Reverse engineering environmental metatranscriptomes clarifies best practices for eukaryotic assembly.

BACKGROUND: Diverse communities of microbial eukaryotes in the global ocean provide a variety of essential ecosystem services, from primary production and carbon flow through trophic transfer to cooperation via symbioses. Increasingly, these communities are being understood through the lens of omics tools, which enable high-throughput processing of diverse communities. Metatranscriptomics offers an understanding of near real-time gene expression in microbial eukaryotic communities, providing a window into community metabolic activity. RESULTS: Here we present a workflow for eukaryotic metatranscriptome assembly, and validate the ability of the pipeline to recapitulate real and manufactured eukaryotic community-level expression data. We also include an open-source tool for simulating environmental metatranscriptomes for testing and validation purposes. We reanalyze previously published metatranscriptomic datasets using our metatranscriptome analysis approach. CONCLUSION: We determined that a multi-assembler approach improves eukaryotic metatranscriptome assembly based on recapitulated taxonomic and functional annotations from an in-silico mock community. The systematic validation of metatranscriptome assembly and annotation methods provided here is a necessary step to assess the fidelity of our community composition measurements and functional content assignments from eukaryotic metatranscriptomes.

DMSP synthesis genes distinguish two types of DMSP producer phenotypes.

Dimethylsulfoniopropionate (DMSP) is an important organic carbon and sulfur source in the surface ocean that fuels microbial activity and significantly impacts Earth's climate. After three decades of research, the cellular role(s) of DMSP and environmental drivers of production remain enigmatic. Recent work suggests that cellular DMSP concentrations, and changes in these concentrations in response to environmental stressors, define two major groups of DMSP producers: high DMSP producers that contain ≥ 50 mM intracellular DMSP and low DMSP producers that contain < 50 mM. Here we show that two recently described DMSP synthesis genes (DSYB and TpMT2) may differentiate these two DMSP phenotypes. A survey of prokaryotic and eukaryotic isolates found a significant correlation between the presence of DSYB and TpMT2 genes and previous measurements of high and low DMSP concentrations, respectively. Phylogenetic analysis demonstrated that DSYB and TpMT2 form two distinct clades. DSYB and TpMT2 were also found to be globally abundant in in situ surface communities, and their taxonomic annotations were similar to those observed for isolates. The strong correlation of the DSYB and TpMT2 synthesis genes with high and low producer phenotypes establishes a foundation for direct quantification of DMSP producers, enabling significantly improved predictions of DMSP in situ.

Transcriptional response of Emiliania huxleyi under changing nutrient environments in the North Pacific Subtropical Gyre.

The widespread coccolithophore Emiliania huxleyi is an abundant oceanic phytoplankton, impacting the global cycling of carbon through both photosynthesis and calcification. Here, we examined the transcriptional responses of populations of E. huxleyi in the North Pacific Subtropical Gyre to shifts in the nutrient environment. Using a metatranscriptomic approach, nutrient-amended microcosm studies were used to track the global metabolism of E. huxleyi. The addition of nitrate led to significant changes in transcript abundance for gene pathways involved in nitrogen and phosphorus metabolism, with a decrease in the abundance of genes involved in the acquisition of nitrogen (e.g. N-transporters) and an increase in the abundance of genes associated with phosphate acquisition (e.g. phosphatases). Simultaneously, after the addition of nitrate, genes associated with calcification and genes unique to the diploid life stages of E. huxleyi significantly increased. These results suggest that nitrogen is a major driver of the physiological ecology of E. huxleyi in this system and further suggest that the addition of nitrate drives shifts in the dominant life-stage of the population. Together, these results underscore the importance of phenotypic plasticity to the success of E. huxleyi, a characteristic that likely underpins its ability to thrive across a variety of marine environments.

Shifting metabolic priorities among key protistan taxa within and below the euphotic zone.

