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.     

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.     

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.     

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.     

Evidence for an acidic compartment in sea urchin eggs (Paracentrotus lividus): role at fertilization

The characteristics of [14C]methylamine accumulation by isolated cortices were measured in eggs from three species of sea urchins: Paracentrotus lividus, Arbacia lixula and Sphaerechinus granularis. In all cases, the results pointed to the existence of an acidic compartment in the cortical zone. In P. lividus eggs, cortical granules did not participate in proton storage which likely took place in pigment granules. [14C]Methylamine accumulation was dramatically reduced by monovalent cation ionophores (monensin and nigericin) and by NH4Cl, but not by valinomycin. ATP depletion only partially affected the isotope uptake. Simultaneous measurements of intracellular pH and of external titratable acidity during ammonia treatment of eggs, indicate that after fertilization, eggs increased their capacity to concentrate hydrogen ions in an intracellular store. Following insemination, cortices from P. lividus eggs exhibited a 3-fold increase in [14C]methylamine accumulation. It is concluded that the egg cortical area contains acidic organelles sequestering hydrogen ions by means of an electrogenic H+ pump, and that this mechanism, enhanced at fertilization, participates in a local alkalinization. The role of such a mechanism is discussed.     

Metal-binding proteins in eggs of various sea urchin species.

Metallothionein presence and amount were determined in the unfertilized eggs of six sea urchin species by silver saturation assay and gel-chromatography of cell extracts. The results showed high levels of metallothionein in the egg cytoplasm of the two Mediterranean species Paracentrotus lividus and Sphaerechinus granularis. No metallothionein was found either in the eggs of Arbacia lixula, or in those of the three Eastern species Strongylocentrotus intermedius, Temnopleurus hardwickii and Clypeaster japonicus. However, the extracts of the latter three species revealed the presence of zinc bound in a macromolecular form, thus suggesting the existence of metal-binding proteins distinct from metallothioneins.     

Response to heat shock of different sea urchin species

It is demonstrated that sea urchin embryos of the species Sphaerechinus granularis are able to respond to heat shock by producing heat shock proteins at the same stage as embryos of Paracentrotus lividus, i.e. after hatching. Arbacia lixula embryos are able to synthesize heat shock proteins already at the stage of 64-128 blastomeres. Embryonic survival is observed if the embryos are heated at the stages at which they can synthesize the heat shock proteins. The inhibition of the bulk protein synthesis after heating at 31 degrees C is never less than 50%.     

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.     

Phenotypic homogeneity of two intertidal snails across a wave exposure gradient in South Australia

Dislodgement by the large drag forces imparted by breaking waves is an important cause of mortality for intertidal snails. The risk of drag-induced dislodgement can be reduced with: (1) a smaller shell of lower maximum projected surface area (MPSA); (2) a streamlined shell shape characterized by a squatter shell; and/or (3) greater adhesive strength attained through a larger foot area or increased foot tenacity. Snails on exposed coasts tend to express traits that increase dislodgement resistance. Such habitat-specific differences could result from direct selection against poorly adapted phenotypes on exposed shores but may reflect gastropod adaptation to high wave action achieved through phenotypic plasticity or genetic polymorphism. With this in mind, we examined the size, shape and adhesive strength of populations of two gastropod species, Austrocochlea constricta (Lamarck) and Nerita atramentosa (Reeve), from two adjacent shores representing extremes in wave exposure. Over a 5 day period, maximum wave forces were more than 10 times greater on the exposed than sheltered shore. Size-frequency distributions indicate that a predator consuming snails within the 1.3-1.8 cm length range regulates sheltered shore populations of both snail species. Although morphological scaling considerations suggest that drag forces should not place physical limits on the size of these gastropods, exposed shore populations of both snails were small relative to the maximum size documented for these species. Therefore, selective forces at the exposed site might favour smaller individuals with increased access to microhabitat refuges. Unexpectedly, however, neither snail species exhibited between-shore differences in shape, foot area or foot tenacity, which are likely to have adaptive explanations. Hence, it is possible that these snails are incapable of adaptive developmental responses to high wave action. Instead, the homogeneous and wave-exposed nature of Australia's southern coastline may have favoured the evolution of generalist strategies in these species.     

Furrowing in altered cell surfaces.

