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A guide to state-of-the-art molecular tools for monitoring and managing the toxigenicity of cyanobacteria Runaway eutrophication and climate change has made the monitoring and management of toxigenic organisms in the world's bodies of water more urgent than ever. In order to influence public policy regarding the detection and quantification of those organisms, it is incumbent upon scientists to raise the awareness of policy makers concerning the increased occurrence of toxigenic cyanobacteria and the threats they pose. As molecular methods can handle many samples in short time and help identify toxigenic organisms, they are reliable, cost-effective tools available for tracking toxigenic cyanobacteria worldwide. This volume arms scientists with the tools they need to track toxigenicity in surface waters and food supplies and, hopefully, to develop new techniques for managing the spread of toxic cyanobacteria. This handbook offers the first comprehensive treatment of molecular tools for monitoring toxigenic cyanobacteria. Growing out of the findings of the landmark European Cooperation in Science and Technology Cyanobacteria project (CYANOCOST), it provides detailed, practical coverage of the full array of available molecular tools and protocols, from water sampling, nucleic acid extraction, and downstream analysis-including PCR and qPCR based methods-to genotyping (DGGE), diagnostic microarrays, and community characterization using next-gen sequencing techniques.-Offers an overview of the latest trends in the field, while providing a foundation for understanding and applying the tools and techniques described -Provides detailed coverage of the full range of molecular tools currently available, with expert guidance on the analysis and interpretation of results -Includes step-by-step guidance on standard operational procedures, including molecular tests used in environmental monitoring, with individual chapters devoted to each procedure -Complements the published Handbook of Cyanobacterial Monitoring and Cyanotoxin Analysis from the CyanoCOST project This handbook is an indispensable working resource for scientists, lab technicians, and water management professionals and an excellent text/reference for graduate students and supervisors who use molecular tools. It will also be of great value to environmental health and protection officials and policy makers.

The harmful cyanobacterium Cylindrospermopsis raciborskii is a widespread species increasingly being recorded in freshwater systems around the world. Studies have demonstrated some key attributes of this species which may explain its global dominance. It has a high level of flexibility with respect to light and nutrients, being capable of growth under low and variable light conditions. However, it is the strategy with respect to nutrient utilization that has received more attention. Unlike many bloom forming species, the dominance of this species is not simply linked to higher nutrient loads. In fact it appears that it is more competitive when phosphorus and nitrogen availability is low and/or variable. An important component of this flexibility appears to be the result of within-population strain variability in responses to nutrients, as well as key physiological adaptations. Strain variability also appears to have an effect on the population-level cell quota of toxins, specifically cylindrospermopsins (CYNs). Field studies in Australia showed that populations had the highest proportion of toxic strains when dissolved inorganic phosphorus was added, resulting in stoichiometrically balanced nitrogen and phosphorus within the cells. These strategies are part of an arsenal of responses to environmental conditions, making it a challenging species to manage. However, our ability to improve bloom prediction will rely on a more detailed understanding of the complex physiology and ecology of this species.

In late May 2016, a cyanobacterial harmful algal bloom (cHAB) was detected in the Maumee River, the largest tributary to Lake Erie, the southernmost lake of the Laurentian Great Lakes system. Testing on 31 May identified Planktothrix agardhii as the dominant cyanobacterium with cell abundance exceeding 1.7×10 9 cells/L and total microcystins (MC) reaching 19 μg/L MC-LR equivalents, a level over 10-fold higher than the 2015 revised U.S. Environmental Protection Agency (EPA) national health advisory levels for drinking water exposure to adults. Low river discharge coincident with negligible precipitation through the latter half of May coincided with an 80% decline in river turbidity that likely favored bloom formation by a low-light adapted P. agardhii population. Also contributing to the cHAB were high initial nutrient loads and an increase of the river temperature from 13°C to 26°C over this same period. The bloom persisted through 5 June with microcystins exceeding 22 μg/L MC-LR equivalents at the bloom peak. By 6 June, the river had returned to its muddy character following a rain event and sampling on 7 June detected only low levels of toxin (<0.6 μg/L) at public water systems located near the bloom origin. The elevated toxin production associated with this early onset bloom was without precedent for the Maumee River and an unique attribute of the cHAB was the high proportion of potentially-toxic genotypes. Whereas Planktothrix spp. is common in lotic environments, and has been previously detected in the Maumee, blooms are not commonly reported. This early onset, microcystin-producing cHAB provided a rare opportunity to glean insights into environmental factors that promote bloom development and dominance by Planktothrix in lotic environments.

