Evaluation of the attachment strength of individuals of Asterina gibbosa (Asteroidea, Echinodermata) during the perimetamorphic period

A turbulent channel flow apparatus was used to determine the adhesion strength of the three perimetamorphic stages of the asteroid Asterina gibbosa, i.e. the brachiolaria larvae, the metamorphic individuals and the juveniles. The mean critical wall shear stresses (wall shear stress required to dislodge 50% of the attached individuals) necessary to detach larvae attached by the brachiolar arms (1.2 Pa) and juveniles attached by the tube feet (7.1 Pa) were one order of magnitude lower than the stress required to dislodge metamorphic individuals attached by the adhesive disc (41 Pa). This variability in adhesion strength reflects differences in the functioning of the adhesive organs for these different life stages of sea stars. Brachiolar arms and tube feet function as temporary adhesion organs, allowing repetitive cycles of attachment to and detachment from the substratum, whereas the adhesive disc is used only once, at the onset of metamorphosis, and is responsible for the strong attachment of the metamorphic individual, which can be described as permanent adhesion. The results confirm that the turbulent water channel apparatus is a powerful tool to investigate the adhesion mechanisms of minute organisms.     

Micro- and nanostructure of the adhesive material secreted by the tube feet of the sea star Asterias rubens

To attach to underwater surfaces, sea stars rely on adhesive secretions produced by specialised organs, the tube feet. Adhesion is temporary and tube feet can also voluntarily become detached. The adhesive material is produced by two types of adhesive secretory cells located in the epidermis of the tube foot disc, and is deposited between the disc surface and the substratum. After detachment, this material remains on the substratum as a footprint. Using LM, SEM, and AFM, we described the fine structure of footprints deposited on various substrata by individuals of Asterias rubens. Ultrastructure of the adhesive layer of attached tube feet was also investigated using TEM. Whatever the method used, the adhesive material appeared as made up of globular nanostructures forming a meshwork deposited on a thin homogeneous film. This appearance did not differ according to whether the footprints were fixed or not, and whether they were observed hydrated or dry. TEM observations suggest that type 2 adhesive cells would be responsible for the release of the material constituting the homogeneous film whereas type 1 adhesive cells would produce the material forming the meshwork. This reticulated pattern would originate from the arrangement of the adhesive cell secretory pores on the disc surface.     

Cloning,Characterization, and Expression Levels of the <Emphasis Type="Italic">Nectin</Emphasis> Gene from the Tube Feet of the Sea Urchin <Emphasis Type="Italic">Paracentrotus Lividus</Emphasis>

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.     

Adaptations to benthic development: functional morphology of the attachment complex of the brachiolaria larva in the sea star Asterina gibbosa

The asteroid Asterina gibbosa lives all its life in close relation to the sea bottom. Indeed, this sea star possesses an entirely benthic, lecithotrophic development. The embryos adhere to the substratum due to particular properties of their jelly coat, and hatching occurs directly at the brachiolaria stage. Brachiolariae have a hypertrophied, bilobed attachment complex comprising two asymmetrical brachiolar arms and a central adhesive disc. This study aims at describing the ultrastructure of the attachment complex and possible adaptations, at the cellular level, to benthic development. Immediately after hatching, early brachiolariae attach by the arms. All along the anterior side of each arm, the epidermis encloses several cell types, such as secretory cells of two types (A and B), support cells, and sensory cells. Like their equivalents in planktotrophic larvae, type A and B secretory cells are presumably involved in a duo-glandular system in which the former are adhesive and the latter de-adhesive in function. Unlike what is observed in planktotrophic larvae, the sensory cells are unspecialized and presumably not involved in substratum testing. During the larval period, the brachiolar arms progressively increase in size and the adhesive disc becomes more prominent. At the onset of metamorphosis, brachiolariae cement themselves strongly to the substratum with the adhesive disc. The disc contains two main cell types, support cells and secretory cells, the latter being responsible for the cement release. During this metamorphosis, the brachiolar arms regress while post-metamorphic structures grow considerably, especially the tube feet, which take over the role of attachment to the substratum. The end of this period corresponds to the complete regression of the external larval structures, which also coincides with the opening of the mouth. This sequence of stages, each possessing its own adhesive strategy, is common to all asteroid species having a benthic development. In A. gibbosa, morphological adaptations to this mode of development include the hypertrophic growth of the attachment complex, its bilobed shape forming an almost completely adhesive sole, and the regression of the sensory equipment.     

