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"Boyer, Gregory L."
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Comparative Metagenomics of Toxic Freshwater Cyanobacteria Bloom Communities on Two Continents
2012
Toxic cyanobacterial blooms have persisted in freshwater systems around the world for centuries and appear to be globally increasing in frequency and severity. Toxins produced by bloom-associated cyanobacteria can have drastic impacts on the ecosystem and surrounding communities, and bloom biomass can disrupt aquatic food webs and act as a driver for hypoxia. Little is currently known regarding the genomic content of the Microcystis strains that form blooms or the companion heterotrophic community associated with bloom events. To address these issues, we examined the bloom-associated microbial communities in single samples from Lake Erie (North America), Lake Tai (Taihu, China), and Grand Lakes St. Marys (OH, USA) using comparative metagenomics. Together the Cyanobacteria and Proteobacteria comprised >90% of each bloom bacterial community sample, although the dominant phylum varied between systems. Relative to the existing Microcystis aeruginosa NIES 843 genome, sequences from Lake Erie and Taihu revealed a number of metagenomic islands that were absent in the environmental samples. Moreover, despite variation in the phylogenetic assignments of bloom-associated organisms, the functional potential of bloom members remained relatively constant between systems. This pattern was particularly noticeable in the genomic contribution of nitrogen assimilation genes. In Taihu, the genetic elements associated with the assimilation and metabolism of nitrogen were predominantly associated with Proteobacteria, while these functions in the North American lakes were primarily contributed to by the Cyanobacteria. Our observations build on an emerging body of metagenomic surveys describing the functional potential of microbial communities as more highly conserved than that of their phylogenetic makeup within natural systems.
Journal Article
A Fresh Perspective on Cyanobacterial Paralytic Shellfish Poisoning Toxins: History, Methodology, and Toxicology
by
Arlinghaus, Kandis M.
,
Boyer, Gregory L.
,
Hapeman, Cathleen J.
in
algal toxins
,
Analysis
,
Animal models
2025
Paralytic shellfish poisoning toxins (PSPTs) are a class of neurotoxins most known for causing illness from consuming contaminated shellfish. These toxins are also present in freshwater systems with the concern that they contaminate drinking and recreational waters. This review provides (1) a complete list of the 84+ known PSPTs and important chemical features; (2) a complete list of all environmental freshwater PSPT detections; (3) an outline of the certified PSPT methods and their inherent weaknesses; and (4) a discussion of PSPT toxicology, the weaknesses in existing data, and existing freshwater regulatory limits. We show ample evidence of production of freshwater PSPTs by cyanobacteria worldwide, but data and method uncertainties limit a proper risk assessment. One impediment is the poor understanding of freshwater PSPT profiles and lack of commercially available standards needed to identify and quantify freshwater PSPTs. Further constraints are the limitations of toxicological data derived from human and animal model exposures. Unassessed mouse toxicity data from 1978 allowed us to calculate and propose toxicity equivalency factors (TEF) for 11-hydroxysaxitoxin (11-OH STX; M2) and 11-OH dcSTX (dcM2). TEFs for the 11-OH STX epimers were calculated to be 0.4 and 0.6 for 11α-OH STX (M2α) and 11β-OH STX (M2β), while we estimate that TEFs for 11α-OH dcSTX (dcM2α) and 11β-OH dcSTX (dcM2β) congeners would be 0.16 and 0.23, respectively. Future needs for freshwater PSPTs include increasing the number of reference materials for environmental detection and toxicity evaluation, developing a better understanding of PSPT profiles and important environmental drivers, incorporating safety factors into exposure guidelines, and evaluating the accuracy of the established no-observed-adverse-effect level.
Journal Article
Molecular prediction of lytic vs lysogenic states for Microcystis phage: Metatranscriptomic evidence of lysogeny during large bloom events
by
Steffen, Morgan M.
,
Boyer, Gregory L.
