Glycoproteins Involved in Sea Urchin Temporary Adhesion.

Biomedical adhesives, despite having been used increasingly in recent years, still face a major technological challenge: strong adhesion in wet environments. In this context, biological adhesives secreted by marine invertebrates have appealing characteristics to incorporate into new underwater biomimetic adhesives: water resistance, nontoxicity and biodegradability. Little is still known about temporary adhesion. Recently, a transcriptomic differential analysis of sea urchin Paracentrotus lividus tube feet pinpointed 16 adhesive/cohesive protein candidates. In addition, it has been demonstrated that the adhesive secreted by this species is composed of high molecular weight proteins associated with N-Acetylglucosamine in a specific chitobiose arrangement. As a follow-up, we aimed to investigate which of these adhesive/cohesive protein candidates were glycosylated through lectin pulldowns, protein identification by mass spectroscopy and in silico characterization. We demonstrate that at least five of the previously identified protein adhesive/cohesive candidates are glycoproteins. We also report the involvement of a third Nectin variant, the first adhesion-related protein to be identified in P. lividus. By providing a deeper characterization of these adhesive/cohesive glycoproteins, this work advances our understanding of the key features that should be replicated in future sea urchin-inspired bioadhesives.

Integrative Transcriptome and Proteome Analysis of the Tube Foot and Adhesive Secretions of the Sea Urchin Paracentrotus lividus.

Echinoderms, such as the rock-boring sea urchin Paracentrotus lividus, attach temporarily to surfaces during locomotion using their tube feet. They can attach firmly to any substrate and release from it within seconds through the secretion of unknown molecules. The composition of the adhesive, as well as the releasing secretion, remains largely unknown. This study re-analyzed a differential proteome dataset from Lebesgue et al. by mapping mass spectrometry-derived peptides to a P. lividus de novo transcriptome generated in this study. This resulted in a drastic increase in mapped proteins in comparison to the previous publication. The data were subsequently combined with a differential RNAseq approach to identify potential adhesion candidate genes. A gene expression analysis of 59 transcripts using whole mount in situ hybridization led to the identification of 16 transcripts potentially involved in bioadhesion. In the future these data could be useful for the production of synthetic reversible adhesives for industrial and medical purposes.

Understanding regeneration through proteomics.

Regeneration is a complex cellular process that, rather than simply forming a scar following injury, the animal forms a new functional tissue. Regeneration is a widespread process among metazoa, although not uniformly. Planaria, starfish, and some worms can regenerate most of their body, whereas many other species can only regenerate parts of specific tissues or fail to accomplish a functional regrowth, as is the case of mammals CNS. Research in regenerative medicine will possibly culminate in the regeneration of organs/tissues originally not prone to this process. Despite the complexity of the interactions and regulatory systems involved, the variety of tissues and organs these cells differentiate into has so far impaired the success of direct transplantation to restore damaged tissues. For this reason, a study, at the molecular level of the regeneration mechanisms developed by different animal models is likely to provide answers to why these processes are not readily activated in mammals. Proteomic-based approaches are being recognized as extremely useful to study of regeneration events, also because there is a relevant contribution of posttranscriptional processes that involve frequently the occurrence of a broad range of PTMs. The present review focuses on the significant knowledge brought up by proteomics in diverse aspects of regeneration research on different animal models, tissues, and organs.

Radial nerve cord protein phosphorylation dynamics during starfish arm tip wound healing events.

