Quantitative Polymerase Chain Reaction (qPCR) is a well-established method for molecular detection and quantification of microorganisms. Molecular detection of toxigenic cyanobacteria using qPCR has also been employed as a useful tool for predicting cyanotoxin production and formation of harmful cyanobacterial blooms (cHABs). Recently, portable thermocyclers and extraction procedures and reagents have been developed for onsite application of qPCR analysis. However, use of portable qPCR analysis for the detection and quantification of toxigenic cyanobacteria has not been evaluated. Here, we provide an evaluation of a field method for DNA extraction and qPCR analysis using the Biomeme Franklin thermocycler for detection of toxigenic cyanobacteria. The results obtained from the portable device provide identification and quantification of toxigenic Microcystis and are comparable with lab-based extraction and analysis. This field method for extraction and qPCR analysis will provide alternative research tools to evaluate source and recreational water for toxic cyanobacteria. This field method allows users to obtain immediate on-site results for identification and quantification of toxigenic Microcystis.

Although most bacteria in water can be effectively inactivated after chlorination during the wastewater treatment process, some may survive and spread waterborne diseases around the world. Their capacity to adapt to stressful conditions may make them even more hazardous. Herein, we investigated the tolerance of chlorine-stressed bacteria under simulated gastrointestinal (GI) conditions and their ability to invade human intestinal epithelial Caco-2 cells to understand the potential human health risk associated with chlorine-treated wastewater discharges. Chlorine-stressed bacterial samples were prepared using bacterial culture from secondary effluent after 30 min of exposure to 0.02 mg/L, 0.09 mg/L, and 0.6 mg/L of free chlorine. Remarkably, while 0.6 mg/L of free chlorine was insufficient for bacterial inactivation, free chlorine concentrations of 0.02-0.09 mg/L significantly promoted bacterial regrowth. Lysinibacillus, Klebsiella, and Morganella have shown low pH adaptability in simulated gastric conditions. Those genera were not observed in gastric conditions in samples with a higher chlorine dose of 0.6 mg/L, but more regrowth of resistant genera occurred in the simulated intestinal condition. Spore-forming bacteria survived even after chlorination and simulated gastric digestion. After disinfection with sublethal chlorine doses of 0.02 and 0.09 mg/L, surviving bacterial populations showed an enhanced ability to invade Caco-2 cells. Escherichia and Bacillus are the most dominant intracellular bacteria, suggesting their superior ability to invade Caco-2 cells. The study emphasized the importance of sufficient chlorination in minimizing microbial risks during wastewater management, reuse, and disposal.

Cyanobacteria harmful algal blooms in lakes are primarily driven by nutrient and temperature conditions, yet the interplay of these abiotic factors with microbial community dynamics during bloom events is complex and challenging to unravel. Despite advances through deep sequencing approaches, the underlying transcriptomic changes occurring within blooming and non-blooming taxa remains an actively expanding area of study. In this work, we examined a spring-summer bloom event in Anderson Lake, WA, which has experienced recurring annual blooms dominated by the filamentous, anatoxin-a producing, diazotroph: Dolichospermum sp. WA102. Our data reveal the overall transcriptional dominance by Dolichospermum sp. WA102 during the bloom, initiated with increasing temperature and light intensity under high available phosphorus but low nitrogen conditions. We find that heterocyst differentiation was already transcriptionally initiated prior to the bloom, facilitating downstream gene cascades necessary for rapid nitrogen fixation and metabolism. As the bloom progresses, phosphorus becomes depleted, necessitating the expression of Pho regulon components in Dolichospermum sp. WA102 and possibly curtailing the bloom itself. We dissect toxin production and the transcriptional subtleties of the anatoxin-a synthesis locus. Additionally, co-occurring taxa exhibited distinct gene expression profiles, with competition for nutrients, light, and potential allelopathic interactions acting as drivers. Overall, our data provide a unique transcriptomic perspective on a single-taxa-driven, anatoxin-producing bloom, highlighting its competitive adaptation to nutrient acquisition and favorable conditions. This deeper understanding of the genetic mechanisms underlying algal bloom events may aid in predicting and preventing future blooms.

