Detection of potential microcystin-producing cyanobacteria in Brazilian reservoirs with a mcyB molecular marker

One of the most serious problems related to water eutrophication is the occurrence of increasingly frequent blooms of toxic cyanobacteria in freshwater ecosystems. Microcystin (MCYST) molecular markers may be used for the detection of toxic cyanobacteria, both cultivated strains and environmental samples, independently of their taxonomic category and production of the toxin at the moment of analysis. Sixty Microcystis spp. strains from 15 water reservoirs of south, southeastern and northeastern Brazil were analyzed by polymerase chain reaction (PCR) with oligonucleotide primers for mcyB gene of the operon that encodes a microcystin synthetase. It was found out that the presence of a unique amplified product of approximately 780 bp in 18 strains, indicated the presence of the microcystin-producing genotype. There was correspondence between the presence of the mcyB gene and microcystin determined by ELISA. Eight reservoirs contained toxic strains, two of these reservoirs being used mainly for public water supply. The coexistence of a mixture of toxic and non-toxic genotypes in populations of several reservoirs was found. Thus, it is evident that Microcystis populations present in blooms compose a mosaic, with genetically different individuals within the same population, each one, possibly, with its own tolerance to environmental factors and with distinct toxicity potential.     

PCR-based identification of microcystin-producing genotypes of different cyanobacterial genera

Microcystins are harmful hepatotoxins produced by many, but not all strains of the cyanobacterial genera Anabaena, Microcystis, Anabaena, Planktothrix, and Nostoc. Waterbodies have to be monitored for the mass development of toxic cyanobacteria; however, because of the close genetic relationship of microcystin-producing and non-producing strains within a genus, identification of microcystin-producers by morphological criteria is not possible. The genomes of microcystin-producing cells contain mcy genes coding for the microcystin synthetase complex. Based on the sequence information of mcy genes from Microcystis and Planktothrix, a primer pair for PCR amplification of a mcyA gene fragment was designed. PCR with this primer pair is a powerful means to identify microcystin-producing strains of the genera Anabaena, Microcystis, and Planktothrix. Moreover, subsequent RFLP analysis of the PCR products generated genus-specific fragments and allowed the genus of the toxin producer to be identified. The assay can be used with DNA from field samples.Abbreviations RFLP Restriction fragment length polymorphism - MALDI-TOF Matrix-assisted laser desorption/ionization-time of flight spectrometry - HPLC High performance liquid chromatography     

Spatial and temporal patterns of morel fruiting

The biotic and abiotic factors conditioning morel fruit body production are incompletely known. We examined spatial and temporal patterns of Morchella esculenta fruiting over five years in a wooded site in Missouri, USA. Fruiting onset was inversely correlated with spring air and soil temperatures, whereas abundance was positively correlated with rain events (>10 mm) during the 30 d preceding fruiting. The two years with the greatest fruiting had the shortest fruiting seasons (6–7 d). Fruiting season length was positively correlated with soil warming, suggesting that a narrow range of optimum soil temperatures favour the explosive production of fruit bodies. All woody stems of at least 1 cm diam were mapped and stem diameter and crown condition were noted. Morel fruit bodies were significantly closer to stems of Carya spp., Tilia americana and Ulmus americana than predicted by the frequencies of these woody species or their contribution to the total basal area on the site. Although intra-annual clustering of fruit bodies was often observed, inter-annual clustering was not. The spatial pattern of M. esculenta fruiting appears to be associated with vegetation pattern, whereas the onset and abundance of fruiting are determined by the interaction of spring temperatures with availability of supporting precipitation.     

