Patrick Flammang | University of Mons (original) (raw)

Papers by Patrick Flammang

Research paper thumbnail of Fine structure of the luminous spines and luciferase detection in the brittle star Amphiura filiformis

Zoologischer Anzeiger, Jul 1, 2017

Amphiura filiformis is a common burrowing brittle star from muddy environments along the European... more Amphiura filiformis is a common burrowing brittle star from muddy environments along the European shores. This species emits a blue light at the level of the arm spine tips when mechanically stimulated. Although the putative luciferase of A. filiformis was recently discovered, the ultrastructure of the luminous cells, i.e. the photocytes, remain unknown for this species as well as for most other bioluminescent echinoderms. In this study, we describe the morphology of the arm and spines in the brittle star A. filiformis and compare it to the luciferase expression pattern revealed by immunohistochemistry. Luciferase is expressed at the base of the spine and at the arm tips within well-defined photocyte clusters. Additionally, we investigated the ultrastructure of spine tissues before and after chemical stimulation of light emission in order to identify photocytes and improve our comprehension of the photogenesis phenomenon in brittle stars. Type II granular cells, found in the basal to mid-basal portion of the spine inner tissues and showing ultrastructural modifications during light-emission, are proposed as photocytes. Putative photocytes appear intimately associated with the spine nerve plexus as well as with mucus cells and presumptive pigment cells. Photocytes are characterised by the presence of specific vesicles with reticulated contents in their cytoplasm. Furthermore, the structure of the calcified spine ossicle has been investigated using microCT scanning.

Research paper thumbnail of Molecular mechanisms mediating stiffening in the mechanically adaptable connective tissues of sea cucumbers

Research Square (Research Square), May 13, 2021

In most animals, connective tissues such as the dermis or tendons present invariant mechanical pr... more In most animals, connective tissues such as the dermis or tendons present invariant mechanical properties, fine-tuned for their structural function. However, echinoderms, a group of sea creatures including sea cucumbers, possess the ability to voluntarily modify the mechanical properties of their connective tissues, which are therefore called mutable collagenous tissues (MCT). Understanding the molecular mechanism underlying MCT mutability is a prerequisite for the development of biomimetic smart dynamic materials. The stiffening and softening cycles of MCTs are made possible by the release of specialized effector proteins. We identified a stiffening factor from the dermis of Holothuria forskali, Hf-(D)Tensilin, and showed that it is localized in the secretory granules of juxtaligamental-like cells, a MCT specific cell type. Using recombinant proteins, we confirmed its effect on the dermis and its aggregation effect on extracted collagen fibrils. A model is proposed for the molecular interactions which mediate collagen fibrils cross-linking by tensilin.

Research paper thumbnail of Convergent Evolution of Attachment Mechanisms in Aquatic Animals

To resist hydrodynamic forces, two main underwater attachment strategies have evolved multiple ti... more To resist hydrodynamic forces, two main underwater attachment strategies have evolved multiple times in aquatic animals: glue-like “bioadhesive secretions” and pressure-driven “suction attachment”. In this review, we use a multi-level approach to highlight convergence in underwater attachment mechanisms across four different length-scales (organism, organ, microscopic and molecular). At the organism level, the ability to attach may serve a variety of functions, the most important being: (i) positional maintenance, (ii) locomotion, (iii) feeding, (iv) building, and (v) defense. Aquatic species that use bioadhesive secretions have been identified in 28 metazoan phyla out of the 34 currently described, while suction organs have a more restricted distribution and have been identified in five phyla. Although biological adhesives are highly diverse, it is possible to categorize them into four main types according to the time scale of operation: permanent, temporary, transitory, and instan...

Research paper thumbnail of Epidemiology of a SKin Ulceration Disease (SKUD) in the sea cucumber Holothuria scabra with a review on the SKUDs in Holothuroidea (Echinodermata)

Scientific Reports, 2020

Aquacultivated sea cucumbers often suffer from SKin Ulceration Diseases (SKUDs). SKUDs have been ... more Aquacultivated sea cucumbers often suffer from SKin Ulceration Diseases (SKUDs). SKUDs have been observed in six holothuroid species from nine countries. All SKUDs present a similar symptom—the skin ulceration—and can be induced by bacteria, viruses, or abiotic factors. We here provide an update on SKUDs in holothuroids and analyse the case of the SKUD observed in Holothuria scabra in Madagascar. Field observations revealed a seasonality of the disease (i.e. wintertime maximum peak). Morphological analyses of integument ulcers showed that sea cucumbers react by forming a collagen fibre plug. Metagenomic analyses revealed a higher proportion of Vibrionaceae (Gammaproteobacteria) in ulcers in comparison to the healthy integument of the same individuals. Experimental infection assays were performed with ulcer crude extracts and bacteria isolated from these extracts (e.g. Vibrio parahaemolyticus) but did not significantly induce skin ulceration. Our results suggest that the disease is n...