ABSTRACT Wild birds are a major source of Campylobacter jejuni, but there is limited information on its virulence, infectivity, and its human health risks associated with exposure to wild bird infested surface waters. This study focused on understanding the virulence of C. jejuni 63A isolated from California gull excreta. Whole-genome analyses revealed its multilocus sequence typing (ST2654) and high similarity of DNA sequences (97%–99%) with other pathogenic Campylobacter spp. strains, particularly those isolated from patients suffering from Guillain–Barre´ syndrome (GBS). Furthermore, analyses revealed that the lipooligosaccharide (LOS) biosynthesis gene cluster, capsular polysaccharide (CPS) gene cluster, and the genes responsible for producing cytolethal distending toxin (CDT) operons/genes (cdtABC) were all present and shared high sequence conservation with the same clinical isolates collected from GBS patients. The unique LOS genes (neuA1, neuB1, neuC1, and cstIII) to GBS patient isolates were identified and categorized 63A as group 1 LOS locus type. The presence of an intact CPS gene cluster and cdtABC genes in 63A along with their high sequence similarity to clinical isolates was strikingly different from many other wild-bird isolates. These findings suggest the high possibility of significant virulence associated with the new isolate. Additionally, 63A may not be limited to only California gull. Phylogenetic analysis of 63A with other hosts including waterfowl such as sandhill cranes shows that the 63A isolate is closely related to a diverse range of hosts acting as reservoirs and/or vectors. Lastly, dose–response analyses of this isolate estimated a median infectious dose of 7.16 × 107 CFU, further suggesting that the 63A isolate has zoonotic potential.IMPORTANCEC. jejuni is one of the most common foodborne zoonotic pathogens worldwide. This study focused on the virulence and infection of C. jejuni derived from California gull. We found that C. jejuni 63A shares high genetic similarity with other pathogenic C. jejuni strains. These strains are particularly associated with patients suffering from GBS. Whole-genome analyses revealed virulence factors like lipooligosaccharide, capsular polysaccharide, and cytolethal distending toxin operons/genes present in 63A. These genes showed high sequence conservation with clinical isolates collected from GBS patients. We also analyzed its presence in other avian feces, like those of sandhill cranes. Our chick infection experiment and dose–response analyses indicate that the 63A isolate has zoonotic potential and could infect humans. These findings highlight the importance of understanding the virulence and potential health risks associated with C. jejuni strains of wild birds.

The excessive growth of harmful cyanobacteria, including Dolichospermum (formerly known as Anabaena), in freshwater bodies has become a pressing global concern. However, detailed information about the role of Dolichospermum in shaping bloom dynamics and producing cyanotoxins is limited. In this study, a bloom event dominated by Dolichospermum spp. at Detroit Lake (Oregon, USA) was examined from 2019 to 2021. In 2019, early summer cyanobacterial community succession reached up to 8.7 % of total phytoplankton abundance. Dolichospermum was the major microcystin (MC)-producing genus, with peak MC levels of 7.34 mu g L-1. The presence of MCs was strongly correlated with the abundance of Dolichospermum (r = 0.84, p < 0.05) and MC synthetase gene, mcyE-Ana (r = 0.63, p < 0.05). Metabolic analyses further showed that the presence of nif/pst genes linked to nitrogen and phosphorus metabolism was dominated by Dolichospermum from the bloom onset until September. In addition, the abundance of Dolichospermum was significantly correlated with the abundance of nitrogen-fixing nif-Ana gene (r = 0.62, p < 0.05). As the lake experienced a longer N and P scarcity period (May to September), the N-2-fixing Dolichospermum was able to dominate over other non-fixing cyanobacteria present, including Microcystis and Planktothrix. Overall, our results facilitate a better understanding of the organism and will help working toward managing/predicting future blooms.

