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Triterpenoids are a group of organic chemicals that consist of over 100 distinct backbones including sterols and saponins (both triterpenoidal- and steroial-glycosides). Saponins includes a variety of specialized defensive, membranolytic metabolites found mainly in plants but also in two classes of Echinoderms : holothuroids (sea cucumbers) and asteroids (sea stars). Saponins are widely studied for their pharmacological properties, attributed to their ability to complex plasma membrane cholesterol, and the formation of pores. However, paradoxically, how sea cucumbers tolerate this membranolytic toxin, the potential biological roles (other than defensive), the biosynthesis and evolution of these highly intriguing molecules in their host sea cucumbers remains enigmatic. Here after we explore these fundamental questions focused around the unique saponinic system of sea cucumbers. Sea cucumbers are thought to produce ∆⁷-sterols instead ∆⁵-cholesterol, in order to have saponin tolerant plasma membranes. The interactions between holothuroid- like ∆⁷-sterols and cholesterol with the holothuroid saponin Frondoside A was explored through the use of complementary biophysical experiments. The saponin caused significantly less permeabilization in liposomes containing the holothuroid sterol than those containing cholesterol and resulted in endothermic interactions versus exothermic interactions with cholesterol containing liposomes. Lipid phase stimulations revealed that Frondoside A has an agglomerating effect on cholesterol domains, however, induced small irregular ∆⁷-sterol clusters. These findings suggest that sterols and saponins have co-evolved in sea cucumbers. This alternate adaptation of a fundamental biological building block (membrane sterols) suggests that having saponins has given a significant evolutionary benefit to sea cucumbers, perhaps more than simple defensive toxin. The high diversity of saponins found in the surrounding water around sea cucumbers further supports the hypothesis that saponin mixtures of holothoroids may play multiple biological roles for them. Statistically significant aggregation was observed in the spatial distribution of the aqua cultivated sea cucumber, Holothuria scabra, both as adults in large sea pens, and as juveniles in aquaria. The role of olfaction and the mechanisms of chemical communication in this behavior was investigated using olfactory experimental assays. In complementation with comparative mass spectrometry a distinct saponin profile/mixture was found to fuction as a sea cucumber pheromone. Saponins are therefore not simple defensive toxins, but a complex mixture of semiochemicals used to communicate with hetero- and conspecifics. These findings suggest that the diversification of saponins and the co-evolution of saponins and sterols in sea cucumbers has been essential to the success of these macroinvertebrates in the benthic marine ecosystems. The common biosynthetic trunk of sterols and saponins from 2,3-oxidosqualene although well described in plants, remains poorly investigated in sea cucumbers. The complex composition of free and conjugated sterols of holothuroid and asteroid tissue (the only two saponin producing echinoderm classes), was highlighted. This composition was integrated with the identification of orthologue and paralogue genes of the biosynthetic pathway. Missing enzymes, and highly divergent sequences suggest a highly distinct triterpenoid pathway in sea cucumbers. The functional expression of the first enzyme in the biosynthesis of sea cucumber triterpenoids, Oxidosqualene Cyclase, revealed the unique production of parkeol in sea cucumbers, and not lanosterol, as found in all other animals, including asteroids. Further suggesting a unique enzymatic adaptation for the biosynthesis of both sterols and saponins. In summary, this PhD brings new light to the biological roles and benefits of the diversification of saponins, and suggests a role of saponins in holothuroids well beyond that of simple toxin. It also highlights for the first time the complexity of the saponin-sterol system of sea cucumbers, and the enzymatic machinery responsible for this unique co-evolution between toxin (saponins) and toxicity mitigation (sterols).

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Depuis plusieurs années, l’algoculture s’est développée et est devenue un moyen de revenus stables dans de nombreux pays dont Madagascar. Les habitants de ce pays cultivent notamment l’algue Kappaphycus alvarezii (Doty) L.M. Liao pour la production de kappa-carraghénanes, une molécule possédant de nombreuses applications alimentaire et pharmaceutique. Une maladie algale épiphytique, l’EFA (Epiphytic Filamentous Algae), peut se retrouver dans les cultures et a pour conséquence la réduction voire la destruction de la production. Les effets de compétition et de parasitisme de ces épiphytes sur l’hôte ont été étudiés pour le carbone et l’azote. Une expérience d’enrichissement en isotopes stables 13C et 15N dans les échantillons de Kappaphycus alvarezii et de ses épiphytes a permis de mesurer l’incorporation relative pour les algues et ses épiphytes puis de mesurer le transfert des algues vers les épiphytes. Dans un premier temps, il en résulte une différence significative dans l’incorporation de ces isotopes. En termes de quantité, les algues incorporent nettement plus de traceurs que leurs épiphytes du fait de leur biomasse très supérieure. Cependant, les épiphytes incorporent plus de carbone et d’azote, par unité de biomasse, que les K. alvarezii. Ceci est notamment lié à l’aspect fonctionnel de ces organismes. Les épiphytes ont des besoins plus rapides pour leur croissance que leurs hôtes qui ont une croissance plus lente. Cela signifie que dans un stade plus avancé du développement épiphytique, ceux-ci peuvent probablement limiter la disponibilité en ressource pour l’hôte. Dans un second temps, nous avons démontré l’occurrence d’un transfert significatif de C et de N entre l’hôte et ses épiphytes. Ce transfert implique que les épiphytes peuvent être considérés, ici, comme des parasites détournant une partie des molécules fabriquées par l’algue hôte. Cette maladie est donc à surveiller de près dans les cultures. Des solutions peuvent cependant être trouvées comme l’enrichissement en sels nutritifs, l’utilisation de l’extrait d’AMPEP (Acadian Marine Plant Extract Powder) de l’algue brune Ascophyllum nodosum comme « vaccin » potentiel ou encore l’utilisation de brouteurs d’épiphytes.

