Nicole Poulton

Recent studies suggest that unidentified prokaryotes fix inorganic carbon at globally significant rates in the immense dark ocean. Using single-cell sorting and whole-genome amplification of prokaryotes from two subtropical gyres, we obtained genomic DNA from 738 cells representing most cosmopolitan lineages. Multiple cells of Deltaproteobacteria cluster SAR324, Gammaproteobacteria clusters ARCTIC96BD-19 and Agg47, and some Oceanospirillales from the lower mesopelagic contained ribulose-1,5-bisphosphate carboxylase-oxygenase and sulfur oxidation genes. These results corroborated community DNA and RNA profiling from diverse geographic regions. The SAR324 genomes also suggested C(1) metabolism and a particle-associated life-style. Microautoradiography and fluorescence in situ hybridization confirmed bicarbonate uptake and particle association of SAR324 cells. Our study suggests potential chemolithoautotrophy in several uncultured Proteobacteria lineages that are ubiquitous in the dark oxygenated ocean and provides new perspective on carbon cycling in the ocean's largest habitat.

Planktonic bacteria dominate surface ocean biomass and influence global biogeochemical processes, but remain poorly characterized owing to difficulties in cultivation. Using large-scale single cell genomics, we obtained insight into the genome content and biogeography of many bacterial lineages inhabiting the surface ocean. We found that, compared with existing cultures, natural bacterioplankton have smaller genomes, fewer gene duplications, and are depleted in guanine and cytosine, noncoding nucleotides, and genes encoding transcription, signal transduction, and noncytoplasmic proteins. These findings provide strong evidence that genome streamlining and oligotrophy are prevalent features among diverse, free-living bacterioplankton, whereas existing laboratory cultures consist primarily of copiotrophs. The apparent ubiquity of metabolic specialization and mixotrophy, as predicted from single cell genomes, also may contribute to the difficulty in bacterioplankton cultivation. Using metagenome fragment recruitment against single cell genomes, we show that the global distribution of surface ocean bacterioplankton correlates with temperature and latitude and is not limited by dispersal at the time scales required for nucleotide substitution to exceed the current operational definition of bacterial species. Single cell genomes with highly similar small subunit rRNA gene sequences exhibited significant genomic and biogeographic variability, highlighting challenges in the interpretation of individual gene surveys and metagenome assemblies in environmental microbiology. Our study demonstrates the utility of single cell genomics for gaining an improved understanding of the composition and dynamics of natural microbial assemblages.

Single-cell genomics is a powerful tool for exploring the genetic makeup of environmental microorganisms, the vast majority of which are difficult, if not impossible, to cultivate with current approaches. Here we present a comprehensive protocol for obtaining genomes from uncultivated environmental microbes via high-throughput single-cell isolation by FACS. The protocol encompasses the preservation and pretreatment of differing environmental samples, followed by the physical separation, lysis, whole-genome amplification and 16S rRNA–based identification of individual bacterial and archaeal cells. The described procedure can be performed with standard molecular biology equipment and a FACS machine. It takes <12 h of bench time over a 4-d time period, and it generates up to 1 μg of genomic DNA from an individual microbial cell, which is suitable for downstream applications such as PCR amplification and shotgun sequencing. The completeness of the recovered genomes varies, with an average of ∼50%.

Microbial hydrolysis of polysaccharides is critical to ecosystem functioning and is of great interest in diverse biotechnological applications, such as biofuel production and bioremediation. Here we demonstrate the use of a new, efficient approach to recover genomes of active polysaccharide degraders from natural, complex microbial assemblages, using a combination of fluorescently labeled substrates, fluorescence-activated cell sorting, and single cell genomics. We employed this approach to analyze freshwater and coastal bacterioplankton for degraders of laminarin and xylan, two of the most abundant storage and structural polysaccharides in nature. Our results suggest that a few phylotypes of Verrucomicrobia make a considerable contribution to polysaccharide degradation, although they constituted only a minor fraction of the total microbial community. Genomic sequencing of five cells, representing the most predominant, polysaccharide-active Verrucomicrobia phylotype, revealed significant enrichment in genes encoding a wide spectrum of glycoside hydrolases, sulfatases, peptidases, carbohydrate lyases and esterases, confirming that these organisms were well equipped for the hydrolysis of diverse polysaccharides. Remarkably, this enrichment was on average higher than in the sequenced representatives of Bacteroidetes, which are frequently regarded as highly efficient biopolymer degraders. These findings shed light on the ecological roles of uncultured Verrucomicrobia and suggest specific taxa as promising bioprospecting targets. The employed method offers a powerful tool to rapidly identify and recover discrete genomes of active players in polysaccharide degradation, without the need for cultivation.

Ocean Sampling Day was initiated by the EU-funded Micro B3 (Marine Microbial Biodiversity, Bioinformatics, Biotechnology) project to obtain a snapshot of the marine microbial biodiversity and function of the world's oceans. It is a simultaneous global mega-sequencing campaign aiming to generate the largest standardized microbial data set in a single day. This will be achievable only through the coordinated efforts of an Ocean Sampling Day Consortium, supportive partnerships and networks between sites. This commentary outlines the establishment, function and aims of the Consortium and describes our vision for a sustainable study of marine microbial communities and their embedded functional traits.

Marine bacteria and archaea play key roles in global biogeochemistry. To improve our understanding of this complex microbiome, we employed single-cell genomics and a randomized, hypothesis-agnostic cell selection strategy to recover 12,715 partial genomes from the tropical and subtropical euphotic ocean. A substantial fraction of known prokaryoplankton coding potential was recovered from a single, 0.4 mL ocean sample, which indicates that genomic information disperses effectively across the globe. Yet, we found each genome to be unique, implying limited clonality within prokaryoplankton populations. Light harvesting and secondary metabolite biosynthetic pathways were numerous across lineages, highlighting the value of single-cell genomics to advance the identification of ecological roles and biotechnology potential of uncultured microbial groups. This genome collection enabled functional annotation and genus-level taxonomic assignments for >80% of individual metagenome reads from the tropical and subtropical surface ocean, thus offering a model to improve reference genome databases for complex microbiomes.

A unique collection of oceanic samples was gathered by the Tara Oceans expeditions (2009-2013), targeting plankton organisms ranging from viruses to metazoans, and providing rich environmental context measurements. Thanks to recent advances in the field of genomics, extensive sequencing has been performed for a deep genomic analysis of this huge collection of samples. A strategy based on different approaches, such as metabarcoding, metagenomics, single-cell genomics and metatranscriptomics, has been chosen for analysis of size-fractionated plankton communities. Here, we provide detailed procedures applied for genomic data generation, from nucleic acids extraction to sequence production, and we describe registries of genomics datasets available at the European Nucleotide Archive (ENA, www.ebi.ac.uk/ena). The association of these metadata to the experimental procedures applied for their generation will help the scientific community to access these data and facilitate their analysis. This paper complements other efforts to provide a full description of experiments and open science resources generated from the Tara Oceans project, further extending their value for the study of the world's planktonic ecosystems.

Abstract Microbial single-cell genomics can be used to provide insights into the metabolic potential, interactions, and evolution of uncultured microorganisms. Here we present WGA-X, a method based on multiple displacement amplification of DNA that utilizes a thermostable mutant of the phi29 polymerase. WGA-X enhances genome recovery from individual microbial cells and viral particles while maintaining ease of use and scalability. The greatest improvements are observed when amplifying high G+C content templates, such as those belonging to the predominant bacteria in agricultural soils. By integrating WGA-X with calibrated index-cell sorting and high-throughput genomic sequencing, we are able to analyze genomic sequences and cell sizes of hundreds of individual, uncultured bacteria, archaea, protists, and viral particles, obtained directly from marine and soil samples, in a single experiment. This approach may find diverse applications in microbiology and in biomedical and forensic studies of humans and other multicellular organisms.

Whereas DNA viruses are known to be abundant, diverse, and commonly key ecosystem players, RNA viruses are insufficiently studied outside disease settings. In this study, we analyzed ≈28 terabases of Global Ocean RNA sequences to expand Earth’s RNA virus catalogs and their taxonomy, investigate their evolutionary origins, and assess their marine biogeography from pole to pole. Using new approaches to optimize discovery and classification, we identified RNA viruses that necessitate substantive revisions of taxonomy (doubling phyla and adding &gt;50% new classes) and evolutionary understanding. “Species”-rank abundance determination revealed that viruses of the new phyla “ Taraviricota ,” a missing link in early RNA virus evolution, and “ Arctiviricota ” are widespread and dominant in the oceans. These efforts provide foundational knowledge critical to integrating RNA viruses into ecological and epidemiological models.

Marine planktonic eukaryotes play critical roles in global biogeochemical cycles and climate. However, their poor representation in culture collections limits our understanding of the evolutionary history and genomic underpinnings of planktonic ecosystems. Here, we used 280 billion Tara Oceans metagenomic reads from polar, temperate, and tropical sunlit oceans to reconstruct and manually curate more than 700 abundant and widespread eukaryotic environmental genomes ranging from 10 Mbp to 1.3 Gbp. This genomic resource covers a wide range of poorly characterized eukaryotic lineages that complement long-standing contributions from culture collections while better representing plankton in the upper layer of the oceans. We performed the first, to our knowledge, comprehensive genome-wide functional classification of abundant unicellular eukaryotic plankton, revealing four major groups connecting distantly related lineages. Neither trophic modes of plankton nor its vertical evolutionary history could completely explain the functional repertoire convergence of major eukaryotic lineages that coexisted within oceanic currents for millions of years.

While several studies have suggested that bacterium-phytoplankton interactions have the potential to dramatically influence harmful algal bloom dynamics, little is known about how bacteria and phytoplankton communities interact at the species composition level. The objective of the current study was to determine whether there are specific associations between diverse phytoplankton and the bacteria that co-occur with them. We determined the phylogenetic diversity of bacterial assemblages associated with 10 Alexandrium strains and representatives of the major taxonomic groups of phytoplankton in the Gulf of Maine. For this analysis we chose xenic phytoplankton cultures that (i) represented a broad taxonomic range, (ii) represented a broad geographic range for Alexandrium spp. isolates, (iii) grew under similar cultivation conditions, (iv) had a minimal length of time since the original isolation, and (v) had been isolated from a vegetative phytoplankton cell. 16S rRNA gene fragments of most Bacteria were amplified from DNA extracted from cultures and were analyzed by denaturing gradient gel electrophoresis and sequencing. A greater number of bacterial species were shared by different Alexandrium cultures, regardless of the geographic origin, than by Alexandrium species and nontoxic phytoplankton from the Gulf of Maine. In particular, members of the Roseobacter clade showed a higher degree of association with Alexandrium than with other bacterial groups, and many sequences matched sequences reported to be associated with other toxic dinoflagellates. These results provide evidence for specificity in bacterium-phytoplankton associations.

Recent discoveries suggest that photoheterotrophs (rhodopsin-containing bacteria (RBs) and aerobic anoxygenic phototrophs (AAPs)) and chemoautotrophs may be significant for marine and freshwater ecosystem productivity. However, their abundance and taxonomic identities remain largely unknown. We used a combination of single-cell and metagenomic DNA sequencing to study the predominant photoheterotrophs and chemoautotrophs inhabiting the euphotic zone of temperate, physicochemically diverse freshwater lakes. Multi-locus sequencing of 712 single amplified genomes, generated by fluorescence-activated cell sorting and whole genome multiple displacement amplification, showed that most of the cosmopolitan freshwater clusters contain photoheterotrophs. These comprised at least 10-23% of bacterioplankton, and RBs were the dominant fraction. Our data demonstrate that Actinobacteria, including clusters acI, Luna and acSTL, are the predominant freshwater RBs. We significantly broaden the known taxonomic range of freshwater RBs, to include Alpha-, Beta-, Gamma- and Deltaproteobacteria, Verrucomicrobia and Sphingobacteria. By sequencing single cells, we found evidence for inter-phyla horizontal gene transfer and recombination of rhodopsin genes and identified specific taxonomic groups involved in these evolutionary processes. Our data suggest that members of the ubiquitous betaproteobacteria Polynucleobacter spp. are the dominant AAPs in temperate freshwater lakes. Furthermore, the RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) gene was found in several single cells of Betaproteobacteria, Bacteroidetes and Gammaproteobacteria, suggesting that chemoautotrophs may be more prevalent among aerobic bacterioplankton than previously thought. This study demonstrates the power of single-cell DNA sequencing addressing previously unresolved questions about the metabolic potential and evolutionary histories of uncultured microorganisms, which dominate most natural environments.