Understanding the process which established the cell division mechanism requires analysis of the role of the responding surface as well as that of stimulatory subsurface structures. Cell surface was altered by the expansion which occurs during exovate formation. Exovates appear on the surface of fertilized Arbacia lixula, Paracentrotus lividus and Echinarachnius parma eggs in response to extreme flattening. They result from cytoplasmic outflow initiated in a very restricted portion of the egg surface. Observations of the formation process in pigmented A. lixula eggs revealed that the original surface may be expanded about 100 fold as the exovate swells. When exovates formed 15-30 minutes after fertilization contain the mitotic apparatus, they divide synchronously with flattened controls. If nucleated exovates are established after the beginning of first cleavage, furrows appear in ten minutes. Exovates established after the beginning of second cleavage develop furrows four minutes after the entrance of the the mitsotic apparatus. Cytoplasm beneath damaged exovate surfaces sometimes develops partial constrictions independently of the surface in the plane the furrow would have occupied. These results suggest that normal surface structure is unnecessary for furrow establishment and function.     

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

Abstract

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.     

Mitochondrial DNA in the sea urchin Arbacia lixula: evolutionary inferences from nucleotide sequence analysis.

From the stirodont Arbacia lixula we determined the sequence of 5,127 nucleotides of mitochondrial DNA (mtDNA) encompassing 18 tRNAs, two complete coding genes, parts of three other coding genes, and part of the 12S ribosomal RNA (rRNA). The sequence confirms that the organization of mtDNA is conserved within echinoids. Furthermore, it underlines the following peculiar features of sea urchin mtDNA: the clustering of tRNAs, the short noncoding regulatory sequence, and the separation by the ND1 and ND2 genes of the two rRNA genes. Comparison with the orthologous sequences from the camarodont species Paracentrotus lividus and Strongylocentrotus purpuratus revealed that (1) echinoids have an extra piece on the amino terminus of the ND5 gene that is probably the remnant of an old leucine tRNA gene; (2) third-position codon nucleotide usage has diverged between A. lixula and the camarodont species to a significant extent, implying different directional mutational pressures; and (3) the stirodont-camarodont divergence occurred twice as long ago as did the P. lividus-S. purpuratus divergence.     

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.     

Microanatomy and ultrastructure of the foot of the infaunal bivalve Tegillarca granosa (Bivalvia: Arcidae)

In this study, the morphology and ultrastructure of the foot of Tegillarca granosa was compared with the bivalves from different habitats. The sediment of habitat of T. granosa is mostly a mixture of sand (68.93%) and mud (24.12%). The foot is wedge-shaped with multiple projections on the surface and covered with ciliary tufts. The epithelial layer is simple and composed of ciliated columnar epithelia and mucous cells. Although the mucous cells are distributed mostly in the epithelial layer, they are developed even in the connective tissues and muscle layers, and the mucous cells mostly contain acidic carboxylated mucosubstances. From the TEM observation, secretory cells are classified into three types. Type A secretory cell has a goblet form and is most widely distributed among the three types. Type B secretory cell has an oval form and the secretory granule has fibrous substance. Type C secretory cell has an elongated elliptic form and membrane-bounded secretory granules. The muscle fiber bundles are composed mainly of smooth muscle fibers. The smooth muscle fibers can be divided into two types. Type A muscle fibers have evenly distributed thick microfilaments between the thin microfilaments of cytoplasm. Type B muscle fiber has cluster of condensed microfilaments in the medulla cytoplasm while the cortical cytoplasm has loose distribution of thin microfilaments.     

An ultrastructural study of the clitellar epithelium of the earthworm Metaphire posthuma (vail.)

The ultrastructure of clitellar epithelium of Metuphire posthuma revealed mainly three types of secretory cells. Most prominent among these are the large slender granular cells which contain a large number of secretory granules filling in the entire columncr region of the cell. The secretory granules are 2-4mu in diameter with a limiting membrane and containing numerous tiny vesicles in a matrix of varying electron density. Basolateral rough endoplasmic reticulum and extensive Golgi cisternae were seen interspersed with the secretory granules. The Golgi cisternae in these cells were quite prominent extending all around the secretory granules. The secretory granules of type 2 cells are spheroid bodies with motley appearance due to varying electron density of the matrix. The immature granules contain fibrillar material. Type 3 cells contained electron lucent membrane-bound mucous like secretory granules which are reticulated with filamentous materials. All the three cell types open to the exterior at the cuticular region which is characterised by the presence of numerous microvilli.     

