Relating target fish DNA concentration to community composition analysis in freshwater fish via metabarcoding.

Metabarcoding has been widely accepted as a useful tool for biodiversity assessment based on eDNA. The method allows for the detection of entire groups of organisms in a single sample, making it particularly applicable in aquatic habitats. The high sensitivity of the molecular approaches is especially beneficial in detecting elusive and rare fish species, improving biodiversity assessments. Numerous biotic and abiotic factors that affect the persistence and availability of fish DNA in surface waters and therefore affecting species detectability, have been identified. However, little is known about the relationship between the total fish DNA concentration and the detectability of differential abundant species. In this study three controlled mock-community DNA samples (56 individual samples) were analyzed by (i) metabarcoding (MiSeq) of 12S rDNA (175 bp) and by (ii) total freshwater fish DNA quantification (via qPCR of 12S rDNA). We show that the fish DNA quantity affects the relative abundance of species-specific sequences and the detectability of rare species. In particular we found that samples with a concentration between 1000 pg/µL down to 10 pg/µL of total fish DNA revealed a stable relative frequency of DNA sequences obtained for a specific fish species, as well as a low variability between replicates. Additionally, we observed that even in complex mock-community DNA samples, a total fish DNA concentration of 23 pg/µL was sufficient to reliably detect all species in every replicate, including three rare species with proportions of ≤0.5 %. We also found that the DNA barcode similarity between species can affect detectability, if evenness is low. Our data suggest that the total DNA concentration of fish is an important factor to consider when analyzing and interpreting relative sequence abundance data. Therefore, the workflow proposed here will contribute to an ecologically and economically efficient application of metabarcoding in fish biodiversity assessment.

High Structural Diversity of Aeruginosins in Bloom-Forming Cyanobacteria of the Genus Planktothrix as a Consequence of Multiple Recombination Events.

Many compounds produced by cyanobacteria act as serine protease inhibitors, such as the tetrapeptides aeruginosins (Aer), which are found widely distributed. The structural diversity of Aer is intriguingly high. However, the genetic basis of this remains elusive. In this study, we explored the genetic basis of Aer synthesis among the filamentous cyanobacteria Planktothrix spp. In total, 124 strains, isolated from diverse freshwater waterbodies, have been compared regarding variability within Aer biosynthesis genes and the consequences for structural diversity. The high structural variability could be explained by various recombination processes affecting Aer synthesis, above all, the acquisition of accessory enzymes involved in post synthesis modification of the Aer peptide (e.g., halogenases, glycosyltransferases, sulfotransferases) as well as a large-range recombination of Aer biosynthesis genes, probably transferred from the bloom-forming cyanobacterium Microcystis. The Aer structural composition differed between evolutionary Planktothrix lineages, adapted to either shallow or deep waterbodies of the temperate climatic zone. Thus, for the first time among bloom-forming cyanobacteria, chemical diversification of a peptide family related to eco-evolutionary diversification has been described. It is concluded that various Aer peptides resulting from the recombination event act in chemical defense, possibly as a replacement for microcystins.

Quantitative relationships among high-throughput sequencing, cyanobacteria toxigenic genotype abundance and microcystin occurrence in bathing waters.

Toxin-producing cyanobacteria pose significant threats to human and animal health if exposed during recreational activities in bathing waters. To better safeguard public health and reduce health risks during the bathing season, an effective monitoring and management strategy is required. Molecular tools used to monitor toxigenic cyanobacteria have been evaluated on the basis of the efficiency and applicability of the method used to (i) establish an early-warning monitoring strategy for EU bathing water sites using both targeted quantitative polymerase chain reaction (qPCR) and non-targeted high-throughput sequencing (HTS) genotype analysis and (ii) to compare the toxigenic potential of cyanobacteria with actual microcystin (MC) occurrence and concentrations. For this purpose, 16 bathing water sites were monitored according to the bathing water directive (BWD) of the European Union (EU) during the bathing season of the summer of 2020 in eastern Austria. The cyanobacterial community composition was analyzed through HTS and qPCR by targeting the microcystin synthetase B gene (mcyB), which indicates MC synthesis within the genera Microcystis and Planktothrix. Within the genus Microcystis, which was identified as the primary MC producer, the mcyB genotypes formed stable subpopulations that increased linearly in correlation with the total Microcystis population. Notably, the HTS cell equivalents assigned to Microcystis and Planktothrix correlated with the corresponding qPCR estimates of genotype abundance, which serves as a confirmation of the suitability of (semi)-quantitative sequencing through HTS. In addition to the elevated trophic state, reduced transparency, increasing water temperatures, as well as cyanobacterial HTS read numbers and Microcystis cell number equivalents per mL estimated through qPCR, were associated with positive MC samples. Therefore, in combination with the monitoring of standard environmental parameters, the use of HTS and qPCR techniques is considered highly useful to ensure the timely identification of health risks to recreational users, as mandated by the BWD.