Research paper thumbnail of Disentangling the Roles of Functional Domains in the Aggregation and Adsorption of the Multimodular Sea Star Adhesive Protein Sfp1

Marine Biotechnology, 2021

Sea stars can adhere to various underwater substrata using an adhesive secretion of which Sfp1 is... more Sea stars can adhere to various underwater substrata using an adhesive secretion of which Sfp1 is a major component. Sfp1 is a multimodular protein composed of four subunits (Sfp1 Alpha, Beta, Delta, and Gamma) displaying different functional domains. We recombinantly produced two fragments of Sfp1 comprising most of its functional domains: the C-terminal part of the Beta subunit (rSfp1 Beta C-term) and the Delta subunit (rSfp1 Delta). Surface plasmon resonance analyses of protein adsorption onto different model surfaces showed that rSfp1 Beta C-term exhibits a significantly higher adsorption than the fibrinogen control on hydrophobic, hydrophilic protein-resistant, and charged self-assembled monolayers, while rSfp1 Delta adsorbed more on negatively charged and on protein-resistant surfaces compared to fibrinogen. Truncated recombinant rSfp1 Beta C-term proteins were produced in order to investigate the role of the different functional domains in the adsorption of this protein. The analysis of their adsorption capacities on glass showed that two mechanisms are involved in rSfp1 Beta C-term adsorption: (1) one mediated by the EGF-like domain and involving Ca2+ and Mg2+ ions, and (2) one mediated by the sequence of Sfp1 Beta with no homology with known functional domain in databases, in the presence of Na+, Ca2+ and Mg2+ ions.

Research paper thumbnail of Structure and composition of the tunic in the sea pineapple Halocynthia roretzi: A complex cellulosic composite biomaterial

Acta Biomaterialia, 2020

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Research paper thumbnail of The cellular basis of bioadhesion of the freshwater polyp Hydra

BMC Zoology, 2016

Background: The freshwater cnidarian Hydra temporarily binds itself to numerous natural substrate... more Background: The freshwater cnidarian Hydra temporarily binds itself to numerous natural substrates encountered underwater, such as stones, leafs, etc. This adhesion is mediated by secreted material from specialized ectodermal modified cells at the aboral end of the animal. The means by which Hydra polyps attach to surface remain unresolved, despite the fact that Hydra is a classic model in developmental and stem cell biology. Results: Here, we present novel observations on the attachment mechanism of Hydra using high pressure transmission electron microscopy, scanning electron microscopy, atomic force microscopy, super-resolution microscopy, and enzyme histochemistry. We analyzed the morphology of ectodermal basal disc cells, studied the secreted material, and its adhesive nature. By electron microscopy we identified four morphologically distinct secretory granules occurring in a single cell type. All the secretory granules contained glycans with different distribution patterns among the granule types. Footprints of the polyps were visualized under dry conditions by atomic force microscopy and found to consist of a meshwork with nanopores occurring in the interstices. Two antibodies AE03 and 3G11, previously used in cell differentiation studies, labelled both, basal disc cells and footprints. Our data suggest that the adhesive components of Hydra are produced, stored and delivered by a single cell type. Video microscopy analysis corroborates a role of muscle contractions for the detachment process. Conclusion: We clearly demonstrated that bioadhesion of Hydra relies on the secreted material. Our data suggest that glycans and/or glycoproteins represent an important fraction of the secreted material. Detachment seems to be initiated by mechanical forces by muscular contractions. Taken together, our study represents the characterization of an unique temporary adhesive system not known in aquatic organisms from other metazoan phyla.

Research paper thumbnail of Chemical characterization of saponins contained in the body wall and the Cuvierian tubules of the sea cucumber Holothuria (Platyperona) sanctori (Delle Chiaje, 1823)

Biochemical Systematics and Ecology, 2016

Holothuria (Platyperona) sanctori is a common sea cucumber of the Mediterranean Sea. Although its... more Holothuria (Platyperona) sanctori is a common sea cucumber of the Mediterranean Sea. Although its ecology and molecular phylogeny have been investigated in previous works, nothing is known about its saponin content, contrarily to other Mediterranean holothuroids. Saponins are bioactive triterpene glycosides that present interesting pharmacological/biological properties, and which can be used as taxonomic markers. This work highlights the different saponins produced by H. sanctori. Mass spectrometry (MALDI-MS and MS/MS) analyses highlighted a total of 18 different saponins, including eight new congeners. Presumptive molecular structures are proposed for all these molecules. A higher diversity of saponins is found in the body wall (12) than in the Cuvierian tubules (8). In addition to this qualitative difference, saponins were also 2e3 fold more concentrated in the Cuvierian tubules than in the integument. Our results demonstrate that H. sanctori, in accordance with its phylogenetic position, is the second species of Holothuria described to date possessing exclusively non-sulfated saponins. The species presents its own mix of saponins, acting as a true chemical signature.

Research paper thumbnail of Enhancing the Membranolytic Activity of Chenopodium quinoa Saponins by Fast Microwave Hydrolysis

Molecules, 2020

Saponins are plant secondary metabolites. There are associated with defensive roles due to their ... more Saponins are plant secondary metabolites. There are associated with defensive roles due to their cytotoxicity and are active against microorganisms. Saponins are frequently targeted to develop efficient drugs. Plant biomass containing saponins deserves sustained interest to develop high-added value applications. A key issue when considering the use of saponins for human healthcare is their toxicity that must be modulated before envisaging any biomedical application. This can only go through understanding the saponin-membrane interactions. Quinoa is abundantly consumed worldwide, but the quinoa husk is discarded due to its astringent taste associated with its saponin content. Here, we focus on the saponins of the quinoa husk extract (QE). We qualitatively and quantitively characterized the QE saponins using mass spectrometry. They are bidesmosidic molecules, with two oligosaccharidic chains appended on the aglycone with two different linkages; a glycosidic bond and an ester function....

