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Little is known about viruses in oxygen-deficient water columns (ODWCs). In surface ocean waters, viruses are known to act as gene vectors among susceptible hosts. Some of these genes may have metabolic functions and are thus termed auxiliary metabolic genes (AMGs). AMGs introduced to new hosts by viruses can enhance viral replication and/or potentially affect biogeochemical cycles by modulating key microbial pathways. Here we identify 748 viral populations that cluster into 94 genera along a vertical geochemical gradient in the Cariaco Basin, a permanently stratified and euxinic ocean basin. The viral communities in this ODWC appear to be relatively novel as 80 of these viral genera contained no reference viral sequences, likely due to the isolation and unique features of this system. We identify viral elements that encode AMGs implicated in distinctive processes, such as sulfur cycling, acetate fermentation, signal transduction, [Fe–S] formation, and N-glycosylation. These AMG-encoding viruses include two putative Mu-like viruses, and viral-like regions that may constitute degraded prophages that have been modified by transposable elements. Our results provide an insight into the ecological and biogeochemical impact of viruses oxygen-depleted and euxinic habitats.

Metagenomics is a powerful method for interpreting the ecological roles and physiological capabilities of mixed microbial communities. Yet, many tools for processing metagenomic data are not designed to consider eukaryotes, nor are they built for an increasing amount of sequence data. EukHeist is an automated pipeline to retrieve eukaryotic and prokaryotic metagenome assembled genomes (MAGs) from large-scale metagenomic datasets. We developed the EukHeist workflow to specifically process large amounts of both metagenomic and/or metatranscriptomic sequence data in an automated and reproducible fashion. Here, we applied EukHeist to the large-size fraction data (0.8-2000µm) from Tara Oceans to recover both eukaryotic and prokaryotic MAGs, which we refer to as TOPAZ (Tara Oceans Particle-Associated MAGs). The TOPAZ MAGs consisted of >900 environmentally-relevant eukaryotic MAGs and >4,000 bacterial and archaeal MAGs. The bacterial and archaeal TOPAZ MAGs expand the known marine phylogenetic diversity through the increase in coverage of likely particle- and host-associated taxa. We also demonstrate an approach to infer the putative functional mode of the recovered eukaryotic MAGs. A global survey of the TOPAZ MAGs enabled the identification of ecological cohorts, driven by specific environmental factors, and putative host-microbe associations.ImportanceDespite the ecological importance of single-celled eukaryotic organisms in marine environments, the majority are difficult to cultivate in the lab. Sequencing genetic material extracted from environmental samples enables researchers to document naturally-occurring protistan communities. However, conventional sequencing methodologies cannot separate out the genomes of individual organisms. To more completely capture the entire genomic content of mixed protistan community, we can create bins of sequences that represent the same organism. We developed a pipeline that enables scientists to bin individual organisms out of metagenomic reads, and show results that provide exciting insights into what protistan communities are present in the ocean and what roles they play in the ecosystem. Here, a global survey of both eukaryotic and prokaryotic MAGs enabled the identification of ecological cohorts, driven by specific environmental factors, and putative host-microbe associations. Accessible and scalable computational tools, such as EukHeist, are likely to accelerate the identification of meaningful genetic signatures from large datasets, ultimately expanding the eukaryotic tree of life.

Microbial eukaryotes (or protists) in marine ecosystems are a link between microbial primary producers and all higher trophic levels. The rate at which heterotrophic protistan grazers consume microbial prey and recycle organic matter is an important factor that influences marine microbial food webs and carbon cycling. At deep-sea hydrothermal vents, chemosynthetic bacteria and archaea form the base of a food web that functions in the absence of sunlight, but the role of protistan grazers in these highly productive ecosystems is largely unexplored. Here, we pair grazing experiments with a molecular survey to quantify protistan grazing and to characterize the composition of vent-associated protists in low-temperature venting fluids from Gorda Ridge in the North East (NE) Pacific Ocean. Results reveal protists exert higher predation pressure at vents compared to the surrounding deep seawater environment and may account for consuming 28-62% of the daily stock of prokaryotic biomass within the hydrothermal vent food web. The vent-associated protistan community was more species rich relative to the background deep sea, and patterns in the distribution and co-occurrence of vent microbes provide additional insights into potential predator-prey interactions. Ciliates, followed by dinoflagellates, Syndiniales, rhizaria, and stramenopiles dominated the vent protist community and included bacterivorous species, species known to host symbionts, and parasites. Our findings provide an estimate of protistan grazing pressure within hydrothermal vent food webs, highlighting the role that diverse deep-sea protistan communities have in carbon cycling.SignificanceHeterotrophic protists are ubiquitous in all aquatic ecosystems and represent an important ecological link because they transfer organic carbon from primary producers to higher trophic levels. Here, we quantify the predator-prey trophic interaction among protistan grazers and microbial prey at multiple sites of hydrothermal venting near the Gorda Ridge spreading center in the NE Pacific Ocean. Grazing pressure was higher at the site of active diffuse flow and was carried out by a highly diverse assemblage of protistan species; elevated grazing rates are attributed to higher concentrations of chemosynthetic microorganisms and biological diversity localized to hydrothermal vent environments.

