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Protist plankton can be divided into three main groups: phytoplankton, zooplankton, and mixoplankton.In situmethods for studying phytoplankton and zooplankton are relatively straightforward since they generally target chlorophyll/photosynthesis or grazing activity, while the integration of both processes within a single cell makes mixoplankton inherently challenging to study. As a result, we understand less about mixoplankton physiology and their role in food webs, biogeochemical cycling, and ecosystems compared to phytoplankton and zooplankton. In this paper, we posit that by merging conventional techniques, such as microscopy and physiological data, with innovative methods likein situsingle-cell sorting and omics datasets, in conjunction with a diverse array of modeling approaches ranging from single-cell modeling to comprehensive Earth system models, we can propel mixoplankton research into the forefront of aquatic ecology. We present eight crucial research questions pertaining to mixoplankton and mixotrophy, and briefly outline a combination of existing methods and models that can be used to address each question. Our intent is to encourage more interdisciplinary research on mixoplankton, thereby expanding the scope of data acquisition and knowledge accumulation for this understudied yet critical component of aquatic ecosystems. 

Rates of microbial processes are fundamental to understanding the significance of microbial impacts on environmental chemical cycling. However, it is often difficult to quantify rates or to link processes to specific taxa or individual cells, especially in environments where there are few cultured representatives with known physiology. Here, we describe the use of the redox-enzyme-sensitive molecular probe RedoxSensor™ Green to measure rates of anaerobic electron transfer physiology (i.e., sulfate reduction and methanogenesis) in individual cells and link those measurements to genomic sequencing of the same single cells. We used this method to investigate microbial activity in hot, anoxic, low-biomass (~10³cells mL⁻¹) groundwater of the Death Valley Regional Flow System, California. Combining this method with electron donor amendment experiments and metatranscriptomics confirmed that the abundant spore formers includingCandidatusDesulforudis audaxviator were actively reducing sulfate in this environment, most likely with acetate and hydrogen as electron donors. Using this approach, we measured environmental sulfate reduction rates at 0.14 to 26.9 fmol cell⁻¹h⁻¹. Scaled to volume, this equates to a bulk environmental rate of ~10³pmol sulfate L⁻¹d⁻¹, similar to potential rates determined with radiotracer methods. Despite methane in the system, there was no evidence for active microbial methanogenesis at the time of sampling. Overall, this method is a powerful tool for estimating species-resolved, single-cell rates of anaerobic metabolism in low-biomass environments while simultaneously linking genomes to phenomes at the single-cell level. We reveal active elemental cycling conducted by several species, with a large portion attributable toCa.Desulforudis audaxviator. 

Abstract The ocean–atmosphere exchange of CO 2 largely depends on the balance between marine microbial photosynthesis and respiration. Despite vast taxonomic and metabolic diversity among marine planktonic bacteria and archaea (prokaryoplankton) 1–3 , their respiration usually is measured in bulk and treated as a ‘black box’ in global biogeochemical models 4 ; this limits the mechanistic understanding of the global carbon cycle. Here, using a technology for integrated phenotype analyses and genomic sequencing of individual microbial cells, we show that cell-specific respiration rates differ by more than 1,000× among prokaryoplankton genera. The majority of respiration was found to be performed by minority members of prokaryoplankton (including the Roseobacter cluster), whereas cells of the most prevalent lineages (including Pelagibacter and SAR86) had extremely low respiration rates. The decoupling of respiration rates from abundance among lineages, elevated counts of proteorhodopsin transcripts in Pelagibacter and SAR86 cells and elevated respiration of SAR86 at night indicate that proteorhodopsin-based phototrophy 3,5–7 probably constitutes an important source of energy to prokaryoplankton and may increase growth efficiency. These findings suggest that the dependence of prokaryoplankton on respiration and remineralization of phytoplankton-derived organic carbon into CO 2 for its energy demands and growth may be lower than commonly assumed and variable among lineages. 

