Viral Production in Seawater Filtered Through 0.2-μm Pore-Size Filters: A Hidden Biogeochemical Cycle in a Neglected Realm

Viral production is a key parameter for assessing virus-mediated biogeochemical cycles. One widely used method for the determination of viral production, called the virus reduction assay, reduces viral abundance, while maintaining bacterial abundance, using 0.2-μm pore-size filters. Viral production is estimated from the increase of viral abundance during incubation. We hypothesized that small-cell-sized bacterial communities can pass through 0.2-μm filters and drive viral production, representing a missing fraction of viral production that is missed by the virus reduction assay. Coastal seawater was filtered through 0.2-μm filters and diluted with virus-free seawater. Viral production in the <0.2-μm filtrate was estimated from changes in viral abundance determined through flow cytometry. We found that viruses were produced in the <0.2-μm communities, which were strongly enriched with low nucleic acid content bacteria. Estimated viral production in the <0.2-μm filtrates accounted for up to 43% of total viral production and 10% of dissolved organic carbon production mediated by viral lysis of bacterial cells. By not considering viral production in these <0.2-μm communities, the virus reduction assay may underestimate viral production. Virus–bacteria interactions in <0.2-μm communities may represent a significant and overlooked role of viruses in marine food webs and carbon fluxes.

Coral Reef Viruses in Kane'ohe Bay, Hawai'i: Abundance, Diversity & Environmental Drivers

<p>Viruses are a ubiquitous component of coral reef ecosystems, with several viral types, from at least seven prokaryotic and 20 eukaryotic virus families currently characterised from the surface mucopolysaccharide layer (SML), coral tissue and the water column. However, little is known about the ecology and function of these viruses. For example, what are the environmental drivers of viral abundance and diversity on coral reefs? In this study, the abundance and distribution of virus-like particles (VLPs) associated with the SML and reef water of the coral Montipora capitata were determined using epifluorescence microscopy, while transmission electron microscopy was employed to determine the morphological diversity of VLPs. Sampling was conducted across the Coconut Island Marine Reserve (CIMR) reef system, Kane’ohe Bay, O’ahu, Hawai’i. Viral abundance was correlated with select environmental drivers and prokaryote abundance, while non-metric multidimensional scaling was used to determine the key environmental drivers of the viral community assemblage. The water column contained high concentrations of VLPs (5.98 × 107 ml-1) and prokaryotes (3.11 × 106 ml-1), consistent with the considerable anthropogenic impacts at this location. In comparison, the SML contained lower concentrations of VLPs (2.61 × 107 ml-1) and prokaryotes (2.08 × 106 ml-1); of note, the densities of viruses and prokaryotes in the SML were strongly coupled while those in the reef water were not. VLP density in the water column varied spatially across the reef, with the most sheltered site and the only one not situated on the reef crest having a greater VLP density than the other sites. Temporal variations in the density of microbes (i.e. viruses and prokaryotes) in the reef water were pronounced, while in the SML microbial densities remained constant. However, no specific environmental drivers of this variability could be identified. In contrast, temperature and water quality were correlated with shifts in the morphological diversity of VLPs across the reef. Small (< 50 nm) polyhedral/spherical VLPs were dominant, and were positively correlated to chlorophyll-a concentration when in the SML. In this same habitat, Fuselloviridae-like VLPs, filamentous VLPs and bead-shaped VLPs were positively correlated to temperature. In the reef water a different pattern was apparent: large (> 100 nm) Podoviridae-like VLPs and elongate Myoviridae-like VLPs, as well as lemon-shaped VLPs of both size classes showed positive associations with turbidity, while large filamentous VLPs, Geminiviridae-like VLPs and rod-shaped VLPs were positively associated with temperature. These results demonstrate that the viral community of Coconut Island’s reef is highly diverse, and subject to spatial and temporal change, especially in the water column. However, while the environmental drivers of viral diversity were partly elucidated, we are still a long way from understanding the drivers of viral abundance. More detailed study, both spatially and temporally, of the CIMR environment is required, as is comprehensive molecular analysis of the viral community of Kane’ohe Bay. Only then can we begin to understand the importance of viruses to the health and function of this, and other reef sites.</p>

Coral Reef Viruses in Kane'ohe Bay, Hawai'i: Abundance, Diversity & Environmental Drivers

