Freshwater Cyanobacterial Blooms Research Articles

What drives the dominance and distribution of Cyanobacteria and Dinoflagellata in reservoirs of Sri Lanka?

Frequent records and health concerns in recent times have directed more attention on freshwater cyanobacterial and dinoflagellate blooms in Sri Lanka. Physico-chemical factors influencing phytoplankton growth are still under debate. This necessitates understanding environmental trends governing the dominance and distribution of algal blooms. Hence this study aimed to assess the dominance of Cyanobacteria and Dinoflagellata in reservoirs of Sri Lanka covering the three major climatic regions of the country, the Wet, Intermediate and Dry Zones. Plankton samples were collected using open type 20 µm plankton net and identified using standard phytoplankton keys. Ninety-one species belonging to three phyla of phytoplankton were identified. Phylum Chlorophyta represented the highest species number followed by Bacillariophyta and Cyanophyta. Fourteen genera of Cyanobacteria were recorded and eleven of them were potentially toxigenic. The widest distribution was shown by Chroococcus and Merismopedia. Cylindrospermopsis raciborskii with the highest mean relative abundance was recorded in Intermediate and Dry Zone reservoirs. Peridinium aciculiferum was the only dinoflagellate species recorded during the study with the highest abundance in the Isinbessagala rocky pond. Except sixteen reservoirs which were mesoeutrophic, all the other studied reservoirs were found to be either eutrophic or hypereutrophic. Iranamadu tank recorded the highest TP concentration (236 µg L-1) value. NO3-:TP ratio revealed the phosphate limited conditions in Wet and Dry Zone reservoirs whereas N was a limited factor in Intermediate Zone. Canonical Correspondence Analysis (CCA) results revealed that Secchi depth and area of water spread are important in determining both site and species variations in reservoirs of Sri Lanka.

Removal of cyanobacteria and cyanotoxins by ferrate from polluted lake water.

Freshwater cyanobacterial blooms are becoming increasingly problematic microbiological pollutants, especially for the water resource and surface natural lakes. Cyanobacterial blooms, which produce toxins and microcystins, negatively affect the quality of water, animal, and human health, and they have also negative impact on recreational activities. The effect of electrochemically prepared potassium ferrate (green oxidation agent) on the water polluted by cyanobacteria and cyanotoxins was studied. The two most frequently occurring cyanobacterial genus Microcystis and Anabaena and the most toxic and abundant microcystin MC-LR were successfully inactivated and treated by ferrate. Potassium ferrates were applied at different conditions, such as varied hydrodynamics flow of samples, pH, and Fe(VI) concentrations. High detected elimination efficiency was consequently tested on the real water matrix from microbiological polluted natural lake Šaštín-Gazárka in Slovakia. The ferrate application leads to the better chemical, biological, microbiological, and ecotoxicological outcomes.

Advances in biological function and molecular regulation of cyanobacterial surface coats in freshwaters

胶被是蓝藻细胞外部的功能性结构，在蓝藻形态适应性策略中发挥重要作用，影响蓝藻水华发生及其优势维持.近年来，对蓝藻胶被的特性、结构组分、影响因子、及其工业应用等方面已有较为丰富且清晰的科学认识.但对胶被生物学功能及相关分子调控机制的研究却相对较少.本文对淡水蓝藻胶被的产生、生物学功能及其分子调控机制进行了综述，总结了胶被4个方面生物学功能：1）保护屏障抵御外界胁迫；2）参与营养物质的传递、吸收和储存：3）参与运动迁移过程；4）促进蓝藻聚集和水华形成；结合近年来分子生物学前沿技术的应用，本文分析蓝藻胶被合成的分子生物学研究现状；探讨蓝藻响应外界不同环境的胶被分子调控机制，并对蓝藻胶被的未来研究热点进行了展望.;As a functional outermost structure surrounding cyanobacteria cells, the surface coats play an important role in morphological adaption, which further influence the dominance and persistence of cyanobacterial blooms in freshwaters. In recent years, there have been extensive studies on the characteristics, structures and components, regulating factors, and industrial applications of cyanobacterial surface coats. The biological function of cyanobacterial surface coats and their molecular regulation mechanisms have still not been clearly understood. In this paper, with the development of analytical technology, we summarized the production, biological functions and especially the molecular response mechanisms of cyanobacterial surface coats in freshwaters. We reviewed the biological functions of surface coats in the following four aspects: 1) serving as a protective barrier against environmental stress, 2) participating in mass transport and storage, 3) motivating the movement, 4) promoting the aggregation and cyanobacterial blooms in freshwaters. In addition, we analyzed current molecular biological research advances on the synthesis of cyanobacterial surface coats. We also discussed the molecular regulation mechanisms of cyanobacteria in response to external environments via surface coats. Finally, we proposed future focus on the cyanobacterial surface coats.