Research paper thumbnail of Heavy metals in Diadema setosum (Echinodermata, Echinoidea) from Singapore coral reefs

Journal of Sea Research, 1997

of Zn, Pb, Cd, Fe, Cr, Cu and Ti were measured in body compartments of the echinoid Diadema setos... more of Zn, Pb, Cd, Fe, Cr, Cu and Ti were measured in body compartments of the echinoid Diadema setosum and in the silt fraction of surface sediment from eight coral reefs in Singapore coastal waters. Metal concentrations in the echinoid varied with the body compartment analysed and with the site of sampling. Amongst the body compartments studied, Zn, Cd, Fe, Cr and Cu were preferentially accumulated in the gonads, whereas Pb and Ti were accumulated mainly in the calcified body compartments. The concentrations of most metals differed according to the reef sampled. A decreasing north-south gradient of metal concentrations was observed in D. setosum populations, correlating to the distance from Singapore. A similar gradient was observed for metal concentrations in sediment. It is suggested that D. setosum could be a valuable bioindicator for assessing heavy metal contamination in coral reef ecosystems of the Indo-West Pacific.

Research paper thumbnail of Challenges and Opportunities

The Journal of Perinatal & Neonatal Nursing, 2002

Research paper thumbnail of Is the adhesive material secreted by sea urchin tube feet species-specific?

Journal of Morphology, 2012

Sea urchin adoral tube feet are highly specialized organs that have evolved to provide efficient ... more 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.

Research paper thumbnail of Comparative histological and immunohistochemical study of sea star tube feet (Echinodermata, Asteroidea)

Journal of Morphology, 2005

Adhesion in sea stars is the function of specialized structures, the tube feet or podia, which ar... more 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., knobending, 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.

Research paper thumbnail of Unusual adhesive production system in the barnacle Lepas anatifera : An ultrastructural and histochemical investigation

Journal of Morphology, 2012

Adhesives that are naturally produced by marine organisms are potential sources of inspiration in... more Adhesives that are naturally produced by marine organisms are potential sources of inspiration in the search for medical adhesives. Investigations of barnacle adhesives are at an early stage but it is becoming obvious that barnacles utilize a unique adhesive system compared to other marine organisms. The current study examined the fine structure and chemis-

Research paper thumbnail of Effects of CO2-induced ocean acidification on physiological and mechanical properties of the starfish Asterias rubens

Journal of Experimental Marine Biology and Ecology, 2013

ABSTRACT The increase in atmospheric CO2 due to anthropogenic activity results in an acidificatio... more ABSTRACT The increase in atmospheric CO2 due to anthropogenic activity results in an acidification of the surface waters of the oceans. Its impact will depend on the considered organisms and ecosystems. The intertidal may harbor organisms pre-adapted to the upcoming changes as they face tidal pH and temperature fluctuations. However, these environments will be more affected as shallow waters will face the highest decrease in seawater pH. In this context, the effects of reduced environmental pH on the physiology and tube feet mechanical properties of the intertidal starfish Asterias rubens¸ a top predator, were investigated during 15 and 27 days. Asterias rubens showed a respiratory acidosis with its coelomic fluid pH always lower than that of seawater. This acidosis was most pronounced at pH 7.4. Notwithstanding, the starfish showed no significant variations in RNA/DNA ratio of different tissues and in tube feet strength. However, respiration rates were significantly lower for individuals maintained at reduced seawater pH. Within the ocean acidification context, the present results suggest that A. rubens withstands the effects of reduced seawater pH, at least for medium term exposures.

Research paper thumbnail of Measurement of the attachment strength of brachiolaria larvae and metamorphic individuals of the sea star Asterina gibbosa by a centrifugation method

Journal of Experimental Marine Biology and Ecology, 2009

Two methods are generally used to measure the adhesive strength of invertebrate larvae: direct me... more Two methods are generally used to measure the adhesive strength of invertebrate larvae: direct measurement with a force transducer connected to the organisms and indirect measurement with a water flow used to dislodge the organisms. Each of these methods, however, has its drawbacks. The present study aimed to design a simple and straightforward method to measure the adhesion strength of marine invertebrate larvae based on centrifugation. This centrifuge technique works in immersed conditions and applies forces acting at 45°to the substratum, therefore mimicking natural conditions. It was tested with three different substrata on two developmental stages of the sea star Asterina gibbosa: the brachiolaria larvae, which use temporary adhesion, and the metamorphic individuals which use permanent adhesion. Measurements were completed by SEM and TEM observations of the larval adhesive organs. The critical detachment force (force required to detach 50% of the larvae) of brachiolaria larvae attached to glass (36 ± 9 µN) and rough PMMA (43 ± 16 µN) were equivalent and both significantly higher than the critical detachment force measured on smooth PMMA (11 ± 8 µN). Most metamorphic individuals, on the other hand, resisted to the highest centrifugation speed used, corresponding to a force of 2.13 mN. For the hydrodynamics of larval settlement and metamorphosis, force is the ecologically relevant factor, and adhesion forces obtained by centrifugation are strikingly similar to forces measured for other marine invertebrate larvae with other methods. This indicates the usefulness of the centrifugation technique to compare adhesion of larvae between different species or development stages, or between different treatments.

