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 because of the lack of a well-established technique to estimate mixotrophy. 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, Marie Skłodowska-Curie grant agreement No 766327, With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)

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, Marie Skłodowska-Curie grant agreement No 766327. [...] With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)

The Live Fluorescently Labelled Algae (LFLA) technique has been used numerous times to estimate micro-zooplankton 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 proto-zooplanktonic 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, Marie Skłodowska-Curie grant agreement No 766327. [...] With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)

Feeding rates, fecal production rates, egg production rates and gross-growth efficiencies of the copepod Paracartia grani offered a variety of phytoplankton, protozooplankton and mixoplankton in monodiet. See Excel file for details, 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

Feeding and egg production rates of the copepods Paracartia grani and Centropages typicus feeding on the dinoflagellate Karlodinium veneficum under different nutrient and trophic conditions. See more details in the Excel file, 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