Echinoderms, as invertebrate deuterostomes, have amazing neuronal intrinsic growth aptitude triggered at any time point during the animal lifespan leading to successful functional tissue regrowth. This trait is known to be in opposition to their mammal close phylogenic relatives that have lost the ability to regenerate their central nervous system. Despite the promising nature of this intrinsic echinoderm trait, it was only recently that this complex biological event started to be unveiled. In the present study, a 2DE gel-based phosphoproteomics approach was used to investigate changes in starfish neuronal protein phosphorylation states at two different wound healing time-graded events following arm tip amputation, 48 h and 13 days. Among the resolved protein spots in 3.0-5.6 NL pH IEF strips, 190, 142, and 124 had a phosphoprotein signal in the control and the two injury experimental groups, respectively. Gel image analysis, highlighted 129 spots with an injury-related protein phosphorylation dynamics, several being exclusively phosphorylated in controls (72 spots), injured nerves (8 spots) or, showing significantly different phosphorylation ratios (37 spots). Within these, a total of 43 proteins were identified with MALDI-TOF/TOF. Altogether, several intervening proteins of important injury-signaling pathways that seem to be modulated through phosphorylation, were identified for the first time in starfish radial nerve cord early regeneration events. These include cytoskeleton re-organization toward the formation of the neuronal growth cones; cell membrane rearrangements, actin filaments, and microtubules dynamics; mRNA binding and transport; lipid signaling; Notch pathway; and neuropeptide processing.

A proteomics study of the induction of somatic embryogenesis in Medicago truncatula using 2DE and MALDI-TOF/TOF.

Medicago truncatula is a model legume, whose genome is currently being sequenced. Somatic embryogenesis (SE) is a genotype-dependent character and not yet fully understood. In this study, a proteomic approach was used to compare the induction and expression phases of SE of both the highly embryogenic line M9-10a of M. truncatula cv. Jemalong and its non-embryogenic predecessor line, M9. The statistical analysis between the lines revealed 136 proteins with significant differential expression (P < 0.05). Of these, 5 had a presence/absence pattern in M9 vs M9-10a and 22 showed an at least twofold difference in terms of spot volume, were considered of particular relevance to the SE process and therefore chosen for identification. Spots were excised in gel digested with trypsin and proteins were identified using matrix-assisted laser desorption ionization-time of flight/time of flight. Identified proteins indicated a higher adaptability of the embryogenic line toward the stress imposed by the inducing culture conditions. Also, some proteins were shown to have a dual pattern of expression: peroxidase, pyrophosphatase and aspartate aminotransferase. These proteins showed higher expression during the induction phases of the M9 line, whereas in the embryogenic line had higher expression at stages coinciding with embryo formation.

Exploring the proteome of an echinoderm nervous system: 2-DE of the sea star radial nerve cord and the synaptosomal membranes subproteome.

We describe the first proteomic characterization of the radial nerve cord (RNC) of an echinoderm, the sea star Marthasterias glacialis. The combination of 2-DE with MS (MALDI-TOF/TOF) resulted in the identification of 286 proteins in the RNC. Additionally, 158 proteins were identified in the synaptosomal membranes enriched fraction after 1-DE separation. The 2-DE RNC reference map is available via the WORLD-2DPAGE Portal (http://www.expasy.ch/world-2dpage/) along with the associated protein identification data which are also available in the PRIDE database. The identified proteins constitute the first high-throughput evidence that seems to indicate that echinoderms nervous transmission relies primarily on chemical synapses which is similar to the synaptic activity in adult mammal's spinal cord. Furthermore, several homologous proteins known to participate in the regeneration events of other organisms were also identified, and thus can be used as targets for future studies aiming to understand the poorly uncharacterized regeneration capability of echinoderms. This "echinoderm missing link" is also a contribution to unravel the mystery of deuterostomian CNS evolution.

Proteome characterization of sea star coelomocytes--the innate immune effector cells of echinoderms.

Sea star coelomic fluid is in contact with all internal organs, carrying signaling molecules and a large population of circulating cells, the coelomocytes. These cells, also known as echinoderm blood cells, are responsible for the innate immune responses and are also known to have an important role in the first stage of regeneration, i.e. wound closure, necessary to prevent disruption of the body fluid balance and to limit the invasion of pathogens. This study focuses on the proteome characterization of these multifunctional cells. The identification of 358 proteins was achieved using a combination of two techniques for protein separation (1-D SDS-PAGE followed by nanoLC and 2-D SDS-PAGE) and MALDI-TOF/TOF MS for protein identification. To our knowledge, the present report represents the first comprehensive list of sea star coelomocyte proteins, constituting an important database to validate many echinoderm-predicted proteins. Evidence for new pathways in these particular echinoderm cells are also described, and thus representing a valuable resource to stimulate future studies aiming to unravel the homology with vertebrate immune cells and particularly the origins of the immune system itself.

