Cell-Death Metabolites from Cocconeis scutellum var. parva Identified by Integrating Bioactivity-Based Fractionation and Non-Targeted Metabolomic Approaches.

Epiphytic diatoms growing in Mediterranean seagrass meadows, particularly those of the genus Cocconeis, are abundant and ecologically significant, even in naturally acidified environments. One intriguing aspect of some benthic diatoms is their production of an unidentified cell-death-promoting compound, which induces destruction of the androgenic gland in Hippolyte inermis Leach, 1816, a shrimp exhibiting protandric hermaphroditism, principally under normal environmental pH levels. The consumption of Cocconeis spp. by this shrimp is vital for maintaining the stability of its natural populations. Although many attempts have been made to reveal the identity of the apoptotic compound, it is still unknown. In this study, we strategically integrated a bioactivity-based fractionation, a metabolomic approach, and two different experimental avenues to identify potential apoptotic metabolites from Cocconeis scutellum var. parva responsible for the sex reversal in H. inermis. Our integrated analysis uncovered two potential candidate metabolites, one putatively identified as a lysophosphatidylglycerol (LPG) (16:1) and the other classified as a fatty acid ester. This is the first time LPG (16:1) has been reported in C. scutellum var. parva and associated with cell-death processes. These candidate metabolites mark substantial progress in elucidating the factors responsible for triggering the removal of the androgenic gland in the early post-larval phases of H. inermis.

Responses of the Macroalga Ulva prolifera Müller to Ocean Acidification Revealed by Complementary NMR- and MS-Based Omics Approaches.

Ocean acidification (OA) is a dramatic perturbation of seawater environments due to increasing anthropogenic emissions of CO2. Several studies indicated that OA frequently induces marine biota stress and a reduction of biodiversity. Here, we adopted the macroalga Ulva prolifera as a model and applied a complementary multi-omics approach to investigate the metabolic profiles under normal and acidified conditions. Our results show that U. prolifera grows at higher rates in acidified environments. Consistently, we observed lower sucrose and phosphocreatine concentrations in response to a higher demand of energy for growth and a higher availability of essential amino acids, likely related to increased protein biosynthesis. In addition, pathways leading to signaling and deterrent compounds appeared perturbed. Finally, a remarkable shift was observed here for the first time in the fatty acid composition of triglycerides, with a decrease in the relative abundance of PUFAs towards an appreciable increase of palmitic acid, thus suggesting a remodeling in lipid biosynthesis. Overall, our studies revealed modulation of several biosynthetic pathways under OA conditions in which, besides the possible effects on the marine ecosystem, the metabolic changes of the alga should be taken into account considering its potential nutraceutical applications.

Groundwater metabolome responds to recharge in fractured sedimentary strata.

Understanding the sources, structure and fate of dissolved organic matter (DOM) in groundwater is paramount for the protection and sustainable use of this vital resource. On its passage through the Critical Zone, DOM is subject to biogeochemical conversions. Therefore, it carries valuable cross-habitat information for monitoring and predicting the stability of groundwater ecosystem services and assessing these ecosystems' response to fluctuations caused by external impacts such as climatic extremes. Challenges arise from insufficient knowledge on groundwater metabolite composition and dynamics due to a lack of consistent analytical approaches for long-term monitoring. Our study establishes groundwater metabolomics to decipher the complex biogeochemical transport and conversion of DOM. We explore fractured sedimentary bedrock along a hillslope recharge area by a 5-year untargeted metabolomics monitoring of oxic perched and anoxic phreatic groundwater. A summer with extremely high temperatures and low precipitation was included in the monitoring. Water was accessed by a monitoring well-transect and regularly collected for liquid chromatography-mass spectrometry (LC-MS) investigation. Dimension reduction of the resulting complex data set by principal component analysis revealed that metabolome dissimilarities between distant wells coincide with transient cross-stratal flow indicated by groundwater levels. Time series of the groundwater metabolome data provides detailed insights into subsurface responses to recharge dynamics. We demonstrate that dissimilarity variability between groundwater bodies with contrasting aquifer properties coincides with recharge dynamics. This includes groundwater high- and lowstands as well as recharge and recession phases. Our monitoring approach allows to survey groundwater ecosystems even under extreme conditions. Notably, the metabolome was highly variable lacking seasonal patterns and did not segregate by geographical location of sampling wells, thus ruling out vegetation or (agricultural) land use as a primary driving factor. Patterns that emerge from metabolomics monitoring give insight into subsurface ecosystem functioning and water quality evolution, essential for sustainable groundwater use and climate change-adapted management.

