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Chironomids are keystone primary benthic consumers with semi‐aquatic life cycles. They support aquatic and terrestrial consumers at higher trophic levels by conveying dietary nutrients, such as fatty acids. In this study, we combined field sampling and laboratory experiments to examine the effects of environmental parameters, including diet, on fatty acid composition and metabolism in chironomid larvae and imagines. Results from 53 lakes showed that lake size, depth, dissolved organic carbon (DOC) concentrations, and trophic state had only marginal effects on the content of long‐chain polyunsaturated fatty acids (LC‐PUFA) in chironomids. Compound‐specific stable hydrogen isotope analyses confirmed that chironomids actively bioconvert dietary fatty acid precursors to LC‐PUFA in all lake types, independent of nutrient or DOC concentrations. Moreover, fatty acid‐specific stable carbon isotope data indicated that the diet of chironomids was subsidized, particularly in oligotrophic lakes in spring, by terrestrial C18 fatty acid precursors that were converted to LC‐PUFA. Data from feeding experiments further confirmed that decreased dietary availability of LC‐PUFA enhanced the conversion of dietary short‐chain precursors to LC‐PUFA. These results suggest that chironomids are PUFA regulators that can sustain LC‐PUFA levels under varying environmental conditions. Furthermore, our results indicate that they bioconvert terrestrial low‐quality material to high‐quality resources, which, via chironomid emergence, support terrestrial food webs. Chironomids are abundant and widespread, and thus, the trophic transfer of LC‐PUFA can have significant implications for the fitness and production of upper trophic level consumers in both aquatic and terrestrial ecosystems. [ABSTRACT FROM AUTHOR]

Understanding the stable hydrogen isotope (δ2H) composition and fractionation in lipid biomolecules of primary producers, such as terrestrial and aquatic plants, is crucial for deciphering past environmental conditions, as well as applying compound-specific stable isotope analysis for the study of metabolic and ecological processes. We conducted a new tracer experiment to explore the δ2H composition of algal fatty acid biomarkers, focusing on freshwater algae, which form the base of aquatic food webs. We selected a range of algal species widely found in freshwater ecosystems and cultivated them under controlled conditions. First, we added 2H2O to ambient water as a tracer to investigate the net hydrogen isotope fractionation during algal lipid synthesis at isotopic equilibrium, which is particularly informative for paleo-geochemical studies. Then, we conducted kinetic experiments to quantify the time needed for algal fatty acids to achieve isotopic steady-state conditions in response to the change in ambient water δ2H values. Our findings revealed substantial variability in hydrogen isotope fractionation among different algal taxa and various fatty acids. Based on taxa, different fatty acids exhibited faster integration of water hydrogen than others, but they were not necessarily in the order of the biosynthetic pathway. This experiment underscores the complexity of hydrogen isotope fractionation and the requirement for controlled laboratory studies to properly apply compound-specific stable H isotope analysis techniques in ecological and paleo-environmental studies. [ABSTRACT FROM AUTHOR]

Acting through a combination of direct and indirect pathogen clearance mechanisms, blood-derived antimicrobial compounds (AMCs) play a pivotal role in innate immunity, safeguarding the host against invading microorganisms. Besides their antimicrobial activity, some AMCs can neutralize endotoxins, preventing their interaction with immune cells and avoiding an excessive inflammatory response. In this study, we aimed to investigate the influence of unfractionated heparin, a polyanionic drug clinically used as anticoagulant, on the endotoxin-neutralizing and antibacterial activity of blood-derived AMCs. Serum samples from healthy donors were pre-incubated with increasing concentrations of heparin for different time periods and tested against pathogenic bacteria (Acinetobacter baumannii, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus) and endotoxins from E. coli, K. pneumoniae, and P. aeruginosa. Heparin dose-dependently decreased the activity of bloodderived AMCs. Consequently, pre-incubation with heparin led to increased activity of LPS and higher values of the pro-inflammatory cytokines tumor necrosis factor a (TNF-a) and interleukin 6 (IL-6). Accordingly, higher concentrations of A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa were observed as well. These findings underscore the neutralizing effect of unfractionated heparin on blood-derived AMCs in vitro and may lead to alternative affinity techniques for isolating and characterizing novel AMCs with the potential for clinical translation. [ABSTRACT FROM AUTHOR]

