Four endogenous products of oxidative bilirubin degradation were isolated and fully characterized. The constitutional isomers belong to the propentdyopents (PDPs). Their structures and further oxidative transformations to biologically active bilirubin oxidation end products (Z-BOXes) are reported. Using liquid chromatography–mass spectrometry protocols, PDPs were detected in human bile and gallstones. Given the recent interest in BOXes as effectors in cerebral vasospasms and liver dysfunction, co-occurring PDPs represent an additional potentially active compound class to be considered.

•New 7-methyl branched glycerol dialkyl glycerol tetraethers (brGDGTs) are described.•New cyclopentane-moiety containing compounds are also described.•These novel brGDGTs were extracted from lake sediments.•Consideration of the new brGDGTs may improve brGDGT-derived proxies.Branched glycerol dialkyl glycerol tetraethers (brGDGTs) are bacterial membrane lipids that are widely used as valuable paleoenvironmental proxies. The recently discovered 6-methyl brGDGTs improved the accuracy of the proxies for temperature “methylation branched tetraethers (MBT)” and soil pH “cyclization branched tetraethers (CBT)”. However, the calibration uncertainties are still substantial for brGDGT-derived proxies (e.g., 5 °C for MBT′5ME). Here we report a series of novel 7-methyl brGDGT isomers that co-eluted with the known 5- and 6-methyl brGDGTs in commonly applied normal phase high performance liquid chromatography (HPLC). Using an optimized HPLC gradient the novel 7-methyl brGDGTs could be structurally characterized and quantified. Their mean relative abundance was in the range of 6% of the total brGDGTs in Chinese and Cameroon lake sediments. The 7-methyl brGDGT IIa7 correlates with sediment pH (R2 = 0.44, root-mean-square error = 0.26 pH unit), a result that motivates the re-analysis of brGDGTs in soils and sediments to further reassess brGDGT-based proxies and to determine the source of 7-methyl brGDGTs. In addition to the 7-methyl brGDGTs, we identified two novel pentamethylated brGDGTs based on the mass spectra of its ether-cleaved hydrocarbon products.

Diatoms are species-rich microalgae that often have a unique life cycle with vegetative cell size reduction followed by size restoration through sexual reproduction of two mating types (MT+ and MT−). In the marine benthic diatom Seminavis robusta, mate-finding is mediated by an l-proline-derived diketopiperazine, a pheromone produced by the attracting mating type (MT−). Here, we investigate the movement patterns of cells of the opposite mating type (MT+) exposed to a pheromone gradient, using video monitoring and statistical modeling. We report that cells of the migrating mating type (MT+) respond to pheromone gradients by simultaneous chemotaxis and chemokinesis. Changes in movement behavior enable MT+ cells to locate the direction of the pheromone source and to maximize their encounter rate towards it.

Covering: up to December 2014 Chemical ecology elucidates the nature and role of natural products as mediators of organismal interactions. The emerging techniques that can be summarized under the concept of metabolomics provide new opportunities to study such environmentally relevant signaling molecules. Especially comparative tools in metabolomics enable the identification of compounds that are regulated during interaction situations and that might play a role as e.g. pheromones, allelochemicals or in induced and activated defenses. This approach helps overcoming limitations of traditional bioassay-guided structure elucidation approaches. But the power of metabolomics is not limited to the comparison of metabolic profiles of interacting partners. Especially the link to other –omics techniques helps to unravel not only the compounds in question but the entire biosynthetic and genetic re-wiring, required for an ecological response. This review comprehensively highlights successful applications of metabolomics in chemical ecology and discusses existing limitations of these novel techniques. It focuses on recent developments in comparative metabolomics and discusses the use of metabolomics in the systems biology of organismal interactions. It also outlines the potential of large metabolomics initiatives for model organisms in the field of chemical ecology.

Metabolomics analysis to unravel secondary metabolite dynamics in microorganisms faces the challenge of immense data sets and complex experimental setups. In this issue, Goodwin et al. present a multiple stimuli approach combined with self-organizing map-based analysis to elucidate variations in the metabolome of Streptomyces coelicolor caused by biotic and environmental perturbations.

