a-straightforward-method-for-glucosinolate-extraction-analysis-with

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography (HPLC)

Glucosinolates are a well-studied and highly diverse class of natural plant compounds. They play important roles in plant resistance, rapeseed oil ...

German Centre for integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig

Friedrich Schiller University Jena

Chemical defense of the invasive red alga Gracilaria vermiculophylla has been studied and compared to that of the noninvasive but related Gracilaria chilensis. Both species rely on a wound-activated chemical defense that makes them less attractive to the herbivorous sea snail Echinolittorina peruviana. The chemical stress response of both species was monitored by LC-ESIMS-based metabolic profiling and revealed commonalities and differences. Both algae rely on a rapid lipoxygenase mediated transformation of arachidonic acid to known and novel oxylipins. Common products are 7,8-dihydroxyeicosatetraenoic acid and a novel eicosanoid with an unusual γ-lactone moiety. Several prostaglandins were predominantly formed by the invasive species. The role of some of these metabolites was investigated by surveying the attachment of E. peruviana on artificial food containing the respective oxylipins. Both algae species are defended against this general herbivore by 7,8-dihydroxyeicosatetraenoic acid, whereas the prostaglandins and the novel oxylipins were inactive at naturally occurring concentrations. The role of different oxylipins in the invasive potential of Gracilaria spp. is discussed.

Conserved and species-specific oxylipin pathways in the wound-activated chemical defense of the noninvasive red alga Gracilaria chilensis and the invasive Gracilaria vermiculophylla.

Investigations of the surface chemistry of marine organisms are essential to understand their chemically mediated interactions with fouling organisms. In this context, the concentration of natural products in the immediate vicinity of algal surfaces, as well as their biological activity, are of particular importance. However, due to lack of appropriate methods, the distribution of compounds within the chemical sphere around marine algae is unknown. This study demonstrates the suitability of confocal resonance Raman microspectroscopy for the determination of metabolites around algal surfaces with a micrometer resolution. The spatial distribution of carotenoids in the diffusion boundary layer of the brown alga Fucus vesiculosus and the green alga Ulva sp. was determined using the disruption-free optical method. A gradient of carotenoids was determined within 0 to 150 μm from the surface of the algae, thereby demonstrating the release of the non-polar metabolites involved in antifouling processes. The differences in the carotenoid composition of the brown and green algae were reflected in the spectra. Resonance Raman microspectroscopy also allowed visualization of the lateral distribution of fucoxanthin on the algal surface and localization of concentration maxima within a 50 × 50 μm(2) area. The results from this work show clearly that established dipping techniques suitable for the extraction of the diffusion boundary layer of macroalgae only provide an average of the local strongly variable concentrations of metabolites on algal surfaces.

Disruption-free imaging by Raman spectroscopy reveals a chemical sphere with antifouling metabolites around macroalgae.

The invasive unicellular green macroalga Caulerpa taxifolia has spread dramatically in the Mediterranean Sea over the last decades. Its success is based on rapid plug formation after wounding, to prevent the loss of cell content. This quick and efficient process involves the rapid transformation of the secondary metabolite caulerpenyne to the reactive 1,4-dialdehyde oxytoxin 2, which acts as a protein crosslinker. The main metabolites of the wound plug were identified as proteins, caulerpenyne derivatives, and sulfated polysaccharides. Because of a methodological deficit, however, the detailed distribution of the compounds within the wound plug of C. taxifolia was unknown. This study demonstrates the suitability of FT-Raman spectroscopy for the noninvasive in vivo determination of caulerpenyne and its derivatives, as well as β-carotene, from signals with special spectral features within the wound plug and the adjacent intact alga tissue, with a resolution of 100 μm. FT-Raman spectra allowed four different zones with distinct chemical compositions around the region of wounds to be characterized. Gradients of the investigated metabolites within the wound plug and the alga could be determined. Moreover, various caulerpenyne derivatives could be identified spectroscopically, and this has led to a mechanistic proposal for the internal and the external wound plug formation.

Raman spectroscopic insights into the chemical gradients within the wound plug of the green alga Caulerpa taxifolia.

Pharmaceuticals consumption by humans and animals is increasing substantially, leading to unprecedented levels of these compounds in aquatic environments worldwide. Recent findings that concentrations reach levels that can directly have negative effects on organisms are important per se, but also sound an alarm for other potentially more pervasive effects that arise from the interconnected nature of ecological communities. Aquatic organisms use chemical cues to navigate numerous challenges, including the location of mates and food, and the avoidance of natural enemies. Low concentrations of pharmaceuticals can disrupt this "smellscape" of information leading to maladaptive responses. Furthermore, direct effects of pharmaceuticals on the traits and abundance of one species can cascade through a community, indirectly affecting other species. We review mechanisms by which pharmaceuticals in surface waters can disrupt natural chemical information flows and species interactions. Pharmaceuticals form a new class of chemical threats, which could have far-reaching implications for ecosystem functioning and conservation management.

Pharmaceuticals May Disrupt Natural Chemical Information Flows and Species Interactions in Aquatic Systems: Ideas and Perspectives on a Hidden Global Change.

The surface chemistry of aquatic organisms determines their biotic interactions. Metabolites in the spatially limited laminar boundary layer mediate processes, such as antifouling, allelopathy and chemical defense against herbivores. However, very few methods are available for the investigation of such surface metabolites. An approach is described in which surfaces are extracted by means of C18 solid phase material. By powdering wet algal surfaces with this material, organic compounds are adsorbed and can be easily recovered for subsequent liquid chromatography/mass spectrometry (LC/MS) and gas chromatography/mass spectrometry (GC/MS) investigations. The method is robust, picks up metabolites of a broad polarity range and is easy to handle. It is more universal compared to established solvent dipping protocols and it does not cause damage to the test organisms. A protocol is introduced for the macroalgae Fucus vesiculosus, Caulerpa taxifolia and Gracilaria vermiculophylla, but it can be easily transferred to other aquatic organisms.

Katharina Grosser

German Centre for integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography (HPLC)