SUMMARYPost-submission note. Since the original submission of this manuscript to bioRxiv, we discovered that some of our results may be impacted by the limitations of some of the comparative genomics tools used in this study. We are working on a revised version of the manuscript.Although horizontal gene transfer is recognized as a major evolutionary process in Bacteria and Archaea, its general patterns remain elusive, due to difficulties tracking genes at relevant resolution and scale within complex microbiomes. To circumvent these challenges, we analyzed a randomized sample of >12,000 genomes of individual cells of Bacteria and Archaea in the tropical and subtropical ocean - a well-mixed, global environment. We found that marine microorganisms form gene exchange networks (GENs) within which transfers of both flexible and core genes are frequent, including the rRNA operon that is commonly used as a conservative taxonomic marker. The data revealed efficient gene exchange among genomes with

Genetic markers and geochemical assays of microbial nitrogen cycling processes, including autotrophic and heterotrophic denitrification, anammox, ammonia oxidation, and nitrite oxidation, were examined across the oxycline, suboxic, and anoxic zones of the Cariaco Basin, Venezuela. Ammonia and nitrite oxidation genes were expressed through the entire gradient. Transcripts associated with autotrophic and heterotrophic denitrifiers were mostly confined to the suboxic zone and below but were also present in particles in the oxycline. Anammox genes and transcripts were detected over a narrow depth range near the bottom of the suboxic zone and coincided with secondary NO2 - maxima and available NH4 + . Dissolved inorganic nitrogen (DIN) amendment incubations and comparisons between our sampling campaigns suggested that denitrifier activity may be closely coupled with NO3 - availability. Expression of denitrification genes at depths of high rates of chemoautotrophic carbon fixation and phylogenetic analyses of nitrogen cycling genes and transcripts indicated a diverse array of denitrifiers, including chemoautotrophs capable of using NO3 - to oxidize reduced sulfur species. Thus, results suggest that the Cariaco Basin nitrogen cycle is influenced by autotrophic carbon cycling in addition to organic matter oxidation and anammox.

The lithified oceanic crust, lower crust gabbros in particular, has remained largely unexplored by microbiologists. Recently, evidence for heterogeneously distributed viable and transcriptionally active autotrophic and heterotrophic microbial populations within low-biomass communities was found down to 750 m below the seafloor at the Atlantis Bank Gabbro Massif, Indian Ocean. Here, we report on the diversity, activity and adaptations of fungal communities in the deep oceanic crust from ~10 to 780 mbsf by combining metabarcoding analyses with mid/high-throughput culturing approaches. Metabarcoding along with culturing indicate a low diversity of viable fungi, mostly affiliated to ubiquitous (terrestrial and aquatic environments) taxa. Ecophysiological analyses coupled with metatranscriptomics point to viable and transcriptionally active fungal populations engaged in cell division, translation, protein modifications and other vital cellular processes. Transcript data suggest possible adaptations for surviving in the nutrient-poor, lithified deep biosphere that include the recycling of organic matter. These active communities appear strongly influenced by the presence of cracks and veins in the rocks where fluids and resulting rock alteration create micro-niches.

The seasonally stratified water column of Saanich Inlet, Vancouver, B.C., Canada provides a natural laboratory for examining how ocean deoxygenation shapes microbial community population dynamics and interactions. Microbial eukaryotes within these communities play significant roles as phototrophs, mixotrophs, symbionts, predators, parasites, and saprotrophs, with population abundances and interactions changing seasonally, and along water column redox gradients. Here, we use time series observations of ribosomal RNA and DNA diversity in combination with co-occurrence correlation and indicator species analyses to observe microbial eukaryote community dynamics over a 12-month period, uncovering significant correlations between parasitic dinoflagellates within Syndiniales and other eukaryotic taxa during months of peak water column stratification. Syndiniales operational taxonomic units (OTUs) were the most abundant indicator OTUs during stratification. Syndiniales OTUs exhibited significant correlations with a broad range of taxa, including members of Apicomplexa, Ciliophora, Dinoflagellata, Chlorophyta, Metazoa and a variety of Stramenopiles including MAST groups. Correlation analyses suggest Syndiniales may influence phytoplankton bloom dynamics in Saanich Inlet as well as abundances of protist and metazoan grazers that are indicator species for peak stratification periods. These observations shed light on the potential role of parasitic protists on organic matter release and bloom termination events during stratification and renewal periods in Saanich Inlet with potential implications for coastal marine foodwebs and fisheries productivity.

A pronounced and relatively stable peak in dark carbon assimilation (DCA) has been observed within the Cariaco Basin's redoxcline over a 19-year observation period, presumably driven largely by sulfur-oxidizing chemoautotrophic bacteria (thioautotrophs). As documented in previous reports, this midwater productivity hotspot is associated with prominent biomass peaks, consisting of prokaryoplankton (Bacteria + Archaea) protists and viruses. Early in the time-series, phylogenetic studies (small subunit ribosomal RNA gene libraries) and fluorescent in situ hybridization (FISH) surveys documented that the chemoautotrophic layer was overwhelmingly populated by e-proteobacteria related to sulfur-oxidizing symbionts from hydrothermal vent systems and β-proteobacteria. However, after May 2009, β- and e-proteobacteria were no longer detected in significant numbers by FISH or by more exhaustive sequencing efforts (454 pyrosequencing and MiSeq iTag libraries). Sulfur oxidation gene quantification (qPCR) also confirmed that e-proteobacteria were rare in samples collected after mid-2009 and that the chemoautotrophic layer became dominated by γ-proteobacterial sulfur-oxidizers (GSOs). Monthly hydrographic and chemical data from the CARIACO Ocean Time-Series were examined for temporal changes that might have driven this shift in the dominant thioautotrophs. Within the redoxcline (250–450 m), significant shifts occurred in water density structure and distributions of H2S, NH4+, O2, and NO2- between the β-/e-proteobacteria-dominated and the γ-proteobacteria-dominated periods. Contrary to observations in other euxinic systems, GSO seem to have a selective advantage over other sulfur-oxidizing lieages during periods of higher and less variable H2S vertical fluxes and higher relative fluxes of H2S to O2 and NO3-. How exactly this changing seascape might have selected for one lineage over another cannot be directly determined. However, spatiotemporal variations in water density and concentration gradients altered vertical fluxes of the major reductant (H2S) and oxidants (O2, NO3-,NO2-), which we postulate provided a more geochemically stable environment favoring GSOs.