Serine protease-mediated host invasion by the parasitic nematode Steinernema carpocapsae.

Steinernema carpocapsae is an insect parasitic nematode used in biological control, which infects insects penetrating by mouth and anus and invading the hemocoelium through the midgut wall. Invasion has been described as a key factor in nematode virulence and suggested to be mediated by proteases. A serine protease cDNA from the parasitic stage was sequenced (sc-sp-1); the recombinant protein was produced in an Escherichia coli system, and a native protein was purified from the secreted products. Both proteins were confirmed by mass spectrometry to be encoded by the sc-sp-1 gene. Sc-SP-1 has a pI of 8.7, a molecular mass of 27.3 kDa, a catalytic efficiency of 22.2 × 10(4) s(-1) m(-1) against N-succinyl-Ala-Ala-Pro-Phe-pNA, and is inhibited by chymostatin (IC 0.07) and PMSF (IC 0.73). Sc-SP-1 belongs to the chymotrypsin family, based on sequence and biochemical analysis. Only the nematode parasitic stage expressed sc-sp-1. These nematodes in the midgut lumen, prepared to invade the insect hemocoelium, expressed higher levels than those already in the hemocoelium. Moreover, parasitic nematode sense insect peritrophic membrane and hemolymph more quickly than they do other tissues, which initiates sc-sp-1 expression. Ex vivo, Sc-SP-1 was able to bind to insect midgut epithelium and to cause cell detachment from basal lamina. In vitro, Sc-SP-1 formed holes in an artificial membrane model (Matrigel), whereas Sc-SP-1 treated with PMSF did not, very likely because it hydrolyzes matrix glycoproteins. These findings highlight the S. carpocapsae-invasive process that is a key step in the parasitism thus opening new perspectives for improving nematode virulence to use in biological control.

Omics-based molecular analyses of adhesion by aquatic invertebrates.

Many aquatic invertebrates are associated with surfaces, using adhesives to attach to the substratum for locomotion, prey capture, reproduction, building or defence. Their intriguing and sophisticated biological glues have been the focus of study for decades. In all but a couple of specific taxa, however, the precise mechanisms by which the bioadhesives stick to surfaces underwater and (in many cases) harden have proved to be elusive. Since the bulk components are known to be based on proteins in most organisms, the opportunities provided by advancing 'omics technologies have revolutionised bioadhesion research. Time-consuming isolation and analysis of single molecules has been either replaced or augmented by the generation of massive data sets that describe the organism's translated genes and proteins. While these new approaches have provided resources and opportunities that have enabled physiological insights and taxonomic comparisons that were not previously possible, they do not provide the complete picture and continued multi-disciplinarity is essential. This review covers the various ways in which 'omics have contributed to our understanding of adhesion by aquatic invertebrates, with new data to illustrate key points. The associated challenges are highlighted and priorities are suggested for future research.

Nanoscale characterization of the temporary adhesive of the sea urchin Paracentrotus lividus.

Background: Unlike the thin homogeneous films that are typical for adhesives produced by humans, biological adhesives present complex hierarchical micro- and nanostructures. Most studies on marine adhesives have focused on permanent adhesives, whereas the nanostructures of nonpermanent, temporary or reversible adhesives have only been examined in some organisms such as marine flatworms, barnacle cyprids, freshwater cnidaria and echinoderms such as sea cucumbers and sea stars. In this study, the first nanoscale characterization of sea urchin temporary adhesives was performed using atomic force microscopy (AFM). Results: The adhesive topography was similar under dry and native (seawater) conditions, which was comprised of a honeycomb-like meshwork of aggregated globular nanostructures. In terms of adhesion forces, higher values were obtained in dry conditions, reaching up to 50 nN. Under native conditions, lower adhesive forces were obtained (up to 500 pN) but the adhesive seemed to behave like a functional amyloid, as evidenced by the recorded characteristic sawtooth force-extension curves and positive thioflavin-T labelling. Conclusion: Our results confirm that like other temporary adhesives, the sea urchin adhesive footprint nanostructure consists of a meshwork of entangled globular nanostructures. Under native conditions, the adhesive footprints of the sea urchin behaved like a functional amyloid, suggesting that among its proteinaceous constituents there are most likely proteins with amyloid quaternary structures or rich in ß-sheets. These results extend our knowledge on sea urchin adhesive composition and mechanical properties essential for the engineering of biomimetic adhesives.