Use of electrospray tandem mass spectrometry for identification of microcystins during a cyanobacterial bloom event

Drastic environmental conditions such as elevated temperature, abrupt pH variation, low turbulence, and high nutrient inputs can enhance the development of toxic cyanobacterial blooms in lakes and reservoirs. This study describes the occurrence of four microcystin variants (MC) in a bloom in the eutrophic reservoir Billings, in S?o Paulo City. The bloom sample was collected in October 2003, and Microcystis were the main genus found. The MC were separated and purified by reverse phase high performance liquid chromatography (RP-HPLC). Their structures were elucidated by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and four MC variants were determined: MC-RR, MC-LR, MC-YR, and MC-hRhR. MC-hRhR is described for the first time as a new variant of MC with two homoarginines at positions 2 and 4 in its structure. ESI-MS/MS analysis thus provides a powerful and convenient tool for the determination of variants of MC. These results represent an important contribution to the knowledge of the biochemistry of toxic cyanobacteria and their toxins, specifically in S?o Paulo State.     

Cyanobacteria and cyanobacterial toxins in the alkaline crater lakes Sonachi and Simbi, Kenya

The phytoplankton communities and the production of cyanobacterial toxins were investigated in two alkaline Kenyan crater lakes, Lake Sonachi and Lake Simbi. Lake Sonachi was mainly dominated by the cyanobacterium Arthrospira fusiformis, Lake Simbi by A. fusiformis and Anabaenopsis abijatae. The phytoplankton biomasses measured were high, reaching up to 3159 mg l⁻¹ in L. Sonachi and up to 348 mg l⁻¹ in L. Simbi. Using HPLC techniques, one structural variant of the hepatotoxin microcystin (microcystin-RR) was found in L. Sonachi and four variants (microcystin-LR, -RR, -LA and -YR) were identified in L. Simbi. The neurotoxin anatoxin-a was found in both lakes. To our knowledge this is the first evidence of cyanobacterial toxins in L. Sonachi and L. Simbi. Total microcystin concentrations varied from 1.6 to 12.0 μg microcystin-LR equivalents g⁻¹ DW in L. Sonachi and from 19.7 to 39.0 μg microcystin-LR equivalents g⁻¹ DW in L. Simbi. Anatoxin-a concentrations ranged from 0.5 to 2.0 μg g⁻¹ DW in L. Sonachi and from 0 to 1.4 μg g⁻¹ DW in L. Simbi. In a monocyanobacterial strain of A. fusiformis, isolated from L. Sonachi, microcystin-YR and anatoxin-a were produced. The concentrations found were 2.2 μg microcystin g⁻¹ DW and 0.3 μg anatoxin-a g⁻¹ DW. This is the first study showing A. fusiformis as producer of microcystins and anatoxin-a. Since A. fusiformis occurs in mass developments in both lakes, a health risk for wildlife can be expected.     

艾比湖湿地芦苇根际黏细菌群落多样性及其时空分布特征

【目的】研究艾比湖湿地芦苇根际土壤未培养黏细菌多样性、群落结构及其时空分布特征,提高对盐碱湿地等极端环境黏细菌资源的认知,为今后开发利用极端环境黏细菌资源奠定基础,也为艾比湖湿地盐漠生态系统修复提供数据支撑。【方法】采集艾比湖湿地10个样地3个月份的芦苇根际土壤,针对16S rRNA基因的V4–V5区使用高通量测序技术研究黏细菌多样性、群落结构及其时空分布特征。【结果】艾比湖湿地黏细菌16S rRNA基因Tags数占细菌的0.22%–3.54%,7月份和样地4的多样性最高,说明属多样性与季节和样地相关。本研究共鉴定到黏球菌目的3个亚目、8个科、14个属,其中海无柄孢囊黏细菌属(Haliangium)为优势属,占10.83%–71.01%。网络共现图表明绝大多数细菌与黏细菌存在相互作用。Spearman分析表明,细菌的Shannon、Chao1和ACE指数影响着黏细菌的多样性和丰富度。冗余分析(redundancy analysis, RDA)表明,土壤无机氮(inorganic nitrogen, IN)、有机质(organic matter, OM)和水溶性镁离子(Mg²⁺)是影响黏细菌多样性、群落结构的主要非生物因子。【结论】艾比湖湿地黏细菌资源丰富,其多样性、群落结构受时空变化影响。生物因素(细菌)和非生物因素(土壤理化性质)共同影响着黏细菌多样性。     

