Targeting a free viral fraction enhances the early alert potential of wastewater surveillance for SARS-CoV-2: a methods comparison spanning the transition between delta and omicron variants in a large urban center.

Introduction: Wastewater surveillance has proven to be a valuable approach to monitoring the spread of SARS-CoV-2, the virus that causes Coronavirus disease 2019 (COVID-19). Recognizing the benefits of wastewater surveillance as a tool to support public health in tracking SARS-CoV-2 and other respiratory pathogens, numerous wastewater virus sampling and concentration methods have been tested for appropriate applications as well as their significance for actionability by public health practices. Methods: Here, we present a 34-week long wastewater surveillance study that covers nearly 4 million residents of the Detroit (MI, United States) metropolitan area. Three primary concentration methods were compared with respect to recovery of SARS-CoV-2 from wastewater: Virus Adsorption-Elution (VIRADEL), polyethylene glycol precipitation (PEG), and polysulfone (PES) filtration. Wastewater viral concentrations were normalized using various parameters (flow rate, population, total suspended solids) to account for variations in flow. Three analytical approaches were implemented to compare wastewater viral concentrations across the three primary concentration methods to COVID-19 clinical data for both normalized and non-normalized data: Pearson and Spearman correlations, Dynamic Time Warping (DTW), and Time Lagged Cross Correlation (TLCC) and peak synchrony. Results: It was found that VIRADEL, which captures free and suspended virus from supernatant wastewater, was a leading indicator of COVID-19 cases within the region, whereas PEG and PES filtration, which target particle-associated virus, each lagged behind the early alert potential of VIRADEL. PEG and PES methods may potentially capture previously shed and accumulated SARS-CoV-2 resuspended from sediments in the interceptors. Discussion: These results indicate that the VIRADEL method can be used to enhance the early-warning potential of wastewater surveillance applications although drawbacks include the need to process large volumes of wastewater to concentrate sufficiently free and suspended virus for detection. While lagging the VIRADEL method for early-alert potential, both PEG and PES filtration can be used for routine COVID-19 wastewater monitoring since they allow a large number of samples to be processed concurrently while being more cost-effective and with rapid turn-around yielding results same day as collection.

Multi-year molecular quantification and 'omics analysis of Planktothrix-specific cyanophage sequences from Sandusky Bay, Lake Erie.

Introduction: Planktothrix agardhii is a microcystin-producing cyanobacterium found in Sandusky Bay, a shallow and turbid embayment of Lake Erie. Previous work in other systems has indicated that cyanophages are an important natural control factor of harmful algal blooms. Currently, there are few cyanophages that are known to infect P. agardhii, with the best-known being PaV-LD, a tail-less cyanophage isolated from Lake Donghu, China. Presented here is a molecular characterization of Planktothrix specific cyanophages in Sandusky Bay. Methods and Results: Putative Planktothrix-specific viral sequences from metagenomic data from the bay in 2013, 2018, and 2019 were identified by two approaches: homology to known phage PaV-LD, or through matching CRISPR spacer sequences with Planktothrix host genomes. Several contigs were identified as having viral signatures, either related to PaV-LD or potentially novel sequences. Transcriptomic data from 2015, 2018, and 2019 were also employed for the further identification of cyanophages, as well as gene expression of select viral sequences. Finally, viral quantification was tested using qPCR in 2015-2019 for PaV-LD like cyanophages to identify the relationship between presence and gene expression of these cyanophages. Notably, while PaV-LD like cyanophages were in high abundance over the course of multiple years (qPCR), transcriptomic analysis revealed only low levels of viral gene expression. Discussion: This work aims to provide a broader understanding of Planktothrix cyanophage diversity with the goals of teasing apart the role of cyanophages in the control and regulation of harmful algal blooms and designing monitoring methodology for potential toxin-releasing lysis events.

Spatio-temporal connectivity of the aquatic microbiome associated with cyanobacterial blooms along a Great Lake riverine-lacustrine continuum.

