Application of sequence-specific labeled 16S rRNA gene oligonucleotide probes for genetic profiling of cyanobacterial abundance and diversity by array hybridization

DNA sequence information for the small-subunit rRNA gene (16S rDNA) obtained from cyanobacterial cultures was used to investigate the presence of cyanobacteria and their abundance in natural habitats. Eight planktonic communities developing in lakes characterized by relatively low algal biomass (mesotrophic) and in lakes with correspondingly high biomass (eutrophic) were selected for the study. The organismal compositions of the water samples were analyzed genetically, using multiplex sequence-specific labeling of oligonucleotide probes targeted to 16S rDNA and subsequent hybridization of the labeled probes to their respective complements spotted onto a solid support (DNA array). Ten probes were established to determine the relative abundances of the discernible cyanobacteria encountered in the selected lakes. The probes were generally specific for their targets, as determined through analyses of clone cultures. Reproducible abundance profiles were established for the lakes investigated in the subsequent analyses of natural cyanobacterial communities. The results from the genetic analyses were then compared with information obtained from standard hydrobiological and hydrochemical analyses. Qualitatively, there were relatively good correlations among the groups of organisms (Nostoc, Microcystis, and Planktothrix species) found in the different lakes. The levels of correlation were lower for the quantitative data. This may, however, be due to differences in sample processing technique. The conclusions from these comparisons are that the genetic abundance profiles may provide a foundation for separating and quantifying genetically distinct groups of cyanobacteria in their natural habitats.     

130 "Plantae" Emended Based on the Common History of the Primary Plastid Endosymbiosis

Travertine terraces have been deposited by calcareous hot springs in Yellowstone from as early as 365,000 years to the present. Most of these porous and non-porous CaCO3 rocks (old or new) contain a 1–2 mm thick greenish band about 1–3 mm below the upper surface. These bands are composed of cyanobacteria and, sometimes, unicellular green algae. Although some moisture may be retained for much of the year, all undergo freezing in winter and desiccation in summer. DGGE (denaturing gradient gel electrophoresis), with subsequent 16S rDNA sequence analyses of bands, has shown that relatively few phylotypes of cyanobacteria are present, but some occur in travertine of very different ages, indicating secondary establishment of the communities. Clonal cultures of predominant types have also been established and sequenced. All those tested are able to survive extreme desiccation. Preliminary sequence analyses of cultures show that some strains are nearly identical to known cyanobacterial strains while others show little similarity. One sequence is 100% identical to the cyanobacterium Cyanobium gracile. This cyanobacterium is known to be distributed worldwide in lakes and brackish seas, but not in a cryptoendolithic environment. Another sequence shows 99% identity to two cyanobacteria isolated from Antarctic freshwater ponds. Both Antarctic ponds and Yellowstone travertine are environments in which adaptations for desiccation and/or freezing tolerance could be crucial. The lack of ecological similarity among some of these strains indicates that genes other than 16S rDNA must be used for differentiation. These results will be discussed along with the ecology of travertine habitats.     

129 Cryptoendolithic Photosynthetic Communities Within Recent and Ancient Travertine in Yellowstone National Park

Travertine terraces have been deposited by calcareous hot springs in Yellowstone from as early as 365,000 years to the present. Most of these porous and non‐porous CaCO3 rocks (old or new) contain a 1–2 mm thick greenish band about 1–3 mm below the upper surface. These bands are composed of cyanobacteria and, sometimes, unicellular green algae. Although some moisture may be retained for much of the year, all undergo freezing in winter and desiccation in summer. DGGE (denaturing gradient gel electrophoresis), with subsequent 16S rDNA sequence analyses of bands, has shown that relatively few phylotypes of cyanobacteria are present, but some occur in travertine of very different ages, indicating secondary establishment of the communities. Clonal cultures of predominant types have also been established and sequenced. All those tested are able to survive extreme desiccation. Preliminary sequence analyses of cultures show that some strains are nearly identical to known cyanobacterial strains while others show little similarity. One sequence is 100% identical to the cyanobacterium Cyanobium gracile. This cyanobacterium is known to be distributed worldwide in lakes and brackish seas, but not in a cryptoendolithic environment. Another sequence shows 99% identity to two cyanobacteria isolated from Antarctic freshwater ponds. Both Antarctic ponds and Yellowstone travertine are environments in which adaptations for desiccation and/or freezing tolerance could be crucial. The lack of ecological similarity among some of these strains indicates that genes other than 16S rDNA must be used for differentiation. These results will be discussed along with the ecology of travertine habitats.     