A metatranscriptome study targeting the protistan community was conducted off the coast of Southern California, at the San Pedro Ocean Time-series station at the surface, 150 m (oxycline), and 890 m to link putative metabolic patterns to distinct protistan lineages. Comparison of relative transcript abundances revealed depth-related shifts in the nutritional modes of key taxonomic groups. Eukaryotic gene expression in the sunlit surface environment was dominated by phototrophs, such as diatoms and chlorophytes, and high abundances of transcripts associated with synthesis pathways (e.g., photosynthesis, carbon fixation, fatty acid synthesis). Sub-euphotic depths (150 and 890 m) exhibited strong contributions from dinoflagellates and ciliates, and were characterized by transcripts relating to digestion or intracellular nutrient recycling (e.g., breakdown of fatty acids and V-type ATPases). These transcriptional patterns underlie the distinct nutritional modes of ecologically important protistan lineages that drive marine food webs, and provide a framework to investigate trophic dynamics across diverse protistan communities.

Metatranscriptome analyses indicate resource partitioning between diatoms in the field.

Diverse communities of marine phytoplankton carry out half of global primary production. The vast diversity of the phytoplankton has long perplexed ecologists because these organisms coexist in an isotropic environment while competing for the same basic resources (e.g., inorganic nutrients). Differential niche partitioning of resources is one hypothesis to explain this "paradox of the plankton," but it is difficult to quantify and track variation in phytoplankton metabolism in situ. Here, we use quantitative metatranscriptome analyses to examine pathways of nitrogen (N) and phosphorus (P) metabolism in diatoms that cooccur regularly in an estuary on the east coast of the United States (Narragansett Bay). Expression of known N and P metabolic pathways varied between diatoms, indicating apparent differences in resource utilization capacity that may prevent direct competition. Nutrient amendment incubations skewed N/P ratios, elucidating nutrient-responsive patterns of expression and facilitating a quantitative comparison between diatoms. The resource-responsive (RR) gene sets deviated in composition from the metabolic profile of the organism, being enriched in genes associated with N and P metabolism. Expression of the RR gene set varied over time and differed significantly between diatoms, resulting in opposite transcriptional responses to the same environment. Apparent differences in metabolic capacity and the expression of that capacity in the environment suggest that diatom-specific resource partitioning was occurring in Narragansett Bay. This high-resolution approach highlights the molecular underpinnings of diatom resource utilization and how cooccurring diatoms adjust their cellular physiology to partition their niche space.

Functional group-specific traits drive phytoplankton dynamics in the oligotrophic ocean.

A diverse microbial assemblage in the ocean is responsible for nearly half of global primary production. It has been hypothesized and experimentally demonstrated that nutrient loading can stimulate blooms of large eukaryotic phytoplankton in oligotrophic systems. Although central to balancing biogeochemical models, knowledge of the metabolic traits that govern the dynamics of these bloom-forming phytoplankton is limited. We used eukaryotic metatranscriptomic techniques to identify the metabolic basis of functional group-specific traits that may drive the shift between net heterotrophy and autotrophy in the oligotrophic ocean. Replicated blooms were simulated by deep seawater (DSW) addition to mimic nutrient loading in the North Pacific Subtropical Gyre, and the transcriptional responses of phytoplankton functional groups were assayed. Responses of the diatom, haptophyte, and dinoflagellate functional groups in simulated blooms were unique, with diatoms and haptophytes significantly (95% confidence) shifting their quantitative metabolic fingerprint from the in situ condition, whereas dinoflagellates showed little response. Significantly differentially abundant genes identified the importance of colimitation by nutrients, metals, and vitamins in eukaryotic phytoplankton metabolism and bloom formation in this system. The variable transcript allocation ratio, used to quantify transcript reallocation following DSW amendment, differed for diatoms and haptophytes, reflecting the long-standing paradigm of phytoplankton r- and K-type growth strategies. Although the underlying metabolic potential of the large eukaryotic phytoplankton was consistently present, the lack of a bloom during the study period suggests a crucial dependence on physical and biogeochemical forcing, which are susceptible to alteration with changing climate.

Vitamin B12 conveys a protective advantage to phycosphere-associated bacteria at high temperatures.