Research paper thumbnail of Morphometry and mechanical design of tube foot stems in sea urchins: a comparative study

Journal of Experimental Marine Biology and Ecology, 2005

ABSTRACT To withstand hydrodynamic forces, sea urchins rely on their adoral tube feet, which are ... more ABSTRACT To withstand hydrodynamic forces, sea urchins rely on their adoral tube feet, which are specialized for attachment. Although it has been often suggested that the degree of development of these tube feet is intimately related to the maximum environmental energy a species can withstand, it has never been demonstrated by mechanical testing. To address this subject, we studied the mechanical properties of the stem of adoral tube feet from three species of sea urchins, Arbacia lixula, Paracentrotus lividus and Sphaerechinus granularis, which have distinct taxonomic, ecological and morphological characteristics. The tube feet of the three species have a very similar morphology. When a tensile force is applied to the tube foot stem, the connective tissue is the only tissue layer bearing the load. The mechanical properties of this tissue give the tube feet an ideal balance of extensibility (139–166%), strength (23–29 MPa) and stiffness (152–328 MPa), which together produce a material with adequate toughness (2.5–2.9 MJ/m3) to absorb the impact of waves and currents, and thus to resist the environmental challenges of the habitats in which sea urchins live. Extended stems of P. lividus were significantly stiffer (328 MPa) than those of the other two species (152 and 183 MPa, for A. lixula and S. granularis, respectively). No interspecific difference was found in terms of extensibility, strength, initial stiffness and toughness between the tube feet from the three species. The difference in local distribution between the species investigated is therefore not only explained by the mechanical properties of their tube feet, but may involve other factors such as tube foot number and arrangement, tube foot disc tenacity or sea urchin size.

Research paper thumbnail of Polyphosphoprotein-Containing Marine Adhesives

The Journal of Adhesion, 2009

... a * , Aurélie Lambert a , Philippe Bailly a & Elise Hennebert a ... During the last decad... more ... a * , Aurélie Lambert a , Philippe Bailly a & Elise Hennebert a ... During the last decade, Herbert Waite and his group have indeed characterized several polyphosphoproteins from the adhesive secretions of two different marine organisms, mussels and tube-building worms. ...

Research paper thumbnail of Biomechanics of Adhesion in Sea Cucumber Cuvierian Tubules (Echinodermata, Holothuroidea)

Integrative and Comparative Biology, 2002

Several species of sea cucumbers, all belonging to a single family, possess a peculiar and specia... more Several species of sea cucumbers, all belonging to a single family, possess a peculiar and specialized defense system, the Cuvierian tubules. It is mobilized when the animal is mechanically stimulated, resulting in the discharge of a few white filaments, the tubules. In seawater, the expelled tubules lengthen considerably and become sticky upon contact with any object. The adhesiveness of their outer epithelium combined with the tensile strength of their collagenous core make Cuvierian tubules very efficient at entangling and immobilizing most potential predators. We have designed a method to measure the adhesion of holothuroid Cuvierian tubules. Tubule adhesive strength was measured in seven species of sea cucumbers belonging to the genera Bohadschia, Holothuria and Pearsonothuria. The tenacities (force per unit area) varied from 30 to 135 kPa, falling within the range reported for marine organisms using non-permanent adhesion. Two species, H. forskali and H. leucospilota, were selected as model species to study the influence of various factors on Cuvierian tubule adhesive strength. Tubule tenacity varied with substratum, temperature and salinity of the seawater, and time following expulsion. These differences give insight into the molecular mechanisms underlying Cuvierian tubule adhesion. Tenacity differences between substrata of varying surface free energy indicate the importance of polar interactions in adhesion. Variation due to temperature and time after expulsion suggests that an increase of tubule rigidity, presumably under enzymatic control, takes place after tubule elongation and reinforces adhesion by minimizing peeling effects.

Research paper thumbnail of Elucidation of molecular diversity and body distribution of saponins in the sea cucumber Holothuria forskali (Echinodermata) by mass spectrometry

Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2009

Sea cucumbers contain triterpene glycosides called saponins. We investigated the complex saponin ... more Sea cucumbers contain triterpene glycosides called saponins. We investigated the complex saponin mixture extracted from the common Mediterranean species Holothuria forskali. Two different body components were analyzed separately: the body wall (which protects the animal and is moreover the most important organ in terms of surface and weight) and the Cuvierian tubules (a defensive organ that can be expelled on predators in response to an attack). MALDI/MS and MALDI/MS/MS were used to detect saponins and describe their molecular structures. As isomers have been found in the Cuvierian tubules, LC/MS and LC/MS/MS were performed to identify each saponin separately. Twelve saponins have been detected in the body wall and 26 in the Cuvierian tubules. All the saponins from the body wall are also present in the Cuvierian tubules but the latter also contain 14 specific saponins. The presence of isomeric saponins complicated structure elucidation . The diversity and organ specificity of the saponins described here are much higher than what had been reported to date in any sea cucumber species.

