The Mixoplankton Database (MDB): Diversity of photo-phago-trophic plankton in form, function, and distribution across the global ocean.

Protist plankton are major members of open-water marine food webs. Traditionally divided between phototrophic phytoplankton and phagotrophic zooplankton, recent research shows many actually combine phototrophy and phagotrophy in the one cell; these protists are the "mixoplankton." Under the mixoplankton paradigm, "phytoplankton" are incapable of phagotrophy (diatoms being exemplars), while "zooplankton" are incapable of phototrophy. This revision restructures marine food webs, from regional to global levels. Here, we present the first comprehensive database of marine mixoplankton, bringing together extant knowledge of the identity, allometry, physiology, and trophic interactivity of these organisms. This mixoplankton database (MDB) will aid researchers that confront difficulties in characterizing life traits of protist plankton, and it will benefit modelers needing to better appreciate ecology of these organisms with their complex functional and allometric predator-prey interactions. The MDB also identifies knowledge gaps, including the need to better understand, for different mixoplankton functional types, sources of nutrition (use of nitrate, prey types, and nutritional states), and to obtain vital rates (e.g. growth, photosynthesis, ingestion, factors affecting photo' vs. phago' -trophy). It is now possible to revisit and re-classify protistan "phytoplankton" and "zooplankton" in extant databases of plankton life forms so as to clarify their roles in marine ecosystems.

A dataset on trophic modes of aquatic protists.

BACKGROUND: An important functional trait of organisms is their trophic mode. It determines their position within food webs, as well as their function within an ecosystem. For the better part of the 20th century, aquatic protist communities were thought to consist mainly of producers (phytoplankton) and consumers (protozooplankton). Phytoplankton cover their energy requirements through photosynthesis (phototrophy), while protozooplankton graze on prey and organic particles (phagotrophy). However, over the past decades, it was shown that another trophic group (mixoplankton) comprise a notable part of aquatic protist communities. Mixoplankton employ a third trophic mode by combining phototrophy and phagotrophy (mixotrophy). Due to the historical dichotomy, it is not straightforward to gain adequate and correct information on the trophic mode of aquatic protists. Long hours of literature research or expert knowledge are needed to correctly assign trophic modes. Additionally, aquatic protists also have a long history of undergoing taxonomic changes which make it difficult to compare past and present literature. While WoRMS, the World Register of Marine Species, keeps track of the taxonomic changes and assigns each species a unique AphiaID that can be linked to its various historic and present taxonomic hierarchy, there is currently no machine-readable database to query aquatic protists for their trophic modes. NEW INFORMATION: This paper describes a dataset that was submitted to WoRMS and links aquatic protist taxa, with a focus on marine taxa, to their AphiaID and their trophic mode. The bulk of the data used for this dataset stems from (routine) monitoring stations in the North Sea and the Baltic Sea. The data were augmented and checked against state-of-the-art knowledge on mixoplankton taxa by consulting literature and experts. Thus, this dataset provides a first attempt to make the trophic mode of aquatic protists easily accessible in both a human- and machine-readable format.

Mixotrophy in Chlorophytes and Haptophytes-Effect of Irradiance, Macronutrient, Micronutrient and Vitamin Limitation.

Chlorophytes and haptophytes are key contributors to global phytoplankton biomass and productivity. Mixotrophic bacterivory has been detected for both groups, but a shortage of studies with cultured representatives hinders a consistent picture of the ecological relevance and regulation of this trophic strategy. Here, the growth, primary production, fraction of feeding cells (acidotropic probes) and bacterivory rates (surrogate prey) are tested for two species of the chlorophyte genus Nephroselmis and the haptophyte Isochrysis galbana under contrasting regimes of light (high vs. low) and nutrients (non-limited and macronutrient-, micronutrient- and vitamin-limited), at low bacterial concentrations (<107 bacteria mL-1). All three species were obligate phototrophs, unable to compensate for low light conditions through feeding. Under nutrient limitation, N. rotunda and I. galbana fed, but growth ceased or was significantly lower than in the control. Thus, mixotrophic bacterivory could be a survival rather than a growth strategy for certain species. In contrast, nutrient-limited N. pyriformis achieved growth rates equivalent to the control through feeding. This strikingly differs with the classical view of chlorophytes as primarily non-feeders and indicates mixotrophic bacterivory can be a significant trophic strategy for green algae, even at the low bacterial concentrations found in oligotrophic open oceans.

