Opsin evolution in the Ambulacraria.

Opsins--G-protein coupled receptors involved in photoreception--have been extensively studied in the animal kingdom. The present work provides new insights into opsin-based photoreception and photoreceptor cell evolution with a first analysis of opsin sequence data for a major deuterostome clade, the Ambulacraria. Systematic data analysis, including for the first time hemichordate opsin sequences and an expanded echinoderm dataset, led to a robust opsin phylogeny for this cornerstone superphylum. Multiple genomic and transcriptomic resources were surveyed to cover each class of Hemichordata and Echinodermata. In total, 119 ambulacrarian opsin sequences were found, 22 new sequences in hemichordates and 97 in echinoderms (including 67 new sequences). We framed the ambulacrarian opsin repertoire within eumetazoan diversity by including selected reference opsins from non-ambulacrarians. Our findings corroborate the presence of all major ancestral bilaterian opsin groups in Ambulacraria. Furthermore, we identified two opsin groups specific to echinoderms. In conclusion, a molecular phylogenetic framework for investigating light-perception and photobiological behaviors in marine deuterostomes has been obtained.

First insights into the biochemistry of tube foot adhesive from the sea urchin Paracentrotus lividus (Echinoidea, Echinodermata).

Sea urchins are common inhabitants of wave-swept shores. To withstand the action of waves, they rely on highly specialized independent adhesive organs, the adoral tube feet. The latter are extremely well-designed for temporary adhesion being composed by two functional subunits: (1) an apical disc that produces an adhesive secretion to fasten the sea urchin to the substratum, as well as a deadhesive secretion to allow the animal to move and (2) a stem that bears the tensions placed on the animal by hydrodynamism. Despite their technological potential for the development of new biomimetic underwater adhesives, very little is known about the biochemical composition of sea urchin adhesives. A characterization of sea urchin adhesives is presented using footprints. The latter contain inorganic residues (45.5%), proteins (6.4%), neutral sugars (1.2%), and lipids (2.5%). Moreover, the amino acid composition of the soluble protein fraction revealed a bias toward six amino acids: glycine, alanine, valine, serine, threonine, and asparagine/aspartic acid, which comprise 56.8% of the total residues. In addition, it also presents higher levels of proline (6.8%) and half-cystine (2.6%) than average eukaryotic proteins. Footprint insolubility was partially overcome using strong denaturing and reducing buffers, enabling the visualization of 13 proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The conjugation of mass spectrometry with homology-database search allowed the identification of six proteins: alpha and beta tubulin, actin, and histones H2B, H3, H2A, and H4, whose location and function in the adhesive are discussed but require further investigation. For the remaining unidentified proteins, five de novo-generated peptide sequences were found that were not present in the available protein databases, suggesting that they might be novel or modified proteins.

Echinoderm adhesive secretions: from experimental characterization to biotechnological applications.

Adhesion is a way of life in echinoderms. Indeed, all the species belonging to this phylum use adhesive secretions extensively for various vital functions. According to the species or to the developmental stage considered, different adhesive systems may be recognized. (1) The tube feet or podia are organs involved in attachment to the substratum, locomotion, feeding or burrowing. Their temporary adhesion relies on a duo-gland adhesive system resorting to both adhesive and de-adhesive secretions. (2) The larval adhesive organs allow temporary attachment of larvae during settlement and strong fixation during metamorphosis. (3) The Cuvierian tubules are sticky defence organs occurring in some holothuroid species. Their efficacy is based on the instantaneous release of a quick-setting adhesive. All these systems rely on different types of adhesion and therefore differ in the way they operate, in their structure and in the composition of their adhesive. In addition to fundamental interests in echinoderm bioadhesives, a substantial impetus behind understanding these adhesives are the potential technological applications that can be derived from their knowledge. These applications cover two broad fields of applied research: design of water-resistant adhesives and development of new antifouling strategies. In this context, echinoderm adhesives could offer novel features or performance characteristics for biotechnological applications. For example, the rapidly attaching adhesive of Cuvierian tubules, the releasable adhesive of tube feet or the powerful adhesive of asteroid larvae could each be useful to address particular bioadhesion problems.