Many marine microbes require vitamin B12 (cobalamin) but are unable to synthesize it, necessitating reliance on other B12-producing microbes. Thus, phytoplankton and bacterioplankton community dynamics can partially depend on the production and release of a limiting resource by members of the same community. We tested the impact of temperature and B12 availability on the growth of two bacterial taxa commonly associated with phytoplankton: Ruegeria pomeroyi, which produces B12 and fulfills the B12 requirements of some phytoplankton, and Alteromonas macleodii, which does not produce B12 but also does not strictly require it for growth. For B12-producing R. pomeroyi, we further tested how temperature influences B12 production and release. Access to B12 significantly increased growth rates of both species at the highest temperatures tested (38 °C for R. pomeroyi, 40 °C for A. macleodii) and A. macleodii biomass was significantly reduced when grown at high temperatures without B12, indicating that B12 is protective at high temperatures. Moreover, R. pomeroyi produced more B12 at warmer temperatures but did not release detectable amounts of B12 at any temperature tested. Results imply that increasing temperatures and more frequent marine heatwaves with climate change will influence microbial B12 dynamics and could interrupt symbiotic resource sharing.

Eukaryotic genomes from a global metagenomic data set illuminate trophic modes and biogeography of ocean plankton.

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 neither 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 sequence data sets. 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 (0.8-2,000 µm) 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 upon the phylogenetic diversity of likely particle- and host-associated taxa. We use these MAGs to demonstrate an approach to infer the putative trophic mode of the recovered eukaryotic MAGs. We also identify ecological cohorts of co-occurring MAGs, which are driven by specific environmental factors and putative host-microbe associations. These data together add to a number of growing resources of environmentally relevant eukaryotic genomic information. Complementary and expanded databases of MAGs, such as those provided through scalable pipelines like EukHeist, stand to advance our understanding of eukaryotic diversity through increased coverage of genomic representatives across the tree of life.IMPORTANCESingle-celled eukaryotes play ecologically significant roles in the marine environment, yet fundamental questions about their biodiversity, ecological function, and interactions remain. Environmental sequencing enables researchers to document naturally occurring protistan communities, without culturing bias, yet metagenomic and metatranscriptomic sequencing approaches cannot separate individual species from communities. To more completely capture the genomic content of mixed protistan populations, we can create bins of sequences that represent the same organism (metagenome-assembled genomes [MAGs]). We developed the EukHeist pipeline, which automates the binning of population-level eukaryotic and prokaryotic genomes from metagenomic reads. We show exciting insight into what protistan communities are present and their trophic roles in the ocean. Scalable computational tools, like EukHeist, may accelerate the identification of meaningful genetic signatures from large data sets and complement researchers' efforts to leverage MAG databases for addressing ecological questions, resolving evolutionary relationships, and discovering potentially novel biodiversity.

Microbiomes of bloom-forming Phaeocystis algae are stable and consistently recruited, with both symbiotic and opportunistic modes.

Phaeocystis is a cosmopolitan, bloom-forming phytoplankton genus that contributes significantly to global carbon and sulfur cycles. During blooms, Phaeocystis species produce large carbon-rich colonies, creating a unique interface for bacterial interactions. While bacteria are known to interact with phytoplankton-e.g., they promote growth by producing phytohormones and vitamins-such interactions have not been shown for Phaeocystis. Therefore, we investigated the composition and function of P. globosa microbiomes. Specifically, we tested whether microbiome compositions are consistent across individual colonies from four P. globosa strains, whether similar microbiomes are re-recruited after antibiotic treatment, and how microbiomes affect P. globosa growth under limiting conditions. Results illuminated a core colonial P. globosa microbiome-including bacteria from the orders Alteromonadales, Burkholderiales, and Rhizobiales-that was re-recruited after microbiome disruption. Consistent microbiome composition and recruitment is indicative that P. globosa microbiomes are stable-state systems undergoing deterministic community assembly and suggests there are specific, beneficial interactions between Phaeocystis and bacteria. Growth experiments with axenic and nonaxenic cultures demonstrated that microbiomes allowed continued growth when B-vitamins were withheld, but that microbiomes accelerated culture collapse when nitrogen was withheld. In sum, this study reveals symbiotic and opportunistic interactions between Phaeocystis colonies and microbiome bacteria that could influence large-scale phytoplankton bloom dynamics and biogeochemical cycles.

Auxotrophic interactions: a stabilizing attribute of aquatic microbial communities?

