Abstract
Despite significant advances in chemical ecology, the biodistribution, temporal changes and ecological function of most marine secondary metabolites remain unknown. One such example is the association between choline esters and Tyrian purple precursors in muricid molluscs. Mass spectrometry imaging (MSI) on nano-structured surfaces has emerged as a sophisticated platform for spatial analysis of low molecular mass metabolites in heterogeneous tissues, ideal for low abundant secondary metabolites. Here we applied desorption-ionisation on porous silicon (DIOS) to examine in situ changes in biodistribution over the reproductive cycle. DIOS-MSI showed muscle-relaxing choline ester murexine to co-localise with tyrindoxyl sulfate in the biosynthetic hypobranchial glands. But during egg-laying, murexine was transferred to the capsule gland, and then to the egg capsules, where chemical ripening resulted in Tyrian purple formation. Murexine was found to tranquilise the larvae and may relax the reproductive tract. This study shows that DIOS-MSI is a powerful tool that can provide new insights into marine chemo-ecology.
Highlights
Despite significant advances in chemical ecology, the biodistribution, temporal changes and ecological function of most marine secondary metabolites remain unknown
Reproducible methodologies that can spatially and temporally detect secondary metabolites in situ will significantly advance the field of chemical ecology[13]
mass spectrometry imaging (MSI) is often based on matrix-assisted laser desorption ionisation (MALDI)[15,16], a soft ionisation technique that relies on aromatic molecules for
Summary
Detection and identification of secondary metabolite patterns on functionalised pSi. To explore the biological roles of muricid choline esters and brominted indoles using DIOS-MSI in the context of egg laying and maternal investment, established spatial patterns based on previous histochemical staining[37] were used as a basis to track changes in secondary metabolite distribution at different phases of the reproductive cycle. The capacity for pSi to desorb and ionise brominated indoles from the tissue samples was confirmed by the detection of isotopic patterns in MS/MS analysis consistent with the crude extracts analysed by LC-MS (Supplementary Figs 2 and 3) and with purified compounds and synthetic 6 bromoisatin and 6,6′-dibromoindigo spotted directly onto the pSi surface[39]. Consistent with our previous studies[25,39], brominated indoles across a broad range of polarities in the low mass region from m/z 256–421 (Fig. 2 and Supplementary Fig. 2), showed affinity to the pSi surface. Stamping allowed low mass metabolite transfer from cryostat sections of the tissue to the pSi (Fig. 2), whilst the hydrophobic pSi surface facilitated the subsequent removal of residual tissue by pipetting with water, with minimal delocalization of the secondary metabolites. The isotopic clusters for mono- and www.nature.com/scientificreports/ Compound murexine choline
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