Guillaume Caulier

The triterpene glycosides of Holothuria forskali: usefulness and efficiency as a chemical defense mechanism against predatory fish

SUMMARY More than 100 triterpene glycosides (saponins) have been characterized in holothuroids in the past several decades. In particular, Holothuria forskali contains 26 saponins in its Cuvierian tubules and 12 in its body wall. This high diversity could be linked to a chemical defense mechanism, the most commonly accepted biological role for these secondary metabolites. We performed an integrated study of the body-wall saponins of H. forskali. The saponins are mainly localized in the epidermis and in the mesothelium of the body wall and appear to be released when the holothuroid is stressed. Among the saponins present in the epidermis, one (holothurinoside G) was detected in the seawater surrounding non-stressed holothuroids and three others (holohurinosides C and F, and desholothurin A) were secreted when the animals were stressed. In addition, two new congeners (detected at m/z 1301 and 1317) were also present in the immediate surroundings of stressed holothuroids. These new saponins do not originate from the epidermis and could come from an internal organ. Quantities of secreted saponins were very low compared with the body wall and Cuvierian tubules concentrations. At natural concentrations, saponins do not represent a threat to the health of predatory fish. The deterrent effect of saponins seems therefore to act as an aposematic signal, warning potential predators of the unpalatability of the holothuroid tissues.

Molecular diversity and body distribution of saponins in the sea star Asterias rubens by mass spectrometry.

Saponins are natural molecules that the common sea star Asterias rubens produces in the form of steroid glycosides bearing a sulfate group attached on the aglycone part. In order to highlight the inter-organ and inter-individual variability, the saponin contents of five distinct body components, namely the aboral body wall, the oral body wall, the stomach, the pyloric caeca and the gonads, from different individuals were separately analyzed by mass spectrometry. MALDI-ToF experiments were selected as the primary tool for a rapid screening of the saponin mixtures, whereas LC-MS and LC-MS/MS techniques were used to achieve chromatographic separation of isomers. First of all, our analyses demonstrated that the diversity of saponins is higher than previously reported. Indeed, nine new congeners were observed in addition to the 17 saponins already described in this species. On the basis of all the collected MS/MS data, we also identified collision-induced key-fragmentations that could be used to reconstruct the molecular structure of both known and unknown saponin ions. Secondly, the comparison of the saponin contents from the five different body components revealed that each organ is characterized by a specific mixture of saponins and that between animals there are also qualitative and quantitative variability of the saponin contents which could be linked to the sex or to the collecting season. Therefore, the observed high variability unambiguously confirms that saponins probably fulfill several biological functions in A. rubens. The current results will pave the way for our future studies that will be devoted to the clarification of the biological roles of saponins in A. rubens at a molecular level.

When a repellent becomes an attractant: harmful saponins are kairomones attracting the symbiotic Harlequin crab

Marine organisms have developed a high diversity of chemical defences in order to avoid predators and parasites. In sea cucumbers, saponins function as repellents and many species produce these cytotoxic secondary metabolites. Nonetheless, they are colonized by numerous symbiotic organisms amongst which the Harlequin crab, Lissocarcinus orbicularis, is one of the most familiar in the Indo-Pacific Ocean. We here identify for the first time the nature of the molecules secreted by sea cucumbers and attracting the symbionts: saponins are the kairomones recognized by the crabs and insuring the symbiosis. The success of this symbiosis would be due to the ability that crabs showed during evolution to bypass the sea cucumber chemical defences, their repellents becoming powerful attractants. This study therefore highlights the complexity of chemical communication in the marine environment.

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

AbstractSaponins 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. Graphical AbstractSaponins are the most abundant secondary metabolites in sea stars. They should therefore participate in important biological activities. Here, MALDI imaging is presented as a powerful method to determine the spatial distribution of saponins within the animal tissues. The inhomogeneity of the intra-organ saponin distribution is highlighted, paving the way for future elegant structure/activity relationship investigations.

Is non-host pollen suitable for generalist bumblebees?