Interest in extracting mineral resources from the seafloor through deep‐sea mining has accelerated in the past decade, driven by consumer demand for various metals like zinc, cobalt, and rare earth elements. While there are ongoing studies evaluating potential environmental impacts of deep‐sea mining activities, these focus primarily on impacts to animal biodiversity. The microscopic spectrum of seafloor life and the services that this life provides in the deep sea are rarely considered explicitly. In April 2018, scientists met to define the microbial ecosystem services that should be considered when assessing potential impacts of deep‐sea mining, and to provide recommendations for how to evaluate and safeguard these services. Here, we indicate that the potential impacts of mining on microbial ecosystem services in the deep sea vary substantially, from minimal expected impact to loss of services that cannot be remedied by protected area offsets. For example, we (1) describe potential major losses of microbial ecosystem services at active hydrothermal vent habitats impacted by mining, (2) speculate that there could be major ecosystem service degradation at inactive massive sulfide deposits without extensive mitigation efforts, (3) suggest minor impacts to carbon sequestration within manganese nodule fields coupled with potentially important impacts to primary production capacity, and (4) surmise that assessment of impacts to microbial ecosystem services at seamounts with ferromanganese crusts is too poorly understood to be definitive. We conclude by recommending that baseline assessments of microbial diversity, biomass, and, importantly, biogeochemical function need to be considered in environmental impact assessments of deep‐sea mining.

Predation by grazing protists in aquatic habitats can influence prokaryotic community structure and provides a source of new, labile organic matter. Due to methodological difficulties associated with studies of deep-sea (below photic zone) microbiota, trophic interactions between eukaryotes and prokaryotes in mesopelagic and bathypelagic realms are largely obscured. Further complicating matters, examinations of trophic interactions using water samples that have been exposed to upwards of hundreds of atmospheres of pressure change prior to initiating experiments can potentially introduce significant artifacts. Here we present results of the first study of protistan grazing in water layers ranging from the euphotic zone to the bathypelagic, utilizing the Microbial Sampler-Submersible Incubation Device (MS-SID) that makes possible in situ studies of microbial activities. Protistan grazing in the mesopelagic and bathypelagic realm of the East Mediterranean Sea was quantified using fluorescently labeled prokaryotes (FLP) prepared from the naturally-occurring prokaryotic assemblages. These studies reveal daily prokaryotic removal due to grazing ranging from 31.3±5.9% at 40 m depth to 0.5±0.3% at 950 m. At 3540 m depth, where a chemocline habitat exists with abundant and active prokaryotes above Urania basin, the daily consumption of prokaryotes by protists was 19.9±6.6% of the in situ abundance.

Obtaining an accurate picture of microbial processes occurring in situ is essential for our understanding of marine biogeochemical cycles of global importance. Water samples are typically collected at depth and returned to the sea surface for processing and downstream experiments. Metatranscriptome analysis is one powerful approach for investigating metabolic activities of microorganisms in their habitat and which can be informative for determining responses of microbiota to disturbances such as the Deepwater Horizon oil spill. For studies of microbial processes occurring in the deep sea, however, sample handling, pressure, and other changes during sample recovery can subject microorganisms to physiological changes that alter the expression profile of labile messenger RNA. Here we report a comparison of gene expression profiles for whole microbial communities in a bathypelagic water column sample collected in the Eastern Mediterranean Sea using Niskin bottle sample collection and a new water column sampler for studies of marine microbial ecology, the Microbial Sampler – In Situ Incubation Device (MS-SID). For some taxa, gene expression profiles from samples collected and preserved in situ were significantly different from potentially more stressful Niskin sampling and preservation on deck. Some categories of transcribed genes also appear to be affected by sample handling more than others. This suggests that for future studies of marine microbial ecology, particularly targeting deep sea samples, an in situ sample collection and preservation approach should be considered.

Deep-sea hypersaline anoxic basins (DHABs) in the Eastern Mediterranean Sea are considered some of the most polyextreme habitats on Earth. In comparison to microbial activities occurring within the haloclines and brines of these unusual water column habitats near the Mediterranean seafloor, relatively little is known about microbial metabolic activities in the underlying sediments. In addition, it is not known whether activities are shaped by the unique chemistries of the different DHAB brines and whether evidence exists for active microbial eukaryotes in those sediments. Metatranscriptome analysis was applied to sediment samples collected using ROV Jason from underneath the haloclines of Urania, Discovery and L'Atalante DHABs and a control site. We report on expression of genes associated with sulfur and nitrogen cycling, putative osmolyte biosynthetic pathways and ion transporters, trace metal detoxification, selected eukaryotic activities (particularly of fungi), microbe–microbe interactions, and motility in sediments underlying the haloclines of three DHABs. Relative to our control sediment sample collected outside of Urania Basin, microbial communities (including eukaryotes) in the Urania and Discovery DHAB sediments showed upregulation of expressed genes associated with nitrogen transformations, osmolyte biosynthesis, heavy metals resistance and metabolism, eukaryotic organelle functions, and cell–cell interactions. Sediments underlying DHAB haloclines that have cumulative physico-chemical stressors within the limits of tolerance for microoorganisms can therefore be hotspots of activity in the deep Mediterranean Sea.