The Impact of Fish Predation and Cyanobacteria on Zooplankton Size Structure in 96 Subtropical Lakes

Zooplankton are relatively small in size in the subtropical regions. This characteristic has been attributed to intense predation pressure, high nutrient loading and cyanobacterial biomass. To provide further information on the effect of predation and cyanobacteria on zooplankton size structure, we analyzed data from 96 shallow aquaculture lakes along the Yangtze River. Contrary to former studies, both principal components analysis and multiple regression analysis showed that the mean zooplankton size was positively related to fish yield. The studied lakes were grouped into three types, namely, natural fishing lakes with low nutrient loading (Type1), planktivorous fish-dominated lakes (Type 2), and eutrophic lakes with high cyanobacterial biomass (Type 3). A marked difference in zooplankton size structure was found among these groups. The greatest mean zooplankton size was observed in Type 2 lakes, but zooplankton density was the lowest. Zooplankton abundance was highest in Type 3 lakes and increased with increasing cyanobacterial biomass. Zooplankton mean size was negatively correlated with cyanobacterial biomass. No obvious trends were found in Type 1 lakes. These results were reflected by the normalized biomass size spectrum, which showed a unimodal shape with a peak at medium sizes in Type 2 lakes and a peak at small sizes in Type 3 lakes. These results indicated a relative increase in medium-sized and small-sized species in Types 2 and 3 lakes, respectively. Our results suggested that fish predation might have a negative effect on zooplankton abundance but a positive effect on zooplankton size structure. High cyanobacterial biomass most likely caused a decline in the zooplankton size and encouraged the proliferation of small zooplankton. We suggest that both planktivorous fish and cyanobacteria have substantial effects on the shaping of zooplankton community, particularly in the lakes in the eastern plain along the Yangtze River where aquaculture is widespread and nutrient loading is high.     

Increased risk of cyanobacterial blooms in northern high‐latitude lakes through climate warming and phosphorus enrichment

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Effects of experimentally induced cyanobacterial blooms on crustacean zooplankton communities

SUMMARY 1. Large in situ enclosures were used to study the effects of experimentally induced cyanobacterial blooms on zooplankton communities. A combination of N and P was added to shallow (2 m) and deep enclosures (5 m) with the goal of reducing the TN : TP ratio to a low level (∼5 : 1) to promote cyanobacterial growth. After nutrient additions, high biomass of cyanobacteria developed rapidly in shallow enclosures reaching levels only observed during bloom events in eutrophic lakes.
2. In the shallow enclosures, particulate phosphorus (PP) was on average 35% higher in comparison with deep enclosures, suggesting that depth plays a key role in P uptake by algae. Phytoplankton communities in both deep and shallow enclosures were dominated by three cyanobacteria species – Aphanizomenon flos-aquae , Anabaena flos-aquae and Microcystis aeruginosa – which accounted for up to 70% of total phytoplankton biomass. However, the absolute biomass of the three species was much higher in shallow enclosures, especially Aphanizomenon flos-aquae . The three cyanobacteria species responded in contrasting ways to nutrient manipulation because of their different physiology.
3. Standardised concentrations of the hepatotoxic microcystin-LR increased as a result of nutrient manipulations by a factor of four in the treated enclosures. Increased biomass of inedible and toxin producing cyanobacteria was associated with a decline in Daphnia pulicaria biomass caused by a reduction in the number of individuals with a body length of >1 mm. Zooplankton biomass did not decline at moderate cyanobacteria biomass, but when cyanobacteria reached high biomass large cladocerans were reduced.
4. Our results demonstrate that zooplankton communities can be negatively affected by cyanobacterial blooms and therefore the potential to use herbivory to reduce algal blooms in such eutrophic lakes appears limited.     