Auxotrophy, or an organism's requirement for an exogenous source of an organic molecule, is widespread throughout species and ecosystems. Auxotrophy can result in obligate interactions between organisms, influencing ecosystem structure and community composition. We explore how auxotrophy-induced interactions between aquatic microorganisms affect microbial community structure and stability. While some studies have documented auxotrophy in aquatic microorganisms, these studies are not widespread, and we therefore do not know the full extent of auxotrophic interactions in aquatic environments. Current theoretical and experimental work suggests that auxotrophy links microbial community members through a complex web of metabolic dependencies. We discuss the proposed ways in which auxotrophy may enhance or undermine the stability of aquatic microbial communities, highlighting areas where our limited understanding of these interactions prevents us from being able to predict the ecological implications of auxotrophy. Finally, we examine an example of auxotrophy in harmful algal blooms to place this often theoretical discussion in a field context where auxotrophy may have implications for the development and robustness of algal bloom communities. We seek to draw attention to the relationship between auxotrophy and community stability in an effort to encourage further field and theoretical work that explores the underlying principles of microbial interactions.

Seasonal and Geographical Transitions in Eukaryotic Phytoplankton Community Structure in the Atlantic and Pacific Oceans.

Much is known about how broad eukaryotic phytoplankton groups vary according to nutrient availability in marine ecosystems. However, genus- and species-level dynamics are generally unknown, although important given that adaptation and acclimation processes differentiate at these levels. We examined phytoplankton communities across seasonal cycles in the North Atlantic (BATS) and under different trophic conditions in the eastern North Pacific (ENP), using phylogenetic classification of plastid-encoded 16S rRNA amplicon sequence variants (ASVs) and other methodologies, including flow cytometric cell sorting. Prasinophytes dominated eukaryotic phytoplankton amplicons during the nutrient-rich deep-mixing winter period at BATS. During stratification ('summer') uncultured dictyochophytes formed ∼35 ± 10% of all surface plastid amplicons and dominated those from stramenopile algae, whereas diatoms showed only minor, ephemeral contributions over the entire year. Uncultured dictyochophytes also comprised a major fraction of plastid amplicons in the oligotrophic ENP. Phylogenetic reconstructions of near-full length 16S rRNA sequences established 11 uncultured Dictyochophyte Environmental Clades (DEC). DEC-I and DEC-VI dominated surface dictyochophytes under stratification at BATS and in the ENP, and DEC-IV was also important in the latter. Additionally, although less common at BATS, Florenciella-related clades (FC) were prominent at depth in the ENP. In both ecosystems, pelagophytes contributed notably at depth, with PEC-VIII (Pelagophyte Environmental Clade) and (cultured) Pelagomonas calceolata being most important. Q-PCR confirmed the near absence of P. calceolata at the surface of the same oligotrophic sites where it reached ∼1,500 18S rRNA gene copies ml-1 at the DCM. To further characterize phytoplankton present in our samples, we performed staining and at-sea single-cell sorting experiments. Sequencing results from these indicated several uncultured dictyochophyte clades are comprised of predatory mixotrophs. From an evolutionary perspective, these cells showed both conserved and unique features in the chloroplast genome. In ENP metatranscriptomes we observed high expression of multiple chloroplast genes as well as expression of a selfish element (group II intron) in the psaA gene. Comparative analyses across the Pacific and Atlantic sites support the conclusion that predatory dictyochophytes thrive under low nutrient conditions. The observations that several uncultured dictyochophyte lineages are seemingly capable of photosynthesis and predation, raises questions about potential shifts in phytoplankton trophic roles associated with seasonality and long-term ocean change.

Keeping it light: (re)analyzing community-wide datasets without major infrastructure.

DNA sequencing technology has revolutionized the field of biology, shifting biology from a data-limited to data-rich state. Central to the interpretation of sequencing data are the computational tools and approaches that convert raw data into biologically meaningful information. Both the tools and the generation of data are actively evolving, yet the practice of re-analysis of previously generated data with new tools is not commonplace. Re-analysis of existing data provides an affordable means of generating new information and will likely become more routine within biology, yet necessitates a new set of considerations for best practices and resource development. Here, we discuss several practices that we believe to be broadly applicable when re-analyzing data, especially when done by small research groups.

Re-assembly, quality evaluation, and annotation of 678 microbial eukaryotic reference transcriptomes.