<p>Viruses are a ubiquitous component of coral reef ecosystems, with several viral types, from at least seven prokaryotic and 20 eukaryotic virus families currently characterised from the surface mucopolysaccharide layer (SML), coral tissue and the water column. However, little is known about the ecology and function of these viruses. For example, what are the environmental drivers of viral abundance and diversity on coral reefs? In this study, the abundance and distribution of virus-like particles (VLPs) associated with the SML and reef water of the coral Montipora capitata were determined using epifluorescence microscopy, while transmission electron microscopy was employed to determine the morphological diversity of VLPs. Sampling was conducted across the Coconut Island Marine Reserve (CIMR) reef system, Kane’ohe Bay, O’ahu, Hawai’i. Viral abundance was correlated with select environmental drivers and prokaryote abundance, while non-metric multidimensional scaling was used to determine the key environmental drivers of the viral community assemblage. The water column contained high concentrations of VLPs (5.98 × 107 ml-1) and prokaryotes (3.11 × 106 ml-1), consistent with the considerable anthropogenic impacts at this location. In comparison, the SML contained lower concentrations of VLPs (2.61 × 107 ml-1) and prokaryotes (2.08 × 106 ml-1); of note, the densities of viruses and prokaryotes in the SML were strongly coupled while those in the reef water were not. VLP density in the water column varied spatially across the reef, with the most sheltered site and the only one not situated on the reef crest having a greater VLP density than the other sites. Temporal variations in the density of microbes (i.e. viruses and prokaryotes) in the reef water were pronounced, while in the SML microbial densities remained constant. However, no specific environmental drivers of this variability could be identified. In contrast, temperature and water quality were correlated with shifts in the morphological diversity of VLPs across the reef. Small (< 50 nm) polyhedral/spherical VLPs were dominant, and were positively correlated to chlorophyll-a concentration when in the SML. In this same habitat, Fuselloviridae-like VLPs, filamentous VLPs and bead-shaped VLPs were positively correlated to temperature. In the reef water a different pattern was apparent: large (> 100 nm) Podoviridae-like VLPs and elongate Myoviridae-like VLPs, as well as lemon-shaped VLPs of both size classes showed positive associations with turbidity, while large filamentous VLPs, Geminiviridae-like VLPs and rod-shaped VLPs were positively associated with temperature. These results demonstrate that the viral community of Coconut Island’s reef is highly diverse, and subject to spatial and temporal change, especially in the water column. However, while the environmental drivers of viral diversity were partly elucidated, we are still a long way from understanding the drivers of viral abundance. More detailed study, both spatially and temporally, of the CIMR environment is required, as is comprehensive molecular analysis of the viral community of Kane’ohe Bay. Only then can we begin to understand the importance of viruses to the health and function of this, and other reef sites.</p>

Coral Reef Viruses in Kane'ohe Bay, Hawai'i: Abundance, Diversity & Environmental Drivers

<p>Viruses are a ubiquitous component of coral reef ecosystems, with several viral types, from at least seven prokaryotic and 20 eukaryotic virus families currently characterised from the surface mucopolysaccharide layer (SML), coral tissue and the water column. However, little is known about the ecology and function of these viruses. For example, what are the environmental drivers of viral abundance and diversity on coral reefs? In this study, the abundance and distribution of virus-like particles (VLPs) associated with the SML and reef water of the coral Montipora capitata were determined using epifluorescence microscopy, while transmission electron microscopy was employed to determine the morphological diversity of VLPs. Sampling was conducted across the Coconut Island Marine Reserve (CIMR) reef system, Kane’ohe Bay, O’ahu, Hawai’i. Viral abundance was correlated with select environmental drivers and prokaryote abundance, while non-metric multidimensional scaling was used to determine the key environmental drivers of the viral community assemblage. The water column contained high concentrations of VLPs (5.98 × 107 ml-1) and prokaryotes (3.11 × 106 ml-1), consistent with the considerable anthropogenic impacts at this location. In comparison, the SML contained lower concentrations of VLPs (2.61 × 107 ml-1) and prokaryotes (2.08 × 106 ml-1); of note, the densities of viruses and prokaryotes in the SML were strongly coupled while those in the reef water were not. VLP density in the water column varied spatially across the reef, with the most sheltered site and the only one not situated on the reef crest having a greater VLP density than the other sites. Temporal variations in the density of microbes (i.e. viruses and prokaryotes) in the reef water were pronounced, while in the SML microbial densities remained constant. However, no specific environmental drivers of this variability could be identified. In contrast, temperature and water quality were correlated with shifts in the morphological diversity of VLPs across the reef. Small (< 50 nm) polyhedral/spherical VLPs were dominant, and were positively correlated to chlorophyll-a concentration when in the SML. In this same habitat, Fuselloviridae-like VLPs, filamentous VLPs and bead-shaped VLPs were positively correlated to temperature. In the reef water a different pattern was apparent: large (> 100 nm) Podoviridae-like VLPs and elongate Myoviridae-like VLPs, as well as lemon-shaped VLPs of both size classes showed positive associations with turbidity, while large filamentous VLPs, Geminiviridae-like VLPs and rod-shaped VLPs were positively associated with temperature. These results demonstrate that the viral community of Coconut Island’s reef is highly diverse, and subject to spatial and temporal change, especially in the water column. However, while the environmental drivers of viral diversity were partly elucidated, we are still a long way from understanding the drivers of viral abundance. More detailed study, both spatially and temporally, of the CIMR environment is required, as is comprehensive molecular analysis of the viral community of Kane’ohe Bay. Only then can we begin to understand the importance of viruses to the health and function of this, and other reef sites.</p>