,
Gao, Guang
in
Ammonium
,
Analysis
,
Bacteriophages - genetics
2017
Microcystis aeruginosa is a freshwater bloom-forming cyanobacterium capable of producing the potent hepatotoxin, microcystin. Despite increased interest in this organism, little is known about the viruses that infect it and drive nutrient mobilization and transfer of genetic material between organisms. The genomic complement of sequenced phage suggests these viruses are capable of integrating into the host genome, though this activity has not been observed in the laboratory. While analyzing RNA-sequence data obtained from Microcystis blooms in Lake Tai (Taihu, China), we observed that a series of lysogeny-associated genes were highly expressed when genes involved in lytic infection were down-regulated. This pattern was consistent, though not always statistically significant, across multiple spatial and temporally distinct samples. For example, samples from Lake Tai (2014) showed a predominance of lytic virus activity from late July through October, while genes associated with lysogeny were strongly expressed in the early months (June-July) and toward the end of bloom season (October). Analyses of whole phage genome expression shows that transcription patterns are shared across sampling locations and that genes consistently clustered by co-expression into lytic and lysogenic groups. Expression of lytic-cycle associated genes was positively correlated to total dissolved nitrogen, ammonium concentration, and salinity. Lysogeny-associated gene expression was positively correlated with pH and total dissolved phosphorous. Our results suggest that lysogeny may be prevalent in Microcystis blooms and support the hypothesis that environmental conditions drive switching between temperate and lytic life cycles during bloom proliferation.
Journal Article
Toxin Dynamics among Populations of the Bioluminescent HAB Species Pyrodinium bahamense from the Indian River Lagoon, FL
2024
Dinoflagellate species that form some of the most frequent toxic blooms are also bioluminescent, yet the two traits are rarely linked when studying bloom development and persistence. P. bahamense is a toxic, bioluminescent dinoflagellate that previously bloomed in Florida with no known record of saxitoxin (STX) production. Over the past 20 years, STX was identified in P. bahamense populations. The goal of this study was to examine toxin dynamics and associated molecular mechanisms in spatially and temporally distinct P. bahamense populations from the Indian River Lagoon, FL. SxtA4 is a key gene required for toxin biosynthesis. SxtA4 genotype analysis was performed on individual cells from multiple sites. Cell abundance, toxin quota cell−1, and sxtA4 and RubisCo (rbcL) transcript abundance were also measured. There was a significant negative correlation between cell abundance and toxin quota cell−1. While the sxtA4+ genotype was dominant at all sites, its frequency varied, but it occurred at 90–100% in many samples. The underlying mechanism for toxin decrease with increased cell abundance remains unknown. However, a strong, statistically significant negative correlation was found between stxA4 transcripts and the sxtA4/rbcL ratio, suggesting cells make fewer sxtA4 transcripts as a bloom progresses. However, the influence of sxtA4− cells must also be considered. Future plans include bioluminescence measurements, normalized to a per cell basis, at sites when toxicity is measured along with concomitant quantification of sxtA4 gene and transcript copy numbers as a means to elucidate whether changes in bloom toxicity are driven more at the genetic (emergence of sxtA4− cells) or transcriptional (repression of sxtA4 in sxtA4+ cells) level. Based on the results of this study, a model is proposed that links the combined traits of toxicity and bioluminescence in P. bahamense bloom development.