BACKGROUND: De novo transcriptome assemblies are required prior to analyzing RNA sequencing data from a species without an existing reference genome or transcriptome. Despite the prevalence of transcriptomic studies, the effects of using different workflows, or "pipelines," on the resulting assemblies are poorly understood. Here, a pipeline was programmatically automated and used to assemble and annotate raw transcriptomic short-read data collected as part of the Marine Microbial Eukaryotic Transcriptome Sequencing Project. The resulting transcriptome assemblies were evaluated and compared against assemblies that were previously generated with a different pipeline developed by the National Center for Genome Research. RESULTS: New transcriptome assemblies contained the majority of previous contigs as well as new content. On average, 7.8% of the annotated contigs in the new assemblies were novel gene names not found in the previous assemblies. Taxonomic trends were observed in the assembly metrics. Assemblies from the Dinoflagellata showed a higher number of contigs and unique k-mers than transcriptomes from other phyla, while assemblies from Ciliophora had a lower percentage of open reading frames compared to other phyla. CONCLUSIONS: Given current bioinformatics approaches, there is no single "best" reference transcriptome for a particular set of raw data. As the optimum transcriptome is a moving target, improving (or not) with new tools and approaches, automated and programmable pipelines are invaluable for managing the computationally intensive tasks required for re-processing large sets of samples with revised pipelines and ensuring a common evaluation workflow is applied to all samples. Thus, re-assembling existing data with new tools using automated and programmable pipelines may yield more accurate identification of taxon-specific trends across samples in addition to novel and useful products for the community.

Transcriptional Shifts Highlight the Role of Nutrients in Harmful Brown Tide Dynamics.

Harmful algal blooms (HABs) threaten ecosystems and human health worldwide. Controlling nitrogen inputs to coastal waters is a common HAB management strategy, as nutrient concentrations often suggest coastal blooms are nitrogen-limited. However, defining best nutrient management practices is a long-standing challenge: in part, because of difficulties in directly tracking the nutritional physiology of harmful species in mixed communities. Using metatranscriptome sequencing and incubation experiments, we addressed this challenge by assaying the in situ physiological ecology of the ecosystem destructive alga, Aureococcus anophagefferens. Here we show that gene markers of phosphorus deficiency were expressed in situ, and modulated by the enrichment of phosphorus, which was consistent with the observed growth rate responses. These data demonstrate the importance of phosphorus in controlling brown-tide dynamics, suggesting that phosphorus, in addition to nitrogen, should be evaluated in the management and mitigation of these blooms. Given that nutrient concentrations alone were suggestive of a nitrogen-limited ecosystem, this study underscores the value of directly assaying harmful algae in situ for the development of management strategies.

Author Correction: Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Transcriptional patterns identify resource controls on the diazotroph Trichodesmium in the Atlantic and Pacific oceans.

The N2-fixing cyanobacterium Trichodesmium is intensely studied because of the control this organism exerts over the cycling of carbon and nitrogen in the low nutrient ocean gyres. Although iron (Fe) and phosphorus (P) bioavailability are thought to be major drivers of Trichodesmium distributions and activities, identifying resource controls on Trichodesmium is challenging, as Fe and P are often organically complexed and their bioavailability to a single species in a mixed community is difficult to constrain. Further, Fe and P geochemistries are linked through the activities of metalloenzymes, such as the alkaline phosphatases (APs) PhoX and PhoA, which are used by microbes to access dissolved organic P (DOP). Here we identified significant correlations between Trichodesmium-specific transcriptional patterns in the North Atlantic (NASG) and North Pacific Subtropical Gyres (NPSG) and patterns in Fe and P biogeochemistry, with the relative enrichment of Fe stress markers in the NPSG, and P stress markers in the NASG. We also observed the differential enrichment of Fe-requiring PhoX transcripts in the NASG and Fe-insensitive PhoA transcripts in the NPSG, suggesting that metalloenzyme switching may be used to mitigate Fe limitation of DOP metabolism in Trichodesmium. This trait may underpin Trichodesmium success across disparate ecosystems.

Transcriptional response of the harmful raphidophyte Heterosigma akashiwo to nitrate and phosphate stress.