Chytrid fungal parasites convert dietary energy and essential dietary molecules, such as long‐chain (LC) polyunsaturated fatty acids (PUFA), from inedible algal/cyanobacteria hosts into edible zoospores. How the improved biochemical PUFA composition of chytrid‐infected diet may extend to zooplankton, linking diet quality to consumer fitness, remains unexplored.Here, we assessed the trophic role of chytrids in supporting dietary energy and PUFA requirements of the crustacean zooplankton Daphnia, when feeding on the filamentous cyanobacterium Planktothrix.Only Daphnia feeding on chytrid‐infected Planktothrix reproduced successfully and had significantly higher survival and growth rates compared with Daphnia feeding on the sole Planktothrix diet. While the presence of chytrids resulted in a two‐fold increase of carbon ingested by Daphnia, carbon assimilation increased by a factor of four, clearly indicating enhanced carbon transfer efficiency with chytrid presence.Bulk carbon (δ13C) and nitrogen (δ15N) stable isotopes did not indicate any treatment‐specific dietary effects on Daphnia, nor differences in trophic position among diet sources and the consumer. Compound‐specific carbon isotopes of fatty acids (δ13CFA), however, revealed that chytrids bioconverted short‐chain to LC‐PUFA, making it available for Daphnia. Chytrids synthesised the ω‐3 PUFA stearidonic acid de novo, which was selectively retained by Daphnia. Values of δ13CFA demonstrated that Daphnia also bioconverted short‐chain to LC‐PUFA.We provide isotopic evidence that chytrids improved the dietary provision of LC‐PUFA for Daphnia and enhanced their fitness. We argue for the existence of a positive feedback loop between enhanced Daphnia growth and herbivory in response to chytrid‐mediated improved diet quality. Chytrids upgrade carbon from the primary producer and facilitate energy and PUFA transfer to primary consumers, potentially also benefitting upper trophic levels of pelagic food webs. [ABSTRACT FROM AUTHOR]

Consumers feeding at the aquatic–terrestrial ecosystem interface may obtain a mixture of aquatic and terrestrial diet resources that vary in nutritional composition. However, in lake riparian spiders, the relative significance of aquatic versus terrestrial diet sources remains to be explored. We investigated the trophic transfer of lipids and polyunsaturated fatty acids (PUFA) from emergent aquatic and terrestrial insects to spiders at varying distances from the shoreline of a subalpine lake in Austria, using differences in fatty acid profiles and compound‐specific stable carbon (δ13C) and hydrogen (δ2H) isotopes. The omega‐3 PUFA content of emergent aquatic insects was higher than that of terrestrial insects. Emergent aquatic insects contained on average 6.6 times more eicosapentaenoic acid (EPA) and 1.2 times more α‐linolenic acid (ALA) than terrestrial insects, whereas terrestrial insects contained on average 2.6 times more linoleic acid (LIN) than emergent aquatic insects. Spiders sampled directly on the lake and in upland habitats had similar EPA contents, but this EPA was derived from different diet sources, depending on the habitat. The δ13CEPA and δ2HEPA values of 'lake spiders' revealed an aquatic diet pathway (i.e. EPA of aquatic origin). In contrast, EPA of spiders collected in terrestrial habitats was depleted in both 13C and 2H compared to any potential food sources, and their ALA isotopic values, suggesting that EPA was partly bioconverted from its dietary precursor ALA (i.e. internal pathway). The δ2H values of fatty acids clearly indicated that diet sources differed depending on the spider's habitat, which was less evident from the δ13C values of the fatty acids. Our data highlight that spiders can use two distinct pathways (trophic versus metabolic) to satisfy their physiological EPA demand, depending on habitat use and dietary availability. [ABSTRACT FROM AUTHOR]

Organisms at the base of aquatic food webs synthesize essential nutrients, such as omega‐3 polyunsaturated fatty acids (n‐3 PUFA), which are transferred to consumers at higher trophic levels. Many consumers, requiring n‐3 long‐chain (LC) PUFA, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have limited ability to biosynthesize them from the essential dietary precursor α‐linolenic acid (ALA) and thus rely on dietary provision of LC‐PUFA.We investigated LC‐PUFA metabolism in freshwater zooplankton using stable hydrogen isotopes (δ2H) of fatty acids as tracers. We conducted feeding experiments with the freshwater keystone grazer Daphnia to quantify changes in the δ2H value of body FA in response to the FA composition of their food and the δ2H value of the ambient water.The isotopic composition of LC‐PUFA changed in Daphnia, depending on the integration of 2H from ambient water during de novo synthesis or bioconversion from dietary precursors, allowing us to distinguish dietary from bioconverted EPA in body tissue. We tested the applicability of these laboratory findings in a field setting by analysing δ2H values of PUFA in primary producers and consumers in eutrophic ponds to track EPA sources of zooplankton.Multilinear regression models that included conversion of ALA to EPA correlated better with zooplankton δ2HEPA than seston δ2HEPA at low dietary EPA supply.This study provides evidence that zooplankton can compensate for low dietary EPA supply by activating LC‐PUFA biosynthesis and shows that herbivorous zooplankton play a crucial role in upgrading FA for higher trophic levels during low dietary EPA supply. A free Plain Language Summary can be found within the Supporting Information of this article. [ABSTRACT FROM AUTHOR]

