17 pages, 4 figures, 1 table, Many protist plankton are mixotrophs, combining phototrophy and phagotrophy. Their role in freshwater and marine ecology has emerged as a major developing feature of plankton research over recent decades. To better aid discussions, we suggest these organisms are termed “mixoplankton”, as “planktonic protist organisms that express, or have potential to express, phototrophy and phagotrophy”. The term “phytoplankton” then describes phototrophic organisms incapable of phagotrophy. “Protozooplankton” describes phagotrophic protists that do not engage in acquired phototrophy. The complexity of the changes to the conceptual base of the plankton trophic web caused by inclusion of mixoplanktonic activities are such that we suggest that the restructured description is termed the “mixoplankton paradigm”. Implications and opportunities for revision of survey and fieldwork, of laboratory experiments and of simulation modelling are considered. The main challenges are not only with taxonomic and functional identifications, and with measuring rates of potentially competing processes within single cells, but with decades of inertia built around the traditional paradigm that assumes a separation of trophic processes between different organisms. In keeping with the synergistic nature of cooperative photo- and phagotrophy in mixoplankton, a comprehensive multidisciplinary approach will be required to tackle the task ahead, This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 766327, Peer reviewed

11 pages, 5 figures, 2 tables, Phagotrophic mixotrophs (mixoplankton) are now widely recognised as important members of food webs, but their role in the functioning of food webs is not yet fully understood. This is due to the lack of a well-established technique to estimate mixotrophic grazing. An immediate step in this direction would be the development of a method that separates mixotrophic from heterotrophic grazing that can be routinely incorporated into the common techniques used to measure microplankton herbivory (e.g., the dilution technique). This idea was explored by the addition of rotenone, an inhibitor of the respiratory electron chain that has been widely used to selectively eliminate metazoans, both in the field and in the laboratory. Accordingly, rotenone was added to auto-, mixo-, and heterotrophic protist cultures in increasing concentrations (ca. 24 h). The results showed that mixotrophs survived better than heterotrophs at low concentrations of rotenone. Nevertheless, their predation was more affected, rendering rotenone unusable as a heterotrophic grazing deterrent. Additionally, it was found that rotenone had a differential effect depending on the growth phase of an autotrophic culture. Altogether, these results suggest that previous uses of rotenone in the field may have disrupted the planktonic food web, This project is a contribution of the Marine Zooplankton Ecology Group from the Generalitat de Catalunya (2017 SGR 87) and has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 766327, With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

14 page, 5 figures, 3 tables, supplementary information https://doi.org/10.1038/s41598-020-69174-w.-- Dataset https://doi.org/10.20350/digitalCSIC/14479.-- It is a contribution of the Marine Zooplankton Ecology Group (2017 SGR 87), Copepod reproductive success largely depends on food quality, which also reflects the prey trophic mode. As such, modelling simulations postulate a trophic enhancement to higher trophic levels when mixotrophy is accounted in planktonic trophodynamics. Here, we tested whether photo-phagotrophic protists (mixoplankton) could enhance copepod gross-growth efficiency by nutrient upgrading mechanisms compared to obligate autotrophs and heterotrophs. To validate the hypothesis, we compared physiological rates of the copepod Paracartia grani under the three functional nutrition types. Ingestion and egg production rates varied depending on prey size and species, regardless of the diet. The gross-growth efficiency was variable and not significantly different across nutritional treatments, ranging from 3 to 25% in the mixoplanktonic diet compared to autotrophic (11–36%) and heterotrophic (8–38%) nutrition. Egg hatching and egestion rates were generally unaffected by diet. Overall, P. grani physiological rates did not differ under the tested nutrition types due to the large species-specific variation within trophic mode. However, when we focused on a single species, Karlodinium veneficum, tested as prey under contrasting trophic modes, the actively feeding dinoflagellate boosted the egestion rate and decreased the copepod gross-growth efficiency compared to the autotrophic ones, suggesting possible involvement of toxins in modulating trophodynamics other than stoichiometric constraints, This research was supported by EC MSCA-ITN 2019 funding to the project MixITiN (Grant Number 766327), With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI), Peer reviewed