Evidence for Recombination in the Microcystin Synthetase (<Emphasis Type="Italic">mcy</Emphasis>) Genes ofToxic Cyanobacteria <Emphasis Type="Italic">Microcystis</Emphasis><Emphasis Type="Bold">spp</Emphasis>

Recombination has been suggested to be an important factor for the genetic variation of bacterial genes, but few studies have dealt with intragenic recombination between the same or closely related species of cyanobacteria. Here we provide strong evidence for recombination in the microcystin synthetase (mcy) gene cluster of the toxic cyanobacteria Microcystis spp. This gene cluster contains 10 genes (mcyA to J) that encode a mixed polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) complex. mcy gene sequences were determined for four selected regions (within mcyA, D, G, and J) within the mcy gene cluster from 1 Canadian and 10 Asian toxic Microcystis and compared with previously published mcy sequences. Split decomposition analysis indicated a reticulate phylogeny of mcyA, and several potential recombination tracts of mcyA were identified by the RDP analysis and a runs test implemented in GENECONV. In contrast, no recombination was detected in the mcyD, G, and J sequences. However, discrepancies among the four mcy gene genealogies were evident from the results of independent split decomposition analyses, which were further supported by incongruence length difference (ILD) tests. Taken together, these findings suggest that both intragenic and intergenic recombination within the mcy gene cluster contributes to the genetic diversity of the mcy genes of Microcystis spp.This article contains online supplementary material.     

Alteration of Ecosystem Function by Zebra Mussels in Oneida Lake: Impacts on Submerged Macrophytes

Dreissenid mussels (the zebra mussel Dreissena polymorpha and the quagga mussel D. bugensis) are ecosystem engineers that modify the physical environment by increasing light penetration. Such a change is likely to affect the distribution and diversity of submerged macrophytes. Filter-feeding by these mussels has been associated with increased water clarity in many North American and European lakes. In this study, we report the increase in water clarity of Oneida Lake, New York, USA, for 1975–2002 and argue that the increase was caused by zebra mussel invasion rather than declines in nutrients. Over the study period, although mean total phosphorus decreased significantly, the main increase in water clarity occurred after the zebra mussel invasion in 1991. The average depth receiving 1% surface light increased from 6.7 m to 7.8 m after the invasion of zebra mussels, representing a 23% areal expansion. The maximum depth of macrophyte colonization, as measured by diver and hydroacoustic surveys, increased from 3.0 m before the invasion of zebra mussels to 5.1 m after their establishment. In addition, macrophyte species richness increased, the frequency of occurrence increased for most species, and the composition of the macrophyte community changed from low-light–tolerant species to those tolerating a wide range of light conditions. Comparisons with observations reported in the literature indicate that increased light penetration alone could explain these changes in macrophyte distribution and diversity. Such changes will increase the importance of benthic primary production over pelagic production in the food web, thereby representing an overall alteration of ecosystem function, a process we refer to as “benthification”.     

Increased Benthic Algal Primary Production in Response to the Invasive Zebra Mussel (Dreissena polymorpha) in a Productive Ecosystem, Oneida Lake, New York

Increased water clarity associated with zebra mussel (Dreissena polymorpha) populations may favor benthic algal primary production in freshwater systems previously dominated by pelagic phytoplankton production. While zebra mussel-mediated water clarity effects on benthic primary production have been implicated in published reports, few production estimates are available. This study estimates benthic primary production in Oneida Lake, NY before and after zebra mussel invasion (1992), using measured photosynthetic parameters ( , α^B and β) from sampled benthic algal communities. In the summers of 2003 and 2004, primary production was measured as O₂ evolution from algal communities on hard (cobble) and soft (sediment) substrate from several depths. We also backcast estimates of benthic primary production from measurements of light penetration since 1975. Estimates of whole-lake epipelic and epilithic algal primary production showed a significant (4%) increase and exhibited significantly less interannual variability subsequent to the establishment of zebra mussels. We applied our model to two lakes of differing trophic status; the model significantly overestimated benthic primary production in a hypereutrophic lake, but there was no significant difference between the actual and predicted primary production values in the oligotrophic lake. The hypereutrophic lake had higher zebra mussel densities than Oneida (224 vs. 41 per sample respectively). Though total community respiration (measured in total darkness) was factored into our model predictions of production, our model may need modification when heterotrophic respiration is a large portion of total community metabolism.     