Drinking water biofilm ecosystems harbor complex and dynamic prokaryotic and eukaryotic microbial communities. However, little is known about the impact of copper corrosion on microbial community functions in metabolisms and resistance. This study was conducted to evaluate the impact of upstream Cu pipe materials on downstream viable community structures, pathogen populations, and metatranscriptomic responses of the microbial communities in drinking water biofilms. Randomly transcribed cDNA was generated and sequenced from downstream biofilm samples of either unplasticized polyvinylchloride (PVC) or Cu coupons. Diverse viable microbial organisms with enriched pathogen-like organisms and opportunistic pathogens were active in those biofilm samples. Cu-influenced tubing biofilms had a greater upregulation of genes associated with potassium (K) metabolic pathways (i.e., K-homeostasis, K-transporting ATPase, and transcriptional attenuator), and a major component of the cell wall of mycobacteria (mycolic acids) compared to tubing biofilms downstream of PVC. Other upregulated genes on Cu influenced biofilms included those associated with stress responses (various oxidative resistance genes), biofilm formation, and resistance to toxic compounds. Downregulated genes included those associated with membrane proteins responsible for ion interactions with potassium; respiration–electron-donating reactions; RNA metabolism in eukaryotes; nitrogen metabolism; virulence, disease, and defense; and antibiotic resistance genes. When combined with our previous identification of biofilm community differences, our studies reveal how microbial biofilms adapt to Cu plumbing conditions by fine-tuning gene expression, altering metabolic pathways, and optimizing their structural organization. This study offers new insights into how copper pipe materials affect the development and composition of biofilms in premise plumbing. Specifically, it highlights copper’s role in inhibiting the growth of many microbes while also contributing to the resistance of some microbes within the drinking water biofilm community.

Phytoplankton overgrowth, which characterizes the eutrophication or trophic status of surface water bodies, threatens ecosystems and public health. Quantitative polymerase chain reaction (qPCR) is promising for assessing the abundance and community composition of phytoplankton. However, applications of qPCR to indicate eutrophication and trophic status, especially in lotic systems, have yet to be comprehensively evaluated. For the first time, this study correlates qPCR-based phytoplankton abundance with chlorophyll a (the most widely used indicator of eutrophication and trophic status) in multiple freshwater rivers. From early summer to late fall in 2017, 2018, and 2019, we evaluated phytoplankton, chlorophyll a, pheophytin a, and the Trophic Level Index (TLI) in twelve large freshwater rivers in three regions (western, midcontinent, and eastern) in the United States. Chlorophyll a concentration had positive allometric correlations with qPCR-based phytoplankton abundance (adjusted R-2 = 0.5437, p-value < 0.001), pheophytin a concentration (adjusted R-2 = 0.3378, p-value <0.001), and TLI (adjusted R-2 = 0.4789, p-value < 0.001). Thus, a greater phytoplankton abundance suggests a higher trophic status. This work also presents the numerical values of qPCR-based phytoplankton abundance defining the boundaries among trophic statuses (e.g., oligotrophic, mesotrophic, and eutrophic) of freshwater rivers. The sampling sites in the midcontinent rivers were more eutrophic because they had significantly higher chlorophyll a concentrations, pheophytin a concentrations, and TLI values than the sites in the western and eastern rivers. The higher phytoplankton abundance at the midcontinent sites confirmed their higher trophic status. By linking qPCR-based phytoplankton abundance to chlorophyll a, this study demonstrates that qPCR is a promising avenue to investigate the population dynamics of phytoplankton and the trophic status (or eutrophication) of freshwater rivers.

Previously, we showed that prophylactic addition of glucose to Harsha Lake water samples could inhibit cyanobacteria growth, at least for a short period of time. The current study tested cyanobacterial control with glucose for the entire Harsha Lake bloom season. Water samples (1000 ml) were collected weekly from Harsha Lake during the algal-bloom season starting June 9 and lasting until August 24, 2022. To each of two 7-liter polypropylene containers, 500 ml of Harsha Lake water was added, and the containers were placed in a controlled environment chamber. To one container labeled "Treated," 0.15 g of glucose was added, and nothing was added to the container labeled "Control." After that, three 25 ml samples from each container were collected and used for 16S rRNA gene sequencing each week. Then 1000 ml of Harsha Lake water was newly collected each week, with 500 ml added to each container, along with the addition of 0.15 g glucose to the "Treated" container. Sequencing data were used to examine differences in the composition of bacterial communities between Treated and Control containers. Treatment with glucose altered the microbial communities by 1) reducing taxonomic diversity, 2) largely eliminating cyanobacterial taxa, and 3) increasing the relative abundance of subsets of non-cyanobacterial taxa (such as Proteobacteria and Actinobacteriota). These effects were observed across time despite weekly inputs derived directly from Lake water. The addition of glucose to a container receiving weekly additions of Lake water suppressed the cyanobacterial populations during the entire summer bloom season. The glucose appears to stimulate the diversity of certain bacterial taxa at the expense of the cyanobacteria.