The attachment complex of brachiolaria larvae of the sea star <Emphasis Type="Italic">Asterias rubens</Emphasis> (Echinodermata): an ultrastructural and immunocytochemical study

The attachment complex of brachiolaria larvae of the asteroid Asterias rubens comprises three brachiolar arms and an adhesive disc located on the preoral lobe. The former are used in temporary attachment and sensory testing of the substratum, whereas the latter is used for permanent fixation to the substratum at the onset of metamorphosis. Brachiolar arms are hollow structures consisting of an extensible stem tipped by a crown of dome-like ciliated papillae. The papilla epidermis is composed of secretory cells (type A, B and C cells), non-secretory ciliated cells, neurosecretory-like cells and support cells. Type A and B secretory cells fill a large part of the papilla epidermis and are always closely associated. They presumably form a duo-gland adhesive system in which type A and B cells are respectively adhesive and de-adhesive in function. The adhesive disc is an epidermal structure mainly composed of secretory cells and support cells. Secretory cells produce the cement, which anchor the metamorphic larva to the substratum until the podia are developed. The relatedness between the composition of the adhesive material in the brachiolaria attachment complex and in the podia of adults was investigated by immunocytochemistry using antibodies raised against podial adhesive secretions of A. rubens. Type A secretory cells were the only immunolabelled cells indicating that their temporary adhesive shares common epitopes with the one of podia. The attachment pattern displayed by the individuals of A. rubens during the perimetamorphic period—temporary, permanent, temporary—is unique among marine non-vertebrate Metazoa.     

Adhesion Strength of Settled Spores of the Green Alga Enteromorpha

Strengths of attachment of spores of the green fouling alga Enteromorpha to glass have been measured using a modified water jet apparatus. Surface pressures of ～250 kPa were required to quantitatively remove attached spores after 4 h contact with a surface. The development of adhesive and cohesive strength is highly time-dependent; after 8 h in contact with a surface spores did not detach, even at pressures in excess of 250 kPa. Spores settled in groups are more resistant to detachment than single spores, which suggests that the adaptive value of gregarious settlement behaviour may lie in the greater resistance of groups to detachment forces in a naturally turbulent environment. The interfacial forces exerted as water impinges on the surface and the derivation of adhesion strength values in terms of wall shear stress are discussed and compared with those obtained by other methods. A surface pressure of 250 kPa approximates to 325 Pa wall shear stress. Calculation using the power-law formula predicts that detachment forces of this magnitude are unlikely to be realized at operating speeds for most vessels and that most Enteromorpha spores would not detach from untreated hulls.     

Treatment of neutrophils with cytochalasins converts rolling to stationary adhesion on P-selectin

We investigated how disruption of the actin cytoskeleton with cytochalasins modified adhesion of neutrophils rolling on a platelet monolayer in vitro at 37°C. When perfused at a wall shear stress of 0.1 Pa over rolling cells, cytochalasin B, cytochalasin D and dihydro-cytochalasin B each induced dose-dependent (∼1–10 μg/ml) conversion to stationary attachment over minutes. Stopping was associated with cell elongation to a teardrop shape. Increased deformability of cytochalasin-treated cells was independently evidenced by more rapid entry into a micropipette. Spherical shape and rolling were reestablished concurrently on washout of the cytochalasins, while increasing the shear stress in the range 0.2 to 1.0 Pa induced tear-drop-shaped cells to restart rolling even in the continued presence of cytochalasin. When cells were pretreated with cytochalasin B, they attached efficiently at 0.1 Pa, rolled initially and only stopped after ∼30 seconds when elongation had been established. Adhesion was selectin-mediated in the presence or absence of cytochalasin B, as judged by inhibition of attachment by antibody against P-selectin and failure of antibody against β2-integrin CD18 to influence adhesion. Cessation of rolling is unlikely to have arisen from an increase in adhesive contact area induced by deformation because stopped cells were found to be attached only at their pointed end. Failure of adhesive bonds to peel may have arisen because selectin ligands freed of cytoskeletal restraint were dragged into this tethered region and clustered there, and because force applied to bonds was influenced by the change in cell shape. These results suggest that cytoskeletal structure is an important modulator of dynamic adhesive responses of leukocytes, via effects on adhesion receptors and cellular mechanics. J. Cell. Physiol. 174:206–216, 1998. © 1998 Wiley-Liss, Inc.     