Lake Erie is subject to recurring events of cyanobacterial harmful algal blooms (cHABs), but measures of nutrients and total phytoplankton biomass seem to be poor predictors of cHABs when taken individually. A more integrated approach at the watershed scale may improve our understanding of the conditions that lead to bloom formation, such as assessing the physico-chemical and biological factors that influence the lake microbial community, as well as identifying the linkages between Lake Erie and the surrounding watershed. Within the scope of the Government of Canada's Genomics Research and Development Initiative (GRDI) Ecobiomics project, we used high-throughput sequencing of the 16S rRNA gene to characterize the spatio-temporal variability of the aquatic microbiome in the Thames River-Lake St. Clair-Detroit River-Lake Erie aquatic corridor. We found that the aquatic microbiome was structured along the flow path and influenced mainly by higher nutrient concentrations in the Thames River, and higher temperature and pH downstream in Lake St. Clair and Lake Erie. The same dominant bacterial phyla were detected along the water continuum, changing only in relative abundance. At finer taxonomical level, however, there was a clear shift in the cyanobacterial community, with Planktothrix dominating in the Thames River and Microcystis and Synechococcus in Lake St. Clair and Lake Erie. Mantel correlations highlighted the importance of geographic distance in shaping the microbial community structure. The fact that a high proportion of microbial sequences found in the Western Basin of Lake Erie were also identified in the Thames River, indicated a high degree of connectivity and dispersal within the system, where mass effect induced by passive transport play an important role in microbial community assembly. Nevertheless, some cyanobacterial amplicon sequence variants (ASVs) related to Microcystis, representing less than 0.1% of relative abundance in the upstream Thames River, became dominant in Lake St. Clair and Erie, suggesting selection of those ASVs based on the lake conditions. Their extremely low relative abundances in the Thames suggest additional sources are likely to contribute to the rapid development of summer and fall blooms in the Western Basin of Lake Erie. Collectively, these results, which can be applied to other watersheds, improve our understanding of the factors influencing aquatic microbial community assembly and provide new perspectives on how to better understand the occurrence of cHABs in Lake Erie and elsewhere.

Corrigendum: Comparative metabolomic analysis of exudates of high-microcystin-producing and low-microcystin-producing Microcystis aeruginosa strains.

[This corrects the article DOI: 10.3389/fmicb.2022.1075621.].

Comparative metabolomic analysis of exudates of microcystin-producing and microcystin-free Microcystis aeruginosa strains.

Cyanobacterial harmful algal blooms (cHABs) dominated by Microcystis aeruginosa threaten the ecological integrity and beneficial uses of lakes globally. In addition to producing hepatotoxic microcystins (MC), M. aeruginosa exudates (MaE) contain various compounds with demonstrated toxicity to aquatic biota. Previously, we found that the ecotoxicity of MaE differed between MC-producing and MC-free strains at exponential (E-phase) and stationary (S-phase) growth phases. However, the components in these exudates and their specific harmful effects were unclear. In this study, we performed untargeted metabolomics based on liquid chromatography-mass spectrometry to reveal the constituents in MaE of a MC-producing and a MC-free strain at both E-phase and S-phase. A total of 409 metabolites were identified and quantified based on their relative abundance. These compounds included lipids, organoheterocyclic compounds, organic acid, benzenoids and organic oxygen compounds. Multivariate analysis revealed that strains and growth phases significantly influenced the metabolite profile. The MC-producing strain had greater total metabolites abundance than the MC-free strain at S-phase, whereas the MC-free strain released higher concentrations of benzenoids, lipids, organic oxygen, organic nitrogen and organoheterocyclic compounds than the MC-producing strain at E-phase. Total metabolites had higher abundance in S-phase than in E- phase in both strains. Analysis of differential metabolites (DMs) and pathways suggest that lipids metabolism and biosynthesis of secondary metabolites were more tightly coupled to growth phases than to strains. Abundance of some toxic lipids and benzenoids DMs were significantly higher in the MC-free strain than the MC-producing one. This study builds on the understanding of MaE chemicals and their biotoxicity, and adds to evidence that non-MC-producing strains of cyanobacteria may also pose a threat to ecosystem health.

Antibiotic pollution promotes dominance by harmful cyanobacteria: A case study examining norfloxacin exposure in competition experiments.

Cyanobacterial harmful algal blooms (cyanoHABs) in freshwater lakes across the globe are often combined with other stressors. Pharmaceutical pollution, especially antibiotics in water bodies, poses a potential hazard in aquatic ecosystems. However, how antibiotics influence the risk of cyanoHABs remains unclear. Here, we investigated the effects of norfloxacin (NOR), one of the most widely used antibiotics globally, to a bloom-forming cyanobacterium (Microcystis aeruginosa) and a common green alga (Scenedesmus quadricauda), under both mono- and coculture conditions. Taxon-specific responses to NOR were evaluated in monoculture. In addition, the growth rate and change in ratio of cyanobacteria to green algae when cocultured with exposure to NOR were determined. In monocultures of Microcystis, exposure to low concentrations of NOR resulted in decreases in biomass, chlorophyll a and soluble protein content, while superoxide anion content and superoxide dismutase activity increased. However, NOR at high concentration only slightly affected Scenedesmus. During the co-culture trials of Microcystis and Scenedesmus, the 5 µg · L-1 NOR treatment increased the ratio of Microcystis to co-cultured Scenedesmus by 47.2%. Meanwhile, although Scenedesmus growth was enhanced by 4.2% under NOR treatment in monoculture, it was conversely inhibited by 63.4% and 38.2% when co-cultured with Microcystis with and without NOR, respectively. Our results indicate that antibiotic pollution has a potential risk to enhance the perniciousness of cyanoHABs by disturbing interspecific interaction between cyanobacteria and green algae. These results reinforce the need for scientists and managers to consider the influence of xenobiotics in shaping the outcome of interactions among multiple species in aquatic ecosystems.

