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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

Growing attention to phytoplankton mixotrophy as a trophic strategy has led to significant revisions of traditional pelagic food web models and ecosystem functioning. Although some empirical estimates of mixotrophy do exist, a much broader set of in situ measurements are required to (i) identify which organisms are acting as mixotrophs in real time and to (ii) assess the contribution of their heterotrophy to biogeochemical cycling. Estimates are needed through time and across space to evaluate which environmental conditions or habitats favour mixotrophy: conditions still largely unknown. We review methodologies currently available to plankton ecologists to undertake estimates of plankton mixotrophy, in particular nanophytoplankton phago-mixotrophy. Methods are based largely on fluorescent or isotopic tracers, but also take advantage of genomics to identify phylotypes and function. We also suggest novel methods on the cusp of use for phago-mixotrophy assessment, including single-cell measurements improving our capacity to estimate mixotrophic activity and rates in wild plankton communities down to the single-cell level. Future methods will benefit from advances in nanotechnology, micromanipulation and microscopy combined with stable isotope and genomic methodologies. Improved estimates of mixotrophy will enable more reliable models to predict changes in food web structure and biogeochemical flows in a rapidly changing world.

Growing attention to phytoplankton mixotrophy as a trophic strategy has led to significant revisions of traditional pelagic food web models and ecosystem functioning. Although some empirical estimates of mixotrophy do exist, a much broader set of in situ measurements are required to (i) identify which organisms are acting as mixotrophs in real time and to (ii) assess the contribution of their heterotrophy to biogeochemical cycling. Estimates are needed through time and across space to evaluate which environmental conditions or habitats favour mixotrophy: conditions still largely unknown. We review methodologies currently available to plankton ecologists to undertake estimates of plankton mixotrophy, in particular nanophytoplankton phago-mixotrophy. Methods are based largely on fluorescent or isotopic tracers, but also take advantage of genomics to identify phylotypes and function. We also suggest novel methods on the cusp of use for phago-mixotrophy assessment, including single-cell measurements improving our capacity to estimate mixotrophic activity and rates in wild plankton communities down to the single-cell level. Future methods will benefit from advances in nanotechnology, micromanipulation and microscopy combined with stable isotope and genomic methodologies. Improved estimates of mixotrophy will enable more reliable models to predict changes in food web structure and biogeochemical flows in a rapidly changing world.

Growing attention to phytoplankton mixotrophy as a trophic strategy has led to significant revisions of traditional pelagic food web models and ecosystem functioning. Although some empirical estimates of mixotrophy do exist, a much broader set of in situ measurements are required to (i) identify which organisms are acting as mixotrophs in real time and to (ii) assess the contribution of their heterotrophy to biogeochemical cycling. Estimates are needed through time and across space to evaluate which environmental conditions or habitats favour mixotrophy: conditions still largely unknown. We review methodologies currently available to plankton ecologists to undertake estimates of plankton mixotrophy, in particular nanophytoplankton phago-mixotrophy. Methods are based largely on fluorescent or isotopic tracers, but also take advantage of genomics to identify phylotypes and function. We also suggest novel methods on the cusp of use for phago-mixotrophy assessment, including single-cell measurements improving our capacity to estimate mixotrophic activity and rates in wild plankton communities down to the single-cell level. Future methods will benefit from advances in nanotechnology, micromanipulation and microscopy combined with stable isotope and genomic methodologies. Improved estimates of mixotrophy will enable more reliable models to predict changes in food web structure and biogeochemical flows in a rapidly changing world.