Structure of the organs of attachment of brachiolaria larvae of Stichaster australis (Verrill) and Coscinasterias calamaria (Gray) (Echinodermata: Asteroidea)

The structure of the brachiolar arms and adhesive disk of the brachiolaria larvae of Stichaster australis (Verrill) and Coscinasterias calamaria (Gray) was determined from light microscopy and from scanning and transmission electron microscopy. The structure of these organs was very similar in both species.The brachiolar arms are comprised of a stem region terminating in a crown of adhesive papillae which are made up of a variety of secretory cell types. Principal among these are elongated cells producing very electron-dense secretory particles, which are released at the free cell surface attached to cilia. Secretory particles appear to be important in temporary attachment of the brachiolar arms to the substratum. Ciliary sense cells, possibly used in the recognition of specific substrata are located at the tip of adhesive papillae.The adhesive disk is comprised of large cells packed with secretory droplets and elongated intracellular fibres. In the attached adhesive disk, secretory droplets are lost, having formed the cement that attaches the disk to the substratum. It appears that adhesive papillae lateral to the adhesive disk hold the disk in position close to the substratum during secretion and hardening of the cement. The intracellular fibres are the principal anchoring structures running from microvilli (locked into the attachment cement) on the surface of the disk to the underlying connective tissue of the attachment stalk.     

Quantitative studies of increased cell-to-substratum adhesion at low temperature

The cell-to-substratum adhesion of an established epithelial cell line cultured for 24 h on glass coverslips was determined at 4 degrees C, 8 degrees C and 37 degrees C using a miniaturised parallel-plate shearing apparatus. The measurements of the minimum shear necessary to dislodge the cells (minimum distraction force, MDF) demonstrated a three- to fourfold increase in the adhesion of the cells at 4 degrees C (6.17 Pa) compared to that at 37 degrees C (1.36 Pa). At 8 degrees C the MDF was 2.31 pascals. Part of the adhesion was resistant to mild trypsinisation. Trypsin-resistant adhesion (TRA) was stabilised by low temperature, and by treatment with concanavalin A (50 micrograms ml-1) or colchicine (200-400 microM). The effects of con A (140 micrograms ml-1) and low temperature (4 degrees C) were additive, giving a combined MDF of greater than 9.27 pascals. On the basis of their different temperature and protease susceptibility it is suggested that trypsin-sensitive adhesion (TSA) and TRA represent separate functional classes of cell-to-substratum attachment corresponding to 'frictional' and 'tractional' adhesion, respectively.     

Adhesive papillae on the brachiolar arms of brachiolaria larvae in two starfishes, Asterina pectinifera and Asterias amurensis, are sensors for metamorphic inducing factor(s)

It has been hypothesized by Barker that starfish brachiolaria larvae initiate metamorphosis by sensing of metamorphic inducing factor(s) with neural cells within the adhesive papillae on their brachiolar arms. We present evidence supporting Barker's hypothesis using brachiolaria larvae of the two species, Asterina pectinifera and Asterias amurensis. Brachiolaria larvae of these two species underwent metamorphosis in response to pebbles from aquaria in which adults were kept. Time-lapse analysis of A. pectinifera indicated that the pebbles were explored with adhesive papillae prior to establishment of a stable attachment for metamorphosis. Microsurgical dissections, which removed adhesive papillae, resulted in failure of the brachiolaria larvae to respond to the pebbles, but other organs such as the lateral ganglia, the oral ganglion, the adhesive disk or the adult rudiment were not required. Immunohistochemical analysis with a neuron-specific monoclonal antibody and transmission electron microscopy revealed that the adhesive papillae contained neural cells that project their processes towards the external surface of the adhesive papillae and they therefore qualify as sensory neural cells.     