Interest in extracting mineral resources from the seafloor through deep-sea mining has accelerated substantially in the past decade, driven by increasing consumer demand for various metals like copper, zinc, manganese, cobalt and rare earth elements. While there are many on-going discussions and studies evaluating potential environmental impacts of deep-sea mining activities, these focus primarily on impacts to animal biodiversity. The microscopic spectrum of life on the seafloor and the services that this microbial realm provides in the deep sea are rarely considered explicitly. In April 2018, a community of scientists met to define the microbial ecosystem services that should be considered when assessing potential impacts of deep-sea mining, and to provide recommendations for how to evaluate these services. Here we show that the potential impacts of mining on microbial ecosystem services in the deep sea vary substantially, from minimal expected impact to complete loss of services that cannot be remedied by protected area offsets. We conclude by recommending that certain types of ecosystems should be “off limits” until initial characterizations can be performed, and that baseline assessments of microbial diversity, biomass, and biogeochemical function need to be considered in environmental impact assessments of all potential instances of deep-sea mining.

Little is known about the relative influence of historic processes and environmental gradients on shaping the diversity of single-celled eukaryotes in marine benthos. By combining pyrosequencing of 18S ribosomal RNA genes with data on multiple environmental factors, we investigated the diversity of microeukaryotes in surficial sediments of three basins of the Yellow Sea Large Marine Ecosystem. A considerable proportion (about 20%) of reads was affiliated with known parasitoid protists. Dinophyta and Ciliophora appeared dominant in terms of relative proportion of reads and operational taxonomic unit (OTU) richness. Overall, OTU richness of benthic microeukaryotes decreased with increasing water depth and decreasing pH. While community composition was significantly different among basins, partial Mantel tests indicated a depth-decay pattern of community similarity, whereby water depth, rather than geographic distance or environment, shaped -diversity of benthic microeukaryotes (including both the abundant and the rare biosphere) on a regional scale. Similar hydrographic and mineralogical factors contributed to the biogeography of both the abundant and the rare OTUs. The trace metal vanadium had a significant effect on the biogeography of the rare biosphere. Our study sheds new light on the composition, diversity patterns and underlying mechanisms of single-celled eukaryote distribution in surficial sediments of coastal oceans.

Niskin bottle rosettes have for years been the workhorse technology for collection of water samples used in biological and chemical oceanography. Studies of marine microbiology and biogeochemical cycling that aim to analyze labile organic molecules including messenger RNA, must take into account factors associated with sampling methodology that obscure an accurate picture of in situ activities/processes. With Niskin sampling, the large and often variable times between sample collection and preservation on deck of a ship, and the sometimes significant physico-chemical changes (e.g., changes in pressure, light, temperature, redox state, etc.) that water samples and organisms are exposed to, are likely to introduce artifacts. These concerns are likely more significant when working with phototrophs, deep-sea microbes, and/or organisms inhabiting low-oxygen or anoxic environments. We report here the development of a new technology for the in situ collection and chemical preservation of particulate microbial samples for a variety of downstream analyses depending on preservative choice by the user. The Fixation Filter Unit, version 3 (FF3) permits filtration of water sample through 47 mm diameter filters of the user׳s choice and upon completion of filtration, chemically preserves the retained sample within 10׳s of seconds. The stand-alone devices can be adapted to hydrocasting or mooring-based platforms.

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.

Using the anoxic Cariaco Basin as a natural laboratory, particle association of bacterial and archaeal taxa was assessed by iTag sequencing and qPCR gene assays of samples spanning an oxic-anoxic-euxinic gradient. A total of 10%-12% of all bacterial and archaeal cells were found in the particle-associated (PA) fraction, operationally defined as prokaryotes captured on 2.7 µm membranes. Both redox condition and size fraction segregated bacterial taxa. Archaeal taxa varied according to redox conditions, but were similar between size fractions. Taxa putatively associated with chemoautotrophic sulfur oxidation and nitrification dominated the free-living (FL) fraction throughout the oxycline (< 1-120 µM O2 ) and upper anoxic layer. Bacteria in the oxycline's PA fraction included taxa known to be aerobic and anaerobic chemoorganotrophs. At shallow anoxic depths, PA taxa were primarily affiliated with anaerobic sulfate ( SO42-)-reducing lineages. PA fractions in the most sulfidic samples were dominated by taxa affiliated with CH4 oxidizing, fermenting and SO42- reducing lineages. Prevalence of particle-associated SO42--reducing taxa and abundant sulfur-oxidizing taxa in both size fractions across the oxic-anoxic interface is consistent with the cryptic sulfur cycling concept. Bacterial assemblage diversity in the PA fraction always exceeded the FL fraction except in the most oxic samples, whereas Archaeal diversity was not consistently different between size fractions. Our results suggest that these particle-associated and free-living bacterial assemblages are functionally different and that the interplay between particle microhabitats and surrounding geochemical regimes is a strong selective force shaping microbial communities throughout the water column.

Little is still known of the impacts of protist grazing on bacterioplankton communities in the dark ocean. Furthermore, the accuracy of assessments of in situ microbial activities, including protist grazing, can be affected by sampling artifacts introduced during sample retrieval and downstream manipulations. Potential artifacts may be increased when working with deep-sea samples or samples from chemically unique water columns such as oxygen minimum zones (OMZs). OMZs are oxygen-depleted regions in the ocean, where oxygen concentrations can drop to

Studies of microbial communities in areas of the world where permanent marine water column oxyclines exist suggest they are "hotspots" of microbial activity, and that these water features and the anoxic waters below them are inhabited by diverse protist taxa, including ciliates. These communities have minimal taxonomic overlap with those in overlying oxic water columns. Some ciliate taxa have been detected in multiple locations where these stable water column oxyclines exist; however, differences in such factors as hydrochemistry in the habitats that have been studied suggest local selection for distinct communities. We compare published data on ciliate communities from studies of deep marine water column oxyclines in Caricao Basin, Venezuela, and the Black Sea, with data from coastal, shallower oxycline waters in Framvaren and Mariager fjords, and from several deep-sea hypersaline anoxic basins in the Eastern Mediterranean Sea. Putative symbioses between Bacteria, Archaea, and ciliates observed along these oxyclines suggests a strategy of cooperative metabolism for survival that includes chemosynthetic autotrophy and exchanges of metabolic intermediates or end products between hosts and their prokaryotic partners.