Predicting the effects of climate change on freshwater cyanobacterial blooms requires consideration of the complete cyanobacterial life cycle

Abstract To date, most research on cyanobacterial blooms in freshwater lakes has focused on the pelagic life stage. However, examining the complete cyanobacterial life cycle—including benthic life stages—may be needed to accurately predict future bloom dynamics. The current expectation, derived from the pelagic life stage, is that blooms will continue to increase due to the warmer temperatures and stronger stratification associated with climate change. However, stratification and mixing have contrasting effects on different life stages: while pelagic cyanobacteria benefit from strong stratification and are adversely affected by mixing, benthic stages can benefit from increased mixing. The net effects of these potentially counteracting processes are not yet known, since most aquatic ecosystem models do not incorporate benthic stages and few empirical studies have tracked the complete life cycle over multiple years. Moreover, for many regions, climate models project both stronger stratification and increased storm-induced mixing in the coming decades; the net effects of those physical processes, even on the pelagic life stage, are not yet understood. We therefore recommend an integrated research agenda to study the dual effects of stratification and mixing on the complete cyanobacterial life cycle—both benthic and pelagic stages—using models, field observations and experiments.

Evaluation of ELISA-based method for total anabaenopeptins determination and comparative analysis with on-line SPE-UHPLC-HRMS in freshwater cyanobacterial blooms

Anabaenopeptins (APs) are bioactive cyanopeptides of emerging concern produced by cyanobacteria. The research for analytical development has recently gained in importance due to their abundance in toxic cyanobacterial blooms. A new commercial enzyme-linked immunosorbent assay kit for the determination of total APs (APtot ELISA) has been released promising a rapid response with good cost efficiency for the routine monitoring of uncommon cyanopeptides. The present study explores the suitability of this new kit in comparison with a validated quantitative analytical method based on liquid chromatography coupled to mass spectrometry (LC-MS). The validation results were comparable with both methods for accuracy, precision, and calibration. Method detection limits were more sensitive using LC-MS specifically evaluated at 0.011 and 0.013 μg L−1 for AP-A and B respectively, compared to APtot ELISA evaluated at 0.10 μg L−1 for total of the two. For APtot ELISA, results were independent from the matrix; however, a systematic signal response was measured in blanks, requiring a blank subtraction in data treatment. Cross-reactivity of APtot ELISA was investigated by analyzing ten cyanopeptides selected for their abundance and diversity. Cyanopeptolin A (CP-A), nodularin-R (NOD), microcystin (MC)-RR, [Asp3]RR, and HilR showed cross-reactivity with an average overestimation going from 25 to 66%. Considering the contribution of cross-reactive cyanopeptides, thirteen lake samples out of fifteen showed higher concentrations using APtot ELISA with overestimation values up to 2261% compared to LC-MS. In light of this study results, LC-MS should still be preconized for the study and monitoring of APs when sensitivity and specificity are needed.

Advancing Knowledge on Cyanobacterial Blooms in Freshwaters

Cyanobacterial blooms have become a frequent phenomenon in freshwaters worldwide; they are a widely known indicator of eutrophication and water quality deterioration. Information and knowledge contributing towards the evaluation of the ecological status of freshwaters, particularly since many are used for recreation, drinking water, and aquaculture, is valuable. This Special Issue, entitled “Advancing Knowledge on Cyanobacterial Blooms in Freshwaters”, includes 11 research papers that will focus on the use of complementary approaches, from the most recently developed molecular-based methods to more classical approaches and experimental and mathematical modelling regarding the factors (abiotic and/or biotic) that control the diversity of not only the key bloom-forming cyanobacterial species, but also their interactions with other biota, either in freshwater systems or their adjacent habitats, and their role in preventing and/or promoting cyanobacterial growth and toxin production.