Cyanobacteria are oxygenic photosynthetic Gram-negative bacteria that can form potentially toxic blooms in eutrophic and slow flowing aquatic ecosystems. Bloom toxicity varies spatially and temporally, but understanding the mechanisms that drive these changes remains largely a mystery. Changes in bloom toxicity may result from changes in intracellular toxin pool sizes of cyanotoxins with differing molecular toxicities, and/or from changes in the cell concentrations of toxic and non-toxic cyanobacterial species or strains within bloom populations. We show here how first-order rate kinetics at the cellular level can be used to explain how environmental conditions drive changes in bloom toxicity at the ecological level. First order rate constants can be calculated for changes in cell concentration ( μ c : specific cell division rate) or the volumetric biomass concentration ( μ g : specific growth rate) between short time intervals throughout the cell cycle. Similar first order rate constants can be calculated for changes in nett volumetric cyanotoxin concentration ( μ tox : specific cyanotoxin production rate) over similar time intervals. How μ c (or μ g ) covaries with μ tox over the cell cycle shows conclusively when cyanotoxins are being produced and metabolised, and how the toxicity of cells change in response to environment stressors. When μ tox / μ c >1, cyanotoxin cell quotas increase and individual cells become more toxic because the nett cyanotoxin production rate is higher than the cell division rate. When μ tox / μ c =1, cell cyanotoxin quotas remains fixed because the nett cyanotoxin production rate matches the cell division rate. When μ tox / μ c <1, the cyanotoxin cell quota decreases because either the nett cyanotoxin production rate is lower than the cell division rate, or metabolic breakdown and/ or secretion of cyanotoxins is occurring. These fundamental equations describe cyanotoxin metabolism dynamics at the cellular level and provide the necessary physiological background to understand how environmental stressors drive changes in bloom toxicity.

Harmful cyanobacterial blooms, especially Microcystis blooms, occur worldwide and draw widespread attention. The dynamics of microcystin-producing Microcystis and competition between microcystin-producing Microcystis and non-microcystin-producing Microcystis are key to predicting and treating Microcystis blooms. Multiplex qPCR is a useful tool to assess such issues. In this study, we developed multiplex qPCR methods with newly-designed probes and primers for the microcystin-synthesis related genes mcyA and mcyE . We used seven toxic Microcystis strains and four non-toxic Microcystis strains to compare the differences in the ratios of toxic and non-toxic Microcystis in mixed cultures, which were calculated using abundances of the genes mcyA, mcyB, mcyD, mcyE and phycocyanin ( PC ). We also compared traditional cell counting and multiplex qPCR. Hierarchical clustering and principal component analysis indicated that mcyD was the most suitable mcy gene for quantification in laboratory experiments. mcyB abundances were always higher; we suggest that the amount of toxic Microcystis measured using mcyB might overestimate the actual percentages.

Microcystins (MCs) are cyclic hepatotoxic peptides produced by the bloom-forming cyanobacterium Microcystis and present a public health hazard to humans and livestock. The removal of MCs from contaminated water with powdered activated carbon (PAC) has been employed as a simple and economic treatment strategy. In this study, PAC-Fe(III) was prepared and utilized for the fast and efficient removal of MCs from water. PAC-Fe(III) exhibited superior microcystin-LR (MC-LR) removal capacity and efficiency compared to the unmodified PAC. The MC-LR removal efficiency of PAC-Fe(III) increased with decreasing pH within the pH range of 4.3 to 9.6. PAC-Fe(III) could be reused for 3 times by methanol elution while the MC-LR removal efficiency was still over 70 percent. The removal efficiency was positively correlated to the ionic strength of water and negatively correlated to alkalinity. Natural organic matter (NOM) such as humic acid (HA) and salicylic acid (SA) generated low interference with MC-LR adsorption by PAC-Fe(III). The complexation reaction between Fe 3+ in PAC-Fe(III) and the functional groups of MCLR was suggested as the key mechanism of MC-LR removal by PAC-Fe(III). The results suggest that Femodified PAC is a promising material for the treatment of MC-contaminated waters.