15 pages, 5 figures, 3 tables, supporting information https://doi.org/10.1002/lno.11948.-- Dataset https://doi.org/10.20350/digitalCSIC/14480.-- It is a contribution of the Marine Zooplankton Ecology Group (2017 SGR 87), Inorganic nutrient limitation affects the stoichiometry and nutritional quality of marine phytoplankton. Mixoplankton, able to photosynthesize and feed simultaneously in the one cell, can compensate shortage of nutrients by phagotrophy, theoretically upgrading their nutritional quality for their predators: the zooplankton. Yet, the additional value that phagotrophy in mixoplankton may provide to support zooplankton growth and recruitment has been poorly explored. Therefore, we investigated the feeding and reproductive performances of the copepods Paracartia grani and Centropages typicus on mono-diets of the dinoflagellate Karlodinium veneficum grown as strict autotroph and as mixotroph, both under N and P depletion, and in nutrient-balanced conditions (f/2 medium; only as autotroph). Feeding and reproduction outputs were generally higher in P. grani than in C. typicus. Both copepod species ingested the mixotrophic K. veneficum at similar rates than the autotrophic ones in either nutrient-limited scenario. However, egg production and recruitment rates generally increased when feeding on mixotrophs, in P. grani on both N- and P-limited diets, and in C. typicus under P limitation. In general, P limitation influenced copepod physiology more than N depletion. Our results show that phagotrophy upgrades nutritional quality in nutrient-limited mixotrophs as prey for copepods compared to the strict autotrophic ones. These findings are among the first reported cases of copepod ingestion in the laboratory on actively feeding mixoplankton, and they highlight the importance of considering the trophic mode of the protist and the response by various zooplanktonic predators when attempting to understand the functioning of marine planktonic food webs, This research was supported by EC MSCA-ITN 2019 funding to the project MixITiN (Grant Number 766327). [...] With the institutional support of the “Severo Ochoa Centre of Excellence” accreditation (CEX2019-000928-S), Peer reviewed

16 pages, 7 figures, 3 tables, supplementary information https://doi.org/10.1038/s41598-021-03176-0.-- Dataser Digital.CSIC https://doi.org/10.20350/digitalCSIC/14484.-- This project is a contribution of the Marine Zooplankton Ecology Group from the Generalitat de Catalunya (2017 SGR 87), It remains unclear as to how mixoplankton (coupled phototrophy and phagotrophy in one cell) affects the estimation of grazing rates obtained from the widely used dilution grazing technique. To address this issue, we prepared laboratory-controlled dilution experiments with known mixtures of phyto-, protozoo-, and mixoplankton, operated under different light regimes and species combinations. Our results evidenced that chlorophyll is an inadequate proxy for phytoplankton when mixoplankton are present. Conversely, species-specific cellular counts could assist (although not fully solve) in the integration of mixoplanktonic activity in a dilution experiment. Moreover, cell counts can expose prey selectivity patterns and intraguild interactions among grazers. Our results also demonstrated that whole community approaches mimic reality better than single-species laboratory experiments. We also confirmed that light is required for protozoo- and mixoplankton to correctly express their feeding activity, and that overall diurnal grazing is higher than nocturnal. Thus, we recommend that a detailed examination of initial and final plankton communities should become routine in dilution experiments, and that incubations should preferably be started at the beginning of both day and night periods. Finally, we hypothesize that in silico approaches may help disentangle the contribution of mixoplankton to the community grazing of a given system, his project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Grant agreement No. 766327. [...] With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), Peer reviewed

12 pages, 6 figures, 2 tables, supplementary data https://doi.org/10.1016/j.marenvres.2022.105558.-- Dataset: Duarte Ferreira, Guilherme; Figueira, Joana; Marques, Sónia Cotrim; Hansen, Per Juel; Calbet, Albert; 2022; The strengths and weaknesses of Live Fluorescently Labelled Algae (LFLA) to estimate herbivory in protozooplankton and mixoplankton [Dataset]; DIGITAL.CSIC; https://doi.org/10.20350/digitalCSIC/14483; http://hdl.handle.net/10261/257594, The Live Fluorescently Labelled Algae (LFLA) technique has been used numerous times to estimate microzooplankton herbivory. Yet, it is unknown how mixoplankton (i.e., single-cell organisms that can combine phototrophy and phagotrophy) affect the outcome of this technique. Hence, we conducted a broad-spectrum assessment of the strengths and weaknesses of the LFLA technique, using several mixoplanktonic and protozooplanktonic grazers. Species from different taxonomic groups and different feeding mechanisms were tested in short-term experiments (ca. 5 h) in the laboratory, at different prey concentrations and during light and dark periods of the day. Overall, our findings suggest that the LFLA technique, due to its short-term nature, is an effective tracker of diel ingestion and digestion rates, and can detect new mixoplanktonic predators. We recommend that, irrespective of the prey concentration, incubations to measure grazing rates with this technique should generally be concluded within 1 h (adaptable to the environmental temperature). Nevertheless, our results also call for caution whenever using LFLA in the field: feeding mechanisms other than direct engulfment (like peduncle feeding) may provide severely biased ingestion rates. Furthermore, size and species selectivity are very hard to circumvent. To reduce the effects of selectivity, we propose the combined use of two distinctly coloured fluorochromes (i.e., distinct emission spectra). With this modification, one could either label different size ranges of prey or account for species-specific interactions in the food web, This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 766327. [...] This project is a contribution of the Marine Zooplankton Ecology Group from the Generalitat de Catalunya (2017 SGR 87) with the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S). Thanks for financial support are also due to MARE (UIDB/04292/2020 + UIDP/04292/2020), to FCT/MEC through national funds, and the co-funding by the FEDER, within the PT2020 Partnership Agreement and Compete 2020, Peer reviewed