Journal Article
Limnological Differences in a Two-Basin Lake Help to Explain the Occurrence of Anatoxin-a, Paralytic Shellfish Poisoning Toxins, and Microcystins
2020
Chautauqua Lake, New York, is a two-basin lake with a deeper, cooler, and less nutrient-rich Northern Basin, and a warmer, shallower, nutrient-replete Southern Basin. The lake is populated by a complex mixture of cyanobacteria, with toxigenic strains that produce microcystins, anatoxins, and paralytic shellfish poisoning toxins (PSTs). Samples collected from 24 sites were analyzed for these three toxin classes over four years spanning 2014–2017. Concentrations of the three toxin groups varied widely both within and between years. During the study, the mean and median concentrations of microcystins, anatoxin-a, and PSTs were 91 and 4.0 μg/L, 0.62 and 0.33 μg/L, and 32 and 16 μg/L, respectively. Dihydro-anatoxin was only detected once in Chautauqua Lake, while homo-anatoxin was never detected. The Northern Basin had larger basin-wide higher biomass blooms with higher concentrations of toxins relative to the more eutrophied Southern Basin, however blooms in the North Basin were infrequent. Chlorophyll concentrations and toxins in the two basins were correlated with different sets of environmental and physical parameters, suggesting that implementing controls to reduce toxin loads may require applications focused on more than reductions in cyanobacterial bloom density (e.g., reduction of phosphorus inputs), and that lake limnological factors and morphology are important determinants in the selection of an appropriate management strategy. Chautauqua Lake is a drinking water source and is also heavily used for recreation. Drinking water from Chautauqua Lake is unlikely to be a significant source of exposure to cyanotoxins due to the location of the intakes in the deeper North Basin, where there were generally low concentrations of toxins in open water; however, toxin levels in many blooms exceeded the US Environmental Protection Agency’s recreational guidelines for exposure to cyanotoxins. Current cyanotoxin monitoring in Chautauqua Lake is focused on microcystins. However, the occurrence of blooms containing neurotoxic cyanotoxins in the absence of the microcystins indicates this restricted monitoring may not be sufficient when aiming to protect against exposure to cyanotoxins. The lake has a large number of tourist visitors; thus, special care should be taken to prevent recreational exposure within this group.
Journal Article
Spatial and Temporal Variation in Paralytic Shellfish Toxin Production by Benthic Microseira (Lyngbya) wollei in a Freshwater New York Lake
by
Martin, Robbie
,
Smith, Zacharias
,
Boyer, Gregory
in
Alkaloids
,
Bacterial Toxins - analysis
,
Chlorophyll
2019
Butterfield Lake is a mesotrophic lake in New York State where residents and pets have experienced unexplained health issues. Microseira wollei (basionym Lyngbya wollei) was found at two of 15 sites in Butterfield Lake and analyzed for microcystins, anatoxins, cylindrospermopsins, and paralytic shellfish poisoning toxins (PSTs). Only PSTs and trace levels of anatoxin-a were detected in these samples. This is the first published report of PSTs within a New York State lake. To evaluate the environmental and temporal drivers leading to the observed toxicity, PST content at the two sites was examined in detail. There were distinct differences in the total PST content, filament nutrient, filament chlorophyll, and relationship to environmental drivers between the sites, as well as distinct differences in the total PST content measured using different analytical techniques. A multivariate model containing site, temperature, and filament chlorophyll explained 85% of the variation in PSTs observed over the growing season. This work emphasizes the importance of proper site selection and choice of analytical technique in the development of monitoring programs to protect lake users from the occurrence of benthic cyanobacteria toxins.
Journal Article
Roles of Nutrient Limitation on Western Lake Erie CyanoHAB Toxin Production
2021
Cyanobacterial harmful algal bloom (CyanoHAB) proliferation is a global problem impacting ecosystem and human health. Western Lake Erie (WLE) typically endures two highly toxic CyanoHABs during summer: a Microcystis spp. bloom in Maumee Bay that extends throughout the western basin, and a Planktothrix spp. bloom in Sandusky Bay. Recently, the USA and Canada agreed to a 40% phosphorus (P) load reduction to lessen the severity of the WLE blooms. To investigate phosphorus and nitrogen (N) limitation of biomass and toxin production in WLE CyanoHABs, we conducted in situ nutrient addition and 40% dilution microcosm bioassays in June and August 2019. During the June Sandusky Bay bloom, biomass production as well as hepatotoxic microcystin and neurotoxic anatoxin production were N and P co-limited with microcystin production becoming nutrient deplete under 40% dilution. During August, the Maumee Bay bloom produced microcystin under nutrient repletion with slight induced P limitation under 40% dilution, and the Sandusky Bay bloom produced anatoxin under N limitation in both dilution treatments. The results demonstrate the importance of nutrient limitation effects on microcystin and anatoxin production. To properly combat cyanotoxin and cyanobacterial biomass production in WLE, both N and P reduction efforts should be implemented in its watershed.