Ultrastructure of the echinoderm cuticle after fast-freezing/freeze substitution and conventional chemical fixations.

The cuticles of the pedicellaria primordia in the sea urchin Paracentrotus lividus and of the tube foot disk in the sea star Asterias rubens were preserved by different methods, viz., glutaraldehyde fixation followed by osmium tetroxide postfixation, glutaraldehyde-ruthenium red fixation followed by osmium tetroxide-ruthenium red postfixation, and two fast freezing / freeze substitution methods (FF/FS). The gross ultrastructure of the cuticle as well as the influence of the preservation method on this ultrastructure were identical for the two tissues studied. The cuticle ultrastructure was poorly preserved after glutaraldehyde fixation / osmium tetroxide postfixation. Its preservation was improved after ruthenium red was added in the fixative and postfixative, but the best preservation was consistently achieved using FF/FS. Both low-pressure freezing (plunge freezing) and high-pressure freezing were tested, the latter giving seemingly better results. With these methods, the cuticle appeared to be composed of a proximal lower cuticle, an intermediate upper cuticle, and a distal fuzzy coat. In particular, cryoimmobilization methods emphasized or revealed the occurrence of a well-developed fibrillar lower cuticle in the pedicellaria, the complexity of the upper cuticle which consisted of several zones, and the importance of the usually poorly preserved fuzzy coat that is actually the thickest layer of the cuticle. These observations bring new insights on the functions of the cuticle, and particularly of the fuzzy coat. According to its preservation characteristics, the fuzzy coat presumably consists mostly of proteoglycans. This composition could give it shock absorption and antifouling properties. Furthermore, its important thickness also implies that molecules detected by the short sensory cilia must diffuse through and could be selected by the fuzzy coat.

Maintaining the line of defense: regeneration of Cuvierian tubules in the sea cucumber Holothuria forskali (Echinodermata, Holothuroidea).

When irritated, individuals of the sea cucumber Holothuria forskali expel a few Cuvierian tubules which lengthen, instantly become sticky, and rapidly immobilize most organisms with which they come into contact. After expulsion, the lost tubules are readily regenerated. When only a few tubules have been expelled, there is often a latent period before the regeneration starts. In contrast, when many tubules have been expelled, the regenerative process starts immediately but proceeds in successive waves of 10 to 30 tubules that begin to regenerate at 10-day intervals. However, in all cases, the complete regeneration of a given tubule takes about 5 weeks and may be divided into three successive phases: an initial repair phase including the overall 48-h post-autotomy period, a true regenerative phase taking about 4 weeks to complete, and a growth phase of about one more week. Initial regeneration events occur by epimorphosis, cell proliferation being essential to the regenerative process, whereas late events occur mainly by morphallaxis, with migration of the newly differentiated cells. The mesothelium is the tissue layer in which cell proliferation is the most precocious and the most important, involving both peritoneocytes and undifferentiated cells (which seem to be dedifferentiated peritoneocytes). As regeneration proceeds, the percentage of undifferentiated cells regularly decreases in parallel with the differentiation of granular (adhesive-secreting) cells and myocytes. The myocytes then separate off from the mesothelium and migrate within the connective tissue layer. Three types of pseudopodial cells follow one another in the tubule connective tissue during regeneration. Type 1 cells have all the characteristics of echinoderm phagocytes and may have a fibroblastic function, cleaning the connective tissue compartment before new collagen synthesis starts. Type 2 cells are rather undifferentiated and divide actively. The presence of type 3 cells is closely associated with the appearance of collagen fibers, and it is suggested that they have a fibroblastic function. In the inner epithelium, cells also divide actively, but only those in which spherules have not yet differentiated in the basal intraconnective processes. It appears, therefore, that in the three tissue layers of the tubules, regeneration proceeds by cell dedifferentiation, then proliferation, and finally by differentiation. Cuvierian tubules thus constitute a very efficient defensive mechanism: their large number, sparing use, and particular regeneration dynamics make them an almost inexhaustible line of defense maintained at limited energy cost.