Identification of a Diagnostic Marker To Detect Freshwater Cyanophages of Filamentous Cyanobacteria

Cyanophages are viruses that infect the cyanobacteria, globally important photosynthetic microorganisms. Cyanophages are considered significant components of microbial communities, playing major roles in influencing host community diversity and primary productivity, terminating cyanobacterial water blooms, and influencing biogeochemical cycles. Cyanophages are ubiquitous in both marine and freshwater systems; however, the majority of molecular research has been biased toward the study of marine cyanophages. In this study, a diagnostic probe was developed to detect freshwater cyanophages in natural waters. Oligonucleotide PCR-based primers were designed to specifically amplify the major capsid protein gene from previously characterized freshwater cyanomyoviruses that are infectious to the filamentous, nitrogen-fixing cyanobacterial genera Anabaena and Nostoc. The primers were also successful in yielding PCR products from mixed virus communities concentrated from water samples collected from freshwater lakes in the United Kingdom. The probes are thought to provide a useful tool for the investigation of cyanophage diversity in freshwater environments.     

Deteriogenic cyanobacteria on historic buildings in Brazil detected by culture and molecular techniques

There are few modern analyses of the cyanobacterial communities in biofilms on external building surfaces. As the classification of cyanobacteria is rapidly changing, we aimed to identify them on historic buildings in Brazil using both established and molecular techniques. In mature biofilms, cyanobacteria of subsections I and II were generally the major biomass; occasionally filamentous genera of the Scytonemataceae, Microchaetaceae and Rivularaceae were dominant. Filamentous organisms of subsections III and IV were more frequently isolated in culture. PCR products using cyanobacteria-specific 16S rDNA primers were sequenced from morphologically identified organisms. Homologies with deposited sequences were generally low. Phylogenetic analysis showed that many isolates were distant from their nearest neighbours, even though they grouped with their appropriate taxa. The majority of cyanobacterial DNA sequences deposited in data banks are aquatic; our results indicate that cyanobacteria from external walls are an ecologically isolated group.     

Linking Cascading Effects of Fish Predation and Zooplankton Grazing to Reduced Cyanobacterial Biomass and Toxin Levels Following Biomanipulation

Eutrophication has been one of the largest environmental problems in aquatic ecosystems during the past decades, leading to dense, and often toxic, cyanobacterial blooms. In a way to counteract these problems many lakes have been subject to restoration through biomanipulation. Here we combine 13 years of monitoring data with experimental assessment of grazing efficiency of a naturally occurring zooplankton community and a, from a human perspective, desired community of large Daphnia to assess the effects of an altered trophic cascade associated with biomanipulation. Lake monitoring data show that the relative proportion of Daphnia spp. grazers in June has increased following years of biomanipulation and that this increase coincides with a drop in cyanobacterial biomass and lowered microcystin concentrations compared to before the biomanipulation. In June, the proportion of Daphnia spp. (on a biomass basis) went from around 3% in 2005 (the first year of biomanipulation) up to around 58% in 2012. During months when the proportion of Daphnia spp. remained unchanged (July and August) no effect on lower trophic levels was observed. Our field grazing experiment revealed that Daphnia were more efficient in controlling the standing biomass of cyanobacteria, as grazing by the natural zooplankton community never even compensated for the algal growth during the experiment and sometimes even promoted cyanobacterial growth. Furthermore, although the total cyanobacterial toxin levels remained unaffected by both grazer communities in the experimental study, the Daphnia dominated community promoted the transfer of toxins to the extracellular, dissolved phase, likely through feeding on cyanobacteria. Our results show that biomanipulation by fish removal is a useful tool for lake management, leading to a top-down mediated trophic cascade, through alterations in the grazer community, to reduced cyanobacterial biomass and lowered cyanobacterial toxin levels. This improved water quality enhances both the ecological and societal value of lakes as units for ecosystem services.     