Journal Article
Variations in the Microcystin Content of Different Fish Species Collected from a Eutrophic Lake
2013
Microcystins produced from cyanobacteria can accumulate in fish tissues. Liquid chromatography coupled with tandem quadrupole mass spectrometry (LC-MS/MS) is an attractive alternative to immunoassays for the determination of low concentrations of microcystins in tissues. Fish taken from Grand Lake St. Marys, a eutrophic lake in Ohio, USA, were analyzed for microcystin-LR in their fillets using LC-MS/MS. Of 129 fish tested for microcystins, only black crappie (Pomoxis nigromaculatus) and common carp (Cyprinus carpio) tested positive for microcystin-LR. Less than 10% of Pomoxis and 7% of Cyprinus samples contained measurable levels of microcystin-LR. Statistical analysis yielded a p-value of 0.07 between Pomoxis and the pooled results of the other four fish species. However, this comparison was complicated by the large difference in sample size between species. Further sampling in Grand Lake St. Marys for microcystin-LR would help determine if microcystin-LR exposure occurs through foodweb transfer.
Journal Article
Effect of salinity stress and nitrogen depletion on growth, morphology and toxin production of freshwater cyanobacterium Microcoleus anatoxicus Stancheva & Conklin
2025
Cyanobacterium
Microcoleus anatoxicus,
isolated from a coastal stream in northern California, produces both anatoxin-a (ATX) and dihydroanatoxin-a (dhATX), responsible for dog deaths, but its environmental preferences are unknown. We tested the effect of environmentally relevant stressors, e.g., salinity enrichment and nitrogen (N) depletion, on mat formation and toxicity of
M. anatoxicus
during the stationary growth phase in culture.
Microcoleus anatoxicus
showed broad salinity tolerance and the potential to enter estuaries and produce toxins in mesohaline conditions. Maximum growth was observed in oligohaline waters with salinity of 4.6 ppt. Moderate salinity stress (up to 7.8 ppt) did not affect dhATX production significantly. In contrast, higher salinity above 9.3 ppt had a detrimental effect on cell growth and significantly suppressed dhATX production. Formation of a common polysaccharide sheath covering multiple filaments was characteristic with increased salinity and may provide protection against osmotic stress.
Microcoleus anatoxicus
grown for 40 days in N-depleted medium formed mats with significantly elevated dhATX and increased ATX concentrations. Phycobilisome degradation was a possible acclimation response to N-limitation, as indicated by distinctly keritomized and pale cells in these cultures. In both experiments, most of the anatoxins were extracellular, probably due to toxin leaking during the stationary growth phase.
Journal Article
Joint analysis of stressors and ecosystem services to enhance restoration effectiveness
by
Smith, Sigrid D. P.
,
Marino, Adrienne L.
,
Steinman, Alan D.
in
Animal, plant and microbial ecology
,
Applied ecology
,
Aquatic ecosystems
2013
With increasing pressure placed on natural systems by growing human populations, both scientists and resource managers need a better understanding of the relationships between cumulative stress from human activities and valued ecosystem services. Societies often seek to mitigate threats to these services through large-scale, costly restoration projects, such as the over one billion dollar Great Lakes Restoration Initiative currently underway. To help inform these efforts, we merged high-resolution spatial analyses of environmental stressors with mapping of ecosystem services for all five Great Lakes. Cumulative ecosystem stress is highest in near-shore habitats, but also extends offshore in Lakes Erie, Ontario, and Michigan. Variation in cumulative stress is driven largely by spatial concordance among multiple stressors, indicating the importance of considering all stressors when planning restoration activities. In addition, highly stressed areas reflect numerous different combinations of stressors rather than a single suite of problems, suggesting that a detailed understanding of the stressors needing alleviation could improve restoration planning. We also find that many important areas for fisheries and recreation are subject to high stress, indicating that ecosystem degradation could be threatening key services. Current restoration efforts have targeted high-stress sites almost exclusively, but generally without knowledge of the full range of stressors affecting these locations or differences among sites in service provisioning. Our results demonstrate that joint spatial analysis of stressors and ecosystem services can provide a critical foundation for maximizing social and ecological benefits from restoration investments.
Journal Article