Accumulation and depuration of microcystin produced by the cyanobacterium <Emphasis Type="Italic">Microcystis</Emphasis> in a freshwater snail

Seasonal changes in microcystin concentrations in a resident snail (Sinotaia histrica) and an edible clam (Corbicula sandai) in Lake Biwa were surveyed. To clarify both the accumulation and depuration of microcystins, experimental studies with microcystin were also carried out on the snail. In the field investigation, microcystin was detected from the hepatopancreas and intestine of S. histrica (up to 3.2µgg^–1 dry weight and 19.5µgg^–1 dry weight, respectively); however, no microcystin was detected in the hepatopancreas of C. sandai. In the laboratory experiment, the microcystin-LR concentration in the hepatopancreas of S. histrica reached a value of 436µgg^–1 dry weight on day 10 of 15 days of uptake, and a high value persisted despite a depuration period of 15 days. The depuration rate constant of microcystin and its biological half-life were 0.0828 day^–1 and 8.4 days, respectively. These results indicate that S. histrica has a high ability to accumulate microcystin in its tissue. Because S. histrica is predated by fish and water fowl, it is likely to play an important role as a vector for microcystin in lakes with dense blooms of toxic cyanobacteria.     

Genetic diversity of Microcystis populations in a bloom and its relationship to the environmental factors in Qinhuai River, China

Cyanobacterial blooms, forming massive scum and various cyanotoxins, increasingly spread in a wide range of freshwater ecosystems. One heavy Microcystis bloom occurred in the entire Qinhuai River basin in 2010 summer for the first time. To determine the Microcystis populations and their spatial distributions along Qinhuai River, a molecular approach was applied by sequencing the DNA library based on the internal transcribed spacer sequences of 16-23S rRNA (ITS). The parsimony network (TCS) analysis showed that 9 groups were formed based on the main 24 genotypes, and each group was dominated by one highly represented root sequence. Marked changes in the composition and proportion of the Microcystis ITS genotype were detected from the upper to the lower reaches. The seed sources forming the bloom were probably located at 4 different locations. Furthermore, it was found that pH was the primary factor affecting the spatial distribution of the main genotype group among samples.     

Spatial and temporal dynamics of phytoplankton communities along a regulated river system, the Nakdong River, Korea

The longitudinal distribution and seasonal fluctuation of phytoplankton communities was studied along the middle to lower part of a regulated river system (Nakdong River, Korea). Phytoplankton biomass decreased sharply in the middle part of the river (182 km upward the estuary dam), and then increased downstream reaching a maximum at the last sampling station (27 km upward the estuary dam). In contrast, there was little downstream fluctuation in species composition, irrespective of pronounced differences in nutrient concentrations (TN, TP, NO₃, NH₄, PO₄) as well as in algal biomass. In the main river channel, small centric diatoms (Stephanodiscus hantzschii, Cyclotella meneghiniana) and pennate diatoms (Synedra, Fragilaria, Nitzschia) were dominant from winter to early spring (November–April). A mixed community of cryptomonads, centric and pennate diatoms, and coenobial greens (Pediastrum, Scenedesmus) was dominant in late spring (May–June). Blue-green algae (Anabaena, Microcystis, Oscillatoria) were dominant in the summer (July–September). A mid-summer Microcystis bloom occurred at all study sites during the dry season, when discharge was low, though the nutrient concentration varied in each study site. Nutrients appeared everywhere to be in excess of algal requirement and apparently did not influence markedly the downstream and seasonal phytoplankton compositional differences in this river.     