Fluids circulating through oceanic crust play important roles in global biogeochemical cycling mediated by their microbial inhabitants, but studying these sites is challenged by sampling logistics and low biomass. Borehole observatories installed at the North Pond study site on the western flank of the Mid-Atlantic Ridge have enabled investigation of the microbial biosphere in cold, oxygenated basaltic oceanic crust. Here we test a methodology that applies redox-sensitive fluorescent molecules for flow cytometric sorting of cells for single cell genomic sequencing from small volumes of low biomass (approximately 10 3 cells ml –1 ) crustal fluid. We compare the resulting genomic data to a recently published paired metagenomic and metatranscriptomic analysis from the same site. Even with low coverage genome sequencing, sorting cells from less than one milliliter of crustal fluid results in similar interpretation of dominant taxa and functional profiles as compared to ‘omics analysis that typically filter orders of magnitude more fluid volume. The diverse community dominated by Gammaproteobacteria, Bacteroidetes, Desulfobacterota, Alphaproteobacteria, and Zetaproteobacteria, had evidence of autotrophy and heterotrophy, a variety of nitrogen and sulfur cycling metabolisms, and motility. Together, results indicate fluorescence activated cell sorting methodology is a powerful addition to the toolbox for the study of low biomass systems or at sites where only small sample volumes are available for analysis. 

A substantial component of phytoplankton production in the oceans is channeled through protistan grazers but understanding what dictates the magnitude of this process on a regional and temporal basis is limited, in part, by a shortage of experimental options. A novel saturation approach based on the functional response of planktonic grazers to increasing prey abundance was developed using laboratory cultures of the predator-prey combination of Ochromonas danica and Micromonas pusilla and tested in the coastal waters of the Gulf of Maine. In incubation series, 2 μm polystyrene microspheres were used as surrogate prey to generate increasing levels of saturation of predator ingestion rates of natural prey, resulting in increased rates of apparent growth of the picophytoplankton populations. The relationship between level of addition of surrogate prey to apparent growth, consistently provided significant estimates of maximal growth in the absence of grazing and grazing mortality for populations of picoeukaryotes and Synechococcus . Estimates of gross growth and grazing mortality were comparable to results from dilution experiments carried out in the same waters. The saturation approach represents an additional tool to investigate predator-prey interactions in planktonic communities. Further investigations may show that it can be used to quantify group-specific grazing mortality and growth rates beyond coastal waters and in multiple size classes of prey. 

In this article, we present Bio-GO-SHIP, a new ocean observing program that will incorporate sustained and consistent global biological ocean observations into the Global Ocean Ship-based Hydrographic Investigations Program (GO-SHIP). The goal of Bio-GO-SHIP is to produce systematic and consistent biological observations during global ocean repeat hydrographic surveys, with a particular focus on the planktonic ecosystem. Ocean plankton are an essential component of the earth climate system, form the base of the oceanic food web and thereby play an important role in influencing food security and contributing to the Blue Economy. Despite its importance, ocean biology is largely under-sampled in time and space compared to physical and chemical properties. This lack of information hampers our ability to understand the role of plankton in regulating biogeochemical processes and fueling higher trophic levels, now and in future ocean conditions. Traditionally, many of the methods used to quantify biological and ecosystem essential ocean variables (EOVs), measures that provide valuable information on the ecosystem, have been expensive and labor- and time-intensive, limiting their large-scale deployment. In the last two decades, new technologies have been developed and matured, making it possible to greatly expand our biological ocean observing capacity. These technologies, including cell imaging, bio-optical sensors and 'omic tools, can be combined to provide overlapping measurements of key biological and ecosystem EOVs. New developments in data management and open sharing can facilitate meaningful synthesis and integration with concurrent physical and chemical data. Here we outline how Bio-GO-SHIP leverages these technological advances to greatly expand our knowledge and understanding of the constituents and function of the global ocean plankton ecosystem.