Toxin Analysis of Freshwater Cyanobacterial and Marine Harmful Algal Blooms on the West Coast of Florida and Implications for Estuarine Environments

Recent marine and freshwater algal and cyanobacterial blooms in Florida have increased public concern and awareness of the risks posed by exposure to these organisms. In 2018, Lake Okeechobee and the Caloosahatchee river, on the west coast of Florida, experienced an extended bloom of Microcystis spp. and a bloom of Karenia brevis in the coastal waters of the Gulf of Mexico that coincided in the Fort Myers area. Samples from the Caloosahatchee at Fort Myers into Pine Island Sound and up to Boca Grande were collected by boat. High concentrations of microcystin-LR were detected in the cyanobacterial bloom along with brevetoxins in the marine samples. Furthermore, β-N-methylamino-L-alanine (BMAA) and isomers N-(2-aminoethyl)glycine (AEG) and 2,4-diaminobuytric acid (DAB) were detected in marine diatoms and dinoflagellates, and cyanobacteria of freshwater origin. High freshwater flows pushed the cyanobacterial bloom to barrier island beaches and Microcystis and microcystins could be detected into the marine environment at a salinity of 41 mS/cm. For comparison, in 2019 collections of Dapis (a new generic segregate from Lyngbya) mats from Sarasota showed high concentrations of BMAA, suggesting the possibility of long-term exposure of residents to BMAA. The findings highlight the potential for multiple, potentially toxic blooms to co-exist and the possible implications for human and animal health.

Growth inhibition of Microcystis aeruginosa and adsorption of microcystin toxin by the yeast Aureobasidium pullulans, with no effect on microalgae.

This study evaluates the inhibitory effect of a yeast strain, Aureobasidium pullulans KKUY0701, isolated from decayed cyanobacterial bloom against harmful cyanobacterium Microcystis aeruginosa and determines the ability of this strain to remove microcystin (MC) toxin from the water. The antialgal activity of this yeast strain was assayed by co-cultivation with M. aeruginosa, diatom, and green algal species. The MC adsorption experiment was conducted in the presence of living and heat-inactivated yeast cells. Both yeast cells and filtrates caused a rapid reduction in the growth of M. aeruginosa, with complete death and cell lysis occurring after 3days. The yeast strain did not exhibit any inhibitory effect on either green algae or diatoms. Both living and heat-inactivated yeast cells were capable of adsorption of MC on their surfaces. Inactivated yeast exhibited higher adsorption capacity and lower intensity than living yeast for the adsorption of MC toxin. The results of this study suggest that this yeast strain could be employed to selectively reduce cyanobacterial blooms in freshwaters. Moreover, the application of heat-inactivated yeast's biomass for toxin adsorption gives new possibilities in drinking water treatment plants.

Alteration of dominant cyanobacteria in different bloom periods caused by abiotic factors and species interactions

Freshwater cyanobacterial blooms have drawn public attention because they threaten the safety of water resources and human health worldwide. Heavy cyanobacterial blooms outbreak in Lake Taihu in summer annually and vanish in other months. To find out the factors impacting the cyanobacterial blooms, the present study measured the physicochemical parameters of water and investigated the composition of microbial community using the 16S rRNA gene and internal transcribed spacer amplicon sequencing in the months with or without bloom. The most interesting finding is that two major cyanobacteria, Planktothrix and Microcystis, dramatically alternated during a cyanobacterial bloom in 2016, which is less mentioned in previous studies. When the temperature of the water began increasing in July, Planktothrix appeared first and showed as a superior competitor for M. aeruginosa in NO3−-rich conditions. Microcystis became the dominant genus when the water temperature increased further in August. Laboratory experiments confirmed the influence of temperature and the total dissolved nitrogen (TDN) form on the growth of Planktothrix and Microcystis in a co-culture system. Besides, species interactions between cyanobacteria and non-cyanobacterial microorganisms, especially the prokaryotes, also played a key role in the alteration of Planktothrix and Microcystis. The present study exhibited the alteration of two dominant cyanobacteria in the different bloom periods caused by the temperature, TDN forms as well as the species interactions. These results helped the better understanding of cyanobacterial blooms and the factors which contribute to them.