•An LC/MS mining approach revealed several sulfated metabolites in Dasycladales.•Two new sulfated phenolic acids from Dasycladales are described.•4-(Sulfooxy)phenylacetic and 4-(sulfooxy)benzoic acid were synthesized and tested.•Their function as precursors of inhibitors of biofilm formation is demonstrated.•Sulfatation/desulfatation allows modulating activity and intracellular storage.Sulfated aromatic acids play a central role as mediators of chemical interactions and physiological processes in marine algae and seagrass. Among others, Dasycladus vermicularis (Scopoli) Krasser 1898 uses a sulfated hydroxylated coumarin derivative as storage metabolite for a protein cross linker that can be activated upon mechanical disruption of the alga. We introduce a comprehensive monitoring technique for sulfated metabolites based on fragmentation patterns in liquid chromatography/mass spectrometry and applied it to Dasycladales. This allowed the identification of two new aromatic sulfate esters 4-(sulfooxy)phenylacetic acid and 4-(sulfooxy)benzoic acid. The two metabolites were synthesized to prove the mass spectrometry-based structure elucidation in co-injections. We show that both metabolites are transformed to the corresponding desulfated phenols by sulfatases of bacteria. In biofouling experiments with Escherichia coli and Vibrio natriegens the desulfated forms were more active than the sulfated ones. Sulfatation might thus represent a measure of detoxification that enables the algae to store inactive forms of metabolites that are activated by settling organisms and then act as defense.Novel sulfated metabolites of Dasycladales are introduced that modulate biofilm formation after desulfatation.

•We introduce a validated quantification of bilirubin degradation end products.•The working ranges are 2.74–163.0 pg/μL (Z-BOX A) and 2.12–162.4 pg/μL (Z-BOX B).•Z-BOX A and Z-BOX B are endogenous serum components of healthy volunteers.•Light exposure leads to photoisomerization of both analytes.Bilirubin oxidation end products (BOXes) appear upon endogenous heme degradation and can be found in the cerebrospinal fluid after hemorrhagic stroke. BOXes are assumed to contribute to delayed cerebral vasospasm and secondary loss of brain tissue. Here, we present a validated LC–ESI-MS/MS method for the sensitive determination of the regio-isomers Z-BOX A and Z-BOX B in human serum. We found that Z-BOX A and Z-BOX B appear in serum of healthy volunteers. The sample preparation includes the addition of 5-bromonicotinamide as internal standard and protein precipitation with acetonitrile. Baseline-separation was achieved on a C-18 column with a binary solvent gradient of formic acid in water/acetonitrile at 1 mL/min within a total analysis time of 17 min. Using single reaction monitoring in the positive ion mode, the linear working ranges were 2.74–163 pg/μL (Z-BOX A) and 2.12–162.4 pg/μL (Z-BOX B) with R2 > 0.995. Intra- and inter-day precisions were <10%. The inherent analyte concentrations of Z-BOX A (14.4  ±   5.1 nM) and Z-BOX B (10.9 ±  3.1 nM) in pooled human serum were determined by standard addition. The photolability of both analytes was demonstrated. This method enables to monitor Z-BOX A and Z-BOX B as a prerequisite to systematically study the biological significance of higher order metabolites of heme degradation.

•Cyclodextrine phases combined with GC and LC cannot resolve the racemate.•After method screening in SFC, separation of both enantiomers is achieved.•Determination of the enantiomeric excess in a complex sample matrix is possible.•Biological derived c(d-Pro–d-Pro) could be detected.The proline derived diketopiperazine has been identified in plants, insects and fungi with unknown function and was recently also reported as the first pheromone from a diatom. Nevertheless the stereochemistry and enantiomeric excess of this natural product remained inaccessible using direct analytical methods. Here we introduce a chiral separation of this metabolite using supercritical fluid chromatography/mass spectrometry. Several chromatographic methods for chiral analysis of the diketopiperazine from the diatom Seminavis robusta and synthetic enantiomers have been evaluated but neither gas chromatography nor high performance liquid chromatography on different chiral cyclodextrin phases were successful in separating the enantiomers. In contrast, supercritical fluid chromatography achieved baseline separation within four minutes of run time using amylose tris(3,5-dimethylphenylcarbamate) as stationary phase and 2-propanol/CO2 as mobile phase. This very rapid chromatographic method in combination with ESI mass spectrometry allowed the direct analysis of the cyclic dipeptide out of the complex sea water matrix after SPE enrichment. The method could be used to determine the enantiomeric excess of freshly released pheromone and to follow the rapid degradation observed in diatom cultures. Initially only trace amounts of c(d-Pro–d-Pro) were found besides the dominant c(l-Pro–l-Pro) in the medium. However the enantiomeric excess decreased upon pheromone degradation within few hours indicating that a preferential conversion and thus inactivation of the l-proline derived natural product takes place.