Phytoplankton alter their biochemical composition according to nutrient availability, such that their bulk elemental composition varies across oceanic provinces. However, the links between plankton biochemical composition and variation in biogeochemical cycling of nutrients remain largely unknown. In a survey of phytoplankton phosphorus stress in the western North Atlantic, we found that phytoplankton in the phosphorus-depleted subtropical Sargasso Sea were enriched in the biochemical polyphosphate (polyP) compared with nutrient-rich temperate waters, contradicting the canonical oceanographic view of polyP as a luxury phosphorus storage molecule. The enrichment in polyP coincided with enhanced alkaline phosphatase activity and substitution of sulfolipids for phospholipids, which are both indicators of phosphorus stress. Further, polyP appeared to be liberated preferentially over bulk phosphorus from sinking particles in the Sargasso Sea, thereby retaining phosphorus in shallow waters. Thus, polyP cycling may form a feedback loop that attenuates the export of phosphorus when it becomes scarce, contributes bioavailable P for primary production, and supports the export of carbon and nitrogen via sinking particles.

Heterotrophic protists are a highly diverse and biogeochemically significant component of marine ecosystems, yet little is known about their species-specific prey preferences and symbiotic interactions in situ. Here we demonstrate how these previously unresolved questions can be addressed by sequencing the eukaryote and bacterial SSU rRNA genes from individual, uncultured protist cells collected from their natural marine environment and sorted by flow cytometry. We detected Pelagibacter ubique in association with a MAST-4 protist, an actinobacterium in association with a chrysophyte and three bacteroidetes in association with diverse protist groups. The presence of identical phylotypes among the putative prey and the free bacterioplankton in the same sample provides evidence for predator-prey interactions. Our results also suggest a discovery of novel symbionts, distantly related to Rickettsiales and the candidate divisions ZB3 and TG2, associated with Cercozoa and Chrysophyta cells. This study demonstrates the power of single cell sequencing to untangle ecological interactions between uncultured protists and prokaryotes.

Recent advances in single-cell genomic and metagenomic techniques have facilitated the discovery of numerous previously unknown, deep branches of the tree of life that lack cultured representatives. Many of these candidate phyla are composed of microorganisms with minimalistic, streamlined genomes lacking some core metabolic pathways, which may contribute to their resistance to growth in pure culture. Here we analyzed single-cell genomes and metagenome bins to show that the "Candidate phylum Rokubacteria", formerly known as SPAM, represents an interesting exception, by having large genomes (6-8 Mbps), high GC content (66%-71%), and the potential for a versatile, mixotrophic metabolism. We also observed an unusually high genomic heterogeneity among individual Rokubacteria cells in the studied samples. These features may have contributed to the limited recovery of sequences of this candidate phylum in prior metagenomic studies. Our analyses suggest that Rokubacteria are distributed globally in diverse terrestrial ecosystems, including soils, the rhizosphere, volcanic mud, oil wells, aquifers and the deep subsurface, with no reports from marine environments to date.

Single-celled eukaryotes (protists) are critical players in global biogeochemical cycling of nutrients and energy in the oceans. While their roles as primary producers and grazers are well appreciated, other aspects of their life histories remain obscure due to challenges in culturing and sequencing their natural diversity. Here, we exploit single-cell genomics and metagenomics data from the circumglobal Tara Oceans expedition to analyze the genome content and apparent oceanic distribution of seven prevalent lineages of uncultured heterotrophic stramenopiles. Based on the available data, each sequenced genome or genotype appears to have a specific oceanic distribution, principally correlated with water temperature and depth. The genome content provides hypotheses for specialization in terms of cell motility, food spectra, and trophic stages, including the potential impact on their lifestyles of horizontal gene transfer from prokaryotes. Our results support the idea that prominent heterotrophic marine protists perform diverse functions in ocean ecology.

This review highlights the concepts and instrumentation of imaging flow cytometry technology and in particular its use for phytoplankton analysis. Imaging flow cytometry, a hybrid technology combining speed and statistical capabilities of flow cytometry with imaging features of microscopy, is rapidly advancing as a cell imaging platform that overcomes many of the limitations of current techniques and contributed significantly to the advancement of phytoplankton analysis in recent years. This review presents the various instrumentation relevant to the field and currently used for assessment of complex phytoplankton communities' composition and abundance, size structure determination, biovolume estimation, detection of harmful algal bloom species, evaluation of viability and metabolic activity and other applications. Also we present our data on viability and metabolic assessment of Aphanizomenon sp. cyanobacteria using Imagestream X Mark II imaging cytometer. Herein, we highlight the immense potential of imaging flow cytometry for microalgal research, but also discuss limitations and future developments.

Abstract. The ratio of two in situ optical measurements – chlorophyll fluorescence (Chl F) and optical particulate backscattering (bbp) – varied with changes in phytoplankton community composition during the North Atlantic Bloom Experiment in the Iceland Basin in 2008. Using ship-based measurements of Chl F, bbp, chlorophyll a (Chl), high-performance liquid chromatography (HPLC) pigments, phytoplankton composition and carbon biomass, we found that oscillations in the ratio varied with changes in plankton community composition; hence we refer to Chl F/bbp as an "optical community index". The index varied by more than a factor of 2, with low values associated with pico- and nanophytoplankton and high values associated with diatom-dominated phytoplankton communities. Observed changes in the optical index were driven by taxa-specific chlorophyll-to-autotrophic carbon ratios and by physiological changes in Chl F associated with the silica limitation. A Lagrangian mixed-layer float and four Seagliders, operating continuously for 2 months, made similar measurements of the optical community index and followed the evolution and later demise of the diatom spring bloom. Temporal changes in optical community index and, by implication, the transition in community composition from diatom to post-diatom bloom communities were not simultaneous over the spatial domain surveyed by the ship, float and gliders. The ratio of simple optical properties measured from autonomous platforms, when carefully validated, provides a unique tool for studying phytoplankton patchiness on extended temporal scales and ecologically relevant spatial scales and should offer new insights into the processes regulating patchiness.

Recent discoveries suggest that the candidate superphyla Patescibacteria and DPANN constitute a large fraction of the phylogenetic diversity of Bacteria and Archaea. Their small genomes and limited coding potential have been hypothesized to be ancestral adaptations to obligate symbiotic lifestyles. To test this hypothesis, we performed cell-cell association, genomic, and phylogenetic analyses on 4,829 individual cells of Bacteria and Archaea from 46 globally distributed surface and subsurface field samples. This confirmed the ubiquity and abundance of Patescibacteria and DPANN in subsurface environments, the small size of their genomes and cells, and the divergence of their gene content from other Bacteria and Archaea. Our analyses suggest that most Patescibacteria and DPANN in the studied subsurface environments do not form specific physical associations with other microorganisms. These data also suggest that their unusual genomic features and prevalent auxotrophies may be a result of ancestral, minimal cellular energy transduction mechanisms that lack respiration, thus relying solely on fermentation for energy conservation.

The role of the Arctic Ocean ecosystem in climate regulation may depend on the responses of marine microorganisms to environmental change. We applied genome-resolved metagenomics to 41 Arctic seawater samples, collected at various depths in different seasons during the Tara Oceans Polar Circle expedition, to evaluate the ecology, metabolic potential and activity of resident bacteria and archaea. We assembled 530 metagenome-assembled genomes (MAGs) to form the Arctic MAGs catalogue comprising 526 species. A total of 441 MAGs belonged to species that have not previously been reported and 299 genomes showed an exclusively polar distribution. Most Arctic MAGs have large genomes and the potential for fast generation times, both of which may enable adaptation to a copiotrophic lifestyle in nutrient-rich waters. We identified 38 habitat generalists and 111 specialists in the Arctic Ocean. We also found a general prevalence of 14 mixotrophs, while chemolithoautotrophs were mostly present in the mesopelagic layer during spring and autumn. We revealed 62 MAGs classified as key Arctic species, found only in the Arctic Ocean, showing the highest gene expression values and predicted to have habitat-specific traits. The Artic MAGs catalogue will inform our understanding of polar microorganisms that drive global biogeochemical cycles.

Phago-mixotrophy, the combination of photoautotrophy and phagotrophy in mixoplankton, organisms that can combine both trophic strategies, have gained increasing attention over the past decade. It is now recognized that a substantial number of protistan plankton species engage in phago-mixotrophy to obtain nutrients for growth and reproduction under a range of environmental conditions. Unfortunately, our current understanding of mixoplankton in aquatic systems significantly lags behind our understanding of zooplankton and phytoplankton, limiting our ability to fully comprehend the role of mixoplankton (and phago-mixotrophy) in the plankton food web and biogeochemical cycling. Here, we put forward five research directions that we believe will lead to major advancement in the field: (i) evolution: understanding mixotrophy in the context of the evolutionary transition from phagotrophy to photoautotrophy; (ii) traits and trade-offs: identifying the key traits and trade-offs constraining mixotrophic metabolisms; (iii) biogeography: large-scale patterns of mixoplankton distribution; (iv) biogeochemistry and trophic transfer: understanding mixoplankton as conduits of nutrients and energy; and (v) in situ methods: improving the identification of in situ mixoplankton and their phago-mixotrophic activity.

Aerobic anoxygenic photoheterotrophic (AAP) bacteria can use both dissolved organic matter and light for energy production, but their photosynthesis does not produce oxygen. We measured AAP bacterial cell and bacteriochlorophyll distributions in the northwest Atlantic, from the coast of the Gulf of Maine to the Sargasso Sea, in October 2001 and March 2002. The abundance of AAPs ranged from 7 x 103 to 9.8 x 104 cells mL−1 (mean, 2.9 x 104 mL−1) in surface waters, or between 1% and 9% (mean, 2.3%) of total bacteria. Mean abundances in October in the Gulf of Maine (6.6 x 104 mL−1) were about five times higher than those measured in March (1.3 x 104 mL−1), whereas the mean Sargasso Sea values were not different between October and March. AAP cells were larger than other bacteria, so AAP biomass ranged from 2% to 13% of total bacterial biomass. AAP cells were higher in abundance, biomass, and proportion of total bacteria in productive coastal and shelf waters than in the Sargasso Sea. Cell quotas of bacteriochlorophyll were low and quite variable, ranging from 0.02 to 0.17 fg cell−1 (mean, 0.08 fg cell−1). Our results indicate possible control by temperature and organic and inorganic nutrients on the distribution of planktonic AAPs, but they do not support the idea that they are specifically adapted to oligotrophic conditions.