The advent of molecular tools in microbial ecology paved the way to exploit the diversity of microbes in extreme environments. Here, we review these tools as applied in one of the most polyextreme habitats known on our planet, namely, deep hypersaline anoxic basins (DHABs), located at ca. 3000–3500 m depth in the Eastern Mediterranean Sea. Molecular gene signatures amplified from environmental DHAB samples identified a high degree of genetic novelty, as well as distinct communities in the DHABs. Canonical correspondence analyses provided strong evidence that salinity, ion composition, and anoxia were the strongest selection factors shaping protistan community structures, largely preventing cross-colonization among the individual basins. Thus, each investigated basin represents a unique habitat (“isolated islands of evolution”), making DHABs ideal model sites to test evolutionary hypotheses. Fluorescence in situ hybridization assays using specifically designed probes revealed that the obtained genetic signatures indeed originated from indigenous polyextremophiles. Electron microscopy imaging revealed unknown ciliates densely covered with prokaryote ectosymbionts, which may enable adaptations of eukaryotes to DHAB conditions. The research reviewed here significantly advanced our knowledge on polyextremophile eukaryotes, which are excellent models for a number of biological research areas, including ecology, diversity, biotechnology, evolutionary research, physiology, and astrobiology.

Randomized, scaled-up single-cell genomics of marine microorganisms reveals a high degree of uniqueness between individual cells, implying limited clonality within populations, and establishes that a large fraction of global genetic diversity can be recovered from a single sample, suggesting effective global dispersal of prokaryoplankton., Simons Foundation (Award 337262), National Science Foundation (Grants OCE-1153588, OCE-1356460, and DBI-0424599)

Carbon fixation by chemoautotrophic microorganisms in the dark ocean has a major impact on global carbon cycling and ecological relationships in the ocean’s interior, but the relevant taxa and energy sources remain enigmatic. We show evidence that nitrite-oxidizing bacteria affiliated with the Nitrospinae phylum are important in dark ocean chemoautotrophy. Single-cell genomics and community metagenomics revealed that Nitrospinae are the most abundant and globally distributed nitrite-oxidizing bacteria in the ocean. Metaproteomics and metatranscriptomics analyses suggest that nitrite oxidation is the main pathway of energy production in Nitrospinae. Microautoradiography, linked with catalyzed reporter deposition fluorescence in situ hybridization, indicated that Nitrospinae fix 15 to 45% of inorganic carbon in the mesopelagic western North Atlantic. Nitrite oxidation may have a greater impact on the carbon cycle than previously assumed.

The deep sedimentary biosphere, extending 100s of meters below the seafloor harbors unexpected diversity of Bacteria, Archaea, and microbial eukaryotes. Far less is known about microbial eukaryotes in subsurface habitats, albeit several studies have indicated that fungi dominate microbial eukaryotic communities and fungal molecular signatures (of both yeasts and filamentous forms) have been detected in samples as deep as 1740 mbsf. Here, we compare and contrast fungal ribosomal RNA gene signatures and whole community metatranscriptomes present in sediment core samples from 6 and 95 mbsf from Peru Margin site 1229A and from samples from 12 and 345 mbsf from Canterbury Basin site U1352. The metatranscriptome analyses reveal higher relative expression of amino acid and peptide transporters in the less nutrient rich Canterbury Basin sediments compared to the nutrient rich Peru Margin, and higher expression of motility genes in the Peru Margin samples. Higher expression of genes associated with metals transporters and antibiotic resistance and production was detected in Canterbury Basin sediments. A poly-A focused metatranscriptome produced for the Canterbury Basin sample from 345 mbsf provides further evidence for active fungal communities in the subsurface in the form of fungal-associated transcripts for metabolic and cellular processes, cell and membrane functions, and catalytic activities. Fungal communities at comparable depths at the two geographically separated locations appear dominated by distinct taxa. Differences in taxonomic composition and expression of genes associated with particular metabolic activities may be a function of sediment organic content as well as oceanic province. Microscopic analysis of Canterbury Basin sediment samples from 4 and 403 mbsf produced visualizations of septate fungal filaments, branching fungi, conidiogenesis, and spores. These images provide another important line of evidence supporting the occurrence and activity of fungi in the deep subseafloor biosphere.

Studies in the center and margin of the Medee Basin, a Mediterranean deep-sea hypersaline anoxic basin, and at a reference site during Penelope cruise (2007), revealed the existence of a 7 m-thick halocline, with high salinity (328 psu), and high sedimentary organic carbon and biopolymer concentrations. The 194 16S rRNA sequences retrieved were grouped into 118 unique phylotypes. Pseudomonas gessardii, dominated in the center, while 33 phylotypes were detected at the margin and 73 at the reference site. The study suggested conditions hostile to bacteria in the sediments of the Medee Basin and preservation of sedimentary labile organic matter.