Research paper thumbnail of Fine structure of the luminous spines and luciferase detection in the brittle star Amphiura filiformis

Zoologischer Anzeiger, Jul 1, 2017

Amphiura filiformis is a common burrowing brittle star from muddy environments along the European... more Amphiura filiformis is a common burrowing brittle star from muddy environments along the European shores. This species emits a blue light at the level of the arm spine tips when mechanically stimulated. Although the putative luciferase of A. filiformis was recently discovered, the ultrastructure of the luminous cells, i.e. the photocytes, remain unknown for this species as well as for most other bioluminescent echinoderms. In this study, we describe the morphology of the arm and spines in the brittle star A. filiformis and compare it to the luciferase expression pattern revealed by immunohistochemistry. Luciferase is expressed at the base of the spine and at the arm tips within well-defined photocyte clusters. Additionally, we investigated the ultrastructure of spine tissues before and after chemical stimulation of light emission in order to identify photocytes and improve our comprehension of the photogenesis phenomenon in brittle stars. Type II granular cells, found in the basal to mid-basal portion of the spine inner tissues and showing ultrastructural modifications during light-emission, are proposed as photocytes. Putative photocytes appear intimately associated with the spine nerve plexus as well as with mucus cells and presumptive pigment cells. Photocytes are characterised by the presence of specific vesicles with reticulated contents in their cytoplasm. Furthermore, the structure of the calcified spine ossicle has been investigated using microCT scanning.

Research paper thumbnail of Molecular mechanisms mediating stiffening in the mechanically adaptable connective tissues of sea cucumbers

Research Square (Research Square), May 13, 2021

In most animals, connective tissues such as the dermis or tendons present invariant mechanical pr... more In most animals, connective tissues such as the dermis or tendons present invariant mechanical properties, fine-tuned for their structural function. However, echinoderms, a group of sea creatures including sea cucumbers, possess the ability to voluntarily modify the mechanical properties of their connective tissues, which are therefore called mutable collagenous tissues (MCT). Understanding the molecular mechanism underlying MCT mutability is a prerequisite for the development of biomimetic smart dynamic materials. The stiffening and softening cycles of MCTs are made possible by the release of specialized effector proteins. We identified a stiffening factor from the dermis of Holothuria forskali, Hf-(D)Tensilin, and showed that it is localized in the secretory granules of juxtaligamental-like cells, a MCT specific cell type. Using recombinant proteins, we confirmed its effect on the dermis and its aggregation effect on extracted collagen fibrils. A model is proposed for the molecular interactions which mediate collagen fibrils cross-linking by tensilin.

Research paper thumbnail of Convergent Evolution of Attachment Mechanisms in Aquatic Animals

To resist hydrodynamic forces, two main underwater attachment strategies have evolved multiple ti... more To resist hydrodynamic forces, two main underwater attachment strategies have evolved multiple times in aquatic animals: glue-like “bioadhesive secretions” and pressure-driven “suction attachment”. In this review, we use a multi-level approach to highlight convergence in underwater attachment mechanisms across four different length-scales (organism, organ, microscopic and molecular). At the organism level, the ability to attach may serve a variety of functions, the most important being: (i) positional maintenance, (ii) locomotion, (iii) feeding, (iv) building, and (v) defense. Aquatic species that use bioadhesive secretions have been identified in 28 metazoan phyla out of the 34 currently described, while suction organs have a more restricted distribution and have been identified in five phyla. Although biological adhesives are highly diverse, it is possible to categorize them into four main types according to the time scale of operation: permanent, temporary, transitory, and instan...

Research paper thumbnail of Epidemiology of a SKin Ulceration Disease (SKUD) in the sea cucumber Holothuria scabra with a review on the SKUDs in Holothuroidea (Echinodermata)

Scientific Reports, 2020

Aquacultivated sea cucumbers often suffer from SKin Ulceration Diseases (SKUDs). SKUDs have been ... more Aquacultivated sea cucumbers often suffer from SKin Ulceration Diseases (SKUDs). SKUDs have been observed in six holothuroid species from nine countries. All SKUDs present a similar symptom—the skin ulceration—and can be induced by bacteria, viruses, or abiotic factors. We here provide an update on SKUDs in holothuroids and analyse the case of the SKUD observed in Holothuria scabra in Madagascar. Field observations revealed a seasonality of the disease (i.e. wintertime maximum peak). Morphological analyses of integument ulcers showed that sea cucumbers react by forming a collagen fibre plug. Metagenomic analyses revealed a higher proportion of Vibrionaceae (Gammaproteobacteria) in ulcers in comparison to the healthy integument of the same individuals. Experimental infection assays were performed with ulcer crude extracts and bacteria isolated from these extracts (e.g. Vibrio parahaemolyticus) but did not significantly induce skin ulceration. Our results suggest that the disease is n...

Research paper thumbnail of Disentangling the Roles of Functional Domains in the Aggregation and Adsorption of the Multimodular Sea Star Adhesive Protein Sfp1

Marine Biotechnology, 2021

Sea stars can adhere to various underwater substrata using an adhesive secretion of which Sfp1 is... more Sea stars can adhere to various underwater substrata using an adhesive secretion of which Sfp1 is a major component. Sfp1 is a multimodular protein composed of four subunits (Sfp1 Alpha, Beta, Delta, and Gamma) displaying different functional domains. We recombinantly produced two fragments of Sfp1 comprising most of its functional domains: the C-terminal part of the Beta subunit (rSfp1 Beta C-term) and the Delta subunit (rSfp1 Delta). Surface plasmon resonance analyses of protein adsorption onto different model surfaces showed that rSfp1 Beta C-term exhibits a significantly higher adsorption than the fibrinogen control on hydrophobic, hydrophilic protein-resistant, and charged self-assembled monolayers, while rSfp1 Delta adsorbed more on negatively charged and on protein-resistant surfaces compared to fibrinogen. Truncated recombinant rSfp1 Beta C-term proteins were produced in order to investigate the role of the different functional domains in the adsorption of this protein. The analysis of their adsorption capacities on glass showed that two mechanisms are involved in rSfp1 Beta C-term adsorption: (1) one mediated by the EGF-like domain and involving Ca2+ and Mg2+ ions, and (2) one mediated by the sequence of Sfp1 Beta with no homology with known functional domain in databases, in the presence of Na+, Ca2+ and Mg2+ ions.