Metatranscriptomic Analyses of Diel Metabolic Functions During a Microcystis Bloom in Western Lake Erie (United States).

This study examined diel shifts in metabolic functions of Microcystis spp. during a 48-h Lagrangian survey of a toxin-producing cyanobacterial bloom in western Lake Erie in the aftermath of the 2014 Toledo Water Crisis. Transcripts mapped to the genomes of recently sequenced lower Great Lakes Microcystis isolates showed distinct patterns of gene expression between samples collected across day (10:00 h, 16:00 h) and night (22:00 h, 04:00 h). Daytime transcripts were enriched in functions related to Photosystem II (e.g., psbA), nitrogen and phosphate acquisition, cell division (ftsHZ), heat shock response (dnaK, groEL), and uptake of inorganic carbon (rbc, bicA). Genes transcribed during nighttime included those involved in phycobilisome protein synthesis and Photosystem I core subunits. Hierarchical clustering and principal component analysis (PCA) showed a tightly clustered group of nighttime expressed genes, whereas daytime transcripts were separated from each other over the 48-h duration. Lack of uniform clustering within the daytime transcripts suggested that the partitioning of gene expression in Microcystis is dependent on both circadian regulation and physicochemical changes within the environment.

Ammonium recycling supports toxic Planktothrix blooms in Sandusky Bay, Lake Erie: Evidence from stable isotope and metatranscriptome data.

Sandusky Bay, Lake Erie, receives high nutrient loadings (nitrogen and phosphorus) from the Sandusky River, which drains an agricultural watershed. Eutrophication and cyanobacterial harmful algal blooms (cyanoHABs) persist throughout summer. Planktothrix agardhii is the dominant bloom-forming species and the main producer of microcystins in Sandusky Bay. Non-N2 fixing cyanobacteria, such as Planktothrix and Microcystis, thrive on chemically reduced forms of nitrogen, such as ammonium (NH4+) and urea. Ammonium regeneration and potential uptake rates and total microbial community demand for NH4+ were quantified in Sandusky Bay. Potential NH4+ uptake rates in the light increased from June to August at all stations. Dark uptake rates also increased seasonally and, by the end of August, were on par with light uptake rates. Regeneration rates followed a similar pattern and were significantly higher in August than June. Ammonium uptake kinetics during a Planktothrix-dominated bloom in Sandusky Bay and a Microcystis-dominated bloom in Maumee Bay were also compared. The highest half saturation constant (Km) in Sandusky Bay was measured in June and decreased throughout the season. In contrast, Km values in Maumee Bay were lowest at the beginning of summer and increased in October. A significant increase in Vmax in Sandusky Bay was observed between July and the end of August, reflective of intense competition for depleted NH4+. Metatranscriptome results from Sandusky Bay show a shift from cyanophycin synthetase (luxury NH4+ uptake; cphA1) expression in early summer to cyanophycinase (intracellular N mobilization; cphB/cphA2) expression in August, supporting the interpretation that the microbial community is nitrogen-starved in late summer. Combined, our results show that, in late summer, when nitrogen concentrations are low, cyanoHABs in Sandusky Bay rely on regenerated NH4+ to support growth and toxin production. Increased dark NH4+ uptake late in summer suggests an important heterotrophic contribution to NH4+ depletion in the phycosphere. Kinetic experiments in the two bays suggest a competitive advantage for Planktothrix over Microcystis in Sandusky Bay due to its higher affinity for NH4+ at low concentrations.

Science meets policy: A framework for determining impairment designation criteria for large waterbodies affected by cyanobacterial harmful algal blooms.