Nitrification is a serious water-quality issue in chloraminated engineered water systems (EWSs). Nitrification is often remediated by a chlorine burn (i.e., a free-chlorine conversion), a short-term switch from chloramination to chlorination in EWSs. Opportunistic pathogens (OPs) are the dominant infectious agents in EWSs. However, the responses of OPs to a chlorine burn are unknown. This study for the first time assessed how a chlorine burn affected OPs in a full-scale EWS. We determined the impact of a 1.5-month chlorine burn on four dominant OPs (Legionella, Mycobacterium, Pseudomonas, and Vermamoeba vermiformis) in a representative full-scale chloraminated EWS in the United States. Legionella and Mycobacterium were the most abundant OPs. In the water main, the summed concentration of the four OPs during the chlorine burn [3.27 +/- 1.58 log(10)(GCN center dot L-1); GCN: genome or gene copy number] was lower (p <= 0.001) than before the burn [4.83 +/- 0.50 log(10)(GCN center dot L-1)]. After the burn, the summed concentration increased to 4.27 +/- 0.68 log(10)(GCN center dot L-1), comparable to before the burn (p > 0.05), indicating a transient effect of the chlorine burn in the water main. At the residential sites, the summed concentrations of the four OPs were comparable (p > 0.05) at 5.50 +/- 0.84, 5.27 +/- 1.44, and 5.08 +/- 0.71 log(10)(GCN center dot L-1) before, during, and after the chlorine burn, respectively. Therefore, the chlorine burn was less effective in suppressing OP (re)growth in the premise plumbing. The low effectiveness might be due to more significant water stagnation and disinfectant residual decay in the premise plumbing. Indeed, for the entire sampling period, the total chlorine residual concentration in the premise plumbing (1.8 mg Cl-2 center dot L-1) was lower than in the water main (2.4 mg Cl-2 center dot L-1). Consequently, for the entire sampling period, the summed concentration of the four OPs in the premise plumbing [5.26 +/- 1.08 log(10)(GCN center dot L-1)] was significantly higher (p < 0.001) than in the water main [4.04 +/- 1.25 log(10)(GCN center dot L-1)]. In addition, the chlorine burn substantially increased the levels of disinfection by-products (DBPs) in the water main. Altogether, a chlorine burn is transient or even ineffective in suppressing OP (re)growth but raises DBP concentrations in chloraminated EWSs. Therefore, the practice of chlorine burns to control nitrification should be optimized, reconsidered, or even replaced.

Previously, we demonstrated that the addition of glucose to lake water could alter the composition of the microbial community so that heterotrophic bacteria came to dominate the cyanobacteria. To target the glucose additions to the euphotic zone, a floating, slow-release glucose source, designated HABS-BLOCKS©, was created. HABS-BLOCKS© consist of blocks of pumice stone, vacuum infiltrated with glucose, and covered in layers of soy wax. In this study, the HABS-BLOCKS© were tested in 7-liter mesocosm vessels (n = 4) that received an initial 750 ml of lake water, followed by weekly additions of 500 ml of freshly collected lake water. Three HABS-BLOCKS© were added to each of two replicate mesocosms. For controls, one mesocosm was left untreated and one mesocosm received three "Dummy" HABS-BLOCKS© (contain no glucose). During a ten-week experiment, 25 ml samples were obtained from each mesocosm weekly, which were then filtered, frozen and latter processed for 16S rRNA sequencing. Planktothrix and Cyanobium were the most abundant cyanobacteria in the lake water. Within three weeks of the start of the experiment, the bacterial community in the HABS-BLOCKS© treated mesocosms became dominated by heterotrophic bacteria, e.g., Asticcacaulis, relative to the control mesocosms. Heterotrophic domination in the HABS-BLOCKS© treated mesocosms continued for the rest of the experiment. HABS-BLOCKS© appears to provide a competitive edge for the heterotrophic bacteria that allows them to dominate the toxin-producing cyanobacteria.