Coral Reef Viruses in Kane'ohe Bay, Hawai'i: Abundance, Diversity & Environmental Drivers

<p>Viruses are a ubiquitous component of coral reef ecosystems, with several viral types, from at least seven prokaryotic and 20 eukaryotic virus families currently characterised from the surface mucopolysaccharide layer (SML), coral tissue and the water column. However, little is known about the ecology and function of these viruses. For example, what are the environmental drivers of viral abundance and diversity on coral reefs? In this study, the abundance and distribution of virus-like particles (VLPs) associated with the SML and reef water of the coral Montipora capitata were determined using epifluorescence microscopy, while transmission electron microscopy was employed to determine the morphological diversity of VLPs. Sampling was conducted across the Coconut Island Marine Reserve (CIMR) reef system, Kane’ohe Bay, O’ahu, Hawai’i. Viral abundance was correlated with select environmental drivers and prokaryote abundance, while non-metric multidimensional scaling was used to determine the key environmental drivers of the viral community assemblage. The water column contained high concentrations of VLPs (5.98 × 107 ml-1) and prokaryotes (3.11 × 106 ml-1), consistent with the considerable anthropogenic impacts at this location. In comparison, the SML contained lower concentrations of VLPs (2.61 × 107 ml-1) and prokaryotes (2.08 × 106 ml-1); of note, the densities of viruses and prokaryotes in the SML were strongly coupled while those in the reef water were not. VLP density in the water column varied spatially across the reef, with the most sheltered site and the only one not situated on the reef crest having a greater VLP density than the other sites. Temporal variations in the density of microbes (i.e. viruses and prokaryotes) in the reef water were pronounced, while in the SML microbial densities remained constant. However, no specific environmental drivers of this variability could be identified. In contrast, temperature and water quality were correlated with shifts in the morphological diversity of VLPs across the reef. Small (< 50 nm) polyhedral/spherical VLPs were dominant, and were positively correlated to chlorophyll-a concentration when in the SML. In this same habitat, Fuselloviridae-like VLPs, filamentous VLPs and bead-shaped VLPs were positively correlated to temperature. In the reef water a different pattern was apparent: large (> 100 nm) Podoviridae-like VLPs and elongate Myoviridae-like VLPs, as well as lemon-shaped VLPs of both size classes showed positive associations with turbidity, while large filamentous VLPs, Geminiviridae-like VLPs and rod-shaped VLPs were positively associated with temperature. These results demonstrate that the viral community of Coconut Island’s reef is highly diverse, and subject to spatial and temporal change, especially in the water column. However, while the environmental drivers of viral diversity were partly elucidated, we are still a long way from understanding the drivers of viral abundance. More detailed study, both spatially and temporally, of the CIMR environment is required, as is comprehensive molecular analysis of the viral community of Kane’ohe Bay. Only then can we begin to understand the importance of viruses to the health and function of this, and other reef sites.</p>

Quantitative PCR of Viral Abundance v1

Protocol for quantifying viral abundance by PCR used in Yoo et al 2021

High viral abundance and low diversity are associated with increased CRISPR-Cas prevalence across microbial ecosystems