First report of microcystins from a Brazilian isolate of the cyanobacteriumMicrocystis aeruginosa

This is the first report on microcystins, cyclic heptapeptide hepatotoxins, from Brazilian water supplies. A colony isolate (NPJB-1) of the colonial cyanobacteriumMicrocystis aeruginosa from Lagoa das Garças, São Paulo, was cultured under non-axenic conditions. Exponential phase cells were harvested, concentrated and lyophilized for mouse bioassays and toxin extraction. The LD₁₀₀ of lyophilized cell suspensions was approximately 31 mg kg^–1 (dry cell weight/animal weight). Isolation, purification and characterization of the toxins were carried out by reversed phase HPLC, HPLC amino acid analysis and fast atom bombardment mass spectrometry. Strain NPJB-1 produces two different hepatotoxic heptapeptide microcystins. The main one was microcystin-LR, the most commonly reported microcystin from cyanobacteria. The other was microcystin-LF, the phenylalanine variant of microcystin-LR. This is the first published report for microcystin-LF.     

Spatial and temporal variation of benthic Cladocera (Crustacea) studied with activity traps in Lake Myvatn, Iceland

Benthic Cladocera were studied with a modified type of an activity trap in Lake Myvatn in 1990–1992. After feasibility experiments, the operation time and the distance of the traps from the benthic substrate were adjusted in order to minimize the effects of diurnal variation and the trapping of planktonic organisms. The trap catches of Eurycercus lamellatus were positively correlated with their abundance at the bottom as estimated by grab sampling (r = 0.910, P < 0.001). The usefulness of the activity trap was demonstrated by: (1) a lake-wide survey of the benthic Cladocera; (2) a study of the seasonal variation in the size distribution, abundance and sex ratio of E. lamellatus; and (3) a study of the seasonal succession of Chydorus sphaericus and Alona affinis. The variation of benthic Cladocera among 21 trap sites distributed on a 1–2-km scale across the lake exceeded the within-site variation. The sampling sites could be divided into five main groups based on cluster analysis. Eurycercus lamellatus was the most common species in the mat of filamentous green algae (Cladophorales). Alonella nana dominated the area of spring water inflow in the north basin and Macrothrix hirsuticornis the area of spring water inflow in the southeastern part of the lake. In other parts of the lake either Chydorus sphaericus or Alona quadrangularis tended to dominate. The size distribution and sex ratio of E. lamellatus was followed at two sites through one summer. In early summer most individuals were females less than 1.45 mm long. Around mid-summer they had grown to 0.69–3.1 mm. By the end of August the size distribution had become bimodal, with a large number of small males and a smaller number of much larger females. The seasonal succession in the abundance of E. lamellatus, A. affinis and C. sphaericus was followed at four sites over two seasons. With some exceptions the abundance of a species followed a similar seasonal trajectory on the different stations in any one year. There was, however, a marked difference between the two years (1991 and 1992), probably related to different temperatures.     

Rapid quantification of mcyB copy numbers on dry chemistry PCR chips and predictability of microcystin concentrations in freshwater environments

Microcystin-producing cyanobacteria cause serious water quality problems worldwide, which has led to growing pressure for more intensive monitoring. Molecular biology methods that are based on identification and enumeration of biosynthetic genes, such as quantitative PCR, show promise in this respect. To be practical in a wide range of settings, these methods need to be usable also by laboratory personnel who do not have previous experience in PCR setup. Here we present a real-time quantitative mcyB dry chemistry PCR assay capable of identifying the three globally most common microcystin-producing cyanobacterial genera, Anabaena, Microcystis and Planktothrix. It minimizes the amount of liquid handling and avoids direct contact with the PCR reagents at the time of analysis. Large quantities of virtually identical chips can be manufactured, improving the comparability of results. Using the dry chemistry PCR chips, freshwater environmental samples from Finnish and Estonian lakes, rivers and reservoirs were analyzed for mcyB. The chip format was found to be highly suitable for water sample analysis due to its ease-of-use, good sensitivity and amplification efficiency. Significant positive correlation (Spearman's rank correlation, ρ > 0.66, P < 0.001) was observed between combined mcyB copy numbers from Microcystis, Anabaena, Planktothrix and total microcystin concentrations, regardless of the method used to measure the toxins (ELISA or LC–MS). Positive correlations were observed also for single lakes.     