Only recently, a novel anoxic hypersaline (thalassic) basin in the eastern Mediterranean was discovered at a depth of 3,258 m. The halite-saturated brine of this polyextreme basin revealed one of the highest salt concentrations ever reported for such an environment (salinity of 348aEuro degrees). Using a eukaryote-specific probe and fluorescence in situ hybridization, we counted 0.6 x 10(4) protists per liter of anoxic brine. SSU rRNA sequence analyses, based on amplification of environmental cDNA identified fungi as the most diverse taxonomic group of eukaryotes in the brine, making deep-sea brines sources of unknown fungal diversity and hotspots for the discovery of novel metabolic pathways and for secondary metabolites. The second most diverse phylotypes are ciliates and stramenopiles (each 20%). The occurrence of closely related ciliate sequences exclusively in other Mediterranean brine basins suggests specific adaptations of the respective organisms to such habitats. Betadiversity-analyses confirm that microeukaryote communities in the brine and the interface are notably different. Several distinct morphotypes in brine samples suggest that the rRNA sequences detected in Thetis brine can be linked to indigenous polyextremophile protists. This contradicts previous assumptions that such extremely high salt concentrations are anathema to eukaryotic life. The upper salinity limits for eukaryotic life remain unidentified.

We investigated the top 30-cm sediment prokaryotic community structure in 5-cm spatial resolution, at an active site of the Amsterdam mud volcano, East Mediterranean Sea, based on the 16S rRNA gene diversity. A total of 339 and 526 sequences were retrieved, corresponding to 25 and 213 unique (≥98% similarity) phylotypes of Archaea and Bacteria, respectively, in all depths. The Shannon-Wiener diversity index H was higher for Bacteria (1.92-4.03) than for Archaea (0.99-1.91) and varied differently between the two groups. Archaea were dominated by anaerobic methanotrophs ANME-1, -2 and -3 groups and were related to phylotypes involved in anaerobic oxidation of methane from similar habitats. The much more complex Bacteria community consisted of 20 phylogenetic groups at the phylum/candidate division level. Proteobacteria, in particular δ-Proteobacteria, was the dominant group. In most sediment layers, the dominant phylotypes of both the Archaea and Bacteria communities were found in neighbouring layers, suggesting some overlap in species richness. The similarity of certain prokaryotic communities was also depicted by using four different similarity indices. The direct comparison of the retrieved phylotypes with those from the Kazan mud volcano of the same field revealed that 40.0% of the Archaea and 16.9% of the Bacteria phylotypes are common between the two systems. The majority of these phylotypes are closely related to phylotypes originating from other mud volcanoes, implying a degree of endemicity in these systems.

We investigated 16S rRNA gene diversity at a high sediment depth resolution (every 5 cm, top 30 cm) in an active site of the Kazan mud volcano, East Mediterranean Sea. A total of 242 archaeal and 374 bacterial clones were analysed, which were attributed to 38 and 205 unique phylotypes, respectively (> or = 98% similarity). Most of the archaeal phylotypes were related to ANME-1, -2 and -3 members originating from habitats where anaerobic oxidation of methane (AOM) occurs, although they occurred in sediment layers with no apparent AOM (below the sulphate depletion depth). Proteobacteria were the most abundant and diverse bacterial group, with the Gammaproteobacteria dominating in most sediment layers and these were related to phylotypes involved in methane cycling. The Deltaproteobacteria included several of the sulphate-reducers related to AOM. The rest of the bacterial phylotypes belonged to 15 known phyla and three unaffiliated groups, with representatives from similar habitats. Diversity index H was in the range 0.56-1.73 and 1.47-3.82 for Archaea and Bacteria, respectively, revealing different depth patterns for the two groups. At 15 and 20 cm below the sea floor, the prokaryotic communities were highly similar, hosting AOM-specific Archaea and Bacteria. Our study revealed different dominant phyla in proximate sediment layers.

We used culture-based and culture-independent approaches to discover diversity and ecology of anaerobic jakobids (Excavata: Jakobida), an overlooked, deep-branching lineage of free-living nanoflagellates related to Euglenozoa. Jakobids are among a few lineages of nanoflagellates frequently detected in anoxic habitats by PCR-based studies, however only two strains of a single jakobid species have been isolated from those habitats. We recovered 712 environmental sequences and cultured 21 new isolates of anaerobic jakobids that collectively represent at least ten different species in total, from which four are uncultured. Two cultured species have never been detected by environmental, PCR-based methods. Surprisingly, culture-based and culture-independent approaches were able to reveal a relatively high proportion of overall species diversity of anaerobic jakobids - 60 % or 80 %, respectively. Our phylogenetic analyses based on SSU rDNA and six protein-coding genes showed that anaerobic jakobids constitute a clade of morphologically similar, but genetically and ecologically diverse protists – Stygiellidae fam. nov. Our investigation combines culture-based and environmental molecular-based approaches to capture a wider extent of species diversity and shows Stygiellidae as a group that ordinarily inhabits anoxic, sulfide- and ammonium-rich marine habitats worldwide.

Oxygen minimum zones (OMZs) caused by water column stratification appear to expand in parts of the world's ocean, with consequences for marine biogeochemical cycles. OMZ formation is often fueled by high surface primary production, and sinking organic particles can be hotspots of interactions and activity within microbial communities. This study investigated the diversity of OMZ protist communities in two biomass size fractions (>30 and 30–1.6 μm filters) from the world's largest permanent OMZ in the Eastern Tropical North Pacific. Diversity was quantified via Illumina MiSeq sequencing of V4 region of 18S SSU rRNA genes in samples spanning oxygen gradients at two stations. Alveolata and Rhizaria dominated the two size fractions at both sites along the oxygen gradient. Community composition at finer taxonomic levels was partially shaped by oxygen concentration, as communities associated with versus anoxic waters shared only ∼32% of operational taxonomic unit (OTU) (97% sequence identity) composition. Overall, only 9.7% of total OTUs were recovered at both stations and under all oxygen conditions sampled, implying structuring of the eukaryotic community in this area. Size-fractionated communities exhibited different taxonomical features (e.g. Syndiniales Group I in the 1.6–30 μm fraction) that could be explained by the microniches created on the surface-originated sinking particles.