Chemically mediated interactions are hypothesized to be essential for ecosystem functioning as co-occurring organisms can influence the performance of each other by metabolic means. A metabolomics approach can support a better understanding of such processes but many problems cannot be addressed due to a lack of appropriate co-culturing and sampling strategies. This is particularly true for planktonic organisms that live in complex but very dilute communities in the open water. Here we present a co-culturing device that allows culturing of microalgae and bacteria that are physically separated but can exchange dissolved or colloidal chemical signals. Identical growth conditions for both partners as well as high metabolite diffusion rates between the culturing chambers are ensured. This setup allowed us to perform a metabolomic survey of the effect of the bacterium Dinoroseobacter shibae on the diatom Thalassiosira pseudonana. GC–MS measurements revealed a pronounced influence of the bacterium on the metabolic profile of T. pseudonana cells with especially intracellular amino acids being up-regulated in co-cultures. Despite the influence on diatom metabolism, the bacterium has little influence on the growth of the algae. This might indicate that the observed metabolic changes represent an adaptive response of the diatoms. Such interactions might be crucial for metabolic fluxes within plankton communities.

We introduce a rigorously validated protocol based on extraction, derivatisation and GC/MS for the analysis of diatom metabolomes. Using this methodology we characterised general patterns of the metabolism of the diatom Skeletonema marinoi during different growth phases. Canonical analysis of principal coordinate revealed clearly that the intracellular metabolites differ between exponential, stationary and declining phase. In addition, diurnal variation during the exponential phase was observed. A detailed analysis of the metabolic changes is presented and discussed in the context of previous physiological studies of diatoms. The observed variability in metabolites has a significant consequence for further physiological and ecological studies. Investigations have to take into account that diatom metabolism is a highly dynamic process and that food quality, chemical defence and also the production of signal molecules might be dependent on different growth phases or diurnal variations. The introduced protocol is in general suitable for the monitoring of microalgae and has also the potential to be applied to complex plankton communities.

Covering: up to August 2010

We addressed the role of the unusual acetylenic fatty acid dicranin as a precursor for volatile oxylipins in the moss Dicranum scoparium. Dicranin is transformed immediately after mechanical wounding of moss tissue to volatile C5- and C6-oxylipins. The transformation of synthetic deuterium labeled dicranin was monitored using LC/MS analysis and multivariate statistics to identify polar metabolites produced during volatile formation. Among the newly formed oxylipins is a highly reactive conjugated C13 allene with similar degrees of labeling compared to the C5 volatiles suggesting that it results as second cleavage product from the biosynthesis of pentenal and pentenone.

