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PLoS One
2010 Nov 10;511:e13923. doi: 10.1371/journal.pone.0013923.
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Localization of secondary metabolites in marine invertebrates: contribution of MALDI MSI for the study of saponins in Cuvierian tubules of H. forskali.
Van Dyck S
,
Flammang P
,
Meriaux C
,
Bonnel D
,
Salzet M
,
Fournier I
,
Wisztorski M
.
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BACKGROUND: Several 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.
RESULTS: These 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.
CONCLUSIONS: All 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.
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21085713
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Figure 1. Histochemical labeling of the Cuvierian tubules of Holothuria forskali.
(A, C, D: paraffin sections; B: cryo-section). A: Cross section through a whole tubule stained with the PAS method; B–D: Transverse sections through the tubule wall labeled with PSA, Con A and LCA, respectively (C and D were counterstained). ac: adluminal cell layer of the mesothelium; cm: circular muscle; ct: connective tissue; g: granular cell layer of the mesothelium; ict: inner connective tissue layer; ie: inner epithelium; lm: longitudinal muscle; m: mesothelium; ml: muscular layer; ncp: neurosecretory-like cell processes; oct: outer connective tissue layer; s: spherulocytes.
Figure 2. Detection of Saponins by MALDI direct tissue analysis.Comparison of an average mass spectrum of the Cuvierian tubules of Holothuria forskali obtained by direct MALDI-MSI (A) and a mass spectrum obtained by classical MALDI-MS analysis after extraction and purification of saponins from the tubules (B). Saponin ions are marked by an asterisk.
Figure 3. Direct tissue MS/MS analysis of the saponin ions at m/z 1287.Characteristic signature peaks of saponins (m/z 507 and m/z 523) are marked by an asterisk.
Figure 4. Molecular structures of saponins from the Cuvierian tubules of Holothuria forskali.Isomeric congeners are presented in columns (a) and (b). Structures and corresponding names are from Van Dyck et al.
[17].
Figure 5. Comparison of saponins composition between stressed and relaxed holothuroids.(A) Pseudogel view of 300 mass spectra from the Cuvierian tubules of Holothuria forskali: spectra from stressed holothuroids (upper part) and from relaxed holothuroids (lower part). (B and C) scores plot of first, second and third principal component considering all the ions (B) or only the saponin ions (C) in the MALDI spectra. Each data point represents one mass spectrum (red, relaxed; green, stressed). (D) Scores plot of first, second and third loading considering only the saponin ions. Each data point represents one m/z ratio.
Figure 6. Repartition of saponin groups depending on the state of stress.(A) Optical image of sections through bundles of Cuvierian tubules. The red dots depict the positions at which each mass spectrum was acquired. (B, C and D) Visualization of signal intensity for the group of saponins at m/z 1125 and 1141 (B); the group of saponins at m/z 1287 and 1303 (C); and the group of saponins at m/z 1433, 1449, 1463 and 1479 (D) in tubule frozen sections from stressed (left part) or relaxed (right part) individuals.
Figure 7. MALDI-Imaging results shown in false colour representation.The color intensity reflects intensities of the selected mass signals: green, ions at m/z 843 used as a counterstain; red, the different saponin ions for which the m/z ratios are indicated below the images.
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