Recent applications of culture-independent, molecular methods have revealed unexpectedly high diversity in a variety of functional and phylogenetic groups of microorganisms in the ocean. However, none of the existing research tools are free from significant limitations, such as PCR and cloning biases, low phylogenetic resolution and others. Here, we employed novel, single-cell sequencing techniques to assess the composition of small (<10 μm diameter), heterotrophic protists from the Gulf of Maine. Single cells were isolated by flow cytometry, their genomes amplified, and 18S rRNA marker genes were amplified and sequenced. We compared the results to traditional environmental PCR cloning of sorted cells. The diversity of heterotrophic protists was significantly higher in the library of single amplified genomes (SAGs) than in environmental PCR clone libraries of the 18S rRNA gene, obtained from the same coastal sample. Libraries of SAGs, but not clones contained several recently discovered, uncultured groups, including picobiliphytes and novel marine stramenopiles. Clone, but not SAG, libraries contained several large clusters of identical and nearly identical sequences of Dinophyceae, Cercozoa and Stramenopiles. Similar results were obtained using two alternative primer sets, suggesting that PCR biases may not be the only explanation for the observed patterns. Instead, differences in the number of 18S rRNA gene copies among the various protist taxa probably had a significant role in determining the PCR clone composition. These results show that single-cell sequencing has the potential to more accurately assess protistan community composition than previously established methods. In addition, the creation of SAG libraries opens opportunities for the analysis of multiple genes or entire genomes of the uncultured protist groups.

Leptothrix ochracea is a common inhabitant of freshwater iron seeps and iron-rich wetlands. Its defining characteristic is copious production of extracellular sheaths encrusted with iron oxyhydroxides. Surprisingly, over 90% of these sheaths are empty, hence, what appears to be an abundant population of iron-oxidizing bacteria, consists of relatively few cells. Because L. ochracea has proven difficult to cultivate, its identification is based solely on habitat preference and morphology. We utilized cultivation-independent techniques to resolve this long-standing enigma. By selecting the actively growing edge of a Leptothrix-containing iron mat, a conventional SSU rRNA gene clone library was obtained that had 29 clones (42% of the total library) related to the Leptothrix/Sphaerotilus group (≤96% identical to cultured representatives). A pyrotagged library of the V4 hypervariable region constructed from the bulk mat showed that 7.2% of the total sequences also belonged to the Leptothrix/Sphaerotilus group. Sorting of individual L. ochracea sheaths, followed by whole genome amplification (WGA) and PCR identified a SSU rRNA sequence that clustered closely with the putative Leptothrix clones and pyrotags. Using these data, a fluorescence in-situ hybridization (FISH) probe, Lepto175, was designed that bound to ensheathed cells. Quantitative use of this probe demonstrated that up to 35% of microbial cells in an actively accreting iron mat were L. ochracea. The SSU rRNA gene of L. ochracea shares 96% homology with its closet cultivated relative, L. cholodnii, This establishes that L. ochracea is indeed related to this group of morphologically similar, filamentous, sheathed microorganisms.

Marine Group I (MGI) Thaumarchaeota are one of the most abundant and cosmopolitan chemoautotrophs within the global dark ocean. To date, no representatives of this archaeal group retrieved from the dark ocean have been successfully cultured. We used single cell genomics to investigate the genomic and metabolic diversity of thaumarchaea within the mesopelagic of the subtropical North Pacific and South Atlantic Ocean. Phylogenetic and metagenomic recruitment analysis revealed that MGI single amplified genomes (SAGs) are genetically and biogeographically distinct from existing thaumarchaea cultures obtained from surface waters. Confirming prior studies, we found genes encoding proteins for aerobic ammonia oxidation and the hydrolysis of urea, which may be used for energy production, as well as genes involved in 3-hydroxypropionate/4-hydroxybutyrate and oxidative tricarboxylic acid pathways. A large proportion of protein sequences identified in MGI SAGs were absent in the marine cultures Cenarchaeum symbiosum and Nitrosopumilus maritimus, thus expanding the predicted protein space for this archaeal group. Identifiable genes located on genomic islands with low metagenome recruitment capacity were enriched in cellular defense functions, likely in response to viral infections or grazing. We show that MGI Thaumarchaeota in the dark ocean may have more flexibility in potential energy sources and adaptations to biotic interactions than the existing, surface-ocean cultures.

Abstract Ocean plankton comprise organisms from viruses to fish larvae that are fundamental to ecosystem functioning and the provision of marine services such as fisheries and CO 2 sequestration. The latter services are partly governed by variations in plankton community composition and the expression of traits such as body size at community-level. While community assembly has been thoroughly studied for the smaller end of the plankton size spectrum, the larger end comprises ectotherms that are often studied at the species, or group-level, rather than as communities. The body size of marine ectotherms decreases with temperature, but controls on community-level traits remain elusive, hindering the predictability of marine services provision. Here, we leverage Tara Oceans datasets to determine how zooplankton community composition and size structure varies with latitude, temperature and productivity-related covariates in the global surface ocean. Zooplankton abundance and median size decreased towards warmer and less productive environments, as a result of changes in copepod composition. However, some clades displayed the opposite relationships, which may be ascribed to alternative feeding strategies. Given that climate models predict increasingly warmed and stratified oceans, our findings suggest that zooplankton communities will shift towards smaller organisms which might weaken their contribution to the biological carbon pump.

The ocean-atmosphere exchange of CO2 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 models4; 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 phototrophy3,5-7 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 CO2 for its energy demands and growth may be lower than commonly assumed and variable among lineages.

Two cases of primary plastid endosymbiosis are known. The first occurred ca. 1.6 billion years ago and putatively gave rise to the canonical plastid in algae and plants. The second is restricted to a genus of rhizarian amoebae that includes Paulinella chromatophora. Photosynthetic Paulinella species gained their plastid from an α-cyanobacterial source and are sister to plastid-lacking phagotrophs such as Paulinella ovalis that ingest cyanobacteria. To study the role of feeding behavior in plastid origin, we analyzed single-cell genome assemblies from six P. ovalis-like cells isolated from Chesapeake Bay, USA. Dozens of contigs in these cell assemblies were derived from prey DNA of α-cyanobacterial origin and associated cyanophages. We found two examples of horizontal gene transfer (HGT) in P. ovalis-like nuclear DNA from cyanobacterial sources. This work suggests the first evidence of a link between feeding behavior in wild-caught cells, HGT and plastid primary endosymbiosis in the monophyletic Paulinella lineage.

Viruses are major pathogens in all biological systems. Virus propagation and downstream analysis remains a challenge, particularly in the ocean where the majority of their microbial hosts remain recalcitrant to current culturing techniques. We used a cultivation-independent approach to isolate and sequence individual viruses. The protocol uses high-speed fluorescence-activated virus sorting flow cytometry, multiple displacement amplification (MDA), and downstream genomic sequencing. We focused on 'giant viruses' that are readily distinguishable by flow cytometry. From a single-milliliter sample of seawater collected from off the dock at Boothbay Harbor, ME, USA, we sorted almost 700 single virus particles, and subsequently focused on a detailed genome analysis of 12. A wide diversity of viruses was identified that included Iridoviridae, extended Mimiviridae and even a taxonomically novel (unresolved) giant virus. We discovered a viral metacaspase homolog in one of our sorted virus particles and discussed its implications in rewiring host metabolism to enhance infection. In addition, we demonstrated that viral metacaspases are widespread in the ocean. We also discovered a virus that contains both a reverse transcriptase and a transposase; although highly speculative, we suggest such a genetic complement would potentially allow this virus to exploit a latency propagation mechanism. Application of single virus genomics provides a powerful opportunity to circumvent cultivation of viruses, moving directly to genomic investigation of naturally occurring viruses, with the assurance that the sequence data is virus-specific, non-chimeric and contains no cellular contamination.

Abstract Marine planktonic protists are critical components of ocean ecosystems and are highly diverse. Molecular sequencing methods are being used to describe this diversity and reveal new associations and metabolisms that are important to how these ecosystems function. We describe here the use of the single cell genomics approach to sample and interrogate the diversity of the smaller (pico- and nano-sized) protists from a range of oceanic samples. We created over 900 single amplified genomes (SAGs) from 8 Tara Ocean samples across the Indian Ocean and the Mediterranean Sea. We show that flow cytometric sorting of single cells effectively distinguishes plastidic and aplastidic cell types that agree with our understanding of protist phylogeny. Yields of genomic DNA with PCR-identifiable 18S rRNA gene sequence from single cells was low (15% of aplastidic cell sorts, and 7% of plastidic sorts) and tests with alternate primers and comparisons to metabarcoding did not reveal phylogenetic bias in the major protist groups. There was little evidence of significant bias against or in favor of any phylogenetic group expected or known to be present. The four open ocean stations in the Indian Ocean had similar communities, despite ranging from 14°N to 20°S latitude, and they differed from the Mediterranean station. Single cell genomics of protists suggests that the taxonomic diversity of the dominant taxa found in only several hundreds of microliters of surface seawater is similar to that found in molecular surveys where liters of sample are filtered.

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 34:263-277 (2004) - doi:10.3354/ame034263 Counting heterotrophic nanoplanktonic protists in cultures and aquatic communities by flow cytometry Julie M. Rose1,*, David A. Caron1, Michael E. Sieracki2, Nicole Poulton2 1Department of Biological Sciences, University of Southern California, 3616 Trousdale Parkway, AHF 301, Los Angeles, California 90089-0371, USA 2Bigelow Laboratory for Ocean Sciences, PO Box 475, 180 McKown Point Road, West Boothbay Harbor, Maine 04575, USA *Email: jrose@usc.edu ABSTRACT: The food vacuole stain LysoTracker Green® was used to enumerate heterotrophic protists on a standard model flow cytometer. Appropriate stain concentration and staining time were determined using cultures of protists. Stained heterotrophic protists consistently formed distinct populations within cytograms of green fluorescence versus forward scatter. Cytometric counts of cultured species were compared to direct counts using light microscopy at cell abundances ranging from 103 to 106 cells ml-1. A regression of these data was highly significant and yielded a slope of 0.95. Stained populations were accurately counted during lag, exponential and early stationary growth phases. Growth rates calculated from cytometric counts were not statistically different from those based on microscopy. The method was applied to 26 natural plankton samples, and general region definitions on the cytograms were established that identified heterotrophic protistan assemblages. A regression of cytometric counts versus direct counts yielded a slope of 1.16. LysoTracker Green® can only be used with live samples because preservation destroys membrane potential, resulting in loss of fluorescence. However, the flow cytometric method employing LysoTracker Green® is highly applicable for monitoring the growth of many heterotrophic protists in cultures and has the potential to be extremely useful for field samples, providing comparable counts to microscopical methods while allowing much faster sample processing. KEY WORDS: Heterotrophic protists · Flow cytometry · Nanoplankton · Fluorescent staining Full article in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 34, No. 3. Online publication date: March 09, 2004 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2004 Inter-Research.

The predominant model of the role of viruses in the marine trophic web is that of the “viral shunt”, where viral infection funnels a substantial fraction of the microbial primary and secondary production back to the pool of dissolved organic matter. Here, we analyzed the composition of non-eukaryotic DNA associated with individual cells of small, planktonic protists in the Gulf of Maine and the Mediterranean Sea. We found viral DNA associated with a substantial fraction cells from the Gulf of Maine (51%) and the Mediterranean Sea (35%). While Mediterranean SAGs contained a larger proportion of cells containing bacterial sequences (49%), a smaller fraction of cells contained bacterial sequences in the Gulf of Maine (19%). In Gulf of Maine cells, nearly identical bacteriophage and ssDNA virus sequences where found across diverse lineages of protists, suggesting many of these viruses are non-infective. The fraction of cells containing viral DNA varied among protistan lineages and reached 100% in Choanozoa and 100% in Picozoa. These two groups also contained significantly higher numbers of viral sequences than other identified taxa. We consider mechanisms that may explain the presence of viral DNA in protistan cells and conclude that protistan predation on free viral particles contributed to the observed patterns. These findings confirm prior experiments with protistan isolates and indicate that the viral shunt is complemented by a viral link in the marine microbial food web. This link may constitute a sink of viral particles in the ocean and has implications for the flow of carbon through the microbial food web.