Current evidence suggests that pollen is both chemically and structurally protected. Despite increasing interest in studying bee–flower networks, the constraints for bee development related to pollen nutritional content, toxicity and digestibility as well as their role in the shaping of bee–flower interactions have been poorly studied. In this study we combined bioassays of the generalist bee Bombus terrestris on pollen of Cirsium, Trifolium, Salix, and Cistus genera with an assessment of nutritional content, toxicity, and digestibility of pollen. Microcolonies showed significant differences in their development, non‐host pollen of Cirsium being the most unfavorable. This pollen was characterized by the presence of quite rare δ7‐sterols and a low digestibility. Cirsium consumption seemed increase syrup collection, which is probably related to a detoxification mixing behavior. These results strongly suggest that pollen traits may act as drivers of plant selection by bees and partly explain why Asteraceae pollen is rare in bee generalist diet.

The diet of the Harlequin crab Lissocarcinus orbicularis , an obligate symbiont of sea cucumbers (holothuroids) belonging to the genera Thelenota , Bohadschia and Holothuria

The present paper characterizes, for the first time, the diet of the Harlequin crab Lissocarcinus orbicularis, an obligate symbiotic crab that associates with sea cucumbers (holothuroids) belonging to the genera Thelenota, Bohadschia and Holothuria. These tropical holothuroids host a rich symbiotic community in the Indo-West Pacific Ocean of which the Harlequin crab is the best known. The diet of L. orbicularis was characterized by analyzing the microscopic, molecular and isotopic signatures obtained from its gastric content. The presence of sea cucumber ossicles in the gastric mills of the crabs suggests that symbionts eat the superficial integument of their host and this was supported by the fact that Holothuroid DNA was detected in the stomach of L. orbicularis after DGGE and sequencing of the 18S rDNA gene. The stable isotopic δ13C and δ15N values of crab tissues were compared with diverse potential food sources including three holothuroids, three algae, one sea grass as well as the organic matter contained in the water column, in the sediment, and the second most abundant symbiont, the polychaete Gastrolepidia clavigera. The low δ15N values of crabs suggests that the crabs do not exclusively feed on sea cucumber tissue but assimilate diverse food sources such as sea grasses and organic matter contained in sediment that have similar δ13C values. There were no differences between the feeding of males and females but there was a positive correlation between the carapace length and the stable isotopic values indicating a shift of the food source as crabs grow larger.

The Roles of Spinochromes in Four Shallow Water Tropical Sea Urchins and Their Potential as Bioactive Pharmacological Agents

Spinochromes are principally known to be involved in sea urchin pigmentation as well as for their potentially interesting pharmacological properties. To assess their biological role in sea urchin physiology, experiments are undertaken on crude extracts from four species and on four isolated spinochromes in order to test their antibacterial, antioxidant, inflammatory and cytotoxic activities. First, the antibacterial assays show that the use of crude extracts as representatives of antibacterial effects of spinochromes are inaccurate. The assays on purified spinochromes showed a decrease in the growth of four strains with an intensity depending on the spinochromes/bacteria system, revealing the participation of spinochromes in the defense system against microorganisms. Secondly, in the 2,2-diphenyl-1-picrylhydrazyl antioxidant assays, spinochromes show an enhanced activity compared to the positive control. This latter observation suggests their involvement in ultraviolet radiation protection. Third, spinochromes present a pro-inflammatory effect on lipopolysaccharide-stimulated macrophages, highlighting their possible implication in the sea urchin immune system. Finally, cytotoxicity assays based on Trypan blue exclusion, performed in view of their possible future applications as drugs, show a weak cytotoxicity of these compounds against human cells. In conclusion, all results confirm the implication of spinochromes in sea urchin defense mechanisms against their external environment and reveal their potential for pharmacological and agronomical industries.

Tackling saponin diversity in marine animals by mass spectrometry: data acquisition and integration