A study of the temporary adhesion of the podia in the sea star asterias rubens (Echinodermata, asteroidea) through their footprints

Sea stars are able to make firm but temporary attachments to various substrata owing to secretions released by their podia. A duo-glandular model has been proposed in which an adhesive material is released by two types of non-ciliated secretory (NCS1 and NCS2) cells and a de-adhesive material is released by ciliated secretory (CS) cells. The chemical composition of these materials and the way in which they function have been investigated by studying the adhesive footprints left by the asteroids each time they adhere to a substratum. The footprints of Asterias rubens consist of a sponge-like material deposited as a thin layer on the substratum. Inorganic residues apart, this material is made up mainly of proteins and carbohydrates. The protein moiety contains significant amounts of both charged (especially acidic) and uncharged polar residues as well as half-cystine. The carbohydrate moiety is also acidic, comprising both uronic acids and sulphate groups. Polyclonal antibodies have been raised against footprint material and were used to locate the origin of footprint constituents in the podia. Extensive immunoreactivity was detected in the secretory granules of both NCS1 and NCS2 cells, suggesting that their secretions together make up the bulk of the adhesive material. No immunoreactivity was detected in the secretory granules of CS cells, and the only other structure strongly labelled was the outermost layer of the cuticle, the fuzzy coat. This pattern of immunoreactivity suggests that the secretions of CS cells are not incorporated into the footprints, but instead might function to jettison the fuzzy coat, thereby allowing the podium to detach.

Fine structure of the dorsal papillae in the holothurioid Holothuria forskali (Echinodermata).

The dorsal surface of the holothurioid Holothuria forskali bears several longitudinal rows of modified podia called papillae. Each papilla consists of a conical stem topped by an hemispherical bud. Their gross tissue stratification is the same all along the papilla being made up of four tissue layers, viz. an inner mesothelium, a connective tissue layer, a nerve plexus and an outer epidermis. The latter is differently organized according to whether it belongs to the stem or to the bud. The epidermis of the bud is built up by ciliated cells that intimately contact the nerve plexus and have the classical structure of echinoderm sensory cells. The papillae are thus sensory organs involved in mechanoreception and possibly chemoreception.

The Role of Podial Secretions in Adhesion in Two Species of Sea Stars (Echinodermata).

Individuals of Asterias rubens and Marthasterias glacialis use their podia in locomotion, anchorage, and feeding. Each podium consists of a stem with a disk at its tip. The stem allows the podium to lengthen, flex, and retract, and the disk allows the podium to adhere to the substratum. Adhesion of sea star podia seems to rely on the epidermal secretions of the disk and not on a mechanical sucker-like operation. The disk epidermis is made up of five cell types: nonciliated secretory cells (NCS cells) of two different types (NCS1 and NCS2), both containing granules that are at least partly mucopolysaccharidic in composition; ciliated secretory cells (CS cells) containing small granules of unknown content; nonsecretory ciliated cells (NCS cells); and support cells. The epidermal cells of the podial disk are presumably functioning as a duogland adhesive system that is involved in an adhesive/deadhesive process. The following model is presented. Adhesive secretions are produced by NCS1 and NCS2 cells (both of them have extruded some of their secretory granules in attached podia). These secretions constitute a layer of adhesive material between the podium and the substratum, this layer being the footprint left by the podium after it has become detached from the substratum. Deadhesion, on the other hand, would be due to CS cell secretions. All these secretions would be controlled by stimuli perceived by the two types of ciliated cells (receptor cells), which presumably interact with the secretory cells via the nerve plexus.