Oceanic protist grazing at mesopelagic and bathypelagic depths, and their subsequent effects on trophic links between eukaryotes and prokaryotes, are not well constrained. Recent studies show evidence of higher than expected grazing activity by protists down to mesopelagic depths. This study provides the first exploration of protist grazing in the bathypelagic North Atlantic Deep Water (NADW). Grazing was measured throughout the water column at three stations in the South Atlantic using fluorescently-labeled prey analogues. Grazing in the deep Antarctic Intermediate water (AAIW) and NADW at all three stations removed 3.79% ± 1.72% to 31.14% ± 8.24% of the standing prokaryote stock. These results imply that protist grazing may be a significant source of labile organic carbon at certain meso- and bathypelagic depths.

The sediment microbiota of the Mediterranean deep-sea anoxic hypersaline basins (DHABs) are understudied relative to communities in the brines and halocline waters. In this study, the active fraction of the prokaryotic community in the halocline sediments of L' Atalante, Urania, and Discovery DHABs was investigated based on extracted total RNA and 454 pyrosequencing of the 16S rRNA gene. Bacterial and archaeal communities were different in the sediments underlying the halocline waters of the three habitats, reflecting the unique chemical settings of each basin. The relative abundance of unique operational taxonomic units (OTUs) was also different between deep-sea control sediments and sediments underlying DHAB haloclines, suggesting adaptation to the steep DHAB chemical gradients. Only a few OTUs were affiliated to known bacterial halophilic and/or anaerobic groups. Many OTUs, including some of the dominant ones, were related to aerobic taxa. Archaea were detected only in few halocline samples, with lower OTU richness relative to Bacteria, and were dominated by taxa associated with methane cycling. This study suggests that, while metabolically active prokaryotic communities appear to be present in sediments underlying the three DHABs investigated, their diversity and activity are likely to be more reduced in sediments underlying the brines.

Background The deep-sea hypersaline anoxic basins (DHABs) of the Mediterranean (water depth ~3500 m) are some of the most extreme oceanic habitats known. Brines of DHABs are nearly saturated with salt, leading many to suspect they are uninhabitable for eukaryotes. While diverse bacterial and protistan communities are reported from some DHAB haloclines and brines, loriciferans are the only metazoan reported to inhabit the anoxic DHAB brines. Our goal was to further investigate metazoan communities in DHAB haloclines and brines. Results We report observations from sediments of three DHAB (Urania, Discovery, L’Atalante) haloclines, comparing these to observations from sediments underlying normoxic waters of typical Mediterranean salinity. Due to technical difficulties, sampling of the brines was not possible. Morphotype analysis indicates nematodes are the most abundant taxon; crustaceans, loriciferans and bryozoans were also noted. Among nematodes, Daptonema was the most abundant genus; three morphotypes were noted with a degree of endemicity. The majority of rRNA sequences were from planktonic taxa, suggesting that at least some individual metazoans were preserved and inactive. Nematode abundance data, in some cases determined from direct counts of sediments incubated in situ with CellTrackerTM Green, was patchy but generally indicates the highest abundances in either normoxic control samples or in upper halocline samples; nematodes were absent or very rare in lower halocline samples. Ultrastructural analysis indicates the nematodes in L’Atalante normoxic control sediments were fit, while specimens from L’Atalante upper halocline were healthy or had only recently died and those from the lower halocline had no identifiable organelles. Loriciferans, which were only rarely encountered, were found in both normoxic control samples as well as in Discovery and L’Atalante haloclines. It is not clear how a metazoan taxon could remain viable under this wide range of conditions. Conclusions We document a community of living nematodes in normoxic, normal saline deep-sea Mediterranean sediments and in the upper halocline portions of the DHABs. Occurrences of nematodes in mid-halocline and lower halocline samples did not provide compelling evidence of a living community in those zones. The possibility of a viable metazoan community in brines of DHABs is not supported by our data at this time. Electronic supplementary material The online version of this article (doi:10.1186/s12915-015-0213-6) contains supplementary material, which is available to authorized users.

Some of the most extreme marine habitats known are the Mediterranean deep hypersaline anoxic basins (DHABs; water depth ∼3500 m). Brines of DHABs are nearly saturated with salt, leading many to suspect they are uninhabitable for eukaryotes. While diverse bacterial and protistan communities are reported from some DHAB water-column haloclines and brines, the existence and activity of benthic DHAB protists have rarely been explored. Here, we report findings regarding protists and fungi recovered from sediments of three DHAB (Discovery, Urania, L' Atalante) haloclines, and compare these to communities from sediments underlying normoxic waters of typical Mediterranean salinity. Halocline sediments, where the redoxcline impinges the seafloor, were studied from all three DHABs. Microscopic cell counts suggested that halocline sediments supported denser protist populations than those in adjacent control sediments. Pyrosequencing analysis based on ribosomal RNA detected eukaryotic ribotypes in the halocline sediments from each of the three DHABs, most of which were fungi. Sequences affiliated with Ustilaginomycotina Basidiomycota were the most abundant eukaryotic signatures detected. Benthic communities in these DHABs appeared to differ, as expected, due to differing brine chemistries. Microscopy indicated that only a low proportion of protists appeared to bear associated putative symbionts. In a considerable number of cases, when prokaryotes were associated with a protist, DAPI staining did not reveal presence of any nuclei, suggesting that at least some protists were carcasses inhabited by prokaryotic scavengers.

