The prokaryotic and eukaryotic microbiome of Pacific oyster spat is shaped by ocean warming but not acidification.

Pacific oysters (Magallana gigas, a.k.a. Crassostrea gigas), the most widely farmed oysters, are under threat from climate change and emerging pathogens. In part, their resilience may be affected by their microbiome, which, in turn, may be influenced by ocean warming and acidification. To understand these impacts, we exposed early-development Pacific oyster spat to different temperatures (18°C and 24°C) and pCO2 levels (800, 1,600, and 2,800 µatm) in a fully crossed design for 3 weeks. Under all conditions, the microbiome changed over time, with a large decrease in the relative abundance of potentially pathogenic ciliates (Uronema marinum) in all treatments with time. The microbiome composition differed significantly with temperature, but not acidification, indicating that Pacific oyster spat microbiomes can be altered by ocean warming but is resilient to ocean acidification in our experiments. Microbial taxa differed in relative abundance with temperature, implying different adaptive strategies and ecological specializations among microorganisms. Additionally, a small proportion (~0.2% of the total taxa) of the relatively abundant microbial taxa were core constituents (>50% occurrence among samples) across different temperatures, pCO2 levels, or time. Some taxa, including A4b bacteria and members of the family Saprospiraceae in the phyla Chloroflexi (syn. Chloroflexota) and Bacteroidetes (syn. Bacteroidota), respectively, as well as protists in the genera Labyrinthula and Aplanochytrium in the class Labyrinthulomycetes, and Pseudoperkinsus tapetis in the class Ichthyosporea were core constituents across temperatures, pCO2 levels, and time, suggesting that they play an important, albeit unknown, role in maintaining the structural and functional stability of the Pacific oyster spat microbiome in response to ocean warming and acidification. These findings highlight the flexibility of the spat microbiome to environmental changes.IMPORTANCEPacific oysters are the most economically important and widely farmed species of oyster, and their production depends on healthy oyster spat. In turn, spat health and productivity are affected by the associated microbiota; yet, studies have not scrutinized the effects of temperature and pCO2 on the prokaryotic and eukaryotic microbiomes of spat. Here, we show that both the prokaryotic and, for the first time, eukaryotic microbiome of Pacific oyster spat are surprisingly resilient to changes in acidification, but sensitive to ocean warming. The findings have potential implications for oyster survival amid climate change and underscore the need to understand temperature and pCO2 effects on the microbiome and the cascading effects on oyster health and productivity.

Deep6: Classification of Metatranscriptomic Sequences into Cellular Empires and Viral Realms Using Deep Learning Models.

Deep6 is a deep learning model that classifies metatranscriptomic sequences as short as 250 nucleotides into prokaryotes, eukaryotes, or one of the four viral realms, using a reference-independent and alignment-free approach. Average accuracies range from 0.87 to 0.97, depending on sequence length.

The Environment and Cyanophage Diversity: Insights From Environmental Sequencing of DNA Polymerase.

Globally distributed and abundant cyanophages in the family Myoviridae have dsDNA genomes with variable gene content, including host-derived auxiliary metabolic genes (AMGs) that potentially can facilitate viral replication. However, it is not well understood how this variation in gene content interacts with environmental variables to shape cyanomyovirus communities. This project correlated the genetic repertoire of cyanomyoviruses with their phyologeny, and investigated cyanomyovirus ecotype distribution as a function of environmental conditions across locations and seasons. Reference cyanomyovirus genomes were compared for their overlap in gene content to infer phyologenetic distances, and these distances were compared to distances calculated based on DNA polymerase (gp43) gene sequences. In turn, gp43 partial gene sequences amplified from natural cyanophage communities were used to describe cyanomyovirus community composition and to assess the relationship between environmental variables. The results showed the following: (1) DNA polymerase gene phylogeny generally correlated with the similarity in gene content among reference cyanomyoviruses, and thus can be used to describe environmental cyanomyovirus communities; (2) spatial and seasonal patterns in cyanomyovirus communities were related to environmental variables; (3) salinity and temperature, combined with nutrient concentration were predictors of cyanomyovirus richness, diversity and community composition. This study shows that environmental variables shape viral communities by drawing on a diverse seed bank of viral genotypes. From these results it is evident that that viral ecotypes with their corresponding genetic repertoires underlie selection pressures. However, the mechanisms involved in selecting for specific viral genotypes remain to be fully understood.