Research paper thumbnail of Structure and composition of the tunic in the sea pineapple Halocynthia roretzi: A complex cellulosic composite biomaterial

Acta Biomaterialia, 2020

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Research paper thumbnail of The cellular basis of bioadhesion of the freshwater polyp Hydra

BMC Zoology, 2016

Background: The freshwater cnidarian Hydra temporarily binds itself to numerous natural substrate... more Background: The freshwater cnidarian Hydra temporarily binds itself to numerous natural substrates encountered underwater, such as stones, leafs, etc. This adhesion is mediated by secreted material from specialized ectodermal modified cells at the aboral end of the animal. The means by which Hydra polyps attach to surface remain unresolved, despite the fact that Hydra is a classic model in developmental and stem cell biology. Results: Here, we present novel observations on the attachment mechanism of Hydra using high pressure transmission electron microscopy, scanning electron microscopy, atomic force microscopy, super-resolution microscopy, and enzyme histochemistry. We analyzed the morphology of ectodermal basal disc cells, studied the secreted material, and its adhesive nature. By electron microscopy we identified four morphologically distinct secretory granules occurring in a single cell type. All the secretory granules contained glycans with different distribution patterns among the granule types. Footprints of the polyps were visualized under dry conditions by atomic force microscopy and found to consist of a meshwork with nanopores occurring in the interstices. Two antibodies AE03 and 3G11, previously used in cell differentiation studies, labelled both, basal disc cells and footprints. Our data suggest that the adhesive components of Hydra are produced, stored and delivered by a single cell type. Video microscopy analysis corroborates a role of muscle contractions for the detachment process. Conclusion: We clearly demonstrated that bioadhesion of Hydra relies on the secreted material. Our data suggest that glycans and/or glycoproteins represent an important fraction of the secreted material. Detachment seems to be initiated by mechanical forces by muscular contractions. Taken together, our study represents the characterization of an unique temporary adhesive system not known in aquatic organisms from other metazoan phyla.

Research paper thumbnail of Chemical characterization of saponins contained in the body wall and the Cuvierian tubules of the sea cucumber Holothuria (Platyperona) sanctori (Delle Chiaje, 1823)

Biochemical Systematics and Ecology, 2016

Holothuria (Platyperona) sanctori is a common sea cucumber of the Mediterranean Sea. Although its... more Holothuria (Platyperona) sanctori is a common sea cucumber of the Mediterranean Sea. Although its ecology and molecular phylogeny have been investigated in previous works, nothing is known about its saponin content, contrarily to other Mediterranean holothuroids. Saponins are bioactive triterpene glycosides that present interesting pharmacological/biological properties, and which can be used as taxonomic markers. This work highlights the different saponins produced by H. sanctori. Mass spectrometry (MALDI-MS and MS/MS) analyses highlighted a total of 18 different saponins, including eight new congeners. Presumptive molecular structures are proposed for all these molecules. A higher diversity of saponins is found in the body wall (12) than in the Cuvierian tubules (8). In addition to this qualitative difference, saponins were also 2e3 fold more concentrated in the Cuvierian tubules than in the integument. Our results demonstrate that H. sanctori, in accordance with its phylogenetic position, is the second species of Holothuria described to date possessing exclusively non-sulfated saponins. The species presents its own mix of saponins, acting as a true chemical signature.

Research paper thumbnail of Enhancing the Membranolytic Activity of Chenopodium quinoa Saponins by Fast Microwave Hydrolysis

Molecules, 2020

Saponins are plant secondary metabolites. There are associated with defensive roles due to their ... more Saponins are plant secondary metabolites. There are associated with defensive roles due to their cytotoxicity and are active against microorganisms. Saponins are frequently targeted to develop efficient drugs. Plant biomass containing saponins deserves sustained interest to develop high-added value applications. A key issue when considering the use of saponins for human healthcare is their toxicity that must be modulated before envisaging any biomedical application. This can only go through understanding the saponin-membrane interactions. Quinoa is abundantly consumed worldwide, but the quinoa husk is discarded due to its astringent taste associated with its saponin content. Here, we focus on the saponins of the quinoa husk extract (QE). We qualitatively and quantitively characterized the QE saponins using mass spectrometry. They are bidesmosidic molecules, with two oligosaccharidic chains appended on the aglycone with two different linkages; a glycosidic bond and an ester function....