Application of advanced HepG2 3D cell model for studying genotoxic activity of cyanobacterial toxin cylindrospermopsin

Cylindrospermopsin (CYN) is an emerging cyanotoxin increasingly being found in freshwater cyanobacterial blooms worldwide. Humans and animals are exposed to CYN through the consumption of contaminated water and food as well as occupational and recreational water activities; therefore, it represents a potential health threat. It exhibits genotoxic effects in metabolically active test systems, thus it is considered as pro-genotoxic. In the present study, the advanced 3D cell model developed from human hepatocellular carcinoma (HepG2) cells was used for the evaluation of CYN cyto-/genotoxic activity. Spheroids were formed by forced floating method and were cultured for three days under static conditions prior to exposure to CYN (0.125, 0.25 and 0.5 μg/mL) for 72 h. CYN influence on spheroid growth was measured daily and cell survival was determined by MTS assay and live/dead staining. The influence on cell proliferation, cell cycle alterations and induction of DNA damage (γH2AX) was determined using flow cytometry. Further, the expression of selected genes (qPCR) involved in the metabolism of xenobiotics, proliferation, DNA damage response, apoptosis and oxidative stress was studied. Results revealed that CYN dose-dependently reduced the size of spheroids and affected cell division by arresting HepG2 cells in G1 phase of the cell cycle. No induction of DNA double strand breaks compared to control was determined at applied conditions. The analysis of gene expression revealed that CYN significantly deregulated genes encoding phase I (CYP1A1, CYP1A2, CYP3A4, ALDH3A) and II (NAT1, NAT2, SULT1B1, SULT1C2, UGT1A1, UGT2B7) enzymes as well as genes involved in cell proliferation (PCNA, TOP2α), apoptosis (BBC3) and DNA damage response (GADD45a, CDKN1A, ERCC4). The advanced 3D HepG2 cell model due to its more complex structure and improved cellular interactions provides more physiologically relevant information and more predictive data for human exposure, and can thus contribute to more reliable genotoxicity assessment of chemicals including cyanotoxins.

Modeling for multi-temporal cyanobacterial bloom dominance and distributions using landsat imagery

Cyanobacterial blooms pose great risks to aquatic systems due to its increase in frequency and severity worldwide. This study developed multi-temporal satellite remote sensing models that accurately portray spatial distribution of freshwater cyanobacterial blooms, which traditional monitoring methods cannot accomplish. The models were developed using multiple linear regression involving in-situ measurements and Landsat imagery in Lake Champlain for cyanobacterial blooms and other water quality variables i.e. chlorophyll-a, total phytoplankton, chlorophyte, chrysophyta, pyrrophyta, secchi disk and temperature. The developed models with Landsat imagery successfully captured the distribution of cyanobacterial blooms and other variables (adjusted R2 = 0.6–0.98). Multi-temporal analysis of remote sensing highlighted the high bloom years with quantified cyanobacterial levels over the entire water body, associated with temperature, indicating the impact of precipitation on cyanobacterial bloom growth. The modeling approach in this study can provide information to improve our understanding of algal dominance and dynamics. This approach can be applied to other freshwater systems, providing a pathway for rapid response systems.

Algal bloom inhibition through cobalt nano particles to control Oscillatoria growth in fresh water lakes

Cobalt nano particles (CoNPs) were synthesized and tested against microalgae isolated from fresh water cyanobacterial blooms by assessing the effects on growth rate, biomass concentration and biochemical parameters. Oscillatoria was identified as the predominant isolate from algal blooms and treated with varying concentrations (1, 2, 3, 4 and 5 mg L-1) of CoNPs. Steady decline in the growth rate of microalgae was observed at the end of 5 days indicating the toxicity of CoNPs on microalgal growth. At the end of cultivation period, 88% of reduction in biomass concentration of Microcystis and Oscillatoria were observed at 5 mg L-1 of CoNPs. Both protein and carbohydrate contents of the microalgae were reduced with increasing concentration of nano particles. Based on the findings, this study demonstrates the efficiency of cobalt nano particles (CoNPs) on inhibition of fresh water algal blooms thereby reducing the eutrophication problem.