Rationale: Compound‐specific stable isotope analysis (CSIA) is a powerful tool for a better understanding of trophic transfer of dietary molecules in and across ecosystems. Hydrogen isotope values (δ2H) in consumer tissues have potential to more clearly distinguish dietary sources than 13C or 15N values within and among habitats, but have not been used at the fatty acid level for ecological purposes. Methods: Here we demonstrate a new online high‐capacity gas chromatography–isotope ratio mass spectrometry technique (2H‐CSIA) that offers accurate and reproducible determination of δ2H values for a range of fatty acids from organisms of aquatic food webs. Results: We show that lipid extracts obtained from aquatic sources, such as biofilms, leaves, invertebrates, or fish muscle tissue, have distinctive δ2H values that can be used to assess sources and trophic interactions, as well as dietary allocation and origin of fatty acids within consumer tissue. Conclusions: The new 2H‐CSIA method can be applied to evaluate sources and trophic dynamics of fatty acids in organisms ranging from food web ecology to migratory connectivity. [ABSTRACT FROM AUTHOR]

Compound-specific stable-isotope analysis (CSIA) of fatty acids is a powerful tool to better understand the trophic transfer of fatty acids and their biochemical fate in and across ecosystems, including tracing animal migration and understanding physiological processes. The non-exchangeable nature of C-H bonds in acyl chains, hydrogen ( δ 2 H) and carbon ( δ 13 C) stable-isotope values of fatty acids (FA) provide independent information about the origins of fatty acids. Several technical obstacles must be overcome to ensure accurate and reproducible measurements of FA-CSIA can be made. This protocol describes the sample preparation process for successful stable-isotope analyses of fatty acids obtained from environmental and biological samples. Numerous techniques for the preanalytical processing of fatty acid samples are available, and these often have minimal impact on δ values. Here, we provide an in-depth guide detailing our well-established laboratory protocols, ranging from the initial sample preparation, lipid extraction, and transmethylation to the instrumental arrangement, data collection, and analysis.•Protocol from obtaining a sample to standardized fatty acid specific δ 2 H and δ 13 C values.•Separate GC analysis procedures for C and H are recommended for optimal performance., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)

Non-exchangeable hydrogen-isotope ( δ 2 H n ) measurements of complex organic samples are used in forensics to determine sample authenticity, traceability, and provenance. However, δ 2 H n assays of organics are usually complicated by uncontrolled "exchangeable hydrogen" and residual moisture contamination; hence, δ 2 H n assays are persistently incomparable amongst laboratories. We introduce a revised technical solution (UniPrep2) to control hydrogen-isotope exchange and for robust online sample drying and vapour equilibration. The UniPrep2 device is coupled to a high-temperature thermochemical elemental analyser and continuous-flow isotope-ratio mass spectrometer. This technical solution empowers isotope analysts to:•Conduct reproducible controlled vapour equilibrations of complex organic samples and standards to determine the δ 2 H n values by controlling hydrogen-isotope exchange.•Conduct online vacuum-oven evacuation with extensive helium drying without exposure to air to reabsorb or exchange hydrogen with ambient water vapour. The protocol describes the operation of the UniPrep2 device and the step-by-step procedures needed to obtain accurate and precise δ 2 H n values for a wide range of organic sample types. Two analytical approaches are described in detail; the Dual-Vapour Equilibration (DVE) approach, intended for determining δ 2 H n for a complex organic environmental sample where matrix equivalent H isotope reference materials are not available, and the Comparative Equilibration (CE) approach, which is intended for routine high-throughput analyses of complex organic samples where at least two matrix-equivalent organic isotope reference materials with consensus δ 2 H n values are being used. These standard operating procedures are envisioned to be a sound basis for advancing hydrogen-isotope analysis for different organic environmental matrices and studies., Competing Interests: The authors declare that they have no known competing monetary interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)