Characterization of Potential Threats from Cyanobacterial Toxins in Lake Victoria Embayments and during Water Treatment.

Africa's water needs are often supported by eutrophic water bodies dominated by cyanobacteria posing health threats to riparian populations from cyanotoxins, and Lake Victoria is no exception. In two embayments of the lake (Murchison Bay and Napoleon Gulf), cyanobacterial surveys were conducted to characterize the dynamics of cyanotoxins in lake water and water treatment plants. Forty-six cyanobacterial taxa were recorded, and out of these, fourteen were considered potentially toxigenic (i.e., from the genera Dolichospermum, Microcystis, Oscillatoria, Pseudanabaena and Raphidiopsis). A higher concentration (ranging from 5 to 10 µg MC-LR equiv. L−1) of microcystins (MC) was detected in Murchison Bay compared to Napoleon Gulf, with a declining gradient from the inshore (max. 15 µg MC-LR equiv. L−1) to the open lake. In Murchison Bay, an increase in Microcystis sp. biovolume and MC was observed over the last two decades. Despite high cell densities of toxigenic Microcystis and high MC concentrations, the water treatment plant in Murchison Bay efficiently removed the cyanobacterial biomass, intracellular and dissolved MC to below the lifetime guideline value for exposure via drinking water (<1.0 µg MC-LR equiv. L−1). Thus, the potential health threats stem from the consumption of untreated water and recreational activities along the shores of the lake embayments. MC concentrations were predicted from Microcystis cell numbers regulated by environmental factors, such as solar radiation, wind speed in the N−S direction and turbidity. Thus, an early warning through microscopical counting of Microcystis cell numbers is proposed to better manage health risks from toxigenic cyanobacteria in Lake Victoria.

Toxic/Bioactive Peptide Synthesis Genes Rearranged by Insertion Sequence Elements Among the Bloom-Forming Cyanobacteria Planktothrix.

It has been generally hypothesized that mobile elements can induce genomic rearrangements and influence the distribution and functionality of toxic/bioactive peptide synthesis pathways in microbes. In this study, we performed in depth genomic analysis by completing the genomes of 13 phylogenetically diverse strains of the bloom-forming freshwater cyanobacteria Planktothrix spp. to investigate the role of insertion sequence (IS) elements in seven pathways. Chromosome size varied from 4.7-4.8 Mbp (phylogenetic Lineage 1 of P. agardhii/P. rubescens thriving in shallow waterbodies) to 5.4-5.6 Mbp (Lineage 2 of P. agardhii/P. rubescens thriving in deeper physically stratified lakes and reservoirs) and 6.3-6.6 Mbp (Lineage 3, P. pseudagardhii/P. tepida including planktic and benthic ecotypes). Although the variation in chromosome size was positively related to the proportion of IS elements (1.1-3.7% on chromosome), quantitatively, IS elements and other paralogs only had a minor share in chromosome size variation. Thus, the major part of genomic variation must have resulted from gene loss processes (ancestor of Lineages 1 and 2) and horizontal gene transfer (HGT). Six of seven peptide synthesis gene clusters were found located on the chromosome and occurred already in the ancestor of P. agardhii/P. rubescens, and became partly lost during evolution of Lineage 1. In general, no increased IS element frequency in the vicinity of peptide synthesis gene clusters was observed. We found a higher proportion of IS elements in ten breaking regions related to chromosomal rearrangements and a tendency for colocalization of toxic/bioactive peptide synthesis gene clusters on the chromosome.

Correction: Morón-Asensio et al. Differential Labeling of Chemically Modified Peptides and Lipids among Cyanobacteria Planktothrix and Microcystis. Microorganisms 2021, 9, 1578.

The authors wish to make the following corrections to this paper [...].

DNA sequence and taxonomic gap analyses to quantify the coverage of aquatic cyanobacteria and eukaryotic microalgae in reference databases: Results of a survey in the Alpine region.