Exploring microcystin-degrading bacteria thriving on recycled membranes during a cyanobacterial bloom

Microcystins (MC) are highly toxic secondary metabolites produced by cyanobacterial blooms in many freshwater ecosystems used for recreational and drinking water purposes. So far, biological processes remain to be optimized for an efficient cyanotoxin removal, and new approaches are necessary to compete with physical-chemical treatments. In previous studies we provided a new concept of membrane biofilm reactor made of recycled material, in which a single MC-degrading bacterial strain was inoculated. The present study evaluates the capacity of bacterial consortia associated with freshwater cyanobacterial blooms to form biofilms on recycled membranes and remove MC. Three different discarded reverse osmosis (RO) membranes, previously used in desalination plants after treating brackish water (BWd), seawater (SWd) and brackish water but transformed into nanofiltration (BWt-NF), were exposed to a cyanobacterial bloom in San Juan reservoir (central Spain). Results showed that the three recycled membranes developed a bacterial community with MC removal capacity. Little differences in bacterial coverage and MC removal efficiency between membranes were observed after their exposure in the reservoir. High-throughput sequencing of 16S rRNA gene analysis showed similar bacterial community composition at the phylum level but dissimilar at the order level between the three membranes. This suggests possible surface selectivity on the attached bacterial community. The mlr− candidates such as Burkholderiales and Methylophilales were highly abundant in BWt-NF and BWd, respectively, while mlr+ candidates (e.g. Sphingomonadales) were low abundant in all membranes. Analysis of mlrA and mlrB genes used as markers for MC degradation following mlr-pathway confirmed the presence of this pathway in all membranes. These results suggest the co-existence of both genotypes in membrane-attached native biofilms. Therefore, this study confirms that recycled membranes are suitable support for many MC-degrading bacteria, thus giving value to discarded membranes for eco-friendly and low-cost biological filters.

Tools for successful proliferation: diverse strategies of nutrient acquisition by a benthic cyanobacterium

Freshwater cyanobacterial blooms have increased worldwide, channeling organic carbon into these systems, and threatening animal health through the production of cyanotoxins. Both toxic and nontoxic Microcoleus proliferations usually occur when there are moderate concentrations of dissolved inorganic nitrogen, but when phosphorus is scarce. In order to understand how Microcoleus establishes thick biofilms (or mats) on riverbeds under phosphorus-limiting conditions, we collected Microcoleus-dominated biofilms over a 19-day proliferation event for proteogenomics. A single pair of nitrogen-dependent Microcoleus species were consistently present in relatively high abundance, although each followed a unique metabolic trajectory. Neither possessed anatoxin gene clusters, and only very low concentrations of anatoxins (~2 µg kg−1) were detected, likely originating from rarer Microcoleus species also present. Proteome allocations were dominated by photosynthesizing cyanobacteria and diatoms, and data indicate biomass was actively recycled by Bacteroidetes and Myxococcales. Microcoleus likely acquired nutrients throughout the proliferation event by uptake of nitrate, urea, and inorganic and organic phosphorus. Both species also harbored genes that could be used for inorganic phosphate solubilization with pyrroloquinoline quinone cofactors produced by cohabiting Proteobacteria. Results indicate that Microcoleus are equipped with diverse mechanisms for nitrogen and phosphorus acquisition, enabling them to proliferate and out-compete others in low-phosphorus waters.

Relationship between cyanobacterial bloom impacted drinking water sources and hepatocellular carcinoma incidence rates