Transcriptional responses of the marine green alga Micromonas pusilla and an infecting prasinovirus under different phosphate conditions.

Prasinophytes are widespread marine algae for which responses to nutrient limitation and viral infection are not well understood. We studied the picoprasinophyte, Micromonas pusilla, grown under phosphate-replete (0.65 ± 0.07 d-1 ) and 10-fold lower (low)-phosphate (0.11 ± 0.04 d-1 ) conditions, and infected by the phycodnavirus MpV-SP1. Expression of 17% of Micromonas genes in uninfected cells differed by >1.5-fold (q < 0.01) between nutrient conditions, with genes for P-metabolism and the uniquely-enriched Sel1-like repeat (SLR) family having higher relative transcript abundances, while phospholipid-synthesis genes were lower in low-P than P-replete. Approximately 70% (P-replete) and 30% (low-P) of cells were lysed 24 h post-infection, and expression of ≤5.8% of host genes changed relative to uninfected treatments. Host genes for CAZymes and glycolysis were activated by infection, supporting importance in viral production, which was significantly lower in slower growing (low-P) hosts. All MpV-SP1 genes were expressed, and our analyses suggest responses to differing host-phosphate backgrounds involve few viral genes, while the temporal program of infection involves many more, and is largely independent of host-phosphate background. Our study (i) identifies genes previously unassociated with nutrient acclimation or viral infection, (ii) provides insights into the temporal program of prasinovirus gene expression by hosts and (iii) establishes cell biological aspects of an ecologically important host-prasinovirus system that differ from other marine algal-virus systems.

Author Correction: Re-examination of the relationship between marine virus and microbial cell abundances.

The original publication of this Article included analysis of virus and microbial cell abundances and virus-to-microbial cell ratios. Data in the Article came from 25 studies intended to be exclusively from marine sites. However, 3 of the studies included in the original unified dataset were erroneously classified as marine sites during compilation. The records with mis-recorded longitude and latitude values were, in fact, taken from inland, freshwater sources. The three inland, freshwater datasets are ELA, TROUT and SWAT. The data from these three studies represent 163 of the 5,671 records in the original publication. In the updated version of the Article, all analyses have been recalculated using the same statistical analysis pipeline released via GitHub as part of the original publication. Removal of the three studies reduces the unified dataset to 5,508 records. Analyses involving all grouped datasets have been updated with changes noted in each figure. All key results remain qualitatively unchanged. All data and scripts used in this correction have been made available as a new, updated GitHub release to reflect the updated dataset and figures.

Nutrients and Other Environmental Factors Influence Virus Abundances across Oxic and Hypoxic Marine Environments.

Virus particles are highly abundant in seawater and, on average, outnumber microbial cells approximately 10-fold at the surface and 16-fold in deeper waters; yet, this relationship varies across environments. Here, we examine the influence of a suite of environmental variables, including nutrient concentrations, salinity and temperature, on the relationship between the abundances of viruses and prokaryotes over a broad range of spatial and temporal scales, including along a track from the Northwest Atlantic to the Northeast Pacific via the Arctic Ocean, and in the coastal waters of British Columbia, Canada. Models of varying complexity were tested and compared for best fit with the Akaike Information Criterion, and revealed that nitrogen and phosphorus concentrations, as well as prokaryote abundances, either individually or combined, had significant effects on viral abundances in all but hypoxic environments, which were only explained by a combination of physical and chemical factors. Nonetheless, multivariate models of environmental variables showed high explanatory power, matching or surpassing that of prokaryote abundance alone. Incorporating both environmental variables and prokaryote abundances into multivariate models significantly improved the explanatory power of the models, except in hypoxic environments. These findings demonstrate that environmental factors could be as important as, or even more important than, prokaryote abundance in describing viral abundance across wide-ranging marine environments.