10 pages, 6 figures, 3 tables, supplementary data https://doi.org/10.1016/j.marenvres.2022.105693, Sudden environmental changes like marine heatwaves will become more intense and frequent in the future. Understanding the physiological responses of mixoplankton and protozooplankton, key members of marine food webs, to temperature is crucial. Here, we studied two dinoflagellates (one protozoo- and one mixoplanktonic), two ciliates (one protozoo- and one mixoplanktonic), and two cryptophytes. We report the acute (24 h) responses on growth and grazing to a range of temperatures (5–34 °C). We also determined respiration and photosynthetic rates for the four grazers within 6 °C of warming. The thermal performance curves showed that, in general, ciliates have higher optimal temperatures than dinoflagellates and that protozooplankton is better adapted to warming than mixoplankton. Our results confirmed that warmer temperatures decrease the cellular volumes of all species. Q10 coefficients suggest that grazing is the rate that increases the most in response to temperature in protozooplankton. Yet, in mixoplankton, grazing decreased in warmer temperatures, whereas photosynthesis increased. Therefore, we suggest that the Metabolic Theory of Ecology should reassess mixoplankton's position for the correct parameterisation of future climate change models. Future studies should also address the multigenerational response to temperature changes, to confirm whether mixoplankton become more phototrophic than phagotrophic in a warming scenario after adaptation, This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 766327. [...] Thanks for financial support are also due to the ERASMUS + traineeship program and to the Grants CTM2017-84288-R and PID2020-118645RB-I00 funded by MCIN/AEI Spain/10.13039/501100011033 and by “FEDER Una manera de hacer Europa”. This work is a contribution of the Marine Zooplankton Ecology Group (2017 SGR 87) with the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX 2019-000928-S), Peer reviewed

18 pages, 6 figures, 3 tables.-- Data Availability: Data sharing is not applicable to this article as no new data were created or analyzed in this study, Marine plankton capable of photosynthesis and predation (“mixoplankton”) comprise up to 50% of protist plankton and include many harmful species. However, marine environmental management policies, including the European Union Marine Strategy Framework Directive (MSFD) and the USEPA, assume a strict dichotomy between autotrophic phytoplankton and heterotrophic zooplankton. Mixoplankton often differ significantly from these two categories in their response to environmental pressures and affect the marine environment in ways we are only beginning to understand. While the management policies may conceptually provide scope for incorporating mixoplankton, such action is rarely implemented. We suggest that the effectiveness of monitoring and management programs could benefit from explicit implementations regarding the ecological roles and impact of mixoplankton. Taking the MSFD as an example of marine management guidelines, we propose appropriate methods to explicitly include mixoplankton in monitoring and marine management. Integr Environ Assess Manag 2024;20:1366–1383. © 2024 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC), The authors thank colleagues in the MixITiN project (https://www.mixotroph.org/mixitin/) for discussions that have improved this work. This project received funding from the European Union's Horizon 2020 Research and Innovation Program Project “MixITiN” under Marie Skłodowska-Curie grant agreement No. 766327 and the Leibniz Institute for Baltic Sea Research Warnemünde (IOW). Open Access funding enabled and organized by Projekt DEAL, With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), Peer reviewed

10 pages, 2 figures, supplemental information https://doi.org/10.1016/j.tim.2025.08.008, Protists comprise the vast majority of eukaryotic genetic and functional diversity. While they have traditionally been difficult to study due to their small size and varied phenotypes, environmental sequencing studies have revealed the stunning diversity and abundance of protists in all ecosystems. Protists are key primary and secondary producers across many biomes, with ecological specializations that range from mutualism to parasitism, complex predation behaviors, mixotrophy, detritivory, and saprotrophy. Current genomic and transcriptomic approaches provide valuable insights into protist diversity at the genetic level, but they fall short in capturing the morphological and behavioral traits critical for understanding the functional roles of protists in ecosystems. This knowledge gap hinders our ability to answer important questions about protist functional diversity, including how protist functional groups will respond to environmental change. In this opinion article, we advocate adopting a traits-based approach for studying protist diversity and developing a trait database for protists to support this goal. By integrating examples of recent work characterizing protist functional diversity using a range of approaches, we emphasize the opportunities that trait databases offer and propose strategies for moving towards a trait-based framework to guide future research in protist ecology and evolution, M.J. was supported by the Swedish Research Council (International Postdoc grant 2022-00351). P.R. was supported by a Marie Skłodowska-Curie Actions – Individual Fellowship (MSCA-IF-2020, project number: 101033648, acronym: FROM) from the European Commission. A.M. was part-funded by the European Commission funded H2020-MSCA-ITN Project MixITiN 766327. L.S. was supported by the US National Science Foundation (award DBI 2334522), This work contributes to the Institut de Ciències del Mar "Severo Ochoa Centre of Excellence" accreditation CEX2024-001494-S funded by AEI 10.13039/501100011033 of the Spanish Ministry of Science and Innovation, Peer reviewed