Mixotrophic Phytoflagellate Bacterivory Field Measurements Strongly Biased by Standard Approaches: A Case Study.

Bacterivory among small (≤20 µm) phytoflagellates (SP) is increasingly recognized as a globally relevant phenomenon, impacting a wide range of aspects from primary production levels to marine fisheries. However, to correctly parametrize mixotrophic SP in biogeochemical and food web models, a better understanding of the magnitude and regulation of in situ SP feeding is urgently needed. Current methods to determine SP bacterivory in the field may introduce biases by treating these organisms as equivalent to heterotrophic nanoflagellates (HNF). In the present case study we experimentally tested two generally employed assumptions of such studies: (A) bacterivory rates of the whole SP community and of distinct SP groups remain constant over 'short' time scales (hours to a day) and (B) SP community ingestion rates approximate the average ingestion rate of all feeding individuals. Food vacuole markers (acidotropic probes), were applied along the diel cycle at three stations in December 2015, and May and June 2016. In December and June, surrogate prey (fluorescently labeled bacteria) were used in parallel at one sampling station. Sampling at different times of day produced an up to fourfold difference in estimates of SP daily bacterivorous impact. In contrast, daily bacterivory estimates for HNF remained constant in almost all cases. The perceived principal SP bacterivorous groups also shifted strongly. As an example, picoeukaryotes dominated total SP bacterivory in daylight hours but completely ceased to feed at night. Finally, a large fraction of the SP community was not feeding at all time points tested. This lead to significant errors in estimated ingestion rates determined using the whole SP community, being up to 16 times lower than those determined solely for actively feeding mixotrophic SP. Overall, this case study indicates that applying the two commonly used premises outlined above can introduce significant biases and considerably alter our perception of mixotrophy in a given system.

Dynamics of Sequestered Cryptophyte Nuclei in Mesodinium rubrum during Starvation and Refeeding.

The marine mixotrophic ciliate Mesodinium rubrum is known to acquire chloroplasts, mitochondria, nucleomorphs, and nucleus from its cryptophyte prey, particularly from species in the genera, Geminigera and Teleaulax. The sequestered prey nucleus and chloroplasts are considered to support photosynthesis of M. rubrum. In addition, recent studies have shown enlargement of the retained prey nucleus in starved M. rubrum and have inferred that enlargement results from the fusion of ingested prey nuclei. Thus far, however, little is known about the mechanism underlying the enlargement of the prey nucleus in M. rubrum. Here, we conducted starvation and refeeding studies to monitor the fate of prey nuclei acquired by M. rubrum when feeding on Teleaulax amphioxeia and to explore the influence of the retained prey nucleus on photosynthesis of M. rubrum. Results indicate that enlargement of the prey nucleus does not result from fusion of nuclei. Furthermore, the enlarged prey nucleus does not appear to divide during cell division of M. rubrum. The presence of a prey nucleus significantly affected photosynthetic performance of M. rubrum, while the number of retained chloroplasts had little influence on rate of carbon fixation. We interpret results within the context of a model that considers the dynamics of ingested prey nuclei during division of M. rubrum.

Photoregulation in a Kleptochloroplastidic Dinoflagellate, Dinophysis acuta.