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Marine Science 5 (2018): 273, doi:10.3389/fmars.2018.00273., Mixotrophic flagellates can comprise significant proportions of plankton biomass in marine ecosystems. Despite the growing recognition of the importance of this ecological strategy, and the identification of major environmental factors controlling phagotrophic behavior (light and nutrients), the physiological and molecular mechanisms underlying mixotrophic behavior are still unclear. In this study, we performed RNA-Seq transcriptomic analysis for two mixotrophic prasinophytes, Micromonas polaris and Pyramimonas tychotreta, under dissolved nutrient regimes that altered their ingestion of bacteria prey. Though the strains examined were polar isolates, both belong to genera with widespread distribution. Our aim was to characterize the transcriptomes of these two non-model phytoflagellates, identify transcripts consistent with phagotrophic activity and assess their differential expression in response to nutrient stress. De novo assembly of the transcriptomes yielded large numbers of novel coding transcripts with no known match within public databases. A summary of the transcripts by Gene Ontology terms showed many expected expression patterns, including genes involved in photosynthetic pathways and enzymes implicated in nutrient uptake pathways. Searches of KEGG databases identified several genes associated with intra-cellular digestive pathways actively transcribed in both prasinophytes. Differential expression analysis showed a larger response in P. tychotreta, where 23,373 genes were up-regulated and 1,752 were down-regulated in the low nutrient treatment when phagotrophy was enhanced. In contrast, in M. polaris, low nutrient treatments resulted in up-regulation of 314 transcripts while down-regulating 371. With respect to phagotrophic-related expression, 37 genes were co-expressed in both P. tychotreta and M. polaris, and although the response was less pronounced in M. polaris, it is consistent with differences in observed ingestion behavior. This study presents the, The Owlsnest Super-Computing Cluster at Temple University is funded by a National Science Foundation Grant CNS-09-58854. The CUNY HPCC is operated by the College of Staten Island and funded, in part, by grants from the City of New York, State of New York, CUNY Research Foundation, and National Science Foundation Grants CNS-0958379, CNS-0855217, and ACI 1126113. Support for this work was also supplied by National Science Foundation grants PLR-1341362 (RG), PLR-1603538 (RS), and PLR-1603833 (RG).

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Marine Science 5 (2018): 273, doi:10.3389/fmars.2018.00273., Mixotrophic flagellates can comprise significant proportions of plankton biomass in marine ecosystems. Despite the growing recognition of the importance of this ecological strategy, and the identification of major environmental factors controlling phagotrophic behavior (light and nutrients), the physiological and molecular mechanisms underlying mixotrophic behavior are still unclear. In this study, we performed RNA-Seq transcriptomic analysis for two mixotrophic prasinophytes, Micromonas polaris and Pyramimonas tychotreta, under dissolved nutrient regimes that altered their ingestion of bacteria prey. Though the strains examined were polar isolates, both belong to genera with widespread distribution. Our aim was to characterize the transcriptomes of these two non-model phytoflagellates, identify transcripts consistent with phagotrophic activity and assess their differential expression in response to nutrient stress. De novo assembly of the transcriptomes yielded large numbers of novel coding transcripts with no known match within public databases. A summary of the transcripts by Gene Ontology terms showed many expected expression patterns, including genes involved in photosynthetic pathways and enzymes implicated in nutrient uptake pathways. Searches of KEGG databases identified several genes associated with intra-cellular digestive pathways actively transcribed in both prasinophytes. Differential expression analysis showed a larger response in P. tychotreta, where 23,373 genes were up-regulated and 1,752 were down-regulated in the low nutrient treatment when phagotrophy was enhanced. In contrast, in M. polaris, low nutrient treatments resulted in up-regulation of 314 transcripts while down-regulating 371. With respect to phagotrophic-related expression, 37 genes were co-expressed in both P. tychotreta and M. polaris, and although the response was less pronounced in M. polaris, it is consistent with differences in observed ingestion behavior. This study presents the, The Owlsnest Super-Computing Cluster at Temple University is funded by a National Science Foundation Grant CNS-09-58854. The CUNY HPCC is operated by the College of Staten Island and funded, in part, by grants from the City of New York, State of New York, CUNY Research Foundation, and National Science Foundation Grants CNS-0958379, CNS-0855217, and ACI 1126113. Support for this work was also supplied by National Science Foundation grants PLR-1341362 (RG), PLR-1603538 (RS), and PLR-1603833 (RG).