Evolution of pedal digit orientation and morphology in relation to acquisition and secondary loss of the adhesive system in geckos

Among geckos, the acquisition of the adhesive system is associated with several morphological changes of the feet that are involved in the operation of the adhesive apparatus. However, analyses using a comparative framework are lacking. We applied traditional morphometrics and geometric morphometric analysis with phylogenetic comparative methods to morphological data, collected from X-ray scans, to examine patterns of morphological evolution of the pes in association with the gain and loss of adhesive capabilities, and with habitat occupancy among 102 species of gecko. Padbearing gecko lineages tend to have shorter digits and greater inter-digital angles than padless ones. Arboreal and saxicolous species have shorter digits than terrestrial species. Our results suggest repeated shifts that converge upon a similar padbearing morphology, with some modifications being associated with the habitat occupied. We demonstrate that functional innovation and habitat can operate on, and influence, different components of foot morphology.     

Systematics of the amphiatlantic Microphthalmus-listensis-species-group (Polychaeta: Hesionidae): Facts and concepts for reconstruction of phylogeny and speciation

Previous studies established the existence of morphologically highly similar amphiatlantic populations of the predominantly interstitial genus Microphthatmus (European coastline - from the German Bay to the Bay of Biscaya; American coastline - from Maine to Massachusetts and at the coast of North Carolina). Originally these three populations were seen as belonging to a single species. By using a broad spectrum of different methodologies for character investigations (especially through the use of electron microscopy) three, distinct species can now be distinguished: M. listensis Westheide 1967, M. nahantensis n. sp. and M. carolinensis n. sp. While habitat structure and external morphology - generally important in polychaete systematics - were found to be rather similar between the three species, this study uncovered distinct differences between the species in their internal organization. Such sharp differences could even be followed down to the ultrastructure of single cells (e. g. copulatory stylets, position and number of ovaries, sperm and secretory granules of male accessory glands). Within population character variability is significantly lower in several features of M. carolinensis than in the same characters of the other two species. Similarities as well as differences have been noted in behavioural features and population dynamics between the species.     

Characterisation of the Carbohydrate Fraction of the Temporary Adhesive Secreted by the Tube Feet of the Sea Star <Emphasis Type="Italic">Asterias rubens</Emphasis>

In sea stars, adhesion takes place at the level of a multitude of small appendages, the tube feet. It involves the secretion of an adhesive material which, after tube foot detachment, remains on the substratum as a footprint. It was previously reported that the two main organic components of this material are proteins and carbohydrates. The carbohydrate moiety of the adhesive secretion of Asterias rubens was investigated using a set of 16 lectins which were used on sections through tube feet, on footprints, and on the proteins extracted from these footprints. After gel electrophoresis, these proteins separate into eight protein bands which were named sea star footprint proteins (Sfps). Eleven lectins label the tube foot epidermis at the level of the adhesive cells, four react with footprints, and eight with two of the extracted footprint proteins, which are therefore classified as glycoproteins. Sfp-290 appears to bear mostly N-linked oligosaccharides and Sfp-210 principally O-linked oligosaccharides. The outer chains of both glycoproteins enclose galactose, N-acetylgalactosamine, fucose, and sialic acid residues. Another part of the carbohydrate fraction of the footprints would be in the form of larger molecules, such as sialylated proteoglycans. These two types of glycoconjugates are presumably key components of the sea star temporary adhesive providing both cohesive and adhesive contributions through electrostatic interactions by the polar and hydrogen-bonding functional groups of their glycan chains.     

Patterns in the binding of cytotoxic neuropharmacologic preparations by early sea urchin embryos]

Early embryos of Arbacia lixula, Paracentrotus lividus and Sphaerechinus granularis intensively bind cytotoxic neuropharmacological drugs, such as antiserotonine indole derivatives, cholinolytics and tricyclic antidepressants. The binding intensity decreases markedly upon quaternization of the drugs. Quantitative analysis indicates that: a)with respect to the drugs, the suspension of living embryos may be described as a single adsorbing system following the Langmuir equation; b) at least two independent binding pools exist in embryos; c) the magnitude of cytotoxic effect of a given drug is not proportional to its binding.