Using an online phycocyanin fluorescence probe for rapid monitoring of cyanobacteria in Macau freshwater reservoir

Monitoring of cyanobacteria and their toxins are traditionally conducted by cell counting, chlorophyll-a (chl-a) determination and cyanotoxin measurements, respectively. These methods are tedious, costly, time consuming, and insensitive to rapid changes in water quality and cyanobacterial abundance. We have applied and tested an online phycocyanin (PC) fluorescence probe for rapid monitoring of cyanobacteria in the Macau Storage Reservoir (MSR) that is experiencing cyanobacterial blooms. The relationships among cyanobacterial abundance, biovolume, cylindrospermopsin concentration, and PC fluorescence were analyzed using both laboratory and in-the-field studies. The performance of the probe was compared with traditional methods, and its advantages and limitations were assessed in pure and mixed cyanobacterial cultures in the laboratory. The proposed techniques successfully estimated the species including Microcystis and Cylindrospermopsis, two toxic species recently observed in the MSR. During February–November, 2010, the PC probe detected high correlations between PC and cell numbers (R ² = 0.71). Unlike the chl-a content, which indicates only the total algal biomass, the PC pigment specifically indicates cyanobacteria. These results support the PC parameter as a reliable estimate of cyanobacterial cell number, especially in freshwater bodies where the phytoplankton community and structure are stable. Thus, the PC probe is potentially applicable to online monitoring of cyanobacteria.     

Targeted deep sequencing reveals high diversity and variable dominance of bloom-forming cyanobacteria in eutrophic lakes

Cyanobacterial blooms in eutrophic lakes are severe environmental problems worldwide. To characterize the spatiotemporal heterogeneity of cyanobacterial blooms, a high-throughput method is necessary for the specific detection of cyanobacteria. In this study, the cyanobacterial composition of three eutrophic waters in China (Taihu Lake, Dongqian Lake, and Dongzhen Reservoir) was determined by pyrosequencing the cpcBA intergenic spacer (cpcBA-IGS) of cyanobacteria. A total of 2585 OTUs were obtained from the normalized cpcBA-IGS sequence dataset at a distance of 0.05. The 238 most abundant OTUs contained 92% of the total sequences and were classified into six cyanobacterial groups. The water samples of Taihu Lake were dominated by Microcystis, mixed Nostocales species, Synechococcus, and unclassified cyanobacteria. Besides, all the samples from Taihu Lake were clustered together in the dendrogram based on shared abundant OTUs. The cyanobacterial diversity in Dongqian Lake was dramatically decreased after sediment dredging and Synechococcus became exclusively dominant in this lake. The genus Synechococcus was also dominant in the surface water of Dongzhen Reservoir, while phylogenetically diverse cyanobacteria coexisted at a depth of 10 m in this reservoir. In summary, targeted deep sequencing based on cpcBA-IGS revealed a large diversity of bloom-forming cyanobacteria in eutrophic lakes and spatiotemporal changes in the composition of cyanobacterial communities. The genus Microcystis was the most abundant bloom-forming cyanobacteria in eutrophic lakes, while Synechococcus could be exclusively dominant under appropriate environmental conditions.     

CYANOBACTERIAL ASSEMBLAGES IN PERMANENT ICE COVERS ON ANTARCTIC LAKES: DISTRIBUTION, GROWTH RATE, AND TEMPERATURE RESPONSE OF PHOTOSYNTHESIS

The proliferation of microalgae in the McMurdo Dry Valleys of Antarctica is intricately linked to the seasonal cycle involving the freezing and melting of water. Anecdotal observations and preliminary sampling have found cyanobacterial cells in ice covers on lakes in the McMurdo Dry Valleys, and several of these ice covers are known to undergo seasonal freeze–thaw cycles. Therefore, we sought to determine the distribution and abundance of cyanobacterial assemblages in several permanent ice covers throughout the McMurdo Dry Valleys and to determine their rates of growth and their photosynthetic physiologies upon encountering liquid water. We found that the majority of the permanent ice covers contained cyanobacterial assemblages in close association with sedimentary material. Cyanobacterial biomass was conspicuously absent in sediment-free ice covers, suggesting that the seasonal interaction between the sediments, ice, and solar radiation present the necessary liquid water environment for cyanobacterial growth. All assemblages exhibited extremely low rates of photosynthesis when first exposed to liquid water. Despite the low rates of photosynthesis, a large proportion (41%) of the photosynthate was incorporated into protein, indicating that the cells were undergoing efficient net cellular growth. The short-term response (24 h) of photosynthesis to a range of temperatures showed optimum rates occurring at temperatures >15° C, which is similar to those of psychrotrophic cyanobacteria isolates from soil and stream habitats, which we believe provides the inoculum for the in- ice habitats.     