Cyanobacterial blooms are a global problem, with their occurrence tightly tied to nutrient loading. We cultured Microcystis aeruginosa FACHB-905 in growth medium with either inorganic (orthophosphate) or organic (β-glycerophosphate or polyphosphate) phosphorus and at different N:P ratios with 50:1, 30:1, 16:1, 4:1 and 1:4, serving as the phosphorus source. Fluorescence parameters were measured to determine the response of cellular responses to nutrient stress. Scanning electron microscopy (SEM) and estimates of antioxidant activity were employed to examine potential mechanisms of physical change. The results demonstrate that inorganic phosphorus was more bioavailable to M. aeruginosa relative to organic phosphorus in culture. The highest cell concentration (2.21×10 6 cells/mL), chlorophyll- a (0.39 pg/cell) and phycocyanin (1.57 pg/cell) quotas and high levels of chlorophyll fluorescence parameters (rETR, E k , α , φ PS II and F v / F m ) were obtained when phosphorus was supplied as K 2 HPO 4 at a N:P ratio of 16–30. Organic sources of phosphorus (β-glycerophosphate and polyphosphate) were bioavailable to M. aeruginosa . In addition, too concentrated orthophosphate (N:P=1:4) resulted in the oxidative stress and lipid peroxidation of cell membrane (identified by the antioxidant system activity), and the photosynthetic activity declined consequently. This study has demonstrated the effects of different phosphorus chemistries and N:P ratios on the cyanobacterial growth, photosynthetic activity and cell physiology, which could be an effective tool for predicting cyanobacterial dominance or N-deficiency in natural lakes (due to the superior ability of cyanobacteria for dissolved N and fix atmospheric N in some cases).

Toxic cyanobacteria (TCB) are well-known worldwide for their adverse impacts on humans. Species compositions and seasonal variations of TCB in reservoirs depend on interactions between physical and chemical factors. This study was conducted to evaluate the water quality in the Aha Reservoir, Southwest China, focusing on cyanobacteria and cyanotoxins. Water samples were collected weekly or biweekly from May to September of 2015 and used to delineate temporal variations in density and distribution of toxic cyanobacteria and cyanotoxins in the reservoir. Toxic cyanobacteria identified consisted of Aphanizomenon flos-aquae, Pseudanabaena limnetica, Cylindrospermopsis sp., and Microcystis sp., with Aphanizomenon flos-aquae and Pseudanabaena limnetica being the most common and significant toxic genera. The total biomass of cyanobacteria was 17.0 mg/L. Identification and quantification of microcystin variants were conducted by high performance liquid chromatography (HPLC) using a system equipped with a photodiode array detector. Microcystin levels were between 0–3.0 μg/L, MC-RR was around 0–3.0 μg/L and MC-LR was approximately 0–0.9 μg/L. Overall, the results of this study indicate that the investigated reservoirs should be monitored regularly to minimize potential health risks to the human population.

Species in the cyanobacterial genus Merismopedia are present in freshwaters at different trophic levels, with some species even as the components of cyanobacterial blooms. However, species diversity in this genus was not fully verified by molecular investigation and polyphasic taxonomic studies. In this study, Merismopedia -like strain tenuissima CHAB 7021 was isolated from Ganjiang River in Jiangxi Province, China, and polyphasic characterization of this strain was performed by morphological observation, ultrastructural examination, chemical detection of pigments and phylogenetic analysis based on 16S rRNA gene sequences. Morphological identification of the strain was supported by the ultrastructural features, as the tiny species Merismopedia tenuissima Lemmermann. The phylogeny based on 16S rRNA gene sequences revealed at least three clades formed by the strains of Merismopedia . The three M. tenuissima strains including M. tenuissima CHAB 7021 was gathered in clade III with distant relationship to the clade I formed by the six Merismopedia strains including the type species M. punctata, and such a genetic distance may propose Merismopedia tenuissima to separate from Merismopedia genus. However intermixture relationship in between strains of M. punctate and M. glauca in the phylogenetic tree still complicated the taxonomic status in the genus Merismopedia. The process for taxonomic revision in the Merismopedia genus still await for examination and further information on more strains of type species M. punctata .

The increasing occurrence of cyanobacterial blooms in water bodies is a serious threat to the environment. Efficient in-lake treatment methods for the control of cyanobacteria proliferation are needed, their in-vivo detection to obtain a real-time response to their presence, as well as the information about their physiological state after the applied treatment. In-vivo fluorescence measurements of photosynthetic pigments have proved to be effective for quantitative and qualitative detection of phytoplankton in a water environment. In the experiment, chlorophyll and phycocyanin fluorescence sensors were used concurrently to detect stress caused by electrochemical oxidation applying an electrolytic cell equipped with borondoped diamond electrodes on a laboratory culture of cyanobacteria Microcystis aeruginosa PCC 7806. The inflicted injuries were reflected in a clear transient increase in the phycocyanin fluorescence signal (for 104 %± 43%) 24 h after the treatment, which was not the case for the chlorophyll fluorescence signal. In the next 72 h of observation, the fluorescence signals decreased (on 40% of the starting signal) indicating a reduction of cell number, which was confirmed by cell count (24% reduction of the starting concentration) and analysis of extracted chlorophyll and phycocyanin pigment. These results demonstrate the viability of the combined application of two sensors as a useful tool for in-vivo detection of induced stress, providing real-time information needed for the evaluation of the efficiency of the in-lake treatment and decision upon the necessity of its repetition. The electrochemical treatment also resulted in a lower free microcystins concentration compared to control.