The taxonomic identification of organisms based on the amplification of specific genetic markers (metabarcoding) implicitly requires adequate discriminatory information and taxonomic coverage of environmental DNA sequences in taxonomic databases. These requirements were quantitatively examined by comparing the determination of cyanobacteria and microalgae obtained by metabarcoding and light microscopy. We used planktic and biofilm samples collected in 37 lakes and 22 rivers across the Alpine region. We focused on two of the most used and best represented genetic markers in the reference databases, namely the 16S rRNA and 18S rRNA genes. A sequence gap analysis using blastn showed that, in the identity range of 99-100%, approximately 30% (plankton) and 60% (biofilm) of the sequences did not find any close counterpart in the reference databases (NCBI GenBank). Similarly, a taxonomic gap analysis showed that approximately 50% of the cyanobacterial and eukaryotic microalgal species identified by light microscopy were not represented in the reference databases. In both cases, the magnitude of the gaps differed between the major taxonomic groups. Even considering the species determined under the microscope and represented in the reference databases, 22% and 26% were still not included in the results obtained by the blastn at percentage levels of identity ≥95% and ≥97%, respectively. The main causes were the absence of matching sequences due to amplification and/or sequencing failure and potential misidentification in the microscopy step. Our results quantitatively demonstrated that in metabarcoding the main obstacles in the classification of 16S rRNA and 18S rRNA sequences and interpretation of high-throughput sequencing biomonitoring data were due to the existence of important gaps in the taxonomic completeness of the reference databases and the short length of reads. The study focused on the Alpine region, but the extent of the gaps could be much greater in other less investigated geographic areas.

Differential Labeling of Chemically Modified Peptides and Lipids among Cyanobacteria Planktothrix and Microcystis.

The cyanoHAB forming cyanobacteria Microcystis and Planktothrix frequently produce high intracellular amounts of microcystins (MCs) or anabaenopeptins (APs). In this study, chemically modified MCs and APs have been localized on a subcellular level in Microcystis and Planktothrix applying copper-catalyzed alkyne-azide cycloaddition (CuACC). For this purpose, three different non-natural amino acids carrying alkyne or azide moieties were fed to individual P. agardhii strains No371/1 and CYA126/8 as well as to M. aeruginosa strain Hofbauer showing promiscuous incorporation of various amino acid substrates during non-ribosomal peptide synthesis (NRPS). Moreover, CYA126/8 peptide knock-out mutants and non-toxic strain Synechocystis PCC6803 were processed under identical conditions. Simultaneous labeling of modified peptides with ALEXA405 and ALEXA488 and lipid staining with BODIPY 505/515 were performed to investigate the intracellular location of the modified peptides. Pearson correlation coefficients (PCC) obtained from confocal images were calculated between the different fluorophores and the natural autofluorescence (AF), and between labeled modified peptides and dyed lipids to investigate the spatial overlap between peptides and the photosynthetic complex, and between peptides and lipids. Overall, labeling of modified MCs (M. aeruginosa) and APs (P. agardhii) using both fluorophores revealed increased intensity in MC/AP producing strains. For Synechocystis lacking NRPS, no labeling using either ALEXA405 or ALEXA488 was observed. Lipid staining in M. aeruginosa and Synechocystis was intense while in Planktothrix it was more variable. When compared with AF, both modified peptides and lipids showed a heterologous distribution. In comparison, the correlation between stained lipids and labeled peptides was not increased suggesting a reduced spatial overlap.

Contrasting endolithic habitats for cyanobacteria in spring calcites of the European Alps.