Quantifying Shear-Induced Deformation and Detachment of Individual Adherent Sickle Red Blood Cells

Vaso-occlusive crisis, a common painful complication of sickle cell disease, is a complex process triggered by intercellular adhesive interactions among blood cells and the endothelium in all human organs (e.g., the oxygen-rich lung as well as hypoxic systems such as liver and kidneys). We present a combined experimental-computational study to quantify the adhesive characteristics of sickle mature erythrocytes (SMEs) and irreversibly sickled cells (ISCs) under flow conditions mimicking those in postcapillary venules. We employed an in vitro microfluidic cell adherence assay, which is coated uniformly with fibronectin. We investigated the adhesion dynamics of SMEs and ISCs in pulsatile flow under well-controlled hypoxic conditions, inferring the cell adhesion strength by increasing the flow rate (or wall shear stress (WSS)) until the onset of cell detachment. In parallel, we performed simulations of individual SMEs and ISCs under shear. We introduced two metrics to quantify the adhesion process, the cell aspect ratio (AR) as a function of WSS and its rate of change (the dynamic deformability index). We found that the AR of SMEs decreases significantly with the increase of WSS, consistent between the experiments and simulations. In contrast, the AR of ISCs remains constant in time and independent of the flow rate. The critical WSS value for detaching a single SME in oxygenated state is in the range of 3.9–5.5 Pa depending on the number of adhesion sites; the critical WSS value for ISCs is lower than that of SMEs. Our simulations show that the critical WSS value for SMEs in deoxygenated state is above 6.2 Pa (multiple adhesion sites), which is greater than their oxygenated counterparts. We investigated the effect of cell shear modulus on the detachment process; we found that for the same cell adhesion spring constant, the higher shear modulus leads to an earlier cell detachment from the functionalized surface. These findings may aid in the understanding of individual roles of sickle cell types in sickle cell disease vaso-occlusion.     

Analysis of the Behaviours Mediating Barnacle Cyprid Reversible Adhesion

When exploring immersed surfaces the cypris larvae of barnacles employ a tenacious and rapidly reversible adhesion mechanism to facilitate their characteristic ‘walking’ behaviour. Although of direct relevance to the fields of marine biofouling and bio-inspired adhesive development, the mechanism of temporary adhesion in cyprids remains poorly understood. Cyprids secrete deposits of a proteinaceous substance during surface attachment and these are often visible as ‘footprints’ on previously explored surfaces. The attachment structures, the antennular discs, of cyprids also present a complex morphology reminiscent of both the hairy appendages used by some terrestrial invertebrates for temporary adhesion and a classic ‘suction cup’. Despite the numerous analytical approaches so-far employed, it has not been possible to resolve conclusively the respective contributions of viscoelastic adhesion via the proteinaceous ‘temporary adhesive’, ‘dry’ adhesion via the cuticular villi present on the disc and the behavioural contribution by the organism. In this study, high-speed photography was used for the first time to capture the behaviour of cyprids at the instant of temporary attachment and detachment. Attachment is facilitated by a constantly sticky disc surface – presumably due to the presence of the proteinaceous temporary adhesive. The tenacity of the resulting bond, however, is mediated behaviourally. For weak attachment the disc is constantly moved on the surface, whereas for a strong attachment the disc is spread out on the surface. Voluntary detachment is by force, requiring twisting or peeling of the bond – seemingly without any more subtle detachment behaviours. Micro-bubbles were observed at the adhesive interface as the cyprid detached, possibly an adaptation for energy dissipation. These observations will allow future work to focus more specifically on the cyprid temporary adhesive proteins, which appear to be fundamental to adhesion, inherently sticky and exquisitely adapted for reversible adhesion underwater.     

A turbulent channel flow apparatus for the determination of the adhesion strength of microfouling organisms

The development of novel, fouling‐release surfaces has led to the need for better test methods to evaluate their performance. A water channel has been designed to measure the adhesion strength of microfouling organisms to test surfaces. The apparatus allows six replicate microscope slides to be mounted in a fully‐developed, turbulent channel flow. Wall shear stress in the test section can be varied from 0.9–30 Pa over a Reynolds number range of 2,800 to 27,000 based on the bulk mean velocity and channel height. Calibration of the device indicates that the accuracy and repeatability in the wall shear stress is within 4% throughout the range. Experiments using the fouling diatom Amphora settled on acid‐washed glass slides are presented. The results show significant differences in the shear stress required to remove Amphora cells with settlement time. No significant differences among the replicate slides were observed, indicating flow uniformity in the test section.     

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. J. Morphol., 2011. © 2011 Wiley Periodicals, Inc.     