The ability to enumerate, classify, and determine biomass of phytoplankton from environmental samples is essential for determining ecosystem function and their role in the aquatic community and microbial food web. Traditional micro-phytoplankton quantification methods using microscopic techniques require preservation and are slow, tedious and very laborious. The availability of more automated imaging microscopy platforms has revolutionized the way particles and cells are detected within their natural environment. The ability to examine cells unaltered and without preservation is key to providing more accurate cell concentration estimates and overall phytoplankton biomass. The FlowCam(®) is an imaging cytometry tool that was originally developed for use in aquatic sciences and provides a more rapid and unbiased method for enumerating and classifying phytoplankton within diverse aquatic environments.

Cytometry Part AVolume 99, Issue 4 p. 311-317 EditorialFree Access Best practices in plant cytometry David Galbraith, Corresponding Author David Galbraith galbraith@email.arizona.edu orcid.org/0000-0003-4020-1635 School of Plant Sciences, BIO5 Institute, Arizona Cancer Center, Department of Biomedical Engineering, University of Arizona, Tucson, Arizona, USA State Key Laboratory of Cotton Biology, Key Laboratory of Plant Stress Biology, Henan University, School of Life Sciences, State Key Laboratory of Crop Stress Adaptation and Improvement, Kaifeng, China Correspondence David Galbraith, School of Plant Sciences, BIO5 Institute, Arizona Cancer Center, Department of Biomedical Engineering, University of Arizona, Tucson, AZ, 85721. Email: galbraith@email.arizona.edu Contribution: Conceptualization, Writing - original draft, Writing - review & editingSearch for more papers by this authorJoão Loureiro, João Loureiro orcid.org/0000-0002-9068-3954 Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Coimbra, Portugal Contribution: Conceptualization, Writing - original draft, Writing - review & editingSearch for more papers by this authorIoanna Antoniadi, Ioanna Antoniadi Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå, Sweden Contribution: Writing - review & editingSearch for more papers by this authorJillian Bainard, Jillian Bainard Swift Current Research and Development Centre, Agriculture and Agri-Food Canada, Swift Current, Saskatchewan, Canada Contribution: Writing - review & editingSearch for more papers by this authorPetr Bureš, Petr Bureš Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno, CZ, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorPetr Cápal, Petr Cápal Institute of Experimental Botany of the Czech Academy of Sciences, Olomouc, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorMariana Castro, Mariana Castro Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Coimbra, Portugal Contribution: Writing - review & editingSearch for more papers by this authorSílvia Castro, Sílvia Castro Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Coimbra, Portugal Contribution: Writing - review & editingSearch for more papers by this authorMartin Čertner, Martin Čertner Department of Botany, Faculty of Science, Charles University, Prague, Czech Republic Czech Academy of Sciences, Institute of Botany, Průhonice, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorDora Čertnerová, Dora Čertnerová Department of Botany, Faculty of Science, Charles University, Prague, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorZuzana Chumová, Zuzana Chumová Department of Botany, Faculty of Science, Charles University, Prague, Czech Republic Czech Academy of Sciences, Institute of Botany, Průhonice, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorJaroslav Doležel, Jaroslav Doležel Institute of Experimental Botany of the Czech Academy of Sciences, Olomouc, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorDebora Giorgi, Debora Giorgi Green Biotechnology Laboratory, Biotechnology and Agroindustry Division, Casaccia Research Center, ENEA - Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Rome, Italy Contribution: Writing - review & editingSearch for more papers by this authorBrian C. Husband, Brian C. Husband Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada Contribution: Writing - review & editingSearch for more papers by this authorFilip Kolář, Filip Kolář Department of Botany, Faculty of Science, Charles University, Prague, Czech Republic Czech Academy of Sciences, Institute of Botany, Průhonice, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorPetr Koutecký, Petr Koutecký Department of Botany, Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorPaul Kron, Paul Kron orcid.org/0000-0002-1734-5019 Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada Contribution: Writing - review & editingSearch for more papers by this authorIlia J. Leitch, Ilia J. Leitch Department of Comparative Plant and Fungal Biology, Royal Botanic Gardens, Richmond, UK Contribution: Writing - review & editingSearch for more papers by this authorKarin Ljung, Karin Ljung Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå, Sweden Contribution: Writing - review & editingSearch for more papers by this authorSara Lopes, Sara Lopes Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Coimbra, Portugal Contribution: Writing - review & editingSearch for more papers by this authorMagdalena Lučanová, Magdalena Lučanová Czech Academy of Sciences, Institute of Botany, Průhonice, Czech Republic Department of Botany, Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorSergio Lucretti, Sergio Lucretti Green Biotechnology Laboratory, Biotechnology and Agroindustry Division, Casaccia Research Center, ENEA - Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Rome, Italy Contribution: Writing - review & editingSearch for more papers by this authorWen Ma, Wen Ma School of Plant Sciences, BIO5 Institute, Arizona Cancer Center, Department of Biomedical Engineering, University of Arizona, Tucson, Arizona, USA State Key Laboratory of Cotton Biology, Key Laboratory of Plant Stress Biology, Henan University, School of Life Sciences, State Key Laboratory of Crop Stress Adaptation and Improvement, Kaifeng, China Contribution: Writing - review & editingSearch for more papers by this authorSusanne Melzer, Susanne Melzer Clinical Trial Centre Leipzig, University Leipzig, Leipzig, Germany LIFE-Leipzig Research Center for Civilization Diseases, University of Leipzig, Leipzig, Germany Contribution: Writing - review & editingSearch for more papers by this authorIstván Molnár, István Molnár Institute of Experimental Botany of the Czech Academy of Sciences, Olomouc, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorOndřej Novák, Ondřej Novák Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå, Sweden Laboratory of Growth Regulators, Institute of Experimental Botany of the Czech Academy of Sciences and Faculty of Science of Palacký University, Olomouc, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorNicole Poulton, Nicole Poulton Center for Aquatic Cytometry, Bigelow Laboratory for Ocean Sciences, East Boothbay, Maine, USA Contribution: Writing - review & editingSearch for more papers by this authorVladimír Skalický, Vladimír Skalický Laboratory of Growth Regulators, Institute of Experimental Botany of the Czech Academy of Sciences and Faculty of Science of Palacký University, Olomouc, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorElwira Sliwinska, Elwira Sliwinska Laboratory of Molecular Biology and Cytometry, Department of Agricultural Biotechnology, UTP University of Science and Technology, Bydgoszcz, Poland Contribution: Writing - review & editingSearch for more papers by this authorPetr Šmarda, Petr Šmarda Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno, CZ, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorTyler W. Smith, Tyler W. Smith Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada Contribution: Writing - review & editingSearch for more papers by this authorGuiling Sun, Guiling Sun School of Plant Sciences, BIO5 Institute, Arizona Cancer Center, Department of Biomedical Engineering, University of Arizona, Tucson, Arizona, USA State Key Laboratory of Cotton Biology, Key Laboratory of Plant Stress Biology, Henan University, School of Life Sciences, State Key Laboratory of Crop Stress Adaptation and Improvement, Kaifeng, China Contribution: Writing - review & editingSearch for more papers by this authorPedro Talhinhas, Pedro Talhinhas LEAF, Linking Landscape, Environment, Agriculture and Food, Instituto Superior de Agronomia, Universidade de Lisboa, Lisbon, Portugal Contribution: Writing - review & editingSearch for more papers by this authorAttila Tárnok, Attila Tárnok LIFE-Leipzig Research Center for Civilization Diseases, University of Leipzig, Leipzig, Germany Department of Precision Instruments, Tsinghua University, Beijing, China Department for Therapy Validation, Fraunhofer Institute for Cell Therapy and Immunology IZI, Leipzig, Germany Contribution: Writing - review & editingSearch for more papers by this authorEva M. Temsch, Eva M. Temsch Department of Botany and Biodiversity Research, University of Vienna, Vienna, Austria Contribution: Writing - review & editingSearch for more papers by this authorPavel Trávníček, Pavel Trávníček Czech Academy of Sciences, Institute of Botany, Průhonice, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorTomáš Urfus, Tomáš Urfus Department of Botany, Faculty of Science, Charles University, Prague, Czech Republic Contribution: Writing - review & editingSearch for more papers by this author David Galbraith, Corresponding Author David Galbraith galbraith@email.arizona.edu orcid.org/0000-0003-4020-1635 School of Plant Sciences, BIO5 Institute, Arizona Cancer Center, Department of Biomedical Engineering, University of Arizona, Tucson, Arizona, USA State Key Laboratory of Cotton Biology, Key Laboratory of Plant Stress Biology, Henan University, School of Life Sciences, State Key Laboratory of Crop Stress Adaptation and Improvement, Kaifeng, China Correspondence David Galbraith, School of Plant Sciences, BIO5 Institute, Arizona Cancer Center, Department of Biomedical Engineering, University of Arizona, Tucson, AZ, 85721. Email: galbraith@email.arizona.edu Contribution: Conceptualization, Writing - original draft, Writing - review & editingSearch for more papers by this authorJoão Loureiro, João Loureiro orcid.org/0000-0002-9068-3954 Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Coimbra, Portugal Contribution: Conceptualization, Writing - original draft, Writing - review & editingSearch for more papers by this authorIoanna Antoniadi, Ioanna Antoniadi Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå, Sweden Contribution: Writing - review & editingSearch for more papers by this authorJillian Bainard, Jillian Bainard Swift Current Research and Development Centre, Agriculture and Agri-Food Canada, Swift Current, Saskatchewan, Canada Contribution: Writing - review & editingSearch for more papers by this authorPetr Bureš, Petr Bureš Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno, CZ, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorPetr Cápal, Petr Cápal Institute of Experimental Botany of the Czech Academy of Sciences, Olomouc, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorMariana Castro, Mariana Castro Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Coimbra, Portugal Contribution: Writing - review & editingSearch for more papers by this authorSílvia Castro, Sílvia Castro Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Coimbra, Portugal Contribution: Writing - review & editingSearch for more papers by this authorMartin Čertner, Martin Čertner Department of Botany, Faculty of Science, Charles University, Prague, Czech Republic Czech Academy of Sciences, Institute of Botany, Průhonice, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorDora Čertnerová, Dora Čertnerová Department of Botany, Faculty of Science, Charles University, Prague, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorZuzana Chumová, Zuzana Chumová Department of Botany, Faculty of Science, Charles University, Prague, Czech Republic Czech Academy of Sciences, Institute of Botany, Průhonice, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorJaroslav Doležel, Jaroslav Doležel Institute of Experimental Botany of the Czech Academy of Sciences, Olomouc, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorDebora Giorgi, Debora Giorgi Green Biotechnology Laboratory, Biotechnology and Agroindustry Division, Casaccia Research Center, ENEA - Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Rome, Italy Contribution: Writing - review & editingSearch for more papers by this authorBrian C. Husband, Brian C. Husband Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada Contribution: Writing - review & editingSearch for more papers by this authorFilip Kolář, Filip Kolář Department of Botany, Faculty of Science, Charles University, Prague, Czech Republic Czech Academy of Sciences, Institute of Botany, Průhonice, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorPetr Koutecký, Petr Koutecký Department of Botany, Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorPaul Kron, Paul Kron orcid.org/0000-0002-1734-5019 Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada Contribution: Writing - review & editingSearch for more papers by this authorIlia J. Leitch, Ilia J. Leitch Department of Comparative Plant and Fungal Biology, Royal Botanic Gardens, Richmond, UK Contribution: Writing - review & editingSearch for more papers by this authorKarin Ljung, Karin Ljung Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå, Sweden Contribution: Writing - review & editingSearch for more papers by this authorSara Lopes, Sara Lopes Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Coimbra, Portugal Contribution: Writing - review & editingSearch