Freshwater cyanobacterial blooms have increased in geographic distribution and intensity in recent decades worldwide. Cyanotoxins produced by many of these blooms, such as microcystins, are observed to play a role in tumor promotion and have been associated with increased liver cancer rates at the population level. Exposure occurs primarily via contaminated water (ingestion, inhalation, dermal contact), either from treated drinking water or during recreation in impacted surface waters; additional sources of exposure include consumption of fresh produce grown in cyanotoxin-contaminated environments or through the consumption of seafood caught in bloom-impacted waters.The current ecological study investigates whether populations served by cyanobacterial bloom-impacted surface waters for their drinking water source have higher hepatocellular carcinoma (HCC) incidence rates than those served by non-impacted surface waters and groundwater. Census tract level cancer incidence in the state of Ohio, United States was modeled using a negative binomial generalized linear model, controlling for differences in demographic composition (e.g. age, race, and income) at the census tract level. Presence of cyanobacterial blooms in surface waters was estimated using satellite multi-spectral remote sensing and in situ public water system cyanotoxin monitoring data. Census tracts estimated to be served by bloom-impacted surface waters had 14.2% higher HCC incidence rates than those served by non-bloom-impacted surface waters (incidence rate ratio, IRR: 1.142; 95% CI: 1.037–1.257). Additionally, these bloom-impacted census tracts had a 17.4% higher HCC incidence rate as compared to those estimated to receive drinking water from a groundwater source (IRR: 1.174; 95% CI: 1.101–1.252). No statistical difference was found in HCC incidence rates when comparing areas presumed to be served by non-bloom-impacted surface waters and those presumed to be served by groundwater sources. An important consideration for environmental justice, areas estimated to be served by bloom-impacted surface waters had higher levels of poverty and included a higher percentage of racial and ethnic minority populations than areas served by groundwater. These findings support the need for additional in-depth research into the potential hepatic carcinogenicity and exposures of cyanotoxins in those areas where severe blooms are chronically observed.

The cyanobacterial saxitoxin exacerbates neural cell death and brain malformations induced by Zika virus.

The northeast (NE) region of Brazil commonly goes through drought periods, which favor cyanobacterial blooms, capable of producing neurotoxins with implications for human and animal health. The most severe dry spell in the history of Brazil occurred between 2012 and 2016. Coincidently, the highest incidence of microcephaly associated with the Zika virus (ZIKV) outbreak took place in the NE region of Brazil during the same years. In this work, we tested the hypothesis that saxitoxin (STX), a neurotoxin produced in South America by the freshwater cyanobacteria Raphidiopsis raciborskii, could have contributed to the most severe Congenital Zika Syndrome (CZS) profile described worldwide. Quality surveillance showed higher cyanobacteria amounts and STX occurrence in human drinking water supplies of NE compared to other regions of Brazil. Experimentally, we described that STX doubled the quantity of ZIKV-induced neural cell death in progenitor areas of human brain organoids, while the chronic ingestion of water contaminated with STX before and during gestation caused brain abnormalities in offspring of ZIKV-infected immunocompetent C57BL/6J mice. Our data indicate that saxitoxin-producing cyanobacteria is overspread in water reservoirs of the NE and might have acted as a co-insult to ZIKV infection in Brazil. These results raise a public health concern regarding the consequences of arbovirus outbreaks happening in areas with droughts and/or frequent freshwater cyanobacterial blooms.

Are laboratory growth rate experiments relevant to explaining bloom-forming cyanobacteria distributions at global scale?

Predicting algal population dynamics using models informed by experimental data has been used as a strategy to inform the management and control of harmful cyanobacterial blooms. We selected toxic bloom-forming species Microcystis spp. and Raphidiopsis raciborskii (basionym Cylindrospermopsis raciborskii) for further examination as they dominate in 78 % and 17 %, respectively, of freshwater cyanobacterial blooms (cyanoHABs) reported globally over the past 30 years. Field measurements of cyanoHABs are typically based on biomass accumulation, but laboratory experiments typically measure growth rates, which are an important variable in cyanoHAB models. Our objective was to determine the usefulness of laboratory studies of these cyanoHAB growth rates for simulating the species dominance at a global scale. We synthesized growth responses of M. aeruginosa and R. raciborskii from 20 and 16 culture studies, respectively, to predict growth rates as a function of two environmental variables, light and temperature. Predicted growth rates of R. raciborskii exceeded those of M. aeruginosa at temperatures ≳ 25 °C and light intensities ≳ 150 μmol photons m−2 s-1. Field observations of biomass accumulation, however, show that M. aeruginosa dominates over R. raciborskii, irrespective of climatic zones. The mismatch between biomass accumulation measured in the field, and what is predicted from growth rate measured in the laboratory, hinders effective use of culture studies to predict formation of cyanoHABs in the natural environment. The usefulness of growth rates measured may therefore be limited, and field experiments should instead be designed to examine key physiological attributes such as colony formation, buoyancy regulation and photoadaptation. Improving prediction of cyanoHABs in a changing climate requires a more effective integration of field and laboratory approaches, and an explicit consideration of strain-level variability.

