Our findings suggest that, in Cuvierian tubules, saponins are the most variable compounds during a stress response. With FlexImaging software, it is possible to visualize, using a color gradient, the distribution of the signal obtained for each group of saponins previously defined after PCA on the direct profiling of Cuvierian tubule sections. tissues of relaxed animals. Finally, saponin ions at Rolapitant 1433, FSHR 1449, 1463 and 1479 were observed in some Ct of stressed holothuroids in the outer part of the connective tissue. The saponin groupm/z14xx seems therefore to be stress-specific and could originate from modifications of the saponins withm/zof 11xx. == Conclusions == All the results taken together indicate a complex chemical defense mechanism with, for a single organ, different Rolapitant 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. == Introduction == Sea cucumbers seem to be vulnerable animals regarding their numerous predators referenced in the literature[1]. However, many authors consider predation on adult holothuroids to be infrequent[2],[3]. Amongst the numerous anti-predation mechanisms developed by these animals, the toxicity of the body wall and Rolapitant the presence of Cuvierian tubules seems to be the most effective against non-specialist predators[4],[5]. Cuvierian tubules are little caeca located in the posterior part of the animal, that can be ejected toward a predator in response to an aggression[6]. Expelled tubules lengthen into sticky white threads susceptible to entangle the predator[7],[8]. Although only some species of holothuroids from the family Holothuriidae, includingHolothuria 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]. Indeed, due to their Rolapitant membranolytic action[10],[11], saponins have a wide range of pharmacological effects such as cytotoxicity to tumor cells, or antifungal and hemolytic actions[12],[13]. Triterpene glycosides are particularly concentrated in the Cuvierian tubules of holothuroids[14], [15]and specific congener mixtures are usually associated with the defensive function of this system[12],[16],[17],[18]. Recent studies demonstrated that mass spectrometry (MS) procedures represent very valuable techniques for the detection and identification of saponins[17][20]. However, classical techniques require the complete pounding of the organs to allow the extraction of their contents. In consequence, the determination of the precise localization of saponins in the tissues is impossible. For 10 years, MALDI-MSI (Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging), also called MALDI-Imaging, has undergone many developments[21],[22]and has achieved a certain maturity, allowing it to be now used in many domains like clinical proteomics[23]or pharmaceutical fields[24]. This procedure permits to detect and localize ions of interest directly on tissue sections and nearly without any preparation. The benefit of this technique is the high sensitivity and the use of a soft ionization technique, like MALDI-MS, which allows a label free molecular imaging of a biological tissue section. The great added value of using MALDI-MS direct tissue analysis is to detect hundred of molecules in one spatially-resolved analysis. By this way, MALDI-MSI or MALDI profiling will open the door for important breakthroughs in the field of chemical ecology. In the present study, we investigated the triterpene glycosides of the Cuvierian tubules in the sea cucumberH. forskaliand attempted to localize saponins in their tissues. We combined classical histochemical labelling to MALDI-MS direct tissue profiling and MALDI-MSI in order to detect saponins and describe their spatial localization in the Cuvierian tubules. A statistical multivariate test using the Principal Component Analysis (PCA) method which allows highlighting groups of close ions from dense and complex data sets was conducted to compare the molecular data of Cuvierian tubules from stressed and relaxed holothuroids. == Results and Discussion == == Use of lectins to localize saponins on tissue sections == As no antibodies to saponins are available, lectins were considered as a tool to detect these molecules on Cuvierian tubule sections. Lectins are proteins or Rolapitant glycoproteins of non-immune origin that are able to bind specific carbohydrate motifs in a way similar to the formation of the antibody-antigen complex[25]. Each lectin recognizes specifically one oligosaccharidic chain but it may also bind to similar oligo- or mono-saccharides, although with a lower affinity. To the best of our knowledge, no commercially available lectin is specific of the saponin carbohydrate moiety which encloses glucose (Glc), 3-O-methylglucose (MeGlc), quinovose (Qui) and xylose (Xyl) residues. However, mannose-binding.