Cloning, Characterization, and Expression Levels of the Nectin Gene from the Tube Feet of the Sea Urchin Paracentrotus Lividus.

Marine bioadhesives perform in ways that manmade products simply cannot match, especially in wet environments. Despite their technological potential, bioadhesive molecular mechanisms are still largely understudied, and sea urchin adhesion is no exception. These animals inhabit wave-swept shores, relying on specialized adhesive organs, tube feet, composed by an adhesive disc and a motile stem. The disc encloses a duo-gland adhesive system, producing adhesive and deadhesive secretions for strong reversible substratum attachment. The disclosure of sea urchin Paracentrotus lividus tube foot disc proteome led to the identification of a secreted adhesion protein, Nectin, never before reported in adult adhesive organs but, that given its adhesive function in eggs/embryos, was pointed out as a putative substratum adhesive protein in adults. To further understand Nectin involvement in sea urchin adhesion, Nectin cDNA was amplified for the first time from P. lividus adhesive organs, showing that not only the known Nectin mRNA, called Nectin-1 (GenBank AJ578435), is expressed in the adults tube feet but also a new mRNA sequence, called Nectin-2 (GenBank KT351732), differing in 15 missense nucleotide substitutions. Nectin genomic DNA was also obtained for the first time, indicating that both Nectin-1 and Nectin-2 derive from a single gene. In addition, expression analysis showed that both Nectins are overexpressed in tube feet discs, its expression being significantly higher in tube feet discs from sea urchins just after collection from the field relative to sea urchin from aquarium. These data further advocate for Nectin involvement in sea urchin reversible adhesion, suggesting that its expression might be regulated according to the hydrodynamic conditions.

Deciphering the molecular mechanisms underlying sea urchin reversible adhesion: A quantitative proteomics approach.

UNLABELLED: Marine bioadhesives have unmatched performances in wet environments, being an inspiration for biomedical applications. In sea urchins specialized adhesive organs, tube feet, mediate reversible adhesion, being composed by a disc, producing adhesive and de-adhesive secretions, and a motile stem. After tube foot detachment, the secreted adhesive remains bound to the substratum as a footprint. Sea urchin adhesive is composed by proteins and sugars, but so far only one protein, Nectin, was shown to be over-expressed as a transcript in tube feet discs, suggesting its involvement in sea urchin adhesion. Here we use high-resolution quantitative mass-spectrometry to perform the first study combining the analysis of the differential proteome of an adhesive organ, with the proteome of its secreted adhesive. This strategy allowed us to identify 163 highly over-expressed disc proteins, specifically involved in sea urchin reversible adhesion; to find that 70% of the secreted adhesive components fall within five protein groups, involved in exocytosis and microbial protection; and to provide evidences that Nectin is not only highly expressed in tube feet discs but is an actual component of the adhesive. These results give an unprecedented insight into the molecular mechanisms underlying sea urchin adhesion, and opening new doors to develop wet-reliable, reversible, and ecological biomimetic adhesives. SIGNIFICANCE: Sea urchins attach strongly but in a reversible manner to substratum, being a valuable source of inspiration for industrial and biomedical applications. Yet, the molecular mechanisms governing reversible adhesion are still poorly studied delaying the engineering of biomimetic adhesives. We used the latest mass spectrometry techniques to analyze the differential proteome of an adhesive organ and the proteome of its secreted adhesive, allowing us to uncover the key players in sea urchin reversible adhesion. We demonstrate, that Nectin, a protein previously pointed out as potentially involved in sea urchin adhesion, is not only highly expressed in tube feet discs, but is a genuine component of the secreted adhesive.