Characterization of the lecithotrophic larval development of the temperate New Zealand asterinid Stegnaster inflatus

In the family Asterinidae, development through a planktonic lecithotrophic brachiolaria larva is common and has evolved independently several times. Here, we describe the lecithotrophic development of the asterinid Stegnaster inflatus, a species endemic to New Zealand. Early development through the blastula and gastrula stages is short, with hatching at the brachiolaria stage occurring within 48 hr. After hatching, larvae are negatively buoyant, and without aeration remain near the bottom of the culture containers. The settled benthic juvenile stage was reached in ~2 weeks. The brachiolaria of S. inflatus shares common characteristics with the planktonic brachiolaria of other asterinids in that the brachiolar attachment apparatus comprises three brachia and a central adhesive disc, although the latter is thin and appears to be reduced. Mortensen (1925, Videns kabelige Meddelelser fra Dansk naturhistorisk Forening i København, 79 (15), 261‐420) had hypothesized that individuals of S. inflatus might brood within the “cave” formed in the interambulacral space between the arms. We found no evidence for brooding, but hypothesize that S. inflatus may have demersal development, on or near the bottom, which has implications for larval dispersal and population structure.     

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.     

Environmental post-processing increases the adhesion strength of mussel byssus adhesive

Marine mussels (Mytilus trossulus) attach to a wide variety of surfaces underwater using a protein adhesive that is cured by the surrounding seawater environment. In this study, the influence of environmental post-processing on adhesion strength was investigated by aging adhesive plaques in a range of seawater pH conditions. Plaques took 8–12 days to achieve full strength at pH 8, nearly doubling in adhesion strength (+94%) and increasing the work required to dislodge (+59%). Holding plaques in low pH conditions prevented strengthening, causing the material to tear more frequently under tension. The timescale of strengthening is consistent with the conversion of DOPA to DOPA-quinone, a pH dependent process that promotes cross-linking between adhesive proteins. The precise arrangement of DOPA containing proteins away from the adhesive-substratum interface emphasizes the role that structural organization can have on function, an insight that could lead to the design of better synthetic adhesives and metal-coordinating hydrogels.     

A gastric-brooding asteroid, Smilasterias multipara

The gastric-brooding asterinid sea star, Smilasterias multipara, broods from late August to early November in the shallow sublittoral zone of southeastern Australia. We observed males and females spawning in the laboratory. They shed gametes through gonopores on the sides of the arms. The eggs were orange, about 1.0 mm in diameter, and heavier than seawater. They were externally fertilized by sperm, and placed into the stomach of the female by the tube feet. Twenty-four hours after fertilization, the first cleavage occurred. Cleavage was equal, total, and radial. Development via a non-feeding lecithotrophic brachiolaria was direct, there being no planktrotrophic bipinnaria or brachiolaria larva. Embryos developed, through wrinkled blastula and gastrula stages, into brachiolariae with arms. All of the surfaces of the brachiolaria were covered by cilia. At metamorphosis, a starfish rudiment appeared on the posterior portion of the larval body, while the anterior portion of the larval body was absorbed. Two months after fertilization, metamorphosis was complete. After metamorphosis, juveniles in the stomach grew six pairs of tube feet in each arm. Juveniles, 3 mm in diameter, emerged from the mouth of the mother in early November. Developmental evidence suggests that this asteroid has evolved mechanisms for the protection of larvae and juveniles from gastric digestion.     

Attachment ability of sawfly larvae to smooth surfaces

Larvae of the sawfly Rhadinoceraea micans adhere properly to the anti-adhesive surface of their host plant Iris pseudacorus by using three pairs of thoracic legs, seven pairs of abdominal prolegs, and pygopodia, all provided with various smooth adhesive pads. Their attachment performance to smooth flat hydrophilic and hydrophobic glass and Plexiglas surfaces was studied in centrifugal force experiments. Obtained safety factors on Plexiglas were up to 25 in friction, and 8 in adhesion. Although larvae attached significantly stronger to the hydrophilic glass, they attached well also to the hydrophobic one. Pygopodia are suggested to dominate attachment force generation in the centrifugal force experiment. Transverse body position on the centrifuge drum was significantly advantageous for friction force generation than was longitudinal body position. Results are discussed in the context of the sawfly biology and provide a profound base for further detailed studies on biomechanics of sawfly larvae–plant interactions.