphous calcium carbonate (ACC) and affect its crystallization pathways. However, little is known about the mechanisms behind these phenomena. Here it is shown that ACC is stabilized by pAsp molecules in the solution rather than by the amount of pAsp incorporated into the ACC bulk, and that the effect of pAsp on the polymorph selection is entirely different at low and high concentration of pAsp. At low concentrations, pAsp is more effective in inhibiting the nucleation and growth of vaterite than calcite. At high concentrations, when calcite formation is prevented, the crystallization of vaterite proceeds via a pseudomorphic transformation of ACC nanospheres, where vaterite nucleates on the surface of ACC nanospheres and grows by a local transformation of the bulk ACC phase. These results shed some light on the function of pAsp during an ACC-mediated biomineralization process and provide an explanation for the presence of metastable vaterite at conditions where calcite is thermodynamically favored.

Nano-channels in the spider fang for the transport of Zn ions to cross-link His-rich proteins pre-deposited in the cuticle matrix.

We identify the presence of multiple vascular channels within the spider fang. These channels seem to serve the transport of zinc to the tip of the fang to cross-link the protein matrix by binding to histidine residues. According to amino acid and elemental analysis of fangs extracted shortly after ecdysis, His-rich proteins are deposited before Zn is incorporated into the cuticle. Microscopic and spectroscopic investigations in the electron microscope and synchrotron radiation experiments suggest that Zn ions are transported through these channels in a liable (yet unidentified) form, and then form stable complexes upon His binding. The resulting cross-linking through the Zn-His complexes is conferring hardness to the fang. Our observations of nano-channels serving the Zn-transport within the His-rich protein matrix of the fibre reinforced spider fang may also support recent bio-inspired attempts to design artificial polymeric vascular materials for self-healing and in-situ curing.

Micromechanical properties of strain-sensitive lyriform organs of a wandering spider (Cupiennius salei).

UNLABELLED: Highly sensitive lyriform organs located on the legs of the wandering spider Cupiennius salei allow the spider to detect nanometer-scale strains in the exoskeleton resulting from locomotion or substrate vibrations. Morphological features of the lyriform organs result in their specialization and selective sensitivity to specific mechanical stimuli, which make them interesting for bioinspired strain sensors. Here we utilize atomic force microscopy (AFM)-based force spectroscopy to probe nano-scale mechanical properties of the covering membrane of two lyriform organs found on Cupiennius salei: the vibration sensitive metatarsal lyriform organ (HS10) and the proprioreceptive tibial lyriform organ (HS8). Force distance curves (FDCs) obtained from AFM measurements displayed characteristic multi-layer structure behavior, with calculated elastic moduli ranging from 150MPa to 500MPa for different regions and indentation depths. In addition, we probed the lyriform organs with a large radius tip, which allowed for probing structural deformation by the application of high forces and large scale deformations without damaging the surface. The viscoelastic behavior of the sensor materials observed in this probing suggests mechanical relaxation times potentially playing a role in the time-dependent behavior of the lyriform organs. STATEMENT OF SIGNIFICANCE: Highly sensitive lyriform organs located on the legs of the wandering spider Cupiennius salei allow the spider to detect nanometer-scale strains in the exoskeleton resulting from locomotion or substrate vibrations. Morphological features of the lyriform organs result in their specialization and selective sensitivity to specific mechanical stimuli, which make them an interesting for bioinspired strain sensors. Here we utilize atomic force microscopy (AFM)-based force spectroscopy to probe nano-scale mechanical properties of the covering membrane of two lyriform organs found on Cupiennius salei: the vibration sensitive metatarsal lyriform organ (HS10) and the proprioreceptive tibial lyriform organ (HS8). Force distance curves (FDCs) obtained from AFM measurements displayed characteristic multi-layer structure behavior, with calculated elastic moduli ranging from 150MPa to 500MPa for different regions and indentation depths. The viscoelastic behavior of the sensor materials observed in this probing suggests mechanical relaxation times playing a role in the time-dependent behavior of the lyriform organs.

A vacuole-like compartment concentrates a disordered calcium phase in a key coccolithophorid alga.

Coccoliths are calcitic particles produced inside the cells of unicellular marine algae known as coccolithophores. They are abundant components of sea-floor carbonates, and the stoichiometry of calcium to other elements in fossil coccoliths is widely used to infer past environmental conditions. Here we study cryo-preserved cells of the dominant coccolithophore Emiliania huxleyi using state-of-the-art nanoscale imaging and spectroscopy. We identify a compartment, distinct from the coccolith-producing compartment, filled with high concentrations of a disordered form of calcium. Co-localized with calcium are high concentrations of phosphorus and minor concentrations of other cations. The amounts of calcium stored in this reservoir seem to be dynamic and at a certain stage the compartment is in direct contact with the coccolith-producing vesicle, suggesting an active role in coccolith formation. Our findings provide insights into calcium accumulation in this important calcifying organism.

Ordering of protein and water molecules at their interfaces with chitin nano-crystals.