Most biogeochemical/ecological models divide planktonic protists between phototrophs (phytoplankton) and heterotrophs (zooplankton). However, a large number of planktonic protists are able to combine several mechanisms of carbon and nutrient acquisition. Not representing these multiple mechanisms in biogeochemical/ecological models describing eutrophied coastal ecosystems can potentially lead to different conclusions regarding ecosystem functioning, especially regarding the success of harmful algae, which are often reported as mixotrophic. This modelling study investigates the implications for trophic dynamics of including 3 contrasting forms of mixotrophy, namely osmotrophy (using alkaline phosphatase activity, APA), non-constitutive mixotrophy (acquired phototrophy by microzooplankton) and also constitutive mixotrophy. The application is in the Southern North Sea, an ecosystem that faced, between 1985 and 2005, a significant increase in the nutrient supply N:P ratio (from 31 to 81 mol N:P). The comparison with a traditional model shows that, when the winter N:P ratio in the Southern North Sea is above 22 molN molP−1 (as occurred from mid-1990s), APA allows a 3–32% increase of annual gross primary production (GPP). In result of the higher GPP, the annual sedimentation increases as well as the bacterial production. By contrast, APA does not affect the export of matter to higher trophic levels because the increased GPP is mainly due to Phaeocystis colonies, which are not grazed by copepods. Under high irradiance, non-constitutive mixotrophy appreciably increases annual GPP, transfer to higher trophic levels, sedimentation, and nutrient remineralisation. In this ecosystem, non-constitutive mixotrophy is also observed to have an indirect stimulating effect on diatoms. Constitutive mixotrophy in nanoflagellates appears to have little influence on this ecosystem functioning. An important conclusion from this work is that contrasting forms of mixotrophy have different imp, SCOPUS: ar.j, info:eu-repo/semantics/published

The origin of the photosynthetic organelle in eukaryotes, the plastid, changed forever the evolutionary trajectory of life on our planet. Plastids are highly specialized compartments derived from a putative single cyanobacterial primary endosymbiosis that occurred in the common ancestor of the supergroup Archaeplastida that comprises the Viridiplantae (green algae and plants), red algae, and glaucophyte algae. These lineages include critical primary producers of freshwater and terrestrial ecosystems, progenitors of which provided plastids through secondary endosymbiosis to other algae such as diatoms and dinoflagellates that are critical to marine ecosystems. Despite its broad importance and the success of algal and plant lineages, the phagotrophic origin of the plastid imposed an interesting challenge on the predatory eukaryotic ancestor of the Archaeplastida. By engulfing an oxygenic photosynthetic cell, the host lineage imposed an oxidative stress upon itself in the presence of light. Adaptations to meet this challenge were thus likely to have occurred early on during the transition from a predatory phagotroph to an obligate phototroph (or mixotroph). Modern algae have recently been shown to employ linear tetrapyrroles (bilins) to respond to oxidative stress under high light. Here we explore the early events in plastid evolution and the possible ancient roles of bilins in responding to light and oxygen.

The origin of the photosynthetic organelle in eukaryotes, the plastid, changed forever the evolutionary trajectory of life on our planet. Plastids are highly specialized compartments derived from a putative single cyanobacterial primary endosymbiosis that occurred in the common ancestor of the supergroup Archaeplastida that comprises the Viridiplantae (green algae and plants), red algae, and glaucophyte algae. These lineages include critical primary producers of freshwater and terrestrial ecosystems, progenitors of which provided plastids through secondary endosymbiosis to other algae such as diatoms and dinoflagellates that are critical to marine ecosystems. Despite its broad importance and the success of algal and plant lineages, the phagotrophic origin of the plastid imposed an interesting challenge on the predatory eukaryotic ancestor of the Archaeplastida. By engulfing an oxygenic photosynthetic cell, the host lineage imposed an oxidative stress upon itself in the presence of light. Adaptations to meet this challenge were thus likely to have occurred early on during the transition from a predatory phagotroph to an obligate phototroph (or mixotroph). Modern algae have recently been shown to employ linear tetrapyrroles (bilins) to respond to oxidative stress under high light. Here we explore the early events in plastid evolution and the possible ancient roles of bilins in responding to light and oxygen.