Dimethylsulfoniopropionate (DMSP) is a metabolite involved in central processes of phytoplankton physiology and ecology. Due to its high abundance, this molecule plays also a major role in global sulfur cycling. DMSP concentrations are usually indirectly determined via the base mediated release of dimethylsulfide (DMS) using gas chromatography (GC). However, because other potential precursors of DMS have been reported from macroalgae and phytoplankton, there is a substantial risk for an overestimation using an indirect method. We improve and validate a protocol for the derivatization and determination of DMSP in phytoplankton cultures. The improved method includes derivatization with 1-pyrenyldiazomethane (PDAM) and analysis via ultra performance liquid chromatography coupled with a mass selective detector (UPLC/MS). The protocol allows simultaneous qualitative and quantitative analysis of DMSP, other putative DMS precursors and osmolytes like glycine betaine (GBT). Detection limits are around 100 fmol on column allowing the quantification of DMSP from plankton field samples as well as from phytoplankton cultures. The DMSP content of several phytoplankton cultures was determined using the method involving derivatization of DMSP as well as an indirect headspace method determining DMS. For several algae strains, significant higher concentrations were obtained with the indirect determination. These results underscore the importance of other potential DMS precursors in phytoplankton samples that are not considered when using indirect methods for DMSP determination.Research Highlights► We improve a derivatization-based method for the determination of DMSP from microalgae. ► We apply this method to microalgal cultures and plankton samples. ► This method is compared to established protocols that detect DMSP indirectly via DMS. ► DMS determination using the indirect GC-based approach leads often to an overestimation of DMSP up to 14%. ► Other DMS sources from phytoplankton than DMSP have to be considered.

The red alga Gracilaria chilensis is commercially farmed for the production of agar hydrocolloids, but some susceptible algae in farms suffer from intense epiphyte growth. We investigated the induced chemical defense response of G. chilensis against epiphytes and demonstrated that an extract of an epiphyte-challenged alga can trigger a defense response. The hormonally active metabolites were purified by RP-HPLC. Treatment with the extract or the purified fraction changed the chemical profile of the alga and increased resistance against epiphyte spores. Semi-quantitative RT-PCR and enzyme assays demonstrated that this metabolic response occurs after an increase in lipoxygenase and phospholipase A2 activity. Although this suggests the involvement of regulatory oxylipins, neither jasmonic acid nor the algal metabolite prostaglandin E2 triggers comparable defense responses.

Oxylipins are metabolites which are derived from the oxidative fragmentation of polyunsaturated fatty acids. These metabolites play central roles in plant hormonal regulation and defense. Here we survey the production of volatile oxylipins in bryophytes and report the production of a high structural variety of C5, C6, C8 and C9 volatiles of mosses. In liverworts and hornworts oxylipin production was not as pronounced as in the 23 screened mosses. A biosynthetic investigation revealed that both, C18 and C20 fatty acids serve as precursors for the volatile oxylipins that are mainly produced after mechanical wounding of the green tissue of mosses.A screening of 23 moss species revealed that these bryophytes release a surprising diversity of C5, C6, C8 and C9 volatile oxylipins upon tissue disruption. Precursors of these oxylipins are both, C18 and C20 fatty acids.

A simple, derivatization free method for the direct determination of dimethylsulfoniopropionate (DMSP) using hydrophilic interaction liquid chromatography (HILIC)/mass spectrometry is introduced. DMSP is a zwitterionic osmolyte which is produced from marine plankton, macro algae and higher plants. Due to its central role in climate relevant geochemical processes as well as in plant physiology and chemical ecology there is a great interest in methods for its quantification. Since DMSP is labile and difficult to extract currently most protocols for quantification are based on indirect methods. Here we show that ultra performance liquid chromatography/mass spectrometry using a HILIC stationary phase is suitable for the direct quantification of DMSP from aqueous samples and microalgal extracts. The protocol requires minimal sample preparation and phytoplankton samples can be investigated after filtration of small volumes. The limit of detection is 20 nM and the calibration curve is linear in the range of 60 nM to 50 μM. The use of [2H6]-DMSP as internal standard allows prolonged sample storage since it is transformed with the same kinetics as natural DMSP. This makes the method suitable for both laboratory and field studies.