Synchrotron X-ray diffraction was applied to study the structure of biogenic α-chitin crystals composing the tendon of the spider Cupiennius salei. Measurements were carried out on pristine chitin crystals stabilized by proteins and water, as well as after their deproteinization and dehydration. We found substantial shifts (up to Δq/q=9% in the wave vector in q-space) in the (020) diffraction peak position between intact and purified chitin samples. However, chitin lattice parameters extracted from the set of reflections (hkl), which did not contain the (020)-reflection, showed no systematic variation between the pristine and the processed samples. The observed shifts in the (020) peak position are discussed in terms of the ordering-induced modulation of the protein and water electron density near the surface of the ultra-thin chitin fibrils due to strong protein/chitin and water/chitin interactions. The extracted modulation periods can be used as a quantitative parameter characterizing the interaction length.

Integrated Cooling-Vacuum-Assisted Non-Fractional 1540 nm Erbium:Glass Laser is Effective in Treating Acne Scars.

INTRODUCTION: Acne scars are a common result of in ammatory acne, affecting many patients worldwide. Among which, atrophic scars are the most prevalent form, presenting as dermal depressions caused by inflammatory degeneration of dermal collagen. Mid-infrared laser skin interaction is characterized by its modest absorption in water and nite penetration to the mid-dermis. Since collagen is a desirable laser target, 1540-nm wavelength is amenable for collagen remodeling within the depressed area of atrophic scars. OBJECTIVES: To evaluate the safety and efficacy of acne scars treatment using an integrated cooling-vacuum-assisted 1540 nm Erbium: Glass Laser. PATIENTS AND METHODS: This interventional prospective study included 25 volunteers (10 men, 15 women) with post acne atrophic scars. Patients were treated with a mid-infrared non-fractional 1540 nm Er:Glass laser (Alma Lasers Ltd. Caesarea, Israel) with integrat- ed cooling- vacuum assisted technology. Acne scars were exposed to 3 stacked laser pulses (400-600 mJ/pulse, 4 mm spot size, frequency of 3 Hz). Patients underwent 3-6 treatment sessions with a 2-3 week interval and were followed-up 1 month and 3 months after the last treatment. Clinical photographs were taken by high resolution digital camera before and after treatment. Clinical evaluation was performed by two independent dermatologists and results were graded on a scale of 0 (exacerbation) to 4 (76%-100% improvement). Patients' and physicians' satisfaction were also recorded (on a 1-5 scale). Pain perception and adverse effects were evaluated as well. RESULTS: Almost all patients (24/25) demonstrated a moderate to significant improvement. Average improvement was 3.9 and 4.1 points on the quartile scale used for outcome assessment 1 and 3 months following the last session, respectively. Patient satisfaction rate was 4.2. Side effects were minimal and transient: erythema, mild transient vesicles, and mild pain or inconvenience. CONCLUSION Cooling-Vacuum-Assisted mid-infrared non-fractional Er:Glass 1540 nm laser is safe and effective in the treatment of atrophic acne scars. J Drugs Dermatol. 2016;15(11):1359-1363..

Histochemical evidence of ß-chitin in parapodial glandular organs and tubes of Spiophanes (Annelida, Sedentaria: Spionidae), and first studies on selected Annelida.

A generic character of the genus Spiophanes (Annelida, Sedentaria: Spionidae) is the presence of parapodial glandular organs. Parapodial glandular organs in Spiophanes species include secretory cells with cup-shaped microvilli, similar to those present in deep-sea inhabiting vestimentiferans and frenulate Siboglinidae. These cells are supposed to secrete ß-chitin for tube-building. In this study, transverse histological and/or ultrathin sections of parapodial glandular organs and tubes of Spiophanes spp. as well as of Glandulospio orestes (Spionidae) and Owenia fusiformis (Oweniidae) were examined. Fluorescent markers together with confocal laser scanning microscopy, and Raman spectroscopy were used to detect chitin in the parapodial glandular organs of Spiophanes and/or in the glands of Owenia and Glandulospio. Tubes of these taxa were tested for chitin to elucidate the use of it for tube-building. The examinations revealed a distinct labelling of the gland contents. Raman spectroscopy documented the presence of ß-chitin in both gland types of Spiophanes. The tubes of Spiophanes were found to have a grid-like structure that seems to be built with this ß-chitin. Tests of tubes of Dipolydora quadrilobata (Spionidae) for chitin were negative. However, the results of our study provide strong evidence that Spiophanes species, O. fusiformis and probably also G. orestes produce chitin and supposedly use it for tube-building. This implies that the production of chitin and its use as a constituent part of tube-building is more widespread among polychaetes as yet known. The histochemical data presented in this study support previous assumptions inferring homology of parapodial glandular organs of Spionidae and Siboglinidae based on ultrastructure. Furthermore, transmission electron microscopy-based evidence of secretory cells with nail-headed microvilli in O. fusiformis suggests homology of parapodial grandular organs across annelids including Sipuncula.