Toxic cyanobacterial harmful algal blooms (cyanoHABs) are one of the most significant threats to the security of Earth's surface freshwaters. In the United States, the Federal Water Pollution Control Act of 1972 (i.e., the Clean Water Act) requires that states report any waterbody that fails to meet applicable water quality standards. The problem is that for fresh waters impacted by cyanoHABs, no scientifically-based framework exists for making this designation. This study describes the development of a data-based framework using the Ohio waters of western Lake Erie as an exemplar for large lakes impacted by cyanoHABs. To address this designation for Ohio's open waters, the Ohio Environmental Protection Agency (EPA) assembled a group of academic, state and federal scientists to develop a framework that would determine the criteria for Ohio EPA to consider in deciding on a recreation use impairment designation due to cyanoHAB presence. Typically, the metrics are derived from on-lake monitoring programs, but for large, dynamic lakes such as Lake Erie, using criteria based on discrete samples is problematic. However, significant advances in remote sensing allows for the estimation of cyanoHAB biomass of an entire lake. Through multiple years of validation, we developed a framework to determine lake-specific criteria for designating a waterbody as impaired by cyanoHABs on an annual basis. While the criteria reported in this manuscript are specific to Ohio's open waters, the framework used to determine them can be applied to any large lake where long-term monitoring data and satellite imagery are available.

Terrestrial Origin for Abundant Riverine Nanoscale Ice-Nucleating Particles.

Ice-nucleating particles (INPs) associated with fresh waters are a neglected, but integral component of the water cycle. Abundant INPs were identified from surface waters of both the Maumee River and Lake Erie with ice nucleus spectra spanning a temperature range from -3 to -15 °C. The majority of river INPs were submicron in size and attributed to biogenic macromolecules, inferred from the denaturation of ice-nucleation activity by heat. In a watershed dominated by row-crop agriculture, higher concentrations of INPs were found in river samples compared to lake samples. Further, ice-nucleating temperatures differed between river and lake samples, which indicated different populations of INPs. Seasonal analysis of INPs that were active at warmer temperatures (≥-10 °C; INP-10) showed their concentration to correlate with river discharge, suggesting a watershed origin of these INPs. A terrestrial origin for INPs in the Maumee River was further supported by a correspondence between the ice-nucleation signatures of river INPs and INPs derived from the soil fungus Mortierella alpina. Aerosols derived from turbulence features in the river carry INP-10, although their potential influence on regional weather is unclear. INP-10 contained within aerosols generated from a weir spanning the river, ranged in concentration from 1 to 11 INP m-3, which represented a fold-change of 3.2 over average INP-10 concentrations sampled from aerosols at control locations.

Effects of increasing nitrogen and phosphorus concentrations on phytoplankton community growth and toxicity during Planktothrix blooms in Sandusky Bay, Lake Erie.

Sandusky Bay experiences annual toxic cyanobacterial blooms dominated by Planktothrix agardhii/suspensa. To further understand the environmental drivers of these events, we evaluated changes in the growth response and toxicity of the Planktothrix-dominated blooms to nutrient amendments with orthophosphate (PO4) and inorganic and organic forms of dissolved nitrogen (N; ammonium (NH4), nitrate (NO3) and urea) over the bloom season (June - October). We complemented these with a metagenomic analysis of the planktonic microbial community. Our results showed that bloom growth and microcystin (MC) concentrations responded more frequently to additions of dissolved N than PO4, and that the dual addition of NH4 + PO4 and Urea + PO4 yielded the highest MC concentrations in 54% of experiments. Metagenomic analysis confirmed that P. agardhii/suspensa was the primary MC producer. The phylogenetic distribution of nifH revealed that both heterocystous cyanobacteria and heterotrophic proteobacteria had the genetic potential for N2 fixation in Sandusky Bay. These results suggest that as best management practices are developed for P reductions in Sandusky Bay, managers must be aware of the negative implications of not managing N loading into this system as N may significantly impact cyanobacterial bloom size and toxicity.

Phylogenies of microcystin-producing cyanobacteria in the lower Laurentian Great Lakes suggest extensive genetic connectivity.

Lake St. Clair is the smallest lake in the Laurentian Great Lakes system. MODIS satellite imagery suggests that high algal biomass events have occurred annually along the southern shore during late summer. In this study, we evaluated these events and tested the hypothesis that summer bloom material derived from Lake St. Clair may enter Lake Erie via the Detroit River and represent an overlooked source of potentially toxic Microcystis biomass to the western basin of Lake Erie. We conducted a seasonally and spatially resolved study carried out in the summer of 2013. Our goals were to: 1) track the development of the 2013 summer south-east shore bloom 2) conduct a spatial survey to characterize the extent of toxicity, taxonomic diversity of the total phytoplankton population and the phylogenetic diversity of potential MC-producing cyanobacteria (Microcystis, Planktothrix and Anabaena) during a high biomass event, and 3) compare the strains of potential MC-producers in Lake St. Clair with strains from Lake Erie and Lake Ontario. Our results demonstrated a clear predominance of cyanobacteria during a late August bloom event, primarily dominated by Microcystis, which we traced along the Lake St. Clair coastline downstream to the Detroit River's outflow at Lake Erie. Microcystin levels exceeded the Province of Ontario Drinking Water Quality Standard (1.5 µg L(-1)) for safe drinking water at most sites, reaching up to five times this level in some areas. Microcystis was the predominant microcystin producer, and all toxic Microcystis strains found in Lake St. Clair were genetically similar to toxic Microcystis strains found in lakes Erie and Ontario. These findings suggest extensive genetic connectivity among the three systems.