Kappaphycus alvarezii --- épiphytes --- maladie algale --- EFA --- isotopes stables --- Sciences du vivant > Sciences aquatiques & océanologie --- Sciences du vivant > Sciences de l'environnement & écologie

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Saponins are a very diverse class of secondary metabolites found in plants and some marine invertebrates. They are amphiphilic molecules composed of a hydrophilic sugar moiety, and a hydrophobic steroid/triterpenic-like part known as the aglycone. Saponins are studied for their pharmacological properties such as their anti-fungal, anti-microbial and anti-tumoral activity. Holothuroids, or sea cucumbers, produce saponins as a chemical defense against predators and parasites, but interestingly, are immune to the cytotoxic nature of these chemicals. This immunity is extremely poorly understood. The standing hypothesis, based purely on observation, is that the rare Δ7 and Δ9(11) sterols that replace cholesterol in the cellular plasma membranes of sea cucumbers are responsible for this immunity, however, the molecular mechanism remains obscure.
The aim of this study was to elucidate the mechanisms behind the immunity of holothuroid to the cytotoxic saponins (e.g. Frondoside A) they produce but also to describe, at a molecular level, the interactions that occur between model plasma membranes and saponins. This investigation was conducted using complementary biophysical tools, using both in silico approaches such as the Hypermatrix, IMPALA and Big Monolayer simulation models and with an in vitro technique called Isothermal Titration Calorimetry (ITC). The Hypermatrix method calculates energies of interaction between a central molecule and a surrounding lipid monolayer, allowing to determine if certain interactions are more favorable than others. The Big Monolayer uses these energies to simulate a monolayer composed of different proportions of lipids and saponin. ITC is a technique used to describe interactions in a thermodynamic framework, and allows for the enthalpy, entropy, free Gibbs energy and the binding constant of a particular interaction to be determined from a recorded thermogram. 
The structural differences of the holothuroid sterols were first described and compared to the mammalian membrane sterol: cholesterol. Structural differences of saponins and sterols were associated with different interaction affinities and mechanisms. Saponin-lipid (both phospholipids and sterols) interactions were mainly apolar in nature. Interactions with phospholipids were more favorable than with sterols, and among the sterols, saponins interacted more favorably with cholesterol than the holothuroid Δ7 and Δ9(11) sterols. Liposomes containing cholesterol resulted in exothermic interactions with Frondoside A whereas liposomes containing the Δ7 sterol were endothermic with the same saponin. Big Monolayer simulations using experimental settings previously developed for plant saponins revealed that the holothuroid saponin Frondoside A has an agglomerating effect on cholesterol domains, similarly to the plant saponin. However when interacting with the Δ7 sterols, the sterol domains were fragmented into small clusters.
The coevolution of a saponin-Δ7 sterol pair may be an adaptation required for holothuroid membranes to inhibit the formation of large membrane disruptive sterol domains in the presence of saponin.

saponin --- Holothuroid --- Membrane interactions --- Sterol --- Biophysics --- Physique, chimie, mathématiques & sciences de la terre > Chimie --- Ingénierie, informatique & technologie > Ingénierie chimique --- Sciences du vivant > Sciences aquatiques & océanologie

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The growing success of molecular methods has challenged traditional views of animal evolution and a large number of alternative hypotheses are hotly debated today. For the deep metazoan phylogeny project, data sets of hitherto unmatched quality and quantity were compiled and analysed with innovative bioinformatics tools. The book begins at the base of the tree of life to discuss the origin of animals and early branches of the phylogenetic tree. The following section presents special data sets gained from mitochondrial genomes and from morphology, with a focus on nervous systems. The final

Metazoa --- Metazoans --- Multicellular animals --- Animals --- Phylogeny. --- Basic Sciences. Biology --- Biosystematics. --- Evolution and Phylogeny. --- Evolution. --- Metazoa. --- Taxonomy. --- Zoology.