Deep-sea hypersaline anoxic basins (DHABs) in the Eastern Mediterranean Sea are considered some of the most hostile environments on Earth. Little is known about the biochemical adaptations of microorganisms living in these habitats. This first metatranscriptome analysis of DHAB samples provides significant insights into shifts in metabolic activities of microorganisms as physicochemical conditions change from deep Mediterranean sea water to brine. The analysis of Thetis DHAB interface indicates that sulfate reduction occurs in both the upper (7.0–16.3% salinity) and lower (21.4–27.6%) halocline, but that expression of dissimilatory sulfate reductase is reduced in the more hypersaline lower halocline. High dark-carbon assimilation rates in the upper interface coincided with high abundance of transcripts for ribulose 1,5-bisphosphate carboxylase affiliated to sulfur-oxidizing bacteria. In the lower interface, increased expression of genes associated with methane metabolism and osmoregulation is noted. In addition, in this layer, nitrogenase transcripts affiliated to uncultivated putative methanotrophic archaea were detected, implying nitrogen fixation in this anoxic habitat, and providing evidence of linked carbon, nitrogen and sulfur cycles.

Phagotrophic protists are an important mortality factor of prokaryotes in most aquatic habitats. However, no study has assessed protistan grazing as loss factor of bacterial biomass across the stratification gradient of a temperate freshwater meromictic lake. Protistan grazing effect was quantified in the mixolimnion, the transition zone, and the sulfidic anoxic monimolimnion of Lake Alatsee (Germany). Grazing experiments were performed using prey analogues from the natural prokaryotic assemblage. Daily grazing effect declined from the mixolimnion to the monimolimnion. Heterotrophic flagellates were phagotrophically active in all three water horizons and the main grazers in the monimolimnion. Pigmented flagellates accounted for 70% of total grazing in the mixolimnion and ciliates only for a small fraction of grazing in each depth. Prokaryotic biomass removal peaked in the interface, but protistan impact on the respective prokaryotic abundance was low. Grazing in the anoxic monimolimnion was negligible, with prokaryotic turnover rate being only 0.4% of standing stock. Our results support the assumption that protistan predation in anoxic waters is lower than in oxygenated ones and identify the interface as a microhabitat that supports high grazer biomass, pinpointing the importance of purple sulfur bacteria as carbon source for the upper mixolimnion and the bottom monimolimnion.

We analyzed the bacterial 16S rRNA gene diversity throughout the major components of the drinking water distribution system of a ca. 52,000-inhabitants city (Trikala City, Greece) in order to describe the changes of the bacterial assemblages and to detect possible bacterial pathogens which are not included in the standard monitoring process. Bacterial DAPI counts and DNA extraction was performed in the water pumping wells, the water treatment tank and tap water from households. Approximately 920 bp of the bacterial 16S rDNA were PCR-amplified, cloned, and sequenced for a total of 191 clones, which belonged to 112 unique phylotypes. The water of the pumping wells harbored a typical subsurface bacterial assemblage, with no human pathogens, dominated by beta-Proteobacteria. Cell abundance in the water treatment tank decreased significantly, close to detection limit, but bacterial diversity remained high. However, the dominance of beta-Proteobacteria decreased considerably, indicating the sensitivity of this group to drinking water disinfection treatment. Tap water from the households hosted a much less diverse, low-cell bacterial assemblage, dominated by Mycobacterium-like phylotypes, related to biofilm bacterial communities.

The type of branching is a morphological character of great significance, separating taxa within the true-branched cyanobacteria. In this paper, true-branched morphospecies of cyanobacteria belonging to genus Iphinoe Lamprinou et Pantazidou, from different and geographically isolated caves of Greece, were investigated by coupling classical approach i.e. microscopy analysis (LM, SEM, TEM, CLSM) and molecular analysis (16S rRNA gene diversity). The morphological study revealed interspecific differences which could be attributed to adaptations to local conditions: Iphinoe cf. spelaeobios from the cave 'Diktaion Andron' (Crete island) exhibit different type of branching (Y- and T-type) and smaller size compared to the type species Iphinoe spelaeobios (T- and V-type of branching) from the caves 'Kastria' and 'Koutouki' (Peloponnese, Attica). Despite their phenotypic differences and geographical isolation molecular analysis showed identical 16S rRNA sequences which indicate that both populations belong to the same genus.

We analysed the 16S rRNA gene diversity within the bacterioplankton community in the water column of the ornamental fish Pterophyllum scalare and Archocentrus nigrofasciatus aquaria during a 60-day growth experiment in order to detect any dominant bacterial species and their possible association with the rearing organisms. The basic physical and chemical parameters remained stable but the bacterial community at 0, 30 and 60 days showed marked differences in bacterial cell abundance and diversity. We found high species richness but no dominant phylotypes were detected. Only few of the phylotypes were found in more than one time point per treatment and always with low relative abundance. The majority of the common phylotypes belonged to the Proteobacteria phylum and were closely related to Acinetobacter junii, Pseudomonas sp., Nevskia ramosa, Vogesella perlucida, Chitinomonas taiwanensis, Acidovorax sp., Pelomonas saccharophila and the rest belonged to the α-Proteobacteria, Bacteroidetes, Actinobacteria, candidate division OP11 and one unaffiliated group. Several of these phylotypes were closely related to known taxa including Sphingopyxis chilensis, Flexibacter aurantiacus subsp. excathedrus and Mycobacterium sp. Despite the high phylogenetic diversity most of the inferred ecophysiological roles of the found phylotypes are related to nitrogen metabolism, a key process for fish aquaria.