Abundance and diversity of ammonia-oxidizing archaea and bacteria in sediments of trophic end members of the Laurentian Great Lakes, Erie and Superior.

Ammonia oxidation is the first step of nitrification carried out by ammonia-oxidizing Archaea (AOA) and Bacteria (AOB). Lake Superior and Erie are part of the Great Lakes system differing in trophic status with Lake Superior being oligotrophic and Lake Erie meso- to eutrophic. Sediment samples were collected from both lakes and used to characterize abundance and diversity of AOA and AOB based on the ammonia monooxygenase (amoA) gene. Diversity was accessed by a pyro-sequencing approach and the obtained sequences were used to determine the phylogeny and alpha and beta diversity of the AOA and AOB populations. In Lake Erie copy numbers of bacterial amoA genes were in the same order of magnitude or even higher than the copy numbers of the archaeal amoA genes, while in Lake Superior up to 4 orders of magnitude more archaeal than bacterial amoA copies were detected. The AOB detected in the samples from Lake Erie belonged to AOB that are frequently detected in freshwater. Differences were detected between the phylogenetic affiliations of the AOA from the two lakes. Most sequences detected in Lake Erie clustered in the Nitrososphaera cluster (Thaumarchaeal soil group I.1b) where as most of the sequences in Lake Superior were found in the Nitrosopumilus cluster (Thaumarchaeal marine group I.1a) and the Nitrosotalea cluster. Pearson correlations and canonical correspondence analysis (CCA) showed that the differences in abundance and diversity of AOA are very likely related to the sampling location and thereby to the different trophic states of the lakes.

Plasticity of Total and Intracellular Phosphorus Quotas in Microcystis aeruginosa Cultures and Lake Erie Algal Assemblages.

Blooms of the potentially toxic cyanobacterium Microcystis are common events globally, and as a result significant resources continue to be dedicated to monitoring and controlling these events. Recent studies have shown that a significant proportion of total cell-associated phosphorus (P) in marine phytoplankton can be surface adsorbed; as a result studies completed to date do not accurately report the P demands of these organisms. In this study we measure the total cell-associated and intracellular P as well as growth rates of two toxic strains of Microcystis aeruginosa Kütz grown under a range of P concentrations. The results show that the intracellular P pool in Microcystis represents a percentage of total cell-associated P (50-90%) similar to what has been reported for actively growing algae in marine systems. Intracellular P concentrations (39-147 fg cell(-1)) generally increased with increasing P concentrations in the growth medium, but growth rate and the ratio of total cell-associated to intracellular P remained generally stable. Intracellular P quotas and growth rates in cells grown under the different P treatments illustrate the ability of this organism to successfully respond to changes in ambient P loads, and thus have implications for ecosystem scale productivity models employing P concentrations to predict algal bloom events.

Seasonal Expression of the Picocyanobacterial Phosphonate Transporter Gene phnD in the Sargasso Sea.

In phosphorus-limited marine environments, picocyanobacteria (Synechococcus and Prochlorococcus spp.) can hydrolyze naturally occurring phosphonates as a P source. Utilization of 2-aminoethylphosphonate (2-AEP) is dependent on expression of the phn genes, encoding functions required for uptake, and C-P bond cleavage. Prior work has indicated that expression of picocyanobacterial phnD, encoding the phosphonate binding protein of the phosphonate ABC transporter, is a proxy for the assimilation of phosphonates in natural assemblages of Synechococcus spp. and Prochlorococcus spp (Ilikchyan et al., 2009). In this study, we expand this work to assess seasonal phnD expression in the Sargasso Sea. By RT-PCR, our data confirm that phnD expression is constitutive for the Prochlorococcus spp. detected, but in Synechococcus spp. phnD transcription follows patterns of phosphorus availability in the mixed layer. Specifically, our data suggest that phnD is repressed in the spring when P is bioavailable following deep winter mixing. In the fall, phnD expression follows a depth-dependent pattern reflecting depleted P at the surface following summertime drawdown, and elevated P at depth.