for more papers by this authorMagdalena Lučanová, Magdalena Lučanová Czech Academy of Sciences, Institute of Botany, Průhonice, Czech Republic Department of Botany, Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorSergio Lucretti, Sergio Lucretti Green Biotechnology Laboratory, Biotechnology and Agroindustry Division, Casaccia Research Center, ENEA - Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Rome, Italy Contribution: Writing - review & editingSearch for more papers by this authorWen Ma, Wen Ma School of Plant Sciences, BIO5 Institute, Arizona Cancer Center, Department of Biomedical Engineering, University of Arizona, Tucson, Arizona, USA State Key Laboratory of Cotton Biology, Key Laboratory of Plant Stress Biology, Henan University, School of Life Sciences, State Key Laboratory of Crop Stress Adaptation and Improvement, Kaifeng, China Contribution: Writing - review & editingSearch for more papers by this authorSusanne Melzer, Susanne Melzer Clinical Trial Centre Leipzig, University Leipzig, Leipzig, Germany LIFE-Leipzig Research Center for Civilization Diseases, University of Leipzig, Leipzig, Germany Contribution: Writing - review & editingSearch for more papers by this authorIstván Molnár, István Molnár Institute of Experimental Botany of the Czech Academy of Sciences, Olomouc, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorOndřej Novák, Ondřej Novák Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå, Sweden Laboratory of Growth Regulators, Institute of Experimental Botany of the Czech Academy of Sciences and Faculty of Science of Palacký University, Olomouc, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorNicole Poulton, Nicole Poulton Center for Aquatic Cytometry, Bigelow Laboratory for Ocean Sciences, East Boothbay, Maine, USA Contribution: Writing - review & editingSearch for more papers by this authorVladimír Skalický, Vladimír Skalický Laboratory of Growth Regulators, Institute of Experimental Botany of the Czech Academy of Sciences and Faculty of Science of Palacký University, Olomouc, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorElwira Sliwinska, Elwira Sliwinska Laboratory of Molecular Biology and Cytometry, Department of Agricultural Biotechnology, UTP University of Science and Technology, Bydgoszcz, Poland Contribution: Writing - review & editingSearch for more papers by this authorPetr Šmarda, Petr Šmarda Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno, CZ, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorTyler W. Smith, Tyler W. Smith Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada Contribution: Writing - review & editingSearch for more papers by this authorGuiling Sun, Guiling Sun School of Plant Sciences, BIO5 Institute, Arizona Cancer Center, Department of Biomedical Engineering, University of Arizona, Tucson, Arizona, USA State Key Laboratory of Cotton Biology, Key Laboratory of Plant Stress Biology, Henan University, School of Life Sciences, State Key Laboratory of Crop Stress Adaptation and Improvement, Kaifeng, China Contribution: Writing - review & editingSearch for more papers by this authorPedro Talhinhas, Pedro Talhinhas LEAF, Linking Landscape, Environment, Agriculture and Food, Instituto Superior de Agronomia, Universidade de Lisboa, Lisbon, Portugal Contribution: Writing - review & editingSearch for more papers by this authorAttila Tárnok, Attila Tárnok LIFE-Leipzig Research Center for Civilization Diseases, University of Leipzig, Leipzig, Germany Department of Precision Instruments, Tsinghua University, Beijing, China Department for Therapy Validation, Fraunhofer Institute for Cell Therapy and Immunology IZI, Leipzig, Germany Contribution: Writing - review & editingSearch for more papers by this authorEva M. Temsch, Eva M. Temsch Department of Botany and Biodiversity Research, University of Vienna, Vienna, Austria Contribution: Writing - review & editingSearch for more papers by this authorPavel Trávníček, Pavel Trávníček Czech Academy of Sciences, Institute of Botany, Průhonice, Czech Republic Contribution: Writing - review & editingSearch for more papers by this authorTomáš Urfus, Tomáš Urfus Department of Botany, Faculty of Science, Charles University, Prague, Czech Republic Contribution: Writing - review & editingSearch for more papers by this author First published: 04 January 2021 https://doi.org/10.1002/cyto.a.24295Citations: 5 David Galbraith and João Loureiro contributed equally to this work. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Flow cytometry (FCM) and flow cytometric sorting (FCS) systems have developed as experimental tools of remarkable power and are enjoying an ever-increasing impact in the general field of biology.1 Application of these tools to plant biology has developed more slowly given that the natural form of plants infrequently resembles that of the single cell suspension, prototypically the hematopoietic system that drove the original development of FCM/FCS. Nevertheless, these systems have had a profound influence at all levels of plant biology, from the study of single cells and subcellular organelles, to the behavior of populations of plants, and ultimately to the performance of ecosystems. It is safe to say their impact has not plateaued, as further applications of this unique technology are increasingly developed by innovative scientists around the world to address questions both in the basic sciences, and to increasingly confront emerging problems in the applied sector. For example, in addressing the challenges of sustainable production of sufficient food resources based on plant breeding involving ploidy-based approaches (e.g., induction of polyploidy)2 for the needs of our future global citizens, FCM, and FCS systems will play central roles in this effort. The degree to which FCM and FCS systems have impacted plant biology and applied agricultural sciences must not be understated. The major applications of DNA FCM are ploidy level and genome size estimations, and cell cycle analysis/endoreplication (with the later included in a lower percentage of studies). Indeed, FCM is currently/extensively and almost exclusively employed as the method of choice for measurement of plant genome sizes.3, 4 Measurements of this type impact agriculture in terms of ploidy estimation, with applications ranging from plant biotechnology, breeding and seed quality testing to taxonomy and population biology. They also impact the fundamental plant sciences in terms of biosystematics, ecology, evolution, genomics, and conservation, among other applications. One of the most startling observations of the angiosperms is the bandwidth occupied by genome size, which spans almost 2400-fold. Flow sorting of higher plant chromosomes has provided invaluable information regarding the organization of DNA sequences within plant species. It has also greatly facilitated the process of whole-genome sequencing by permitting subdivision of large genomes into samples comprising entire chromosomes or chromosome arms.5 FCS methods applied to wall-less cells (protoplasts) expressing fluorescent proteins (FPs) in a cell type-specific manner have allowed elucidation of patterns of co-regulated gene expression and plant hormone gradients identification6, 7 within organized tissues, such as roots.8, 9 The trigger to develop this virtual issue came from the publication, in 2017, of an article entitled “Guidelines for the use of flow cytometry and cell sorting in immunological studies” in the European Journal of Immunology.10 As noted in that article, one of the advantages of FCM/FCS systems is that they are relatively simple to implement, with some qualifications, which coupled with the development of user-friendly devices and software during the last 15 years led to increasing applications in other areas, such as plant sciences. However, it is also simple to implement and operate the instruments inappropriately. This calls for a comprehensive and collective summary of the best practices when applying FCM/FCS to plants, as was done for immunology. The first consideration addresses the problem that plants, particularly the vascular plants, in their commonly recognized and utilized forms, exist not as single cell suspensions (typical of immunology) but as complex three-dimensional tissues comprising cells of irregular shapes, different types and functions, that collectively cooperate to produce the final plant form. Optimal methods for producing suspensions of cells, subcellular organelles and other components appropriate for FCM/FCS from these plant tissues and organs, are therefore one of the challenges discussed in this virtual issue. We are fully aware of the mantra that “junk in equals junk out” and having samples of the highest quality prior to FCM/FCS is a critical concern we also addressed here. The second consideration relates to the vast variety of different plant species found globally, and the recognition of the consummate ability of plants to produce secondary metabolites/products, affecting DNA staining and resulting fluorescence. Again, methods for recognizing and handling the different challenges provided to FCM/FCS methods by the biochemistries of the source samples are required. The third consideration focuses on the problem of addressing the non-critical application of FCM/FCS methods developed for mammalian cell systems (typically hematopoietic) to plants without careful consideration of their appropriateness. As it will be detailed in this virtual issue, application of FCM/FCS methods to mammalian cell systems almost exclusively occurs in the context of analysis of samples that comprise a majority, often close to 100%, of single cells in suspension. For plants, particularly when using these instruments and methods for the analysis of organelles in tissue homogenates, the objects of interest comprise a very minor subpopulation of the total particles passing through the instrument. Concepts such as placing initial gates around populations defined by forward scatter (FS) versus side scatter (SS), as routinely used to define leukocytes or other mammalian cells in culture, are at best meaningless and at worst can seriously hamper proper use of the instruments to provide meaningful results. Again, plants are sources of many forms of autofluorescence; in vascular plants, chloroplasts are intensely fluorescent in the red due to the presence of chlorophyll. Phycoerythrin, a red protein-pigment complex from the light-harvesting phycobiliprotein family found in red algae and cryptophytes, is commercially employed as a fluorescent label for antibodies in cytometry. The presence of autofluorescence can restrict the wavelength bandwidths for fluorescence excitation and emission, and this can affect how best to set up FCM/FCS instruments. In order to define and enunciate best practices, we drew together a network of volunteer authors, experienced in the application of FCM/FCS to plants. We have attempted to make this network as comprehensive as possible, to allow recommendations spanning all relevant life-forms, from the simplest photosynthetic microbes, to the more complex lower and vascular plants, and encompassing also the fungi. In this endeavor, we gratefully acknowledge the support of Wiley and Attila Tarnok, EIC of Cytometry. As indicated for the Guidelines in Immunology article,9 we do wish to keep our recommendations updated. Therefore, please send us your critical comments, new ideas, practical suggestions regarding best practices, and new articles that could be useful for possible future versions of this virtual issue. To end, we would like to remember that this virtual issue reflects the vision and dream of the late Jan Suda. He has been an inspiration for all of us, and, most certainly, he left us too soon. We are sure that his legacy will persist, not only in his home country, the Czech Republic, but also across the world. We sincerely hope this virtual issue of Cytometry Part A provides an appropriate tribute. 1 SETTING THE STAGE Describing how FCCS can be optimally applied to plants requires information in two general areas (a) concerning the samples being prepared and analyzed, in our case focusing on the relevant physical features of plants as organisms, and (b) concerning the instrumentation being used for this analysis, centering on sample requirements imposed by engineering design and implementation. 2 VASCULAR AND NONVASCULAR PLANTS Green plants (Viridiplantae) constitute a monophyletic clade within the tree of life and comprise oxygenic photosynthetic eukaryotes.11 The group encompasses green algae and land plants, and further splits into major clades: the Chlorophyta,12 comprising only algae, and Streptophyta formed by several algal groups (such as Zygnematophyceae and Charophyceae;,)13, 14 and the land plants (Embryophyta). Land plants further split into several groups: the possibly paraphyletic assemblage of three bryophyte lineages (Bryophyta—mosses, Marchantiophyta—liverworts, and Anthocerotophyta—hornworts) and three sequentially-splitting lineages of vascular plants: lycopods (Lycopodiophyta), ferns and horsetails (Monilophyta) and seed plants (Spermatophyta). The latter group further splits into gymnosperms (Gymnospermae; i.e., conifers, cycads, Ginkgo, and gnetophytes) and angiosperms (Angiospermae;).15-17 The current review is primarily but not exclusively focused on flow cytometric applications in flowering plants, as they represent the most diverse and economically important, and therefore best studied, group of green plants. However, we mention the other green plant lineages where necessary and we also include other organisms that are found in various parts of the Tree of Life (algae in the traditional sense, fungi) and that share certain features of body organization and life style with plants (such as complex tissues or photosynthesis), and have for a long time