Variation in the Genetic Repertoire of Viruses Infecting Micromonas pusilla Reflects Horizontal Gene Transfer and Links to Their Environmental Distribution.

Prasinophytes, a group of eukaryotic phytoplankton, has a global distribution and is infected by large double-stranded DNA viruses (prasinoviruses) in the family Phycodnaviridae. This study examines the genetic repertoire, phylogeny, and environmental distribution of phycodnaviruses infecting Micromonas pusilla, other prasinophytes and chlorophytes. Based on comparisons among the genomes of viruses infecting M. pusilla and other phycodnaviruses, as well as the genome from a host isolate of M. pusilla, viruses infecting M. pusilla (MpVs) share a limited set of core genes, but vary strongly in their flexible pan-genome that includes numerous metabolic genes, such as those associated with amino acid synthesis and sugar manipulation. Surprisingly, few of these presumably host-derived genes are shared with M. pusilla, but rather have their closest non-viral homologue in bacteria and other eukaryotes, indicating horizontal gene transfer. A comparative analysis of full-length DNA polymerase (DNApol) genes from prasinoviruses with their overall gene content, demonstrated that the phylogeny of DNApol gene fragments reflects the gene content of the viruses; hence, environmental DNApol gene sequences from prasinoviruses can be used to infer their overall genetic repertoire. Thus, the distribution of virus ecotypes across environmental samples based on DNApol sequences implies substantial underlying differences in gene content that reflect local environmental conditions. Moreover, the high diversity observed in the genetic repertoire of prasinoviruses has been driven by horizontal gene transfer throughout their evolutionary history, resulting in a broad suite of functional capabilities and a high diversity of prasinovirus ecotypes.

Re-examination of the relationship between marine virus and microbial cell abundances.

Marine viruses are critical drivers of ocean biogeochemistry, and their abundances vary spatiotemporally in the global oceans, with upper estimates exceeding 10(8) per ml. Over many years, a consensus has emerged that virus abundances are typically tenfold higher than microbial cell abundances. However, the true explanatory power of a linear relationship and its robustness across diverse ocean environments is unclear. Here, we compile 5,671 microbial cell and virus abundance estimates from 25 distinct marine surveys and find substantial variation in the virus-to-microbial cell ratio, in which a 10:1 model has either limited or no explanatory power. Instead, virus abundances are better described as nonlinear, power-law functions of microbial cell abundances. The fitted scaling exponents are typically less than 1, implying that the virus-to-microbial cell ratio decreases with microbial cell density, rather than remaining fixed. The observed scaling also implies that viral effect sizes derived from 'representative' abundances require substantial refinement to be extrapolated to regional or global scales.

A comprehensive method for amplicon-based and metagenomic characterization of viruses, bacteria, and eukaryotes in freshwater samples.

BACKGROUND: Studies of environmental microbiota typically target only specific groups of microorganisms, with most focusing on bacteria through taxonomic classification of 16S rRNA gene sequences. For a more holistic understanding of a microbiome, a strategy to characterize the viral, bacterial, and eukaryotic components is necessary. RESULTS: We developed a method for metagenomic and amplicon-based analysis of freshwater samples involving the concentration and size-based separation of eukaryotic, bacterial, and viral fractions. Next-generation sequencing and culture-independent approaches were used to describe and quantify microbial communities in watersheds with different land use in British Columbia. Deep amplicon sequencing was used to investigate the distribution of certain viruses (g23 and RdRp), bacteria (16S rRNA and cpn60), and eukaryotes (18S rRNA and ITS). Metagenomic sequencing was used to further characterize the gene content of the bacterial and viral fractions at both taxonomic and functional levels. CONCLUSION: This study provides a systematic approach to separate and characterize eukaryotic-, bacterial-, and viral-sized particles. Methodologies described in this research have been applied in temporal and spatial studies to study the impact of land use on watershed microbiomes in British Columbia.

Rising CO2 levels will intensify phytoplankton blooms in eutrophic and hypertrophic lakes.