Eutrophication mediates rapid clonal evolution in Daphnia pulicaria

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Diversity of the cyanobacterial microflora of the northern part of James Ross Island,NW Weddell Sea,Antarctica

The diversity and ecological distribution of cyanobacteria in the northern, deglaciated part of James Ross Island were studied during the Antarctic summer season 2005–2006. Seventy-five cyanobacterial morphotypes were observed in various habitats of this area. The identified cyanobacterial taxa belong to the characteristic and dominant types of coastal Antarctica, and majority of them appeared connected to special habitats and formed distinct populations and ecologically delimited communities. The results are compared and discussed with respect to phenotypically characterised cyanobacterial microflora of maritime Antarctica and to recent molecular analyses of cyanobacterial strains from different Antarctic regions. The existence of a specificity and characteristic composition of Antarctic cyanobacterial communities was demonstrated.     

16S rDNA analyses of the cyanobacterial microbiota through the water-column in a boreal lake with a metalimnic Planktothrix population

The Planktothrix population in Lake Steinsfjord has attracted particular attention, due to the potential development of toxic blooms. This population is special in the sense that mass developments of Planktothrix occur in the metalimnion. We investigated the distribution of Planktothrix, as well as other cyanobacteria, through the water-column during a Planktothrix mass development at 10-16 m depth. The analyses were done by chlorophyll measurements, microscopy, and by a recently developed 16S rDNA array-based method.These analyses showed that Planktothrix dominated the cyanobacterial community at 11 m, while cyanobacteria belonging to the order Nostocales were predominant at 4 m. The combination of analytical methods presented in this work provides a powerful tool to analyze cyanobacterial communities. We have developed a concept that enables both relative (16S rDNA array analyses) and absolute quantification (chlorophyll a measurements) of cyanobacteria through water-columns. Such approaches will be important in better understanding cyanobacterial microbiota and bloom dynamics.     

Community-level assessment of the effects of the broad-spectrum antimicrobial chlorhexidine on the outcome of river microbial biofilm development

Chlorhexidine is a common-use antibacterial agent found in a range of personal-care products. We used rotating annular reactors to cultivate river biofilms under the influence of chlorhexidine or its molar equivalent in nutrients. Studies of the degradation of [(14)C]chlorhexidine demonstrated that no mineralization of the compound occurred. During studies with 100 microg liter(-1) chlorhexidine, significant changes were observed in the protozoan and micrometazoan populations, the algal and cyanobacterial biomass, the bacterial biomass, and carbon utilization. Denaturing gradient gel electrophoresis (DGGE) in combination with statistical analyses showed that the communities developing under control and 100 microg liter(-1) chlorhexidine were significantly different. At 10 microg liter(-1) chlorhexidine, there was significantly increased algal and cyanobacterial biomass while the bacterial biomass was not significantly affected (P < 0.05). No significant effects on protozoan or metazoan grazing were detected at the 10-microg liter(-1) chlorhexidine level. Fluorescent in situ hybridization indicated a significant reduction in the abundance of betaproteobacteria and gammaproteobacteria (P < 0.05). Archaeal cell counts were significantly reduced by both chlorhexidine and nutrient treatments. DGGE and statistical analyses indicated that 10 microg liter(-1) chlorhexidine and molar equivalent nutrient treatments were significantly different from control communities. In contrast to community level observations, toxicological testing with a panel of cyanobacteria, algae, and protozoa indicated no detectable effects at 10, 50, and 100 microg liter(-1) chlorhexidine. Thus, community level assessment indicated a risk of low levels of chlorhexidine in aquatic habitats while conventional approaches did not.     