Harmful cyanobacterial blooms (HCBs) are of growing global concern due to their production of toxic compounds, which threaten ecosystems and human health. Saxitoxins (STXs), commonly known as paralytic shellfish poison, are a neurotoxic alkaloid produced by some cyanobacteria. Although many field studies indicate a widespread distribution of STX, it is understudied relative to other cyanotoxins such as microcystins (MCs). In this study, we assessed eleven U.S. urban lakes using qPCR, sxtA gene-targeting sequencing, and 16S rRNA gene sequencing to understand the spatio-temporal variations in cyanobacteria and their potential role in STX production. During the blooms, qPCR analysis confirmed the presence of the STX-encoding gene sxtA at all lakes. In particular, the abundance of the sxtA gene had a strong positive correlation with STX concentrations in Big 11 Lake in Kansas City, which was also the site with the highest quantified STX concentration. Sequencing analysis revealed that potential STX producers, such as Aphanizomenon, Dolichospermum, and Raphidiopsis, were present. Further analysis targeting amplicons of the sxtA gene identified that Aphanizomenon and/or Dolichospermum are the primary STX producer, showing a significant correlation with sxtA gene abundances and STX concentrations. In addition, Aphanizomenon was associated with environmental factors, such as conductivity, sulfate, and orthophosphate, whereas Dolichospermum was correlated with temperature and pH. Overall, the results herein enhance our understanding of the STX-producing cyanobacteria and aid in developing strategies to control HCBs.

When cyanobacterial phytoplankton form harmful cyanobacterial blooms (HCBs), the toxins they produce threaten freshwater ecosystems. Hydrogen peroxide is often used to control HCBs, but it is broadly toxic and dangerous to handle. Previously, we demonstrated that glucose addition to lake water could suppress the abundance of cyanobacteria. In this study, glucose was compared to hydrogen peroxide for the treatment of cyanobacterial phytoplankton communities. The six-week study was conducted in the large mesocosms facility at Grand Valley State University’s Annis Water Resources Institute in Michigan. To 1000 L of Muskegon Lake water, glucose was added at either 150 mg or 30 mg glucose/L. Hydrogen peroxide was added at 3 mg/L to two 1000 L mesocosms. And two mesocosms were left untreated as controls. Triplicate 100 mL samples were collected weekly from each mesocosm, which were then filtered and frozen at −80 °C for 16S rRNA amplicon sequencing. The 16S rRNA amplicon sequencing results revealed that hydrogen peroxide treatment quickly reduced the relative abundance of the cyanobacteria compared to the control mesocosms, but the cyanobacteria population returned over the course of the 6-week study. On the other hand, both glucose concentrations caused a rapid proliferation of multiple low abundance proteobacterial and bacteroidotal taxa resulting in notable increases in taxonomic richness over the duration of the study and reducing the relative abundance of cyanobacteria. Although hydrogen peroxide quickly suppressed the cyanobacteria, the population later returned to near starting levels. The glucose suppressed the cyanobacterial phytoplankton apparently by promoting competitive heterotrophic bacteria.

Legionella pneumophila is a persistent opportunistic pathogen that poses a significant threat to domestic water systems. Previous studies suggest that copper (Cu) is an effective antimicrobial in water systems. A rapid and sensitive quantification method is desired to optimize the conditions of L. pneumophila treatment by Cu and to better understand the interaction mechanisms between Cu and cells. In this study, we developed a highly sensitive single cell (SC)-ICP-MS method to monitor L. pneumophila cell concentration and track their uptake of Cu. The SC-ICP-MS method showed excellent sensitivity (with a cell concentration detection limit of 1000 cells/mL), accuracy (good agreement with conventional hemocytometry method), and precision (relative standard deviation < 5

Mitigating cyanotoxin production is essential to protecting aquatic ecosystems and public health. However, current harmful cyanobacterial bloom (HCB) control strategies have significant shortcomings. Because predicting HCBs is difficult, current HCB control strategies are employed when heavy HCBs have already occurred. Our pilot study developed an effective HCB prediction approach that is employed before exponential cyanobacterial growth and massive cyanotoxin production can occur. We used a quantitative polymerase chain reaction (qPCR) assay targeting the toxin-encoding gene mcyA to signal the timing of treatment. When control measures were applied at an early growth stage or one week before the exponential growth of Microcystis aeruginosa (predicted by qPCR signals), both hydrogen peroxide (H2O2) and the adsorbent hydroxyapatite (HAP) effectively stopped M. aeruginosa growth and microcystin (MC) production. Treatment with either H2O2 (10 mg·L−1) or HAP (40 µm particles at 2.5 g·L−1) significantly reduced both mcyA gene copies and MC levels compared with the control in a dose-dependent manner. While both treatments reduced MC levels similarly, HAP showed a greater ability to reduce mcyA gene abundance. Under laboratory culture conditions, H2O2 and HAP also prevented MC production when applied at the early stages of the bloom when mcyA gene abundance was below 105 copies·mL−1.