Harmful algal blooms threaten the water quality of many eutrophic and hypertrophic lakes and cause severe ecological and economic damage worldwide. Dense blooms often deplete the dissolved CO2 concentration and raise pH. Yet, quantitative prediction of the feedbacks between phytoplankton growth, CO2 drawdown and the inorganic carbon chemistry of aquatic ecosystems has received surprisingly little attention. Here, we develop a mathematical model to predict dynamic changes in dissolved inorganic carbon (DIC), pH and alkalinity during phytoplankton bloom development. We tested the model in chemostat experiments with the freshwater cyanobacterium Microcystis aeruginosa at different CO2 levels. The experiments showed that dense blooms sequestered large amounts of atmospheric CO2, not only by their own biomass production but also by inducing a high pH and alkalinity that enhanced the capacity for DIC storage in the system. We used the model to explore how phytoplankton blooms of eutrophic waters will respond to rising CO2 levels. The model predicts that (1) dense phytoplankton blooms in low- and moderately alkaline waters can deplete the dissolved CO2 concentration to limiting levels and raise the pH over a relatively wide range of atmospheric CO2 conditions, (2) rising atmospheric CO2 levels will enhance phytoplankton blooms in low- and moderately alkaline waters with high nutrient loads, and (3) above some threshold, rising atmospheric CO2 will alleviate phytoplankton blooms from carbon limitation, resulting in less intense CO2 depletion and a lesser increase in pH. Sensitivity analysis indicated that the model predictions were qualitatively robust. Quantitatively, the predictions were sensitive to variation in lake depth, DIC input and CO2 gas transfer across the air-water interface, but relatively robust to variation in the carbon uptake mechanisms of phytoplankton. In total, these findings warn that rising CO2 levels may result in a marked intensification of phytoplankton blooms in eutrophic and hypertrophic waters.

Contrasting effects of rising CO2 on primary production and ecological stoichiometry at different nutrient levels.

Although rising CO2 concentrations are thought to promote the growth and alter the carbon : nutrient stoichiometry of primary producers, several studies have reported conflicting results. To reconcile these contrasting results, we tested the following hypotheses: rising CO2 levels (1) will increase phytoplankton biomass more at high nutrient loads than at low nutrient loads, but (2) will increase their carbon : nutrient stoichiometry more at low than at high nutrient loads. We formulated a mathematical model to predict dynamic changes in phytoplankton population density, elemental stoichiometry and inorganic carbon chemistry in response to rising CO2 . The model was tested in chemostat experiments with the freshwater cyanobacterium Microcystis aeruginosa. The model predictions and experimental results confirmed the hypotheses. Our findings provide a novel theoretical framework to understand and predict effects of rising CO2 concentrations on primary producers and their nutritional quality as food for herbivores under different nutrient conditions.

Reversal in competitive dominance of a toxic versus non-toxic cyanobacterium in response to rising CO2.

Climate change scenarios predict a doubling of the atmospheric CO(2) concentration by the end of this century. Yet, how rising CO(2) will affect the species composition of aquatic microbial communities is still largely an open question. In this study, we develop a resource competition model to investigate competition for dissolved inorganic carbon in dense algal blooms. The model predicts how dynamic changes in carbon chemistry, pH and light conditions during bloom development feed back on competing phytoplankton species. We test the model predictions in chemostat experiments with monocultures and mixtures of a toxic and non-toxic strain of the freshwater cyanobacterium Microcystis aeruginosa. The toxic strain was able to reduce dissolved CO(2) to lower concentrations than the non-toxic strain, and became dominant in competition at low CO(2) levels. Conversely, the non-toxic strain could grow at lower light levels, and became dominant in competition at high CO(2) levels but low light availability. The model captured the observed reversal in competitive dominance, and was quantitatively in good agreement with the results of the competition experiments. To assess whether microcystins might have a role in this reversal of competitive dominance, we performed further competition experiments with the wild-type strain M. aeruginosa PCC 7806 and its mcyB mutant impaired in microcystin production. The microcystin-producing wild type had a strong selective advantage at low CO(2) levels but not at high CO(2) levels. Our results thus demonstrate both in theory and experiment that rising CO(2) levels can alter the community composition and toxicity of harmful algal blooms.