It is often difficult to decide which cyanobacteria found in endolithic habitats of calcite spring-tufa deposits are present as ephemeral components of the biota or are persistent, structural elements. To answer this question, we repeatedly studied two microhabitats of contrasting calcareous tufa springs in the European Alps. Pigment extracts, fluorescence probe measurements of in situ samples and traditional microscopy confirmed the dominance of cyanobacteria over eukaryotic algae and their viability in both microhabitats. Spring Site 1 (Laas, Northern Italy) is characterized by a highly variable, moist to dry and sun-exposed waterfall tufa consisting of fibrous calcite. A segment of these deposits in the lateral flank of a grotto contained dark endolithic layers in dim light, 1-2 mm below the surface, where aggregated cyanobacterial cells were dominant but not directly attached to calcites, a potential sign of gentle endolithic dissolution rather than calcite precipitation induced by cyanobacteria. Site 2 (Mühlau, Austria), in contrast, is a moss-tufa microhabitat associated with a seepage spring situated in a shady gorge, where the targeted stromatolites consisted of bark-like sheets of friable, orange to light-brown when wet (drying violet) 'styrofoam'- like aggregates of minute crystallites on the day-light exposed surfaces. These calcites were observed to nucleate directly on external sheaths of viable cyanobacteria trichomes. A polyphasic approach including LM, SEM, TEM exhibited a number of identical but also some divergent cyanobacteria of which two key taxa were specific for each of the two microhabitats (Nostoc and Pseudoscytonema at Sites 1 and 2 respectively). Both cyanobacterial communities characterised, by the cloning of 16S rDNA showed a dominance of mostly unknown and partly divergent filamentous cyanobacteria assigned to the order of Synechococcales. Our microhabitat study of alpine crenal calcites highlights the rather divergent biotic responses of cyanobacteria within spring tufa deposits.

Toward Disentangling the Multiple Nutritional Constraints Imposed by Planktothrix: The Significance of Harmful Secondary Metabolites and Sterol Limitation.

The harmful bloom-forming cyanobacterium Planktothrix is commonly considered to be nutritionally inadequate for zooplankton grazers, resulting in limited top-down control. However, interactions between Planktothrix and zooplankton grazers are poorly understood. The food quality of Planktothrix is potentially constrained by morphological properties (i.e., filament formation), the production of harmful secondary metabolites, and a deficiency in essential lipids (i.e., primarily sterols). Here, we investigated the relative significance of toxin production (microcystins, carboxypeptidase A inhibitors, protease inhibitors) and sterol limitation for the performance of Daphnia feeding on one Planktothrix rubescens and one P. agardhii wild-type/microcystin knock-out mutant pair. Our data suggest that the poor food quality of both Planktothrix spp. is due to deleterious effects mediated by various harmful secondary metabolites and that the impact of sterol limitation is partially or completely superimposed by toxicity. The significance of the different factors seems to depend on the metabolite profile of the considered Planktothrix strain and the Daphnia clone that is used for the experiments. The toxin-responsive gene expression (transporter genes, gpx, and trypsin) and enzyme activity patterns revealed strain-specific food quality constraints and that Daphnia is capable of modulating its physiological responses according to the ingested Planktothrix strain. Future studies need to consider that Planktothrix-grazer interactions are simultaneously modulated by multiple factors to improve our understanding of top-down influences on Planktothrix bloom formation.

A rigorous assessment and comparison of enumeration methods for environmental viruses.

Determining exact viral titers in a given sample is essential for many environmental and clinical applications, e.g., for studying viral ecology or application of bacteriophages for food safety. However, virus quantification is not a simple task, especially for complex environmental samples. While clonal viral isolates can be quantified with relative high accuracy using virus-specific methods, i.e., plaque assay or quantitative real-time PCR, these methods are not valid for complex and diverse environmental samples. Moreover, it is not yet known how precisely laser-based methods, i.e., epifluorescence microscopy, flow cytometry, and nanoparticle tracking analysis, quantify environmental viruses. In the present study, we compared five state-of-the-art viral quantification methods by enumerating four model viral isolates of different genome and size characteristics as well as four different environmental water samples. Although Nanoparticle tracking analysis combined with gentle staining at 30 °C could be confirmed by this study to be a reliable quantification technique for tested environmental samples, environmental samples still lack an universally applicable and accurate quantification method. Special attention has to be put on optimal sample concentrations as well as optimized sample preparations, which are specific for each method. As our results show the inefficiency when enumerating small, or single-stranded DNA or RNA viruses, the global population of viruses is presumably higher than expected.

Chemically labeled toxins or bioactive peptides show a heterogeneous intracellular distribution and low spatial overlap with autofluorescence in bloom-forming cyanobacteria.

Harmful algal blooms formed by colony-forming cyanobacteria deteriorate water resources by producing cyanotoxins, which frequently occur at high intracellular concentrations. We aimed to localize toxic microcystins (MCs) and bioactive anabaenopeptins (APs) at the subcellular level under noninvasive conditions. Since both metabolites are synthesized nonribosomally, the relaxed specificity of key enzymes catalyzing substrate activation allowed chemical labeling