Water quality improvement and phytoplankton response in the drinking water source in Meiliang Bay of Lake Taihu, China

Water quality experienced changes throughout the 3-year ecological engineering experiment in the drinking water source in Meiliang Bay of Lake Taihu. Average concentrations of TN, TP, NH₄⁺, BOD₅ and transparency in the drinking water source during the period of July–December 2005 were 1.85, 0.13, 0.23, 3.03 mg L⁻¹ and 27.5 cm, respectively, decreasing by 47.9%, 21.2%, 83.3%, 54.4% and 24.2%, compared to concentrations from the same period in 2003. Concentrations of chlorophyll a and COD were 89.9 μg L⁻¹ and 6.45 mg L⁻¹, increasing by 27.9% and 17.7%, compared to the values in 2003. Cyanobacteria (mainly Microcystis) dominated the phytoplankton community in the ecological engineering area during July–December 2005. Densities of cyanobacteria and Microcystis were higher in 2005 than in 2004 and higher inside the engineering area than outside. Density percentages of cyanobacteria and Microcystis to total algae were above 90% and 60% during the bloom period. Average density of flagellate algae was higher during July–December 2005 than in 2004. Changes in water quality in the engineering area resulted mainly from the weakening of waves, decrease in concentrations of suspended solids, and assimilation of mass algae and periphytons. In spite of initial improvement of water quality, cyanobacterial bloom still determined the phytoplankton dynamics and variations. Additionally, nutrient concentration still remained at a high level without control of external loading. Therefore, a more holistic approach and long-term management should be adopted in Lake Taihu.     

Nutrient limitation of Microcystis aeruginosa in northern California Klamath River reservoirs

Nutrient limitations were investigated in Copco and Iron Gate Reservoirs, on the Klamath River in California, where blooms of the toxin-producing cyanobacterium Microcystis aeruginosa were first reported in 2005. Nutrient enrichment experiments conducted in situ in June and August, 2007 and 2008, determined responses in phytoplankton biomass, Microcystis abundance and microcystin concentration to additions of phosphorus and different forms of nitrogen (NH₄⁺, NO₃, and urea). Microcystis abundance was determined using quantitative PCR targeting the phycocyanin intergenic spacer cpcBA.Total phytoplankton biomass increased with additions of N both before and during Microcystis blooms, with no primary effects from P, suggesting overall N limitation for phytoplankton growth during the summer season. NH₄⁺ generally produced the greatest response in phytoplankton growth, while Microcystis abundance increased in response to all forms of N. Microcystis doubling time in the in situ experiments was 1.24–1.39 days when N was not limiting growth. The results from this study suggest availability of N during the summer is a key growth-limiting factor for the initiation and maintenance of toxic Microcystis blooms in Copco and Iron Gate Reservoirs in the Klamath River.     

Spatial distribution and morphologic diversity of virioplankton in Lake Donghu, China

The spatial distribution and morphological diversity of virioplankton were determined in Lake Donghu which contains three trophic regions: hypertrophic, eutrophic and mesotrophic region. Virioplankton abundance measured by transmission electron microscope (TEM) ranged from 7.7 × 10⁸ to 3.0 × 10⁹ ml^–1, being among the highest observed in any natural aquatic system examined so far. The spatial distribution of virioplankton was correlated significantly with chlorophyll a concentration (r = 0.847; P < 0.01) at the sampling sites in Lake Donghu. 76 morphotypes were observed. Most morphotypes have tails, belonging to Siphoviridae, Myoviridae and Podoviridae. The majority of tailed phages in the lake were Myoviridae. Morphotypes which were rarely reported, such as prolate-headed virus-like particles, lemon-shaped virus-like particle, and viruses resembling Tectiviridae and Corticoviridae were all observed in the lake. It is concluded that the high viral abundance might be associated with high density of phytoplankton including algae and cyanobacteria. There was high viral diversity in this eutrophic shallow lake. In addition, cyanophage represented an important fraction of the virioplankton community in Lake Donghu.