Aquifer system and depth specific chemical patterns in fractured-rock groundwater from the Critical Zone revealed by untargeted LC-MS-based metabolomics.

In the Earth's Critical Zone, water plays an essential role as a collector and transporter of metabolites and their transformation products. It is generally believed that the chemical profiles of groundwater are strongly impacted by land use. However, predictors for the effects of above-ground natural and anthropogenic activities on below-ground chemistry are rare. We reasoned that comparing groundwater metabolomes from different land-use sites and depths can give insight into this coupling of above and below-ground processes in the Critical Zone. This study used an LC-MS-based untargeted metabolomic approach to identify links between groundwater metabolomes from monitoring wells in fractured carbonate-/siliciclastic alternations along a hillslope of the Hainich Critical Zone Exploratory (CZE) in Thuringia, Germany. Our results identify the land-use type, aquifer system, and sampling depth as critical factors determining the differences among groundwater metabolomes. We established five groundwater metabolic clusters and correlated these to the aquifer systems, hydrogeochemistry, and microbial community composition. Our untargeted metabolomic approach reveals the limited connectivity of groundwater chemical profiles with above-ground activities and illustrates how deep the input signals can travel.

Microbial community functioning during plant litter decomposition.

Microbial life in soil is fueled by dissolved organic matter (DOM) that leaches from the litter layer. It is well known that decomposer communities adapt to the available litter source, but it remains unclear if they functionally compete or synergistically address different litter types. Therefore, we decomposed beech, oak, pine and grass litter from two geologically distinct sites in a lab-scale decomposition experiment. We performed a correlative network analysis on the results of direct infusion HR-MS DOM analysis and cross-validated functional predictions from 16S rRNA gene amplicon sequencing and with DOM and metaproteomic analyses. Here we show that many functions are redundantly distributed within decomposer communities and that their relative expression is rapidly optimized to address litter-specific properties. However, community changes are likely forced by antagonistic mechanisms as we identified several natural antibiotics in DOM. As a consequence, the decomposer community is specializing towards the litter source and the state of decomposition (community divergence) but showing similar litter metabolomes (metabolome convergence). Our multi-omics-based results highlight that DOM not only fuels microbial life, but it additionally holds meta-metabolomic information on the functioning of ecosystems.

Untargeted Metabolomics Unveil Changes in Autotrophic and Mixotrophic Galdieria sulphuraria Exposed to High-Light Intensity.

The thermoacidophilic red alga Galdieria sulphuraria has been optimizing a photosynthetic system for low-light conditions over billions of years, thriving in hot and acidic endolithic habitats. The growth of G. sulphuraria in the laboratory is very much dependent on light and substrate supply. Here, higher cell densities in G. sulphuraria under high-light conditions were obtained, although reductions in photosynthetic pigments were observed, which indicated this alga might be able to relieve the effects caused by photoinhibition. We further describe an extensive untargeted metabolomics study to reveal metabolic changes in autotrophic and mixotrophic G. sulphuraria grown under high and low light intensities. The up-modulation of bilayer lipids, that help generate better-ordered lipid domains (e.g., ergosterol) and keep optimal membrane thickness and fluidity, were observed under high-light exposure. Moreover, high-light conditions induced changes in amino acids, amines, and amide metabolism. Compared with the autotrophic algae, higher accumulations of osmoprotectant sugars and sugar alcohols were recorded in the mixotrophic G. sulphuraria. This response can be interpreted as a measure to cope with stress due to the high concentration of organic carbon sources. Our results indicate how G. sulphuraria can modulate its metabolome to maintain energetic balance and minimize harmful effects under changing environments.

