Abstract
BackgroundSeveral species of sea cucumbers of the family Holothuriidae possess a particular mechanical defense system called the Cuvierian tubules (Ct). It is also a chemical defense system as triterpene glycosides (saponins) appear to be particularly concentrated in Ct. In the present study, the precise localization of saponins in the Ct of Holothuria forskali is investigated. Classical histochemical labeling using lectin was firstly performed but did not generate any conclusive results. Thus, MALDI mass spectrometry Imaging (MALDI-MSI) was directly applied and completed by statistical multivariate tests. A comparison between the tubules of relaxed and stressed animals was realized.ResultsThese analyses allowed the detection of three groups of ions, corresponding to the isomeric saponins of the tubules. Saponins detected at m/z 1287 and 1303 were the most abundant and were apparently localized in the connective tissue of the tubules of both relaxed and stressed individuals. Saponins at m/z 1125 and 1141 were detected in lower amount and were present in tissues of relaxed animals. Finally, saponin ions at 1433, 1449, 1463 and 1479 were observed in some Ct of stressed holothuroids in the outer part of the connective tissue. The saponin group m/z 14xx seems therefore to be stress-specific and could originate from modifications of the saponins with m/z of 11xx.ConclusionsAll the results taken together indicate a complex chemical defense mechanism with, for a single organ, different sets of saponins originating from different cell populations and presenting different responses to stress. The present study also reflects that MALDI-MSI is a valuable tool for chemical ecology studies in which specific chemical signalling molecules like allelochemicals or pheromones have to be tracked. This report represents one of the very first studies using these tools to provide a functional and ecological understanding of the role of natural products from marine invertebrates.
Highlights
Sea cucumbers seem to be vulnerable animals regarding their numerous predators referenced in the literature [1]
Three lectins specific to glucose- and mannose-containing oligosaccharides (Con A, Lens culinaris agglutinin (LCA) and Pisum sativum agglutinin (PSA)) were selected. To confirm that these lectins are able to recognize the saccharidic chain of the saponins of H. forskali, we used a lectin-binding assay based on the technique developed by Smith [26] to detect cell surface glycolipids of human and bovine erythrocytes on thin layer chromatograms (TLC)
The three lectins labeled the saponin spots, concanavaline A (Con A) presented the strongest and fastest reaction compared to LCA and PSA which were both characterized by a much lighter staining
Summary
Sea cucumbers seem to be vulnerable animals regarding their numerous predators referenced in the literature [1]. Only some species of holothuroids from the family Holothuriidae, including Holothuria forskali, possess Cuvierian tubules, all the sea cucumbers contain saponins in their body wall and viscera. Saponins of sea cucumbers are secondary metabolites having a triterpene glycoside structure They have long been suggested to play a role in the defense of these animals as a toxin [4,9]. Several species of sea cucumbers of the family Holothuriidae possess a particular mechanical defense system called the Cuvierian tubules (Ct). It is a chemical defense system as triterpene glycosides (saponins) appear to be concentrated in Ct. In the present study, the precise localization of saponins in the Ct of Holothuria forskali is investigated. A comparison between the tubules of relaxed and stressed animals was realized
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