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.

The dinoflagellate Alexandrium fundyense is a common, recurring harmful algal bloom (HAB) species in the Gulf of Maine. To date, most physiological measurements of phytoplankton in the field provide data on the entire community, yet efforts to obtain species-specific data are particularly important for understanding the ecological and physiological dynamics of HAB species, such as, Alexandrium. Alexandrium spp., do not usually dominate the planktonic community in the Gulf of Maine, but are of great interest due to the potent toxins produced. In order to determine the nutritional status of Alexandrium spp. in natural populations, indicators of nutrient deprivation need to be identified that are specific to that one species. To date, the saxitoxin content of A. fundyense is known to vary under different environmental conditions such as nitrogen and phosphorous limitation. However, in batch culture the composition of the toxin (the relative amounts of each saxitoxin derivative per cell) appears to be a stable quantity and thus is sometimes viewed as a biochemical marker of individual strains. In more recent studies, toxin composition has been shown to vary during progressive N- and P- limitation, once the cells are given time to achieve steady state in semi-continuous, nutrient-limited cultures. Using both the absolute toxin concentrations and relative proportion (mole % total toxin) of each toxin derivative, N- and P-limitation can be distinguished based on the observed trends in the different saxitoxin derivatives. In this study, we examine the toxin content and composition in natural A. fundyense populations during a spring bloom in Casco Bay, ME from April–June of 1998. This allows us to examine whether A. fundyense populations in the western Gulf of Maine are sufficiently homogenous to permit the detection of toxin composition and toxin content differences through time and space, and if so, to determine whether those changes are indicative of a particular nutritional state (e.g., N-limitation). Using both toxin composition and toxin ratios determined from field samples during an A. fundyense bloom, the ratios generally correlated with N-limitation in the Casco Bay region.

A massive outbreak of Karenia brevis that had been ongoing for several months along the southwestern coast of Florida was sampled in early September 2005 off Sanibel Island to assess the utility of bio-optical features and ataxonomic analysis (quantification of eukaryotic and cyanobacterial picoplankton) by flow cytometry in monitoring red tide blooms. Sea-surface sampling followed aircraft visual location of discolored water. Within the most concentrated area of the bloom, chlorophyll a values exceeded 500 μg l−1, and concentrations of nitrate (0.3 μM ± 0.0) and ammonium (<0.2 μM) were depleted compared to high concentrations of total dissolved nitrogen, total dissolved phosphorus, and soluble reactive phosphorus (141 ± 34 μM, 16.5 ± 2.5 μM, and 6.44 ± 0.57 μM, respectively). Low water clarity in the bloom (Secchi depth transparency 0.3 m, Kd estimated at 4.83 m−1) was strongly influenced by attenuation from dinoflagellates as well as chromophoric dissolved organic matter (CDOM). The fact that the K. brevis bloom occurred in lower-salinity (30 psu), high-nutrient waters implicates riverine transport of land-based nutrients as a source of nutrient supplies that fueled or sustained the bloom. Throughout ongoing efforts to advance modeling and technological capabilities that presently lack reliable predictive capability, bio-optical remote sensing via aerial flyovers along with in-water sensor data can continue to provide accurate coverage of relatively large temporal and spatial features. Flow cytometry can provide conservative (because of some cell lysis), rapid, near-real-time validation of bloom components. The concentration and position of the organisms, along with water mass scalars, can also help to diagnose factors promoting K. brevis bloom development and dispersion.

The marine lithospheric subsurface is one of the largest biospheres on Earth; however, little is known about the identity and ecological function of microorganisms found in low abundance in this habitat, though these organisms impact global-scale biogeochemical cycling. Here, we describe the diversity and metabolic potential of sediment and endolithic (within rock) microbial communities found in ultrasmall amounts (101 to 104 cells cm-3) in the subsurface of the Atlantis Massif, an oceanic core complex on the Mid-Atlantic Ridge that was sampled on International Ocean Discovery Program (IODP) Expedition 357. This study used fluorescence-activated cell sorting (FACS) to enable the first amplicon, metagenomic, and single-cell genomic study of the shallow (<20 m below seafloor) subsurface of an actively serpentinizing marine system. The shallow subsurface biosphere of the Atlantis Massif was found to be distinct from communities observed in the nearby Lost City alkaline hydrothermal fluids and chimneys, yet similar to other low-temperature, aerobic subsurface settings. Genes associated with autotrophy were rare, although heterotrophy and aerobic carbon monoxide and formate cycling metabolisms were identified. Overall, this study reveals that the shallow subsurface of an oceanic core complex hosts a biosphere that is not fueled by active serpentinization reactions and by-products. IMPORTANCE The subsurface rock beneath the ocean is one of the largest biospheres on Earth, and microorganisms within influence global-scale nutrient cycles. This biosphere is difficult to study, in part due to the low concentrations of microorganisms that inhabit the vast volume of the marine lithosphere. In spite of the global significance of this biosphere, little is currently known about the microbial ecology of such rock-associated microorganisms. This study describes the identity and genomic potential of microorganisms in the subsurface rock and sediment at the Atlantis Massif, an underwater mountain near the Mid-Atlantic Ridge. To enable our analyses, fluorescence-activated cell sorting (FACS) was used as a means to concentrate cells from low biomass environmental samples for genomic analyses. We found distinct rock-associated microorganisms and found that the capacity for microorganisms to utilize organic carbon was the most prevalent form of carbon cycling. We additionally identified a potential role for carbon monoxide metabolism in the subsurface.

Coccolithophores are typically thought of as photoautotrophs, yet a few genera inhabit sub-euphotic environments with insufficient light for photosynthesis, suggesting that other carbon acquisition strategies are likely. Field experiments were performed in the northwest Atlantic (a region with potentially abundant coccolithophores). Phytoplankton populations were incubated with 14 C-labeled dissolved organic carbon (DOC) compounds, acetate, mannitol, and glycerol. Coccolithophores were sorted from these populations 24 hours later using flow cytometry, and DOC uptake was measured. DOC uptake rates were as high as 10 −15 moles cell −1 day −1 , slow relative to photosynthesis rates (10 −12 moles cell −1 day −1 ). Growth rates on the organic compounds were low, suggesting that osmotrophy plays more of a survival strategy in low-light situations. Assimilated DOC was found in both particulate organic carbon and calcite coccoliths (particulate inorganic carbon), suggesting that osmotrophic uptake of DOC into coccolithophore calcite is a small but notable part of the biological carbon pump and alkalinity pump paradigms.

Satellite remote sensing is a powerful tool to monitor the global dynamics of marine plankton. Previous research has focused on developing models to predict the size or taxonomic groups of phytoplankton. Here, we present an approach to identify community types from a global plankton network that includes phytoplankton and heterotrophic protists and to predict their biogeography using global satellite observations. Six plankton community types were identified from a co-occurrence network inferred using a novel rDNA 18 S V4 planetary-scale eukaryotic metabarcoding dataset. Machine learning techniques were then applied to construct a model that predicted these community types from satellite data. The model showed an overall 67% accuracy in the prediction of the community types. The prediction using 17 satellite-derived parameters showed better performance than that using only temperature and/or the concentration of chlorophyll a. The constructed model predicted the global spatiotemporal distribution of community types over 19 years. The predicted distributions exhibited strong seasonal changes in community types in the subarctic-subtropical boundary regions, which were consistent with previous field observations. The model also identified the long-term trends in the distribution of community types, which suggested responses to ocean warming.

High-throughput sequencing of ocean biomes has revealed vast eukaryotic microbial diversity, a significant proportion of which remains uncharacterized. Here we use a temporal approach to understanding eukaryotic diversity at the Scripps Pier, La Jolla, California, USA, via high-throughput amplicon sequencing of the 18S rRNA gene, the abundances of both Synechococcus and Synechococcus grazers, and traditional oceanographic parameters. We also exploit our ability to track operational taxonomic units (OTUs) temporally to evaluate the ability of 18S sequence-based OTU assignments to meaningfully reflect ecological dynamics. The eukaryotic community is highly dynamic in terms of both species richness and composition, although proportional representation of higher-order taxa remains fairly consistent over time. Synechococcus abundance fluctuates throughout the year. OTUs unique to dates of Synechococcus blooms and crashes or enriched in Synechococcus addition incubation experiments suggest that the prasinophyte Tetraselmis sp. and Gymnodinium-like dinoflagellates are likely Synechococcus grazers under certain conditions, and may play an important role in their population fluctuations.

Cell abundances of Prochlorococcus, Synechococcus, and autotrophic picoeukaryotes were estimated in surface waters using principal component analysis (PCA) of hyperspectral and multispectral remote-sensing reflectance data. This involved the development of models that employed multilinear correlations between cell abundances across the Atlantic Ocean and a combination of PCA scores and sea surface temperatures. The models retrieve high Prochlorococcus abundances in the Equatorial Convergence Zone and show their numerical dominance in oceanic gyres, with decreases in Prochlorococcus abundances towards temperate waters where Synechococcus flourishes, and an emergence of picoeukaryotes in temperate waters. Fine-scale in-situ sampling across ocean fronts provided a large dynamic range of measurements for the training dataset, which resulted in the successful detection of fine-scale Synechococcus patches. Satellite implementation of the models showed good performance (R2 > 0.50) when validated against in-situ data from six Atlantic Meridional Transect cruises. The improved relative performance of the hyperspectral models highlights the importance of future high spectral resolution satellite instruments, such as the NASA PACE mission's Ocean Color Instrument, to extend our spatiotemporal knowledge about ecologically relevant phytoplankton assemblages.

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 (~10 3 cells 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 including Candidatus Desulforudis 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 −1 h −1 . Scaled to volume, this equates to a bulk environmental rate of ~10 3 pmol sulfate L −1 d −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 to Ca. Desulforudis audaxviator.

Discriminating infected from healthy cells is the first step to understanding the mechanisms and ecological implications of viral infection. We have developed a method for detecting, sorting, and performing molecular analysis of individual, infected cells of the important microalga Emiliania huxleyi, based on known physiological responses to viral infection. Of three fluorescent dyes tested, FM 1-43 (for detecting membrane blebbing) gave the most unequivocal and earliest separation of cells. Furthermore, we were able to amplify the genomes of single infected cells using Multiple Displacement Amplification. This novel method to reliably discriminate infected from healthy cells in cultures will allow researchers to answer numerous questions regarding the mechanisms and implications of viral infection of E. huxleyi. The method may be transferable to other virus-host systems.

Many optical and biogeochemical data sets, crucial for algorithm development and satellite data validation, are collected using underway seawater systems over the course of research cruises. Phytoplankton and particle size distribution (PSD) in the ocean is a key measurement, required in oceanographic research and ocean optics. Using a data set collected in the North Atlantic, spanning different oceanic water types, we outline the differences observed in concurrent samples collected from two different flow-through systems: a permanently plumbed science seawater supply with an impeller pump, and an independent system with shorter, clean tubing runs and a diaphragm pump. We observed an average of 40% decrease in phytoplankton counts, and significant changes to the PSD in 10-45 µm range, when comparing impeller and diaphragm pump systems. Change in PSD seems to be more dependent on the type of the phytoplankton, than the size, with photosynthetic ciliates displaying the largest decreases in cell counts (78%). Comparison of chlorophyll concentrations across the two systems demonstrated lower sensitivity to sampling system type. Observed changes in several measured biogeochemical parameters (associated with phytoplankton size distribution) using the two sampling systems, should be used as a guide towards building best practices when it comes to the deployment of flow-through systems in the field for examining optics and biogeochemistry. Using optical models, we evaluated potential impact of the observed change in measured phytoplankton size spectra onto scattering measurements, resulting in significant differences between modeled optical properties across systems (~40%). Researchers should be aware of the methods used with previously collected data sets, and take into consideration the potentially significant and highly variable ecosystem-dependent biases in designing field studies in the future.

Planktonic photosynthetic organisms of the class Mamiellophyceae include the smallest eukaryotes (less than 2 µm), are globally distributed and form the basis of coastal marine ecosystems. Eight complete fully annotated 13–22 Mb genomes from three genera, Ostreococcus , Bathycoccus and Micromonas , are available from previously isolated clonal cultured strains and provide an ideal resource to explore the scope and challenges of analysing single cell amplified genomes (SAGs) isolated from a natural environment. We assembled data from 12 SAGs sampled during the Tara Oceans expedition to gain biological insights about their in situ ecology, which might be lost by isolation and strain culture. Although the assembled nuclear genomes were incomplete, they were large enough to infer the mating types of four Ostreococcus SAGs. The systematic occurrence of sequences from the mitochondria and chloroplast, representing less than 3% of the total cell's DNA, intimates that SAGs provide suitable substrates for detection of non-target sequences, such as those of virions. Analysis of the non-Mamiellophyceae assemblies, following filtering out cross-contaminations during the sequencing process, revealed two novel 1.6 and 1.8 kb circular DNA viruses, and the presence of specific Bacterial and Oomycete sequences suggests that these organisms might co-occur with the Mamiellales. This article is part of a discussion meeting issue ‘Single cell ecology’.