Proteomic dataset of the sea urchin Paracentrotus lividus adhesive organs and secreted adhesive.

Sea urchins have specialized adhesive organs called tube feet, which mediate strong but reversible adhesion. Tube feet are composed by a disc, producing adhesive and de-adhesive secretions for substratum attachment, and a stem for movement. After detachment the secreted adhesive remains bound to the substratum as a footprint. Recently, a label-free quantitative proteomic approach coupled with the latest mass-spectrometry technology was used to analyze the differential proteome of Paracentrotus lividus adhesive organ, comparing protein expression levels in the tube feet adhesive part (the disc) versus the non-adhesive part (the stem), and also to profile the proteome of the secreted adhesive (glue). This data article contains complementary figures and results related to the research article "Deciphering the molecular mechanisms underlying sea urchin reversible adhesion: a quantitative proteomics approach" (Lebesgue et al., 2016) [1]. Here we provide a dataset of 1384 non-redundant proteins, their fragmented peptides and expression levels, resultant from the analysis of the tube feet differential proteome. Of these, 163 highly over-expressed tube feet disc proteins (>3-fold), likely representing the most relevant proteins for sea urchin reversible adhesion, were further annotated in order to determine the potential functions. In addition, we provide a dataset of 611 non-redundant proteins identified in the secreted adhesive proteome, as well as their functional annotation and grouping in 5 major protein groups related with adhesive exocytosis, and microbial protection. This list was further analyzed to identify the most abundant protein groups and pinpoint putative adhesive proteins, such as Nectin, the most abundant adhesive protein in sea urchin glue. The obtained data uncover the key proteins involved in sea urchins reversible adhesion, representing a step forward to the development of new wet-effective bio-inspired adhesives.

Comparative histological and immunohistochemical study of sea star tube feet (Echinodermata, Asteroidea).

Adhesion in sea stars is the function of specialized structures, the tube feet or podia, which are the external appendages of the water-vascular system. Adhesive secretions allow asteroid tube feet to perform multiple functions. Indeed, according to the sea star species considered, the tube feet may be involved in locomotion, fixation, or burrowing. Different tube foot shapes usually correspond to this variety of function. In this study, we investigated the variability of the morphology of sea star tube feet as well as the variability of the composition of their adhesive secretions. This second aspect was addressed by a comparative immunohistochemical study using antibodies raised against the adhesive material of the forcipulatid Asterias rubens. The tube feet from 14 sea star species representing five orders and 10 families of the Class Asteroidea were examined. The histological study revealed three main tube foot morphotypes, i.e., knob-ending, simple disc-ending, and reinforced disc-ending. Analysis of the results suggests that tube foot morphology is influenced by species habitat, but within limits imposed by the evolutionary lineage. In immunohistochemistry, on the other hand, the results were very homogeneous. In every species investigated there was a very strong immunolabeling of the adhesive cells, independently of the taxon considered, of the tube foot morphotype or function, or of the species habitat. This indicates that the adhesives in all the species considered are closely related, probably sharing many identical molecules or, at least, many identical epitopes on their constituents.

Experimental strategies for the identification and characterization of adhesive proteins in animals: a review.

Adhesive secretions occur in both aquatic and terrestrial animals, in which they perform diverse functions. Biological adhesives can therefore be remarkably complex and involve a large range of components with different functions and interactions. However, being mainly protein based, biological adhesives can be characterized by classical molecular methods. This review compiles experimental strategies that were successfully used to identify, characterize and obtain the full-length sequence of adhesive proteins from nine biological models: echinoderms, barnacles, tubeworms, mussels, sticklebacks, slugs, velvet worms, spiders and ticks. A brief description and practical examples are given for a variety of tools used to study adhesive molecules at different levels from genes to secreted proteins. In most studies, proteins, extracted from secreted materials or from adhesive organs, are analysed for the presence of post-translational modifications and submitted to peptide sequencing. The peptide sequences are then used directly for a BLAST search in genomic or transcriptomic databases, or to design degenerate primers to perform RT-PCR, both allowing the recovery of the sequence of the cDNA coding for the investigated protein. These sequences can then be used for functional validation and recombinant production. In recent years, the dual proteomic and transcriptomic approach has emerged as the best way leading to the identification of novel adhesive proteins and retrieval of their complete sequences.