Modelling lake cyanobacterial blooms: Disentangling the climate‐driven impacts of changing mixed depth and water temperature

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Identification of a diagnostic marker to detect freshwater cyanophages of filamentous cyanobacteria

Cyanophages are viruses that infect the cyanobacteria, globally important photosynthetic microorganisms. Cyanophages are considered significant components of microbial communities, playing major roles in influencing host community diversity and primary productivity, terminating cyanobacterial water blooms, and influencing biogeochemical cycles. Cyanophages are ubiquitous in both marine and freshwater systems; however, the majority of molecular research has been biased toward the study of marine cyanophages. In this study, a diagnostic probe was developed to detect freshwater cyanophages in natural waters. Oligonucleotide PCR-based primers were designed to specifically amplify the major capsid protein gene from previously characterized freshwater cyanomyoviruses that are infectious to the filamentous, nitrogen-fixing cyanobacterial genera Anabaena and Nostoc. The primers were also successful in yielding PCR products from mixed virus communities concentrated from water samples collected from freshwater lakes in the United Kingdom. The probes are thought to provide a useful tool for the investigation of cyanophage diversity in freshwater environments.     

Climate change amplifies the risk of potentially toxigenic cyanobacteria

Cyanobacterial blooms pose a significant threat to water security, with anthropogenic forcing being implicated as a key driver behind the recent upsurge and global expansion of cyanobacteria in modern times. The potential effects of land-use alterations and climate change can lead to complicated, less-predictable scenarios in cyanobacterial management, especially when forecasting cyanobacterial toxin risks. There is a growing need for further investigations into the specific stressors that stimulate cyanobacterial toxins, as well as resolving the uncertainty surrounding the historical or contemporary nature of cyanobacterial-associated risks. To address this gap, we employed a paleolimnological approach to reconstruct cyanobacterial abundance and microcystin-producing potential in temperate lakes situated along a human impact gradient. We identified breakpoints (i.e., points of abrupt change) in these time series and examined the impact of landscape and climatic properties on their occurrence. Our findings indicate that lakes subject to greater human influence exhibited an earlier onset of cyanobacterial biomass by 40 years compared to less-impacted lakes, with land-use change emerging as the dominant predictor. Moreover, microcystin-producing potential increased in both high- and low-impact lakes around the 1980s, with climate warming being the primary driver. Our findings chronicle the importance of climate change in increasing the risk of toxigenic cyanobacteria in freshwater resources.     

Distribution and diversity of natural product genes in marine and freshwater cyanobacterial cultures and genomes

Natural products are a functionally diverse class of biochemically synthesized compounds, which include antibiotics, toxins, and siderophores. In this paper, we describe both the detection of natural product activities and the sequence identification of gene fragments from two molecular systems that have previously been implicated in natural product production, i.e., nonribosomal peptide synthetases (NRPSs) and modular polyketide synthases (PKSs), in diverse marine and freshwater cyanobacterial cultures. Using degenerate PCR and the sequencing of cloned products, we show that NRPSs and PKSs are common among the cyanobacteria tested. Our molecular data, when combined with genomic searches of finished and progressing cyanobacterial genomes, demonstrate that not all cyanobacteria contain NRPS and PKS genes and that the filamentous and heterocystous cyanobacteria are the richest sources of these genes and the most likely sources of novel natural products within the phylum. In addition to validating the use of degenerate primers for the identification of PKS and NRPS genes in cyanobacteria, this study also defines numerous gene fragments that will be useful as probes for future studies of the synthesis of natural products in cyanobacteria. Phylogenetic analyses of the cyanobacterial NRPS and PKS fragments sequenced in this study, as well as those from the cyanobacterial genome projects, demonstrate that there is remarkable diversity and likely novelty of these genes within the cyanobacteria. These results underscore the potential variety of novel products being produced by these ubiquitous organisms.