AbstractSaponin analysis by mass spectrometry methods is nowadays progressively supplementing other analytical methods such as nuclear magnetic resonance (NMR). Indeed, saponin extracts from plant or marine animals are often constituted by a complex mixture of (slightly) different saponin molecules that requires extensive purification and separation steps to meet the requirement for NMR spectroscopy measurements. Based on its intrinsic features, mass spectrometry represents an inescapable tool to access the structures of saponins within extracts by using LC-MS, MALDI-MS, and tandem mass spectrometry experiments. The combination of different MS methods nowadays allows for a nice description of saponin structures, without extensive purification. However, the structural characterization process is based on low kinetic energy CID which cannot afford a total structure elucidation as far as stereochemistry is concerned. Moreover, the structural difference between saponins in a same extract is often so small that coelution upon LC-MS analysis is unavoidable, rendering the isomeric distinction and characterization by CID challenging or impossible. In the present paper, we introduce ion mobility in combination with liquid chromatography to better tackle the structural complexity of saponin congeners. When analyzing saponin extracts with MS-based methods, handling the data remains problematic for the comprehensive report of the results, but also for their efficient comparison. We here introduce an original schematic representation using sector diagrams that are constructed from mass spectrometry data. We strongly believe that the proposed data integration could be useful for data interpretation since it allows for a direct and fast comparison, both in terms of composition and relative proportion of the saponin contents in different extracts. Graphical AbstractA combination of state-of-the-art mass spectrometry methods, including ion mobility spectroscopy, is developed to afford a complete description of the saponin molecules in natural extracts

Effects of Holothuroid Ichtyotoxic Saponins on the Gills of Free-Living Fishes and Symbiotic Pearlfishes

Several carapid fishes, known as pearlfishes, are endosymbiotic in holothuroids and asteroids. These echinoderms contain a strong concentration of saponins that are efficient membranolytic repellents to predators. We compared the effects of exposure to saponins from the sea cucumber body wall and from the Cuvierian tubules on the behavior and gill ultrastructure of pearlfishes and free-living fishes. Saponins were extracted from the body wall of two holothuroids, the Mediterranean Holothuria forskali and the tropical Bohadschia atra, and from the water surrounding the Cuvierian tubules of B. atra. Five species of carapids that live in symbiosis with holothuroids and seven species of free-living fishes were exposed to these extracts. The free-living fishes exhibited a stress response and died about 45 times faster than pearlfishes when exposed to the same quantity of saponins. Cuvierian tubules and saponins extracted from the body wall were lethal to the free-living fishes, whereas the carapids were much less sensitive. The carapids did not exhibit a stress response. The high toxicity shown by Cuvierian tubules was not explained by the nature of the saponins that were identified by mass spectrometry, but it is likely due to the higher concentration of saponins in the tubules. Histology and scanning and transmission electron microscopy of the gills of the free-living fishes and pearlfishes showed that saponins act at the level of the secondary lamellae where they induce the detachment of the epithelia, create edema at the level of the epithelia, and induce pores in the epithelial cells that lead to their destruction and the invasion of inner cells (pillar cells and red blood cells). This sequence of events happens 5 min after saponin exposure in free-living fishes and after 1 h in carapids.

Ion mobility mass spectrometry of saponin ions

RATIONALE Saponins are natural compounds presenting a high structural diversity whose structural characterization remains extremely challenging. Ideally, saponin structures are best established using nuclear magnetic resonance experiments conducted on isolated molecules. However, saponins are also increasingly characterized using tandem mass spectrometry (MSMS) coupled with liquid chromatography, even if CID experiments are often quite limited in accurately determining the saponin structure. METHODS We consider here ion mobility mass spectrometry (IMMS) as an orthogonal tool for the structural characterization of saponin isomers by comparing the experimental collision cross sections of saponin ions with theoretical CCS for candidate ion structures. Indeed, state-of-the-art theoretical calculations perfectly complement the experimental results, allowing deciphering ion structures at the molecular level. RESULTS We demonstrate that ion mobility can contribute to the structural characterization of saponins because different saponin ions present significantly distinct collisional cross sections. Depending on the nature of the cation (in the positive ion mode), the CCS differences can also be exacerbated, optimizing the gas-phase separation. When associated with Molecular Dynamics simulations, the CCS data can be used to describe the interactions between the cations, i.e. H+ , Na+ and K+ , and the saponin molecules at a molecular level. CONCLUSIONS Our work contributes to resolve the relation between the primary and secondary structures of saponin ions. However, it is obvious that the structural diversity and complexity of the saponins cannot be definitively unraveled by measuring a single numerical value, here the CCS. Consequently, the structural characterization of unknown saponins will be difficult to achieve based on ion mobility mass spectrometry alone. Nevertheless, we demonstrated that monodesmosidic and bidesmosidic saponins can be distinguished via IMMS.