Some phagotrophic organisms can retain chloroplasts of their photosynthetic prey as so-called kleptochloroplasts and maintain their function for shorter or longer periods of time. Here we show for the first time that the dinoflagellate Dinophysis acuta takes control over "third-hand" chloroplasts obtained from its ciliate prey Mesodinium spp. that originally ingested the cryptophyte chloroplasts. With its kleptochloroplasts, D. acuta can synthesize photosynthetic as well as photoprotective pigments under long-term starvation in the light. Variable chlorophyll fluorescence measurements showed that the kleptochloroplasts were fully functional during 1 month of prey starvation, while the chlorophyll a-specific inorganic carbon uptake decreased within days of prey starvation under an irradiance of 100 µmol photons m(-2) s(-1). While D. acuta cells can regulate their pigmentation and function of kleptochloroplasts they apparently lose the ability to maintain high inorganic carbon fixation rates.

Defining Planktonic Protist Functional Groups on Mechanisms for Energy and Nutrient Acquisition: Incorporation of Diverse Mixotrophic Strategies.

Arranging organisms into functional groups aids ecological research by grouping organisms (irrespective of phylogenetic origin) that interact with environmental factors in similar ways. Planktonic protists traditionally have been split between photoautotrophic "phytoplankton" and phagotrophic "microzooplankton". However, there is a growing recognition of the importance of mixotrophy in euphotic aquatic systems, where many protists often combine photoautotrophic and phagotrophic modes of nutrition. Such organisms do not align with the traditional dichotomy of phytoplankton and microzooplankton. To reflect this understanding, we propose a new functional grouping of planktonic protists in an eco-physiological context: (i) phagoheterotrophs lacking phototrophic capacity, (ii) photoautotrophs lacking phagotrophic capacity, (iii) constitutive mixotrophs (CMs) as phagotrophs with an inherent capacity for phototrophy, and (iv) non-constitutive mixotrophs (NCMs) that acquire their phototrophic capacity by ingesting specific (SNCM) or general non-specific (GNCM) prey. For the first time, we incorporate these functional groups within a foodweb structure and show, using model outputs, that there is scope for significant changes in trophic dynamics depending on the protist functional type description. Accordingly, to better reflect the role of mixotrophy, we recommend that as important tools for explanatory and predictive research, aquatic food-web and biogeochemical models need to redefine the protist groups within their frameworks.

Metabolic and physiological changes in Prymnesium parvum when grown under, and grazing on prey of, variable nitrogen:phosphorus stoichiometry.

Mixotrophy is found in almost all classes of phytoplankton in a wide range of aquatic habitats ranging from oligotrophic to eutrophic marine and freshwater systems. Few studies have addressed how the nutritional status of the predator and/or the prey affects mixotrophic metabolism despite the realization that mixotrophy is important ecologically. Laboratory experiments were conducted to examine changes in growth rates and physiological states of the toxic haptophyte Prymnesium parvum when fed Rhodomonas salina of varying nutritional status. Haemolytic activity of P. parvum and prey mortality of R. salina were also measured. P. parvum cultures grown to be comparatively low in nitrogen (low-N), phosphorus (low-P) or low in both nutrients (low-NP) were mixed with low-NP, low-N, and low-P R. salina in all possible combinations, i.e., a 3×3 factorial design. N deficiency was obtained in the low-N cultures, while true P deficiency may not have been obtained in the low-P cultures. Mortality rates of R. salina (both due to ingestion and/or cell rupture as a function of grazing or toxic effects) were higher when R. salina cells were low-P, N-rich, regardless of the nutritional state of P. parvum. Mortality rates were, however, directly related to the initial prey:predator cell ratios. On the other hand, growth of the predator was a function of nutritional status and a significant positive correlation was observed between growth rates of P. parvum and cell-specific depletion rates of N, whereas no such relationship was found between P. parvum growth rates and depletion rates of P. In addition, the greatest changes in chlorophyll content and stoichiometric ratios of P. parvum were observed in high N:P conditions. Therefore, P. parvum may show enhanced success under conditions of higher inorganic N:P, which are likely favored in the future due to increases in eutrophication and altered nutrient stoichiometry driven by anthropogenic nutrient loads that are increasingly enriched in N relative to P.