Proteolytic events are relevant cellular responses during nervous system regeneration of the starfish Marthasterias glacialis.

The molecular pathways that trigger the amazing intrinsic regenerative ability of echinoderm nervous system are still unknown. In order to approach this subject, a 2D-DIGE proteomic strategy was used, to screen proteome changes during neuronal regeneration in vivo, using starfish (Asteroidea, Echinodermata) as a model. A total of 528 proteins showed significant variations during radial nerve cord regeneration in both soluble and membrane protein-enriched fractions. Several functional classes of proteins known to be involved in axon regeneration events in other model organisms, such as chordates, were identified for the first time in the regenerating echinoderm nervous system. Unexpectedly, most of the identified proteins presented a molecular mass either higher or lower than expected. Such results suggest a functional modulation through protein post-translational modifications, such as proteolysis. Among these are proteins involved in cytoskeleton and microtubule regulators, axon guidance molecules and growth cone modulators, protein de novo synthesis machinery, RNA binding and transport, transcription factors, kinases, lipid signaling effectors and proteins with neuroprotective functions. In summary, the impact of proteolysis during regeneration events is here shown, although requiring further studies to detail on the mechanisms involving this post-transcriptional event on nervous system regeneration. BIOLOGICAL SIGNIFICANCE: The nervous systems of some organisms present a complete inability of neurons to regrow across a lesion site, which is the case of the adult mammalian central nervous system (CNS). Expanding our knowledge on how other animals regenerate their nervous system offers great potential for groundbreaking biomedical applications towards the enhancement of mammalian CNS regeneration. In order to approach this subject, a 2D-DIGE proteomic strategy was used for the first time, to screen the proteome changes during neuronal regeneration in vivo, using starfish (Asteroidea, Echinodermata) as a model. We strongly believe in the relevance of our results and have clear evidences that this work constitutes a solid basis for new research on starfish regenerating nerve cord. We also believe this work will have a significant impact not only on the general scientific community as we present here an alternative animal model to neurobiology, but also on the scientific community that works with echinoderms or closely related marine invertebrates, which are constantly searching for specific protein markers of several tissues, thus constituting an important advance towards the improvement of large scale protein information of unsequenced, but yet not less important organisms.

Mapping sea urchins tube feet proteome--a unique hydraulic mechano-sensory adhesive organ.

Marine organisms secrete adhesives for substrate attachment that to be effective require functional assembly underwater and displacement of water, ions, and weakly bound polyions that are ubiquitous in seawater. Therefore, understanding the characteristics of these protein/carbohydrate-based marine adhesives is imperative to decipher marine adhesion and also, to accelerate the development of new biomimetic underwater adhesives and anti-fouling agents. The present study, aims at mapping the proteome of the sea urchin Paracentrotus lividus adhesive organs using a combination of complementary protein separation techniques (1-D-nanoLC and 2-DE), databases and search algorithms. This strategy resulted in the identification of 328 non-redundant proteins, constituting the first comprehensive list of sea urchin tube feet proteins. Given the known importance of phosphorylation and glycosylation in marine adhesion, the 2DE proteome was re-analyzed with specific fluorescent stains for these two PTMs, resulting in the identification of 69 non-redundant proteins. The obtained results demonstrate that tube feet are unique mechano-sensory adhesive organs and highlight putative adhesive proteins, that although requiring further confirmation, constitute a step forward in the quest to decipher sea urchins temporary adhesion.

Is the adhesive material secreted by sea urchin tube feet species-specific?

