Exploring Metabolic Signals of Mixotrophy in Marine Protists Using Compound-Specific Hydrogen Isotopes

Despite its central role in marine food webs, nutrient cycling, and carbon export, mixotrophy remains difficult to quantify, largely because robust tools for resolving the relative contributions of autotrophic and heterotrophic metabolism in plankton are still lacking. Mixotrophic strategies blur traditional functional classifications and could hypothetically confer ecological advantages under variable environmental regimes. Yet current approaches often rely on bulk physiological rates or grazing experiments that provide only partial or indirect insights into trophic behaviour. As highlighted by Millette et al.(2024), these methodological limitations hinder the integration of mixotrophy into ecosystem and biogeochemical models, underscoring the need for novel, process-based tracers capable of resolving trophic behaviour at the level of cellular metabolism.Hydrogen isotope ratios (δ²H) in lipids and carbohydrates from aquatic and terrestrial organisms, as well as from sedimentary archives, are widely employed to reconstruct past hydroclimatic conditions. Emerging evidence, however, indicates that δ²H values in these biomolecules also encode metabolic signals in addition to climatic ones (Holloway-Phillips et al., 2025). Such influences complicate straightforward climatic reconstructions and highlight the need to better identify the processes that determine δ²H variability in organic matter. Yet, once these contributions are disentangled, the metabolic information embedded in δ²H values may itself become a valuable tracer for unresolved ecophysiological processes—among them, marine mixotrophyPrevious experimental work has revealed that lipid δ²H values in bacteria (Zhang et al., 2009) and green algae (Cormier et al., 2022) respond specifically to their trophic metabolism. Building on these findings, we present initial experiments with protists designed to test whether δ²H & δ13C values of different biomolecules (including fatty acids, phytols and sterols) similarly reflect shifts in central metabolic pathways. Two complementary experimental systems are compared: continuous cultures of Chlorella under osmo-heterotrophic conditions, and batch cultures of mixoplankton feeding on prey.These new compound-specific isotope measurements were obtained using gas chromatography–isotope ratio mass spectrometry on the aforementioned compounds from these systems alongside RNA-sec, pigment and physiological data. Our data suggest that lipid δ²H values are indeed sensitive to the degree of heterotrophic growth in diverse protist lineages, pointing to their potential as indicators of metabolic flexibility.If these relationships can be confirmed and quantitatively calibrated, compound-specific hydrogen isotope analysis could provide a powerful new tool for investigating the prevalence and dynamics of mixotrophy.References:Zhang, X. et al. (2009). PNAS. doi:10.1073/pnas.0903030106Cormier, M.-A. et al. (2022). New Phytologist. doi:10.1111/nph.18023Millette, N. C. et al. (2024). Journal of Plankton Research. doi:10.1093/plankt/fbad020Holloway-Phillips, M. et al. (2026). New Phytologist. doi:10.1111/nph.70845