Ocean acidification with (de)eutrophication will alter future phytoplankton growth and succession.

Human activity causes ocean acidification (OA) though the dissolution of anthropogenically generated CO2 into seawater, and eutrophication through the addition of inorganic nutrients. Eutrophication increases the phytoplankton biomass that can be supported during a bloom, and the resultant uptake of dissolved inorganic carbon during photosynthesis increases water-column pH (bloom-induced basification). This increased pH can adversely affect plankton growth. With OA, basification commences at a lower pH. Using experimental analyses of the growth of three contrasting phytoplankton under different pH scenarios, coupled with mathematical models describing growth and death as functions of pH and nutrient status, we show how different conditions of pH modify the scope for competitive interactions between phytoplankton species. We then use the models previously configured against experimental data to explore how the commencement of bloom-induced basification at lower pH with OA, and operating against a background of changing patterns in nutrient loads, may modify phytoplankton growth and competition. We conclude that OA and changed nutrient supply into shelf seas with eutrophication or de-eutrophication (the latter owing to pollution control) has clear scope to alter phytoplankton succession, thus affecting future trophic dynamics and impacting both biogeochemical cycling and fisheries.

Prymnesium parvum revisited: relationship between allelopathy, ichthyotoxicity, and chemical profiles in 5 strains.

Bioassay-guided discovery of ichthyotoxic algal compounds using in vivo fish assays is labor intensive, costly, and highly regulated. Since the mode of action of most known algal-mediated fish-killing toxins is damage to the cell membranes in the gills, various types of cell-based bioassays are often used for bioassay guided purification of new ichthyotoxins. Here we tested the hypothesis that allelopathy is related to ichthyotoxicity and thus that a microalgal bioassay can be used as a proxy for ichthyotoxicity by comparing the toxicity of five strains of Prymnesium parvum toward rainbow trout (Oncorhynchus mykiss, 10 g) and the microalga Teleaulax acuta. No relationship between median effective concentrations (EC50s) on fish and median lethal concentrations (LC50s) on algae was observed in the 5 strains showing that a microalgal bioassay cannot be used as a proxy for ichthyotoxicity. Fish were more sensitive to P. parvum with EC50s ranging from 6×10(3) to 40×10(3) cells ml(-1), compared to the test alga where LC50s ranged from 30×10(3) to nearly non-toxic at 500×10(3) cells ml(-1). In addition, the cellular concentrations of two recently suggested ichthyotoxins produced by P. parvum, the "golden algae toxins", GAT 512 and a novel GAT 510, did not show any relationship to either ichthyotoxicity or allelopathy, and are not the biologically relevant toxins, but are simply lipids found in algal chloroplasts. Finally, we demonstrate that the recently suggested ichthyotoxin, oleamide, could not be detected in any of the five P. parvum strains above the limit of detection, nor was it found in a (13)C-labeled strain. Instead we document that oleamide can easily be extracted from plastic materials, which may have been the source of oleamide reported previously.

Studies on the genus Mesodinium II. Ultrastructural and molecular investigations of five marine species help clarifying the taxonomy.

We provide a detailed study of four marine Mesodinium species and compare the data to the companion article on Mesodinium chamaeleon and other available studies on Mesodinium, to shed some light on the taxonomy of the genus. Micrographs of two red phototrophic Mesodinium species, Mesodinium rubrum and Mesodinium major n. sp., as well as the first published micrographs of two heterotrophic species, M. pulex and M. pupula are presented in combination with molecular analyses based on the ribosomal genes. The main conclusion of this study is the invalidity of the genus Myrionecta based on the arrangements of the basal bodies forming the cirri and the separation of species formerly known as M. rubrum resulting in an emended description of M. rubrum and the description of a related new species M. major n. sp.