Induced chemical defense reactions are widespread in marine brown algae. Despite the evidence that the biosynthesis of defense metabolites can be up-regulated upon herbivory, we do not know how this regulation of biosynthetic pathways to secondary metabolites is achieved in brown algae. In higher plants, the phytohormone jasmonic acid (JA) is crucial for the mediation of induced chemical defenses, and several findings of this metabolite from marine sources have been reported. We tested the hypothesis that JA or related metabolites play a role in induced brown algal defense. Quantification of oxylipins with a detection limit around 20 ng g−1 algal tissue did not reveal the presence of JA in the seven examined brown algal species Dictyota dichotoma, Colpomenia peregrina, Ectocarpus fasciculatus, Fucus vesiculosus, Himanthalia elongata, Saccharina latissima (formerly Laminaria saccharina), and Sargassum muticum. Moreover, treatment with ecologically relevant concentrations of JA and methyl jasmonate did not lead to a significant change in the profile of medium- and non-polar metabolites of the tested algae. Only when high concentrations of ≥500 μg ml−1 medium of the phytohormones were applied that a metabolic response which could be attributed to unspecific stress was observed. Bioassays with D. dichotoma that focused on medium- and non-polar compounds confirmed the lack of a biological role of JA and methyl jasmonate in the induction of algal induced chemical defenses. The phytohormone-treated samples did not exhibit any increased defense potential towards the amphipod Ampithoe longimana and the isopod Paracerceis caudata. JA and related phytohormones, known to be active in higher plants, thus appear to play no role in brown algae for induction of the defense chemicals studied here.

The chemical defence potential against herbivores of certain Dictyotalean brown algae increases after tissue disruption. This wound activated defence has been explored in bioassays, but the metabolic pathways behind it are unknown. Here we describe a metabolic profiling approach to identify the activated defence metabolites. Before and after tissue damage of Dictyota dichotoma modified diterpenes, non-volatile medium polar metabolites as well as volatile compounds were profiled. While comparison of extracted intact and mechanically wounded algae revealed no significant differences in structure and distribution of semi-volatile and reversed phase LC/MS detectable metabolites, a strong release of gaseous volatiles was observed. Solid phase micro extraction (SPME) and GC/MS were used for identification and quantification of these biogenic gases. This showed that D. dichotoma released elevated amounts of trimethylamine (TMA) and dimethylsulphide (DMS) after mechanical tissue damage. To study the ecological significance of compounds released post injury and of the biosynthetically connected non-volatile acrylate, choice assays were performed with the amphipod Amphithoe longimana. Behavioural assays on artificial diets did not reveal any repellent role for the single isolated metabolites. In strong contrast, a mixture of TMA, DMS and acrylate significantly reduced the association of the herbivores with the treated food pellets.This shows that mixtures of these biogenic gases and acrylate are recognized by the herbivores and influence food selection.

The sulfur metabolite dimethylsulfoniopropionate (DMSP) is the most important precursor of the climate relevant metabolite dimethylsulfide (DMS). It has thus gained interest in the context of climate change and several studies investigated the influence of elevated temperature and/or CO2 on DMSP in complex plankton communities. However, only little information about changes in response to these factors in single species is available. Therefore, we analyzed DMSP in different phytoplankton cultures (Thalassiosira pseudonana, Phaeodactylum tricornutum, Emiliania huxleyi) under the influence of increased temperature by 6 °C and elevated CO2 to 790 ppmv. In addition, we addressed glycine betaine (GBT) that fulfills a similar function as osmolyte like DMSP. In all cultures GBT concentrations increased at higher temperature and decreased at elevated CO2. In contrast, diatoms and prymnesiophytes revealed opposite trends for DMSP. In diatoms increased CO2 and temperature led to decreased DMSP concentrations, while rather elevated levels of this metabolite under the influence of these parameters were observed for E. huxleyi.Highlights► We monitored the influence of T and CO2 on the marine osmolytes DMSP and GBT. ► Diatoms and prymnesiophytes showed a uniform pattern for GBT regulation. ► The regulation of DMSP production is more complex with strain dependent variability. ► Synergetic effects of elevated temperature and CO2 were observed for DMSP production. ► The study has major impact on considerations of the ocean of the future.