Sea urchin adoral tube feet are highly specialized organs that have evolved to provide efficient attachment to the substratum. They consist of a disk and a stem that together form a functional unit. Tube foot disk tenacity (adhesive force per unit area) and stem mechanical properties (e.g., stiffness) vary between species but are apparently not correlated with sea urchin taxa or habitats. Moreover, ultrastructural studies of sea urchin disk epidermis pointed out differences in the internal organization of the adhesive secretory granules among species. This prompted us to look for interspecific variability in the composition of echinoid adhesive secretions, which could explain the observed variability in adhesive granule ultrastructure and disk tenacity. Antisera raised against the footprint material of Sphaerechinus granularis (S. granularis) were first used to locate the origin of adhesive footprint constituents in tube feet by taking advantage of the polyclonal character of the generated antibodies. Immunohistochemical assays showed that the antibodies specifically labeled the adhesive secretory cells of the disk epidermis in the tube feet of S. granularis. The antibodies were then used on tube foot histological sections from seven other sea urchin species to shed some light on the variability of their adhesive substances by looking for antibody cross-reactivity. Surprisingly, no labeling was observed in any of the species tested. These results indicate that unlike the adhesive secretions of asteroids, those of echinoids do not share common epitopes on their constituents and thus would be "species-specific." In sea urchins, variations in the composition of adhesive secretions could therefore explain interspecific differences in disk tenacity and in adhesive granule ultrastructure.

Morphology and tenacity of the tube foot disc of three common European sea urchin species: a comparative study.

The variation in tenacity of single tube feet from three sea urchin species with contrasted habitats was assessed and correlated with the ultrastructure of their adhesive secretory granules. The tube feet of Arbacia lixula and Sphaerechinus granularis have larger discs and more complex adhesive granules than those of Paracentrotus lividus, but A. lixula attaches to glass with significantly lower tenacity (0.05-0.09 MPa) than the other two species (0.10-0.20 and 0.11 -0.29 MPa, respectively). However, the estimated maximal attachment force one tube foot can produce is similar for all three species investigated. No clear relationship between tube foot size, tenacity, adhesive secretory granule ultrastructure and species habitat can therefore be established. For P. lividus the tenacity of single tube foot discs on four different smooth substrata was also compared, which showed that both the total surface energy and the ratio of polar to non-polar forces at the surface influence tube foot attachment strength. This influence of the surface characteristics of the substratum appears to affect the cohesiveness of the adhesive secretion more than its adhesiveness.

Adhesion of echinoderm tube feet to rough surfaces.

Echinoderms attach strongly and temporarily to the substratum by means of specialized organs, the podia or tube feet. The latter consist of a basal extensible cylinder, the stem, which bears an apical flattened disc. The disc repeatedly attaches to and detaches from the substratum through adhesive and de-adhesive secretions. In their activities, echinoderms have to cope with substrata of varying degrees of roughness as well as with changing hydrodynamic conditions, and therefore their tube feet must adapt their attachment strength to these environmental constraints. This study is the first attempt to evaluate the influence of substratum roughness on the temporary adhesion of echinoderm tube feet and to investigate the material properties of their contact surface. It was demonstrated that tube foot discs are very soft (E-modulus of 6.0 and 8.1 kPa for sea stars and sea urchins, respectively), have viscoelastic properties and adapt their surface to the substratum profile. They also show increased adhesion on a rough substratum in comparison to its smooth counterpart, which is due mostly to an increase in the geometrical area of contact between the disc and the surface. Tenacity (force per unit area) increases with roughness [e.g. 0.18 and 0.34 MPa on smooth polymethyl-methacrylate (PMMA), 0.21 and 0.47 MPa on rough PMMA for sea stars and sea urchins, respectively] if only the projected surface area of the adhesive footprint is considered. However, if this tenacity is corrected to take into account the actual substratum 3-D profile, surface roughness no longer influences significantly the corrected adhesion strength (e.g. 0.18 and 0.34 MPa on smooth PMMA, 0.19 and 0.42 MPa on rough PMMA for sea stars and sea urchins, respectively). It can be hypothesized that, under slow self-imposed forces, disc material behaves viscously to adapt to substratum roughness while the adhesive fills out only very small surface irregularities (in the nanometer range). It is deposited as a thin film ideal for generation of strong adhesion. Under short pulses of wave-generated forces, attached discs probably behave elastically, distributing the stress along the entire contact area, in order to avoid crack generation and thus precluding disc peeling and tube foot detachment.