Stable-isotope dilution LC-MS/MS method for quantitative determination of microcystin conjugates with cysteine and glutathione in biotic matrices.

Microcystins are cyclic peptide toxins with hepatotoxic and tumor-promoting properties, which are produced in significant quantities (up to tens of μg/L) in freshwater cyanobacterial water blooms. Several studies reported microcystin accumulation in fish with possible food transfer to humans. These compounds are further metabolized to cysteine and glutathione conjugates which can be present in tissues in significant concentrations. In this study, we focused on the development and evaluation of robust and highly sensitive SPE-LC-MS/MS method for the analysis of microcystin conjugates in fish tissue samples. For the first time, we demonstrate the use of isotopically labeled internal standards which are essential for accurate and precise determination of analytes in complex biotic matrices. LLOQs of respective microcystin conjugates (signal-to-noise ratio; S/N > 10, peak-to-peak method) ranged from 3.3 to 5.0ng/g of tissue fresh weight (FW). The calibration was linear within a range of concentrations from 1 to 70ng/mL for all analyzed conjugates. The precision and repeatability of the method were very good with recoveries in the range of 88.5-107.6% and relative standard deviations between 8.8 and 13.2% for all analytes. In the follow-up study, fully validated method was used for the determination of microcystin conjugate levels in common carp exposed to microcystin-containing cyanobacterial biomass under controlled conditions. Significant amounts of microcystin conjugates (up to 55ng/g) were found in the tissues of fish after 7weeks of exposure. Our method was shown to be robust, sensitive, selective, and suitable for the determination of trace levels of microcystin conjugates in fish tissues.

High‐resolution imaging particle analysis of freshwater cyanobacterial blooms

AbstractEffective assessment of the health risk of cyanobacterial blooms requires an early warning system, which enables rapid detection of species of concern and determination of whether their cell concentrations exceed advisory guidelines. Advanced digital flow cytometry using FlowCam® (Fluid Imaging Technologies) in combination with light microscopy is a solid prospect for tracking cyanobacterial communities in a timely manner. However, implementation of such a method poses several challenges for the user. We first address sample preparation, instrumentation, taxonomic enumeration, and trouble‐shooting to facilitate high throughput of analyses of water samples for total cyanobacterial cell counts and their species composition. Preservation and initial screening of samples using light microscopy to estimate community size structure are endorsed to insure their archival quality and avoid clogging of the flow cell. We show that the highest magnification (×20 objective) is needed to achieve representative total and species‐specific cell enumerations. We also report that total cyanobacterial cell counts for samples analyzed using FlowCam vs. inverted light microscopy show significant positive correlation, as do those for preserved vs. live samples. Quantification of community composition using FlowCam vs. light microscopy also shows strong concordance. Although our FlowCam method performs well in the context of the World Health Organization advisory threshold of a total cyanobacterial count of 100,000 cells mL−1, it remains a work in progress in terms of reliably automated species‐level identifications.

Microbial Diversity and Toxin Risk in Tropical Freshwater Reservoirs of Cape Verde.

The Cape Verde islands are part of the African Sahelian arid belt that possesses an erratic rain pattern prompting the need for water reservoirs, which are now critical for the country’s sustainability. Worldwide, freshwater cyanobacterial blooms are increasing in frequency due to global climate change and the eutrophication of water bodies, particularly in reservoirs. To date, there have been no risk assessments of cyanobacterial toxin production in these man-made structures. We evaluated this potential risk using 16S rRNA gene amplicon sequencing and full metagenome sequencing in freshwater reservoirs of Cape Verde. Our analysis revealed the presence of several potentially toxic cyanobacterial genera in all sampled reservoirs. Faveta potentially toxic and bloom-forming Microcystis sp., dominated our samples, while a Cryptomonas green algae and Gammaproteobacteria dominated Saquinho and Poilão reservoirs. We reconstructed and assembled the Microcystis genome, extracted from the metagenome of bulk DNA from Faveta water. Phylogenetic analysis of Microcystis cf. aeruginosa CV01’s genome revealed its close relationship with other Microcystis genomes, as well as clustering with other continental African strains, suggesting geographical coherency. In addition, it revealed several clusters of known toxin-producing genes. This survey reinforces the need to better understand the country’s microbial ecology as a whole of water reservoirs on the rise.