Mild winters are modifying the plankton spring bloom composition so that diatoms are decreasing and dinoflagellates increasing. We used two common spring bloom phytoplankton species, a diatom and a dinoflagellate to study the effects of changing bloom composition on the reproduction of the calanoid copepod Acartia bifilosa Giesbrecht, a dominant species in the northern Baltic Sea. Egg production was significantly higher when copepods were fed with Scrippsiella hangoei (Schiller) Larsen dinoflagellates or a mixture of Scrippsiella and Skeletonema marinoi Sarno and Zingone diatoms than when they were provided with Skeletonema only. This effect was observed despite the fact that the Skeletonema strain did not produce polyunsaturated aldehydes (PUAs) and its nutritional quality was high according to polyunsaturated fatty acid (PUFAs) and sterol measurements, and moderate according to mineral (C:N and C:P) measurements. When offered mixtures of Skeletonema and Scrippsiella, copepods ingested both, even when the other one was rare. This indicates potential positive effect of multispecies diets not verified in this study. Here we show that increasing dinoflagellate:diatom ratio might have a positive effect on copepod reproduction.► Copepod reproduction in relation to food quality on diatom and dinoflagellate diets. ► Egg production higher on dinoflagellate diet or on a mixture of both. ► Both species eaten when offered as mixtures, even when the other one was rare. ► Copepods may benefit from changing spring bloom composition.

Dimethylsulfoniopropionate (DMSP), a sulfur containing osmolyte and antioxidant, is suggested to take over the role of nitrogen-containing osmolytes under nitrate-depleted conditions. But other factors like time of day or limitation by nutrients can influence the cellular DMSP content as well. Especially in diatoms the complex dynamics of this central metabolite are still poorly understood. Therefore, we monitored the DMSP content in batch cultures of the diatom Skeletonema marinoi over the entire culture development, using a detailed sampling protocol on a diurnal basis to investigate the influence of the day/night cycle and limitation by nitrogen and other nutrients on cellular DMSP. Cultures were inoculated with initial high nitrate (N +) or low nitrate (N −) concentrations to evaluate the effects of nitrogen availability. The DMSP content per cell varied significantly with a 4-fold increase over the entire experiment. For N + conditions a pronounced diurnal pattern was found with increasing DMSP contents per cell during the light and decreasing DMSP contents during the dark period. However, when DMSP was normalized to cell volume, the highest concentrations occurred near the beginning of the light period. For N − cultures no such pronounced pattern was found. DMSP values of N − cultures increased within a few hours after the medium became nitrate depleted, while no such connection between nitrogen-concentration and DMSP could be observed in the N + cultures, where possibly an overflow mechanism or limitation of other nutrients such as Si might affect DMSP concentrations. Based on this data set we can conclude that DMSP formation in S. marinoi is under a pronounced diurnal control and depends in a rather complex manner on other factors such as growth limitation by nutrients.Highlights►Particulate DMSP in diatom batch cultures with high and low nitrate concentrations. ►High sampling rate to analyze influence of daytime, growth phase, nutrient depletion. ►DMSP is under pronounced diurnal control and depends on multiple (limiting) factors. ►Multiple physiological implementations can be deduced from the results. ►Alternative DMS-precursors become more prominent during culture development.

In phytoplankton a high species diversity of microalgae co-exists at a given time. But diversity is not only reflected by the species composition. Within these species different life phases as well as different metabolic states can cause additional diversity. One important example is the coccolithophore Emiliania huxleyi. Diploid cells play an important role in marine ecosystems since they can form massively abundant algal blooms but in addition the less abundant haploid life phase of E. huxleyi occurs in lower quantities. Both life phases may fulfill different functions in the plankton. We hypothesize that in addition to the functional diversity caused by this life phase transition the growth stage of cells can also influence the metabolic composition and thus the ecological impact of E. huxleyi. Here we introduce a metabolomic survey in dependence of life phases as well as different growth phases to reveal such changes. The comparative metabolomic approach is based on the extraction of intracellular metabolites from intact microalgae, derivatization and analysis by gas chromatography coupled to mass spectrometry (GC–MS). Automated data processing and statistical analysis using canonical analysis of principal coordinates (CAP) revealed unique metabolic profiles for each life phase. Concerning the correlations of metabolites to growth phases, complex patterns were observed. As for example the saccharide mannitol showed its highest concentration in the exponential phase, whereas fatty acids were correlated to stationary and sterols to declining phase. These results are indicative for specific ecological roles of these stages of E. huxleyi and are discussed in the context of previous physiological and ecological studies.