Untargeted Metabolomics Approach Reveals Differences in Host Plant Chemistry Before and After Infestation With Different Pea Aphid Host Races.

The pea aphid (Acyrthosiphon pisum), a phloem-sucking insect, has undergone a rapid radiation together with the domestication and anthropogenic range expansion of several of its legume host plants. This insect species is a complex of at least 15 genetically different host races that can all develop on the universal host plant Vicia faba. However, each host race is specialized on a particular plant species, such as Medicago sativa, Trifolium pratense, or Pisum sativum, which makes it an attractive model insect to study ecological speciation. Previous work revealed that pea aphid host plants produce a specific phytohormone profile depending on the host plant - host race combination. Native aphid races induce lower defense hormone levels in their host plant than non-native pea aphid races. Whether these changes in hormone levels also lead to changes in other metabolites is still unknown. We used a mass spectrometry-based untargeted metabolomic approach to identify plant chemical compounds that vary among different host plant-host race combinations and might therefore, be involved in pea aphid host race specialization. We found significant differences among the metabolic fingerprints of the four legume species studied prior to aphid infestation, which correlated with aphid performance. After infestation, the metabolic profiles of M. sativa and T. pratense plants infested with their respective native aphid host race were consistently different from profiles after infestation with non-native host races and from uninfested control plants. The metabolic profiles of P. sativum plants infested with their native aphid host race were also different from plants infested with non-native host races, but not different from uninfested control plants. The compounds responsible for these differences were putatively identified as flavonoids, saponins, non-proteinogenic amino acids and peptides among others. As members of these compound classes are known for their activity against insects and aphids in particular, they may be responsible for the differential performance of host races on native vs. non-native host plants. We conclude that the untargeted metabolomic approach is suitable to identify candidate compounds involved in the specificity of pea aphid - host plant interactions.

Modulation of Legume Defense Signaling Pathways by Native and Non-native Pea Aphid Clones.

The pea aphid (Acyrthosiphon pisum) is a complex of at least 15 genetically different host races that are native to specific legume plants, but can all develop on the universal host plant Vicia faba. Despite much research, it is still unclear why pea aphid host races (biotypes) are able to colonize their native hosts while other host races are not. All aphids penetrate the plant and salivate into plant cells when they test plant suitability. Thus plants might react differently to the various pea aphid host races. To find out whether legume species vary in their defense responses to different pea aphid host races, we measured the amounts of salicylic acid (SA), the jasmonic acid-isoleucine conjugate (JA-Ile), other jasmonate precursors and derivatives, and abscisic acid (ABA) in four different species (Medicago sativa, Trifolium pratense, Pisum sativum, V. faba) after infestation by native and non-native pea aphid clones of various host races. Additionally, we assessed the performance of the clones on the four plant species. On M. sativa and T. pratense, non-native clones that were barely able to survive or reproduce, triggered a strong SA and JA-Ile response, whereas infestation with native clones led to lower levels of both phytohormones. On P. sativum, non-native clones, which survived or reproduced to a certain extent, induced fluctuating SA and JA-Ile levels, whereas the native clone triggered only a weak SA and JA-Ile response. On the universal host V. faba all aphid clones triggered only low SA levels initially, but induced clone-specific patterns of SA and JA-Ile later on. The levels of the active JA-Ile conjugate and of the other JA-pathway metabolites measured showed in many cases similar patterns, suggesting that the reduction in JA signaling was due to an effect upstream of OPDA. ABA levels were downregulated in all aphid clone-plant combinations and were therefore probably not decisive factors for aphid-plant compatibility. Our results suggest that A. pisum clones manipulate plant-defense signaling to their own advantage, and perform better on their native hosts due to their ability to modulate the SA- and JA-defense signaling pathways.