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 575:43-56 (2017) - DOI: https://doi.org/10.3354/meps12212 Zooplankton grazing and egestion shifts particle size distribution in natural communities Karen Stamieszkin1,*, Nicole J. Poulton2, Andrew J. Pershing3 1Darling Marine Center, 193 Clark’s Cove Road, Walpole, Maine 04573, USA 2Bigelow Laboratory for Ocean Sciences, 60 Bigelow Drive, East Boothbay, Maine 04544, USA 3Gulf of Maine Research Institute, 350 Commercial Street, Portland, Maine 04101, USA *Corresponding author: karen.stamieszkin@maine.edu ABSTRACT: Marine plankton communities can be viewed in terms of their size structure rather than their taxonomic composition, revealing how allometric relationships affect the functioning of the community. Oceanic particle size spectra can be used to explain and predict variability in carbon export efficiency, because large particles generally sink faster than small particles. Since plankton trophic interactions impact particle size in the surface ocean, this size-structured view is a useful simplification for connecting plankton ecology with biogeochemistry. We conducted a series of grazing experiments to test the hypothesis that mesozooplankton shift particle size spectra toward larger particles, in a predictable manner that reflects their community size structure, through grazing and egestion of fecal pellets. These experiments were carried out over several months, and used natural communities of mesozooplankton and their microplankton prey collected in the coastal Gulf of Maine. After incubation, we analyzed the samples to determine size distribution and taxonomic information. Our results show that mesozooplankton grazing impacts microplankton in proportion to their abundance. Size relationships between plankton predators and prey that have been established at the individual level do not linearly translate to the community level. Further, while grazing itself does not significantly alter the particle size spectrum, repackaging of prey into mesozooplankton fecal pellets shifts particle size spectra toward larger particles. KEY WORDS: Plankton grazing · Egestion · Particle size distribution · Imaging cytometry · Carbon flux Full text in pdf format PreviousNextCite this article as: Stamieszkin K, Poulton NJ, Pershing AJ (2017) Zooplankton grazing and egestion shifts particle size distribution in natural communities. Mar Ecol Prog Ser 575:43-56. https://doi.org/10.3354/meps12212 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 575. Online publication date: July 20, 2017 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2017 Inter-Research.

The original version of this Article contained errors in the units of concentration of three reagents listed in the Methods. These errors have all been corrected in both the PDF and HTML versions of the Article.

This technical manual guides the user through the process of creating a data table for the submission of taxonomic and morphological information for plankton and other particles from images to a repository. Guidance is provided to produce documentation that should accompany the submission of plankton and other particle data to a repository, describes data collection and processing techniques, and outlines the creation of a data file. Field names include scientificName that represents the lowest level taxonomic classification (e.g., genus if not certain of species, family if not certain of genus) and scientificNameID, the unique identifier from a reference database such as the World Register of Marine Species or AlgaeBase. The data table described here includes the field names associatedMedia, scientificName/ scientificNameID for both automated and manual identification, biovolume, area_cross_section, length_representation and width_representation. Additional steps that instruct the user on how to format their data for a submission to the Ocean Biodiversity Information System (OBIS) are also included. Examples of documentation and data files are provided for the user to follow. The documentation requirements and data table format are approved by both NASA’s SeaWiFS Bio-optical Archive and Storage System (SeaBASS) and the National Science Foundation’s Biological and Chemical Oceanography Data Management Office (BCO-DMO).

Abstract. The ratio of two in situ optical measurements, chlorophyll fluorescence (Chl F) and optical particulate backscattering (bbp), varied with changes in phytoplankton community composition during the North Atlantic Bloom experiment in the Iceland Basin in 2008. Using ship-based measurements of Chl F, bp, chlorophyll a (Chl), HPLC pigments, phytoplankton composition and carbon biomass, we found that oscillations in the ratio varied with changes in plankton community composition; hence we refer to Chl F/bp as an "optical community index". The index varied by more than a factor of two, with low values associated with pico- and nanophytoplankton and high values associated with diatom dominated phytoplankton communities. A Lagrangian mixed-layer float and four Seagliders, operating continuously for two months, made similar measurements of the optical community index and followed the evolution and later demise of the diatom spring bloom. Temporal changes in optical community index and, by implication the transition in community composition from diatom to post-diatom bloom communities, were not simultaneous over the spatial domain surveyed by the ship, float and gliders. Not only phytoplankton biomass, but also community composition was patchy at the submesoscale. The ratio of simple optical properties measured from autonomous platforms, when carefully validated, provides a tool for studying phytoplankton patchiness on extended temporal scales and ecological relevant spatial scales, and should offer new insights into the processes regulating patchiness.

Abstract Recent discoveries suggest that the candidate superphyla Patescibacteria and DPANN constitute a large fraction of the phylogenetic diversity of Bacteria and Archaea. Their small genomes and limited coding potential have been hypothesized to be ancestral adaptations to obligate symbiotic lifestyles. To test this hypothesis, we performed cell-cell association, genomic, and phylogenetic analyses on 4,829 individual cells of Bacteria and Archaea from 46 globally distributed surface and subsurface field samples. This confirmed the ubiquity and abundance of Patescibacteria and DPANN in subsurface environments, the small size of their genomes and cells, and the divergence of their gene content from other Bacteria and Archaea. Our analyses suggest that most Patescibacteria and DPANN in the studied subsurface environments do not form specific physical associations with other microorganisms. These data also suggest that their unusual genomic features and prevalent auxotrophies may be a result of minimal cellular energy transduction mechanisms that potentially precede the evolution of respiration, thus relying solely on fermentation for energy conservation.

Quantum dots (Qdots) are semiconducting nanocrystals composed of periodic elements with different intrinsic band‐gap energies that yield unique fluorescent signatures. Unlike conventional organic fluorophores, Qdots are photo‐chemically stable and have a wide absorption spectrum, but a narrow, tunable emission spectrum. Multiple colors can be imaged from a single excitation wavelength allowing for labeling of many different target sites (e.g., membrane proteins) simultaneously. We conjugated Qdots to primary antibodies specific for the soluble enzyme nitrate reductase (NR) and a light‐harvesting structural protein localized in the chloroplast, the fucoxanthin chlorophyll a/c protein (FCP), in the marine diatom Skeletonema costatum . By fluorescence microscopy, we successfully detected NR and FCP in single cells of S. costatum with a clarity and definition that was not obtainable with conventional organic fluorophores. Biotinylated cells labeled with Qdot‐strepavidin conjugate and Qdot‐FCP immuno‐labeled cells were detected by flow cytometry. Qdot bioconjugates provide an alternative photostable probe for surface or intracellular protein immuno‐localization in the study of marine bacteria and phytoplankton metabolism and physiology.

Abstract : One challenge in phytoplankton ecology is to measure species-specific physiological responses to changes in environmental conditions. Of importance are species such as the toxic dinoflagellate Alexandrium fundyense which inhabit coastal regions and are not usually dominant. The aim of this thesis was to identify physiological and behavioral diagnostics of A. fundyense from the Gulf of Maine, and use these indicators to evaluate field populations. Using a surface-specific monoclonal-antibody two methods, flow cytometry and immunomagnetic bead-separation, were developed to identify and separate A. fundyense. Flow-cytometric techniques successfully detected and separated Alexandrium from co-occurring organisms. Using immunomagnetic-separation live A. fundyense cell were separated from spiked field samples and then used to obtain accurate chlorophyll, protein and biomass estimates. Laboratory water columns, nitrogen-(N)-starved batch cultures, and N-limited, semi-continuous cultures, were used to identify indicators of different N-nutritional states. Prolonged N-stress caused dramatic changes intracellular biochemistry, specifically toxin content and composition, however behavioral modifications were not observed. The indicators, specifically toxin ratios of different derivatives, were then used to examine toxin variability in field populations. Toxin compositional changes did occur that were generally consistent with N-stress as the bloom progressed. Overall, these results suggest that these approaches could provide valuable species-specific physiological information.

The most probable number (MPN) assay is used to enumerate viable phytoplankton in the 10–50 μm size range for Type Approval of ballast water management systems. However, its validation has been questioned by the United States Coast Guard, which does not accept it as an alternative to its required method. To address key elements of method validation, three research laboratories used the same experimental protocols to enumerate viable organisms from uniformly viable and heat-killed cultures of three species of phytoplankton. Statistical analysis of the ratio of MPN estimates of viable organisms to measurements from flow cytometry was consistent with predictions based on perfect adherence to the method’s assumptions. These include reliable detection of all viable organisms and no false-positive results. The validation confirmed established estimates of the MPN method’s precision and demonstrated reproducibility, having been transferred to and implemented by two of the laboratories for the first time.

The sequencing of the Crassostrea virginica genome has brought back the interest for gene delivery and editing methodologies. Here, we report the expression in oyster hemocytes of two heterologous expression vectors under the CMV promoter delivered with dendrimers. Expression was monitored using confocal microscopy, flow cytometry, and immunofluorescence assay. C. virginica hemocytes were able to express the green fluorescence protein and Crassostrea gigas vascular endothelial growth factor under CMV viral promoter both in vivo and in vitro. These results provide the bases for interrogating the genome and adapting genome editing methodologies.

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Abstract Microplastic particles (&lt; 5 mm) are now found throughout earth's ecosystems, with smaller microplastics often showing greater impacts on organismal health than larger ones. Unfortunately, there are no readily available analytical approaches that can couple microplastics enumeration and polymer determination for smaller microplastics (&lt; 10 μ m), and 1–20 μ m particles are difficult to quantify with existing techniques. This study presents a method using Nile red (NR) staining and flow cytometry (FCM) to quantify and isolate small microplastic particles for subsequent identification by pyrolysis gas chromatography–mass spectrometry (pyGCMS). Results using standard plastic particles showed that FCM sorting can provide sufficient material for pyGCMS analyses; the polymer composition remains identifiable after the processing steps. The post‐sorting concentration step yielded recovery of 58%–83% of the original plastic polymer mass. Analysis of a mixed plastic standard solution showed no significant difference in plastic counts obtained by microscopy and FCM, although blank correction reduces the FCM counts to 62% of the microscopy counts. The applicability of NR staining and FCM was demonstrated through analysis of small microplastic particles (5–45 μ m) from Lake Superior surface water samples, which showed particle abundances two to three orders of magnitude higher than particles &gt; 100 μ m that were counted using FTIR microscopy. PyGCMS analysis of a test lake sample showed the presence of polyethylene in this small size fraction. Careful attention to blanks and longer FCM sorting times (&gt; 2 h) are recommended for successful analysis of natural aquatic samples processed by this approach.

The purpose of this document is to provide guidance for establishing and maintaining growth and development of flow cytometry shared resource laboratories. While the best practices offered in this manuscript are not intended to be universal or exhaustive, they do outline key goals that should be prioritized to achieve operational excellence and meet the needs of the scientific community. Additionally, this document provides information on available technologies and software relevant to shared resource laboratories. This manuscript builds on the work of Barsky et al. 2016 published in Cytometry Part A and incorporates recent advancements in cytometric technology. A flow cytometer is a specialized piece of technology that require special care and consideration in its housing and operations. As with any scientific equipment, a thorough evaluation of the location, space requirements, auxiliary resources, and support is crucial for successful operation. This comprehensive resource has been written by past and present members of the International Society for Advancement of Cytometry (ISAC) Shared Resource Laboratory (SRL) Emerging Leaders Program https://isac-net.org/general/custom.asp?page=SRL-Emerging-Leaders with extensive expertise in managing flow cytometry SRLs from around the world in different settings including academia and industry. It is intended to assist in establishing a new flow cytometry SRL, re-purposing an existing space into such a facility, or adding a flow cytometer to an individual lab in academia or industry. This resource reviews the available cytometry technologies, the operational requirements, and best practices in SRL staffing and management.