The marine eukaryotic alga Heterosigma akashiwo (Raphidophyceae) is known for forming ichthyotoxic harmful algal blooms (HABs). In the past 50 years, H. akashiwo blooms have increased, occurring globally in highly eutrophic coastal and estuarine systems. These systems often incur dramatic physicochemical changes, including macronutrient (nitrogen and phosphorus) enrichment and depletion, on short timescales. Here, H. akashiwo cultures grown under nutrient replete, low N and low P growth conditions were examined for changes in biochemical and physiological characteristics in concert with transcriptome sequencing to provide a mechanistic perspective on the metabolic processes involved in responding to N and P stress. There was a marked difference in the overall transcriptional pattern between low N and low P transcriptomes. Both nutrient stresses led to significant changes in the abundance of thousands of contigs related to a wide diversity of metabolic pathways, with limited overlap between the transcriptomic responses to low N and low P. Enriched contigs under low N included many related to nitrogen metabolism, acquisition, and transport. In addition, metabolic modules like photosynthesis and carbohydrate metabolism changed significantly under low N, coincident with treatment-specific changes in photosynthetic efficiency and particulate carbohydrate content. P-specific contigs responsible for P transport and organic P use were more enriched in the low P treatment than in the replete control and low N treatment. These results provide new insight into the genetic mechanisms that distinguish how this HAB species responds to these two common nutrient stresses, and the results can inform future field studies, linking transcriptional patterns to the physiological ecology of H. akashiwo in situ.

Conserved Transcriptional Responses to Nutrient Stress in Bloom-Forming Algae.

The concentration and composition of bioavailable nitrogen (N) and phosphorus (P) in the upper ocean shape eukaryotic phytoplankton communities and influence their physiological responses. Phytoplankton are known to exhibit similar physiological responses to limiting N and P conditions such as decreased growth rates, chlorosis, and increased assimilation of N and P. Are these responses similar at the molecular level across multiple species? To interrogate this question, five species from biogeochemically important, bloom-forming taxa (Bacillariophyta, Dinophyta, and Haptophyta) were grown under similar low N, low P, and replete nutrient conditions to identify transcriptional patterns and associated changes in biochemical pools related to N and P stress. Metabolic profiles, revealed through the transcriptomes of these taxa, clustered together based on species rather than nutrient stressor, suggesting that the global metabolic response to nutrient stresses was largely, but not exclusively, species-specific. Nutrient stress led to few transcriptional changes in the two dinoflagellates, consistent with other research. An orthologous group analysis examined functionally conserved (i.e., similarly changed) responses to nutrient stress and therefore focused on the diatom and haptophytes. Most conserved ortholog changes were specific to a single nutrient treatment, but a small number of orthologs were similarly changed under both N and P stress in 2 or more species. Many of these orthologs were related to photosynthesis and may represent generalized stress responses. A greater number of orthologs were conserved across more than one species under low P compared to low N. Screening the conserved orthologs for functions related to N and P metabolism revealed increased relative abundance of orthologs for nitrate, nitrite, ammonium, and amino acid transporters under N stress, and increased relative abundance of orthologs related to acquisition of inorganic and organic P substrates under P stress. Although the global transcriptional responses were dominated by species-specific changes, the analysis of conserved responses revealed functional similarities in resource acquisition pathways among different phytoplankton taxa. This overlap in nutrient stress responses observed among species may be useful for tracking the physiological ecology of phytoplankton field populations.

Virus-host relationships of marine single-celled eukaryotes resolved from metatranscriptomics.

Establishing virus-host relationships has historically relied on culture-dependent approaches. Here we report on the use of marine metatranscriptomics to probe virus-host relationships. Statistical co-occurrence analyses of dsDNA, ssRNA and dsRNA viral markers of polyadenylation-selected RNA sequences from microbial communities dominated by Aureococcus anophagefferens (Quantuck Bay, NY), and diatoms (Narragansett Bay, RI) show active infections by diverse giant viruses (NCLDVs) associated with algal and nonalgal hosts. Ongoing infections of A. anophagefferens by a known Mimiviridae (AaV) occur during bloom peak and decline. Bloom decline is also accompanied by increased activity of viruses other than AaV, including (+) ssRNA viruses. In Narragansett Bay, increased temporal resolution reveals active NCLDVs with both 'boom-and-bust' and 'steady-state infection'-like ecologies that include known as well as novel virus-host interactions. Our approach offers a method for screening active viral infections and develops links between viruses and their potential hosts in situ. Our observations further demonstrate that previously unknown virus-host relationships in marine systems are abundant.