Research paper thumbnail of Heavy metals in Diadema setosum (Echinodermata, Echinoidea) from Singapore coral reefs

Journal of Sea Research, 1997

of Zn, Pb, Cd, Fe, Cr, Cu and Ti were measured in body compartments of the echinoid Diadema setos... more of Zn, Pb, Cd, Fe, Cr, Cu and Ti were measured in body compartments of the echinoid Diadema setosum and in the silt fraction of surface sediment from eight coral reefs in Singapore coastal waters. Metal concentrations in the echinoid varied with the body compartment analysed and with the site of sampling. Amongst the body compartments studied, Zn, Cd, Fe, Cr and Cu were preferentially accumulated in the gonads, whereas Pb and Ti were accumulated mainly in the calcified body compartments. The concentrations of most metals differed according to the reef sampled. A decreasing north-south gradient of metal concentrations was observed in D. setosum populations, correlating to the distance from Singapore. A similar gradient was observed for metal concentrations in sediment. It is suggested that D. setosum could be a valuable bioindicator for assessing heavy metal contamination in coral reef ecosystems of the Indo-West Pacific.

Research paper thumbnail of Challenges and Opportunities

The Journal of Perinatal & Neonatal Nursing, 2002

Research paper thumbnail of Is the adhesive material secreted by sea urchin tube feet species-specific?

Journal of Morphology, 2012

Sea urchin adoral tube feet are highly specialized organs that have evolved to provide efficient ... more 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.

Research paper thumbnail of Comparative histological and immunohistochemical study of sea star tube feet (Echinodermata, Asteroidea)

Journal of Morphology, 2005

Adhesion in sea stars is the function of specialized structures, the tube feet or podia, which ar... more 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., knobending, 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.

Research paper thumbnail of Unusual adhesive production system in the barnacle Lepas anatifera : An ultrastructural and histochemical investigation

Journal of Morphology, 2012

Adhesives that are naturally produced by marine organisms are potential sources of inspiration in... more Adhesives that are naturally produced by marine organisms are potential sources of inspiration in the search for medical adhesives. Investigations of barnacle adhesives are at an early stage but it is becoming obvious that barnacles utilize a unique adhesive system compared to other marine organisms. The current study examined the fine structure and chemis-

Research paper thumbnail of Effects of CO2-induced ocean acidification on physiological and mechanical properties of the starfish Asterias rubens

Journal of Experimental Marine Biology and Ecology, 2013

ABSTRACT The increase in atmospheric CO2 due to anthropogenic activity results in an acidificatio... more ABSTRACT The increase in atmospheric CO2 due to anthropogenic activity results in an acidification of the surface waters of the oceans. Its impact will depend on the considered organisms and ecosystems. The intertidal may harbor organisms pre-adapted to the upcoming changes as they face tidal pH and temperature fluctuations. However, these environments will be more affected as shallow waters will face the highest decrease in seawater pH. In this context, the effects of reduced environmental pH on the physiology and tube feet mechanical properties of the intertidal starfish Asterias rubens¸ a top predator, were investigated during 15 and 27 days. Asterias rubens showed a respiratory acidosis with its coelomic fluid pH always lower than that of seawater. This acidosis was most pronounced at pH 7.4. Notwithstanding, the starfish showed no significant variations in RNA/DNA ratio of different tissues and in tube feet strength. However, respiration rates were significantly lower for individuals maintained at reduced seawater pH. Within the ocean acidification context, the present results suggest that A. rubens withstands the effects of reduced seawater pH, at least for medium term exposures.

Research paper thumbnail of Measurement of the attachment strength of brachiolaria larvae and metamorphic individuals of the sea star Asterina gibbosa by a centrifugation method

Journal of Experimental Marine Biology and Ecology, 2009

Two methods are generally used to measure the adhesive strength of invertebrate larvae: direct me... more Two methods are generally used to measure the adhesive strength of invertebrate larvae: direct measurement with a force transducer connected to the organisms and indirect measurement with a water flow used to dislodge the organisms. Each of these methods, however, has its drawbacks. The present study aimed to design a simple and straightforward method to measure the adhesion strength of marine invertebrate larvae based on centrifugation. This centrifuge technique works in immersed conditions and applies forces acting at 45°to the substratum, therefore mimicking natural conditions. It was tested with three different substrata on two developmental stages of the sea star Asterina gibbosa: the brachiolaria larvae, which use temporary adhesion, and the metamorphic individuals which use permanent adhesion. Measurements were completed by SEM and TEM observations of the larval adhesive organs. The critical detachment force (force required to detach 50% of the larvae) of brachiolaria larvae attached to glass (36 ± 9 µN) and rough PMMA (43 ± 16 µN) were equivalent and both significantly higher than the critical detachment force measured on smooth PMMA (11 ± 8 µN). Most metamorphic individuals, on the other hand, resisted to the highest centrifugation speed used, corresponding to a force of 2.13 mN. For the hydrodynamics of larval settlement and metamorphosis, force is the ecologically relevant factor, and adhesion forces obtained by centrifugation are strikingly similar to forces measured for other marine invertebrate larvae with other methods. This indicates the usefulness of the centrifugation technique to compare adhesion of larvae between different species or development stages, or between different treatments.