Cyanobacteria and cyanotoxin producing cyanobacterial blooms are a trending focus of current research. Many studies focus on bloom events in lentic environments such as lakes or ponds. Comparatively few studies have explored lotic environments and fewer still have examined the cyanobacterial communities and potential cyanotoxin producers during ambient, non-bloom conditions. Here we used a metagenomics-based approach to profile non-bloom microbial communities and cyanobacteria in 12 major U.S. rivers at multiple time points during the summer months of 2019. Our data show that U.S. rivers possess microbial communities that are taxonomically rich, yet largely consistent across geographic location and time. Within these communities, cyanobacteria often comprise significant portions and frequently include multiple species with known cyanotoxin producing strains. We further characterized these potential cyanotoxin producing taxa by deep sequencing amplicons of the microcystin E ( mcyE ) gene. We found that rivers containing the highest levels of potential cyanotoxin producing cyanobacteria consistently possess taxa with the genetic potential for cyanotoxin production and that, among these taxa, the predominant genus of origin for the mcyE gene is Microcystis . Combined, these data provide a unique perspective on cyanobacteria and potential cyanotoxin producing taxa that exist in large rivers across the U.S. and can be used to better understand the ambient conditions that may precede bloom events in lotic freshwater ecosystems.

Populations of resident, non-migratory Canada geese are rapidly increasing. Canada geese are known to transmit viral and bacterial diseases, posing a possible threat to human health. The most prevalent pathogens vectored by geese are Campylobacter species, yet the current understanding of the identity and virulence of these pathogens is limited. In our previous study, we observed a high prevalence of Campylobacter spp. in the Banklick Creek wetland-a constructed treatment wetland (CTW) located in northern KY (USA) used to understand sources of fecal contamination originating from humans and waterfowl frequenting the area. To identify the types of Campylobacter spp. found contaminating the CTW, we performed genetic analyses of Campylobacter 16s ribosomal RNA amplified from CTW water samples and collected fecal material from birds frequenting those areas. Our results showed a high occurrence of a Campylobacter canadensis-like clade from the sampling sites. Whole-genome sequence analyses of an isolate from Canada goose fecal material, called MG1, were used to confirm the identity of the CTW isolates. Further, we examined the phylogenomic position, virulence gene content, and antimicrobial resistance gene profile of MG1. Lastly, we developed an MG1-specific real-time PCR assay and confirmed the presence of MG1 in Canada goose fecal samples surrounding the CTW. Our findings reveal that the Canada goose-vectored Campylobacter sp. MG1 is a novel isolate compared to C. canadensis that possesses possible zoonotic potential, which may be of human health concern.

Phytoplankton is the essential primary producer in fresh surface water ecosystems. However, excessive phytoplankton growth due to eutrophication significantly threatens ecologic, economic, and public health. Therefore, phytoplankton identification and quantification are essential to understanding the productivity and health of freshwater ecosystems as well as the impacts of phytoplankton overgrowth (such as Cyanobacterial blooms) on public health. Microscopy is the gold standard for phytoplankton assessment but is time-consuming, has low throughput, and requires rich experience in phytoplankton morphology. Quantitative polymerase chain reaction (qPCR) is accurate and straightforward with high throughput. In addition, qPCR does not require expertise in phytoplankton morphology. Therefore, qPCR can be a useful alternative for molecular identification and enumeration of phytoplankton. Nonetheless, a comprehensive study is missing which evaluates and compares the feasibility of using qPCR and microscopy to assess phytoplankton in fresh water. This study 1) compared the performance of qPCR and microscopy in identifying and quantifying phytoplankton and 2) evaluated qPCR as a molecular tool to assess phytoplankton and indicate eutrophication. We assessed phytoplankton using both qPCR and microscopy in twelve large freshwater rivers across the United States from early summer to late fall in 2017, 2018, and 2019. qPCR- and microscope-based phytoplankton abundance had a significant positive linear correlation (adjusted R2 = 0.836, p-value < 0.001). Phytoplankton abundance had limited temporal variation within each sampling season and over the three years studied. The sampling sites in the midcontinent rivers had higher phytoplankton abundance than those in the eastern and western rivers. For instance, the concentration (geometric mean) of Bacillariophyta, Cyanobacteria, Chlorophyta, and Dinoflagellates at the sampling sites in the midcontinent rivers was approximately three times that at the sampling sites in the western rivers and approximately 18 times that at the sampling sites in the eastern rivers. Welch's analysis of variance indicates that phytoplankton abundance at the sampling sites in the midcontinent rivers was significantly higher than that at the sampling sites in the eastern rivers (p-value = 0.013) but was comparable to that at the sampling sites in the western rivers (p-value = 0.095). The higher phytoplankton abundance at the sampling sites in the midcontinent rivers was presumably because these rivers were more eutrophic. Indeed, low phytoplankton abundance occurred in oligotrophic or low trophic sites, whereas eutrophic sites had greater phytoplankton abundance. This study demonstrates that qPCR-based phytoplankton abundance can be a useful numerical indicator of the trophic conditions and water quality in freshwater rivers.