CRISPR-Cas are adaptive immune systems that protect their hosts against viruses and other parasitic mobile genetic elements. Consequently, selection from viruses and other genetic parasites is often assumed to drive the acquisition and maintenance of these immune systems in nature, but this remains untested. Here, we analyse the abundance of CRISPR arrays in natural environments using metagenomic datasets from 332 terrestrial, aquatic and host-associated ecosystems. For each metagenome we quantified viral abundance and levels of viral community diversity to test whether these variables can explain variation in CRISPR-Cas abundance across ecosystems. We find a strong positive correlation between CRISPR-Cas abundance and viral abundance. In addition, when controlling for differences in viral abundance, we found that the CRISPR-Cas systems are more abundant when viral diversity is low. We also found differences in relative CRISPR-Cas abundance among environments, with environmental classification explaining ~24% of variation in CRISPR-Cas abundance. However, the correlations with viral abundance and diversity are broadly consistent across diverse natural environments. These results indicate that viral abundance and diversity are major ecological factors that drive the selection and maintenance of CRISPR-Cas in microbial ecosystems.

Assessing the involvement of the placental microbiome and virome in preeclampsia using non coding RNA sequencing

Objectives Preeclampsia is a dangerous pregnancy complication. The source of preeclampsia is unknown, though the placenta is believed to have a central role in its pathogenesis. An association between maternal infection and preeclampsia has been demonstrated, yet the involvement of the placental microbiome in the etiology of preeclampsia has not been determined. In this study, we examined whether preeclampsia is associated with an imbalanced microorganism composition in the placenta. Methods To this end, we developed a novel method for the identification of bacteria/viruses based on sequencing of small non-coding RNA, which increases the microorganism-to-host ratio, this being a major challenge in microbiome methods. We validated the method on various infected tissues and demonstrated its efficiency in detecting microorganisms in samples with extremely low bacterial/viral biomass. We then applied the method to placenta specimens from preeclamptic and healthy pregnancies. Since the placenta is a remarkably large and heterogeneous organ, we explored the bacterial and viral RNA at each of 15 distinct locations. Results Bacterial RNA was detected at all locations and was consistent with previous studies of the placental microbiome, though without significant differences between the preeclampsia and control groups. Nevertheless, the bacterial RNA composition differed significantly between various areas of the placenta. Viral RNA was detected in extremely low quantities, below the threshold of significance, thus viral abundance could not be determined. Conclusions Our results suggest that the bacterial and viral abundance in the placenta may have only limited involvement in the pathogenesis of preeclampsia. The evidence of a heterogenic bacterial RNA composition in the various placental locations warrants further investigation to capture the true nature of the placental microbiome.

Seasonal Variation in Viral Infection Rates and Cell Sizes of Infected Prokaryotes in a Large and Deep Freshwater Lake (Lake Biwa, Japan)

As viruses regulate prokaryotic abundance and the carbon cycle by infecting and lysing their prokaryotic hosts, the volume of infected prokaryotes is an important parameter for understanding the impact of viruses on aquatic environments. However, literature regarding the seasonal and spatial variations in the cell volume of infected prokaryotes is limited, despite the volume of the prokaryotic community varying dynamically with season and water column depth. Here, we conducted a field survey for two annual cycles in a large and deep freshwater lake (Lake Biwa, Japan), where large prokaryotes inhabit the deeper layer during the stratified period. We used transmission electron microscopy to reveal the seasonal and spatial variation in the frequency of viral infection and cell volume of infected prokaryotes. We found that the viral infection rate in the surface layer increased when estimated contact rates increased during the middle of the stratified period, whereas the infection rate in the deeper layer increased despite low estimated contact rates during the end of the stratified period. In addition, in the deeper layer, the fraction of large prokaryotes in the total and infected prokaryotic communities increased progressively while the number of intracellular viral particles increased. We suggest different ways in which the viral abundance is maintained in the two water layers. In the surface layer, it is speculated that viral abundance is supported by the high viral infection rate because of the high activity of prokaryotes, whereas in the deeper layer, it might be supported by the larger number of intracellular viral particles released from large prokaryotes. Moreover, large prokaryotes could contribute as important sources of organic substrates via viral lysis in the deeper layer, where labile dissolved organic matter is depleted.