Research paper thumbnail of Morphometry and mechanical design of tube foot stems in sea urchins: a comparative study

Journal of Experimental Marine Biology and Ecology, 2005

ABSTRACT To withstand hydrodynamic forces, sea urchins rely on their adoral tube feet, which are ... more ABSTRACT To withstand hydrodynamic forces, sea urchins rely on their adoral tube feet, which are specialized for attachment. Although it has been often suggested that the degree of development of these tube feet is intimately related to the maximum environmental energy a species can withstand, it has never been demonstrated by mechanical testing. To address this subject, we studied the mechanical properties of the stem of adoral tube feet from three species of sea urchins, Arbacia lixula, Paracentrotus lividus and Sphaerechinus granularis, which have distinct taxonomic, ecological and morphological characteristics. The tube feet of the three species have a very similar morphology. When a tensile force is applied to the tube foot stem, the connective tissue is the only tissue layer bearing the load. The mechanical properties of this tissue give the tube feet an ideal balance of extensibility (139–166%), strength (23–29 MPa) and stiffness (152–328 MPa), which together produce a material with adequate toughness (2.5–2.9 MJ/m3) to absorb the impact of waves and currents, and thus to resist the environmental challenges of the habitats in which sea urchins live. Extended stems of P. lividus were significantly stiffer (328 MPa) than those of the other two species (152 and 183 MPa, for A. lixula and S. granularis, respectively). No interspecific difference was found in terms of extensibility, strength, initial stiffness and toughness between the tube feet from the three species. The difference in local distribution between the species investigated is therefore not only explained by the mechanical properties of their tube feet, but may involve other factors such as tube foot number and arrangement, tube foot disc tenacity or sea urchin size.

Research paper thumbnail of Polyphosphoprotein-Containing Marine Adhesives

The Journal of Adhesion, 2009

... a * , Aurélie Lambert a , Philippe Bailly a & Elise Hennebert a ... During the last decad... more ... a * , Aurélie Lambert a , Philippe Bailly a & Elise Hennebert a ... During the last decade, Herbert Waite and his group have indeed characterized several polyphosphoproteins from the adhesive secretions of two different marine organisms, mussels and tube-building worms. ...

Research paper thumbnail of Biomechanics of Adhesion in Sea Cucumber Cuvierian Tubules (Echinodermata, Holothuroidea)

Integrative and Comparative Biology, 2002

Several species of sea cucumbers, all belonging to a single family, possess a peculiar and specia... more Several species of sea cucumbers, all belonging to a single family, possess a peculiar and specialized defense system, the Cuvierian tubules. It is mobilized when the animal is mechanically stimulated, resulting in the discharge of a few white filaments, the tubules. In seawater, the expelled tubules lengthen considerably and become sticky upon contact with any object. The adhesiveness of their outer epithelium combined with the tensile strength of their collagenous core make Cuvierian tubules very efficient at entangling and immobilizing most potential predators. We have designed a method to measure the adhesion of holothuroid Cuvierian tubules. Tubule adhesive strength was measured in seven species of sea cucumbers belonging to the genera Bohadschia, Holothuria and Pearsonothuria. The tenacities (force per unit area) varied from 30 to 135 kPa, falling within the range reported for marine organisms using non-permanent adhesion. Two species, H. forskali and H. leucospilota, were selected as model species to study the influence of various factors on Cuvierian tubule adhesive strength. Tubule tenacity varied with substratum, temperature and salinity of the seawater, and time following expulsion. These differences give insight into the molecular mechanisms underlying Cuvierian tubule adhesion. Tenacity differences between substrata of varying surface free energy indicate the importance of polar interactions in adhesion. Variation due to temperature and time after expulsion suggests that an increase of tubule rigidity, presumably under enzymatic control, takes place after tubule elongation and reinforces adhesion by minimizing peeling effects.

Research paper thumbnail of Elucidation of molecular diversity and body distribution of saponins in the sea cucumber Holothuria forskali (Echinodermata) by mass spectrometry

Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2009

Sea cucumbers contain triterpene glycosides called saponins. We investigated the complex saponin ... more Sea cucumbers contain triterpene glycosides called saponins. We investigated the complex saponin mixture extracted from the common Mediterranean species Holothuria forskali. Two different body components were analyzed separately: the body wall (which protects the animal and is moreover the most important organ in terms of surface and weight) and the Cuvierian tubules (a defensive organ that can be expelled on predators in response to an attack). MALDI/MS and MALDI/MS/MS were used to detect saponins and describe their molecular structures. As isomers have been found in the Cuvierian tubules, LC/MS and LC/MS/MS were performed to identify each saponin separately. Twelve saponins have been detected in the body wall and 26 in the Cuvierian tubules. All the saponins from the body wall are also present in the Cuvierian tubules but the latter also contain 14 specific saponins. The presence of isomeric saponins complicated structure elucidation . The diversity and organ specificity of the saponins described here are much higher than what had been reported to date in any sea cucumber species.

Research paper thumbnail of Inter- and intra-organ spatial distributions of sea star saponins by MALDI imaging

Saponins are secondary metabolites that are abundant and diversified in echinoderms. Mass spectro... more Saponins are secondary metabolites that are abundant
and diversified in echinoderms. Mass spectrometry is
increasingly used not only to identify saponin congeners within
animal extracts but also to decipher the structure/biological
activity relationships of these molecules by determining their
inter-organ and inter-individual variability. The usual method
requires extensive purification procedures to prepare saponin
extracts compatible with mass spectrometry analysis. Here,
we selected the sea star Asterias rubens as a model animal to
prove that direct analysis of saponins can be performed on
tissue sections.We also demonstrated that carboxymethyl cellulose
can be used as an embedding medium to facilitate the
cryosectioning procedure. Matrix-assisted laser desorption/
ionization (MALDI) imaging was also revealed to afford interesting
data on the distribution of saponin molecules within
the tissues. We indeed highlight that saponins are located not
only inside the body wall of the animals but also within the
mucus layer that probably protects the animal against external
aggressions.