To ensure drinking-water safety, it is necessary to understand the factors that regulate harmful cyanobacterial blooms (HCBs) and the toxins they produce. One controlling factor might be any relationship between fungi and the cyanobacteria. To test this possibility, water samples were obtained from Harsha Lake in southwestern Ohio during the 2015, 2016, and 2017 bloom seasons, i.e., late May through September. In each water sample, the concentration of the filamentous fungus Cladosporium cladosporioides was determined by quantitative PCR (qPCR) assay, and Microcystis aeruginosa microcystin-gene transcript copy number (McyG TCN) was quantified by reverse-transcriptase qPCR (RT-qPCR) analyses. The results showed that during each bloom season, the C. cladosporioides concentration and McyG TCN appeared to be interrelated. Therefore, C. cladosporioides concentrations were statistically evaluated via regression on McyG TCN in the water samples for lag times of 1 to 7 days. The regression equation developed to model the relationship demonstrated that a change in the C. cladosporioides concentration resulted in an opposing change in McyG TCN over an approximately 7-day interval. Although the interaction between C. cladosporioides and McyG TCN was observed in each bloom season, the magnitude of each component varied yearly. To better understand this possible interaction, outdoor Cladosporium spore-count data for the Harsha Lake region were obtained for late May through September of each year from the South West Ohio Air Quality Agency. The average Cladosporium spore count in the outdoor air samples was significantly greater in 2016 than in either 2015 or 2017, and the M. aeruginosa McyG TCN was significantly lower in Harsha Lake water samples in 2016 compared to 2015 or 2017. These results suggest that there might be a “balanced antagonism” between C. cladosporioides and M. aeruginosa during the bloom season.

To mitigate harmful cyanobacterial blooms (HCBs), toxic algicides have been used, but alternative methods of HCB prevention are needed. Our goal was to test the prophylactic addition of glucose to inhibit HCB development, using Microcystis and the toxin microcystin as the HCB model. Water samples were collected weekly, from 4 June to 2 July, from Harsha Lake in southwestern Ohio during the 2021 algal bloom season. From each weekly sample, a 25 mL aliquot was frozen for a 16S rRNA gene sequencing analysis. Then, 200 mL of Harsha Lake water was added to each of the three culture flasks, and glucose was added to create concentrations of 0 mM (control), 1.39 mM, or 13.9 mM glucose, respectively. The microcystin concentration in each flask was measured after 1 and 2 weeks of incubation. The results showed an 80 to 90% reduction in microcystin concentrations in glucose-treated water compared to the control. At the end of the second week of incubation, a 25 mL sample was also obtained from each of the culture flasks for molecular analysis, including a 16S rRNA gene sequencing and qPCR-based quantification of Microcystis target genes. Based on these analyses, the glucose-treated water contained significantly lower Microcystis and microcystin producing gene (mcy) copy numbers than the control. The 16S rRNA sequencing analysis also revealed that Cyanobacteria and Proteobacteria were initially the most abundant bacterial phyla in the Harsha Lake water, but as the summer progressed, Cyanobacteria became the dominant phyla. However, in the glucose-treated water, the Cyanobacteria decreased and the Proteobacteria increased in weekly abundance compared to the control. This glucose-induced proteobacterial increase in abundance was driven primarily by increases in two distinct families of Proteobacteria: Devosiaceae and Rhizobiaceae. In conclusion, the prophylactic addition of glucose to Harsha Lake water samples reduced Cyanobacteria's relative abundance, Microcystis numbers and microcystin concentrations and increased the relative abundance of Proteobacteria compared to the control.