We examined the effect of light attenuation, due to floating plants, on the community structure of the main phagotrophic protists and their grazing rates in a wetland in the Lower Paraná Basin. Ingestion experiments (winter and summer) were conducted at 2 sites in the same shallow lake that had contrasting light scenarios: open waters (light) and under profuse macrophyte coverage (dark: light attenuation ~97%). We compared the rates at which protists ingested 3 types of tracer prey: fluorescently labelled heterotrophic bacteria (FLB), picocyanobacteria (FLC) and picoeukaryotic algae (FLA). Light influenced both the structure of the microbial communities and the protistan grazing rates. Heterotrophic flagellates (HF) were more abundant under the macrophytes, whereas mixotrophic algae (cryptophytes) and autotrophic and heterotrophic picoplankton populations attained higher abundances in open waters. Specific grazing rates (SGRs) of mixotrophs on heterotrophic bacteria (HB) were higher in the light (7.9 to 15.5 prey cells grazer−1 h−1), than in darkness (0.1 to 5.1 prey cells grazer−1 h−1); the same trend was observed on picocyanobacteria (Pcy) (1.1 and 0.2 prey cells grazer−1 h−1, light and dark). SGRs of HF were 1.0 to 7.3 cells grazer−1 h−1 (on HB) and 0.01 to 1.8 prey cells grazer−1 h−1 (on Pcy), with highest values in summer and no pattern in relation to light. SGRs of ciliates were higher in summer and in darkness. Clearance rates (CR) on Pcy were higher than on HB, for both HF and mixotrophic algae. In winter, cryptophytes contributed up to 93% of the microbial grazing in the light, whereas HF were more important in darkness; in summer, bacterivory was dominated by heterotrophs in both light scenarios. Our experimental results highlight the importance of light conditions in structuring bacterial grazing by protists

A new species of the genus Fragilidium, F. duplocampanaeforme sp. nov., is described from examinations by LM and SEM. This species has been recorded in summer on the French Atlantic coast, over a number of years. It was never abundant in the plankton and was very often associated with Fragilidium subglobosum, Pyrophacus horologium and also with toxigenic species of the genera Alexandrium and Dinophysis. Phagotrophy of F. duplocampanaeforme on Dinophysis prey is shown, and sexual reproduction is suggested by the observation of gamete-like small forms. The size and the peculiar shape of its cells do not correspond to any known taxon, but the plate arrangement fits the genus Fragilidium. The plate formula is Po, Pc, 4', 8 '', 10c, 6s?, 7"', 2 '''', lp. A close examination of the plate morphology reveals an apical closing platelet Pc and significant differences from known Fragilidium species. Plate ornamentation is complex. A longitudinal fold and an unusual optional pore are seen on the antapical plate 2 ''''. Other distinctive morphological features are emphasized which discriminate this new species from others of the genus Fragilidium.

A new species of the genus Fragilidium, F. duplocampanaeforme sp. nov., is described from examinations by LM and SEM. This species has been recorded in summer on the French Atlantic coast, over a number of years. It was never abundant in the plankton and was very often associated with Fragilidium subglobosum, Pyrophacus horologium and also with toxigenic species of the genera Alexandrium and Dinophysis. Phagotrophy of F. duplocampanaeforme on Dinophysis prey is shown, and sexual reproduction is suggested by the observation of gamete-like small forms. The size and the peculiar shape of its cells do not correspond to any known taxon, but the plate arrangement fits the genus Fragilidium. The plate formula is Po, Pc, 4', 8 '', 10c, 6s?, 7"', 2 '''', lp. A close examination of the plate morphology reveals an apical closing platelet Pc and significant differences from known Fragilidium species. Plate ornamentation is complex. A longitudinal fold and an unusual optional pore are seen on the antapical plate 2 ''''. Other distinctive morphological features are emphasized which discriminate this new species from others of the genus Fragilidium.

A new species of the genus Fragilidium, F. duplocampanaeforme sp. nov., is described from examinations by LM and SEM. This species has been recorded in summer on the French Atlantic coast, over a number of years. It was never abundant in the plankton and was very often associated with Fragilidium subglobosum, Pyrophacus horologium and also with toxigenic species of the genera Alexandrium and Dinophysis. Phagotrophy of F. duplocampanaeforme on Dinophysis prey is shown, and sexual reproduction is suggested by the observation of gamete-like small forms. The size and the peculiar shape of its cells do not correspond to any known taxon, but the plate arrangement fits the genus Fragilidium. The plate formula is Po, Pc, 4', 8 '', 10c, 6s?, 7"', 2 '''', lp. A close examination of the plate morphology reveals an apical closing platelet Pc and significant differences from known Fragilidium species. Plate ornamentation is complex. A longitudinal fold and an unusual optional pore are seen on the antapical plate 2 ''''. Other distinctive morphological features are emphasized which discriminate this new species from others of the genus Fragilidium.

Beisner, B. E., Grossart, H.-P., & Gasol, J. M. (2019). A guide to methods for estimating phago-mixotrophy in nanophytoplankton. Journal of Plankton Research, 41(2), 77–89. Available here