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.

ABSTRACT Recent advances in single-cell genomic and metagenomic techniques have facilitated the discovery of numerous previously unknown, deep branches of the tree of life that lack cultured representatives. Many of these candidate phyla are composed of microorganisms with minimalistic, streamlined genomes lacking some core metabolic pathways, which may contribute to their resistance to growth in pure culture. Here we analyzed single-cell genomes and metagenome bins to show that the “Candidate phylum SPAM” represents an interesting exception, by having large genomes (6-8 Mbps), high GC content (66%-71%), and the potential for a versatile, mixotrophic metabolism. We also observed an unusually high genomic heterogeneity among individual SPAM cells in the studied samples. These features may have contributed to the limited recovery of sequences of this candidate phylum in prior metagenomic studies. Based on these observations, we propose renaming SPAM to “Candidate phylum Solagigasbacteria”. Current evidence suggests that Solagigasbacteria are distributed globally in diverse terrestrial ecosystems, including soils, the rhizosphere, volcanic mud, oil wells, aquifers and the deep subsurface, with no reports from marine environments to date.

Imaging flow cytometry was first introduced into algal research in the late 1990s. The ability to enumerate, classify and determine biomass of phytoplankton from environmental samples is essential for determining ecosystem function, carbon density, and the role phytoplankton play in both freshwater and marine microbial food webs. Traditional micro-phytoplankton quantification methods use microscopic techniques that require preservation and are slow, tedious and very laborious. The availability of automated imaging microscopy platforms has revolutionized the way particles and cells are detected within their natural environment. The ability to examine cells unaltered and without preservation is key to providing more accurate cell concentration estimates and phytoplankton biomass. Currently many imaging cytometry tools are available for use in aquatic sciences and provide a more rapid and unbiased method for enumerating and classifying phytoplankton within diverse aquatic environments. Imaging flow cytometry, combines the speed and statistical capabilities of flow cytometry with imaging features of microscopy, and has contributed significantly to the advancement of phytoplankton analysis in recent years. This presentation discusses various instrumentation and applications relevant tophytoplankton communities.

Quantum dots (Qdots) are semiconducting nanocrystals composed of periodic elements with different intrin- sic band-gap energies that yield unique fluorescent signatures. Unlike conventional organic fluorophores, Qdots are photo-chemically stable and have a wide absorption spectrum, but a narrow, tunable emission spectrum. Multiple colors can be imaged from a single excitation wavelength allowing for labeling of many different tar- get sites (e.g., membrane proteins) simultaneously. We conjugated Qdots to primary antibodies specific for the soluble enzyme nitrate reductase (NR) and a light- harvesting structural protein localized in the chloroplast, the fucoxanthin chlorophyll a/c protein (FCP), in the marine diatom Skeletonema costatum. By fluorescence microscopy, we successfully detected NR and FCP in single cells of S. costatum with a clarity and definition that was not obtainable with conventional organic fluorophores. Biotinylated cells labeled with Qdot-strepavidin conjugate and Qdot-FCP immuno-labeled cells were detected by flow cytometry. Qdot bioconjugates provide an alternative photostable probe for surface or intracellular protein immuno-localization in the study of marine bacteria and phytoplankton metabolism and physiology.

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.

This protocol describes the sequencing and data quality control for theTaraOceans expedition and is part ofViral to metazoan marine plankton nucleotide sequences from theTaraOceans expedition. Figure 3:Overview of experimental pipeline from nucleic acids to sequences. (Red crosses highlight QC steps where experiments can be stopped.)

This protocol describes thenucleic acids preparations for theTaraOceans expedition and is part ofViral to metazoan marine plankton nucleotide sequences from theTaraOceans expedition. Figure 1: Overview of -omics analysis strategy applied on Tara Oceans samples.

Summary Marine bacteria and archaea play key roles in global biogeochemistry. To improve our understanding of this complex microbiome, we employed single-cell genomics and a randomized, hypothesis-agnostic cell selection strategy to recover 12,715 partial genomes from the tropical and subtropical euphotic ocean. A substantial fraction of known prokaryoplankton coding potential was recovered from a single, 0.4 mL ocean sample, which indicates that genomic information disperses effectively across the globe. Yet, we found each genome to be unique, implying limited clonality within prokaryoplankton populations. Light harvesting and secondary metabolite biosynthetic pathways were numerous across lineages, highlighting the value of single-cell genomics to advance the identification of ecological roles and biotechnology potential of uncultured microbial groups. This genome collection enabled functional annotation and genus-level taxonomic assignments for >80% of individual metagenome reads from the tropical and subtropical surface ocean, thus offering a model to improve reference genome databases for complex microbiomes.

The protocols in this collection are from the Alberti A., et al manuscript (Alberti A. 2017, Scientific Data). These protocols provide detailed procedures applied for genomic data generation, from nucleic acids extraction to sequence production, and we describe registries of genomics datasets available at the European Nucleotide Archive (ENA,www.ebi.ac.uk/ena). This collection complements other efforts to provide a full description of experiments and open science resources generated from theTaraOceans project, further extending their value for the study of the world’s planktonic ecosystems.' From the Methods section: 'The generation of information-rich data from marine plankton samples presents unique challenges that are inherent to the particular sampling conditions at sea and the wide spectrum of organisms included in that environment. All processing steps, including biomass collection, sample preservation, nucleic acids extractions and sequencing library preparation, are critical and require specific protocols and robust methods in order to ensure comparability of results and limit potential biases. Our methods were either developed specifically forTaraOceans samples or carefully selected among existing ones in order to meet the requirements of our sequencing strategy and to produce optimized datasets for downstream bioinformatics analyses, as for example the production of overlapping reads from metagenomics libraries to facilitate assembly. They are presented in five sub-sections, starting with a brief description of how samples were handled between the research vessel and the processing laboratories (protocol 1). Protocol 2reports on DNA and RNA extractions procedures for -omics analyses, including the generation of amplified genomic DNA from uncultured isolated unicellular eukaryotes. The generation of 18S and 16S rRNA amplicons from DNA of specific size-fractions is described in protocol 3 and Illumina libraries preparation in protocol 4. Sequencing procedures and post-sequencing data processing are described in protocol 5. For details on the onboard sampling protocols, see Pesantet al.'

Planktonic photosynthetic organisms of the class Mamiellophyceae include the smallest eukaryotes (less than 2 µm), are globally distributed and form the basis of coastal marine ecosystems. Eight complete fully annotated 13–22 Mb genomes from three genera, <i>Ostreococcus</i>, <i>Bathycoccus</i> and <i>Micromonas</i>, are available from previously isolated clonal cultured strains and provide an ideal resource to explore the scope and challenges of analysing Single Cell Amplified Genomes (SAGs) isolated from a natural environment. We assembled data from 12 SAGs sampled during the Tara Oceans expedition to gain biological insights about their <i>in situ</i> ecology, which might be lost by isolation and strain culture. Although the assembled nuclear genomes were incomplete, they were large enough to infer the mating types of four <i>Ostreococcus</i> SAGs. The systematic occurrence of sequences from the mitochondria and chloroplast, representing less than 3% of the total cell's DNA, intimates that SAGs provide suitable substrates for detection of non-target sequences, such as those of virions. Analysis of the non-Mamiellophyceae assemblies, following filtering out cross-contaminations during the sequencing process, revealed two novel 1.6 and 1.8 kb circular DNA viruses, and the presence of specific Bacterial and Oomycete sequences suggests that these organisms might co-occur with the Mamiellales.This article is part of the discussion meeting issue ‘Single cell ecology’.

Planktonic photosynthetic organisms of the class Mamiellophyceae include the smallest eukaryotes (less than 2 µm), are globally distributed and form the basis of coastal marine ecosystems. Eight complete fully annotated 13–22 Mb genomes from three genera, <i>Ostreococcus</i>, <i>Bathycoccus</i> and <i>Micromonas</i>, are available from previously isolated clonal cultured strains and provide an ideal resource to explore the scope and challenges of analysing Single Cell Amplified Genomes (SAGs) isolated from a natural environment. We assembled data from 12 SAGs sampled during the Tara Oceans expedition to gain biological insights about their <i>in situ</i> ecology, which might be lost by isolation and strain culture. Although the assembled nuclear genomes were incomplete, they were large enough to infer the mating types of four <i>Ostreococcus</i> SAGs. The systematic occurrence of sequences from the mitochondria and chloroplast, representing less than 3% of the total cell's DNA, intimates that SAGs provide suitable substrates for detection of non-target sequences, such as those of virions. Analysis of the non-Mamiellophyceae assemblies, following filtering out cross-contaminations during the sequencing process, revealed two novel 1.6 and 1.8 kb circular DNA viruses, and the presence of specific Bacterial and Oomycete sequences suggests that these organisms might co-occur with the Mamiellales.This article is part of the discussion meeting issue ‘Single cell ecology’.

Planktonic photosynthetic organisms of the class Mamiellophyceae include the smallest eukaryotes (less than 2 µm), are globally distributed and form the basis of coastal marine ecosystems. Eight complete fully annotated 13–22 Mb genomes from three genera, <i>Ostreococcus</i>, <i>Bathycoccus</i> and <i>Micromonas</i>, are available from previously isolated clonal cultured strains and provide an ideal resource to explore the scope and challenges of analysing Single Cell Amplified Genomes (SAGs) isolated from a natural environment. We assembled data from 12 SAGs sampled during the Tara Oceans expedition to gain biological insights about their <i>in situ</i> ecology, which might be lost by isolation and strain culture. Although the assembled nuclear genomes were incomplete, they were large enough to infer the mating types of four <i>Ostreococcus</i> SAGs. The systematic occurrence of sequences from the mitochondria and chloroplast, representing less than 3% of the total cell's DNA, intimates that SAGs provide suitable substrates for detection of non-target sequences, such as those of virions. Analysis of the non-Mamiellophyceae assemblies, following filtering out cross-contaminations during the sequencing process, revealed two novel 1.6 and 1.8 kb circular DNA viruses, and the presence of specific Bacterial and Oomycete sequences suggests that these organisms might co-occur with the Mamiellales.This article is part of the discussion meeting issue ‘Single cell ecology’.

Planktonic photosynthetic organisms of the class Mamiellophyceae include the smallest eukaryotes (less than 2 µm), are globally distributed and form the basis of coastal marine ecosystems. Eight complete fully annotated 13–22 Mb genomes from three genera, <i>Ostreococcus</i>, <i>Bathycoccus</i> and <i>Micromonas</i>, are available from previously isolated clonal cultured strains and provide an ideal resource to explore the scope and challenges of analysing Single Cell Amplified Genomes (SAGs) isolated from a natural environment. We assembled data from 12 SAGs sampled during the Tara Oceans expedition to gain biological insights about their <i>in situ</i> ecology, which might be lost by isolation and strain culture. Although the assembled nuclear genomes were incomplete, they were large enough to infer the mating types of four <i>Ostreococcus</i> SAGs. The systematic occurrence of sequences from the mitochondria and chloroplast, representing less than 3% of the total cell's DNA, intimates that SAGs provide suitable substrates for detection of non-target sequences, such as those of virions. Analysis of the non-Mamiellophyceae assemblies, following filtering out cross-contaminations during the sequencing process, revealed two novel 1.6 and 1.8 kb circular DNA viruses, and the presence of specific Bacterial and Oomycete sequences suggests that these organisms might co-occur with the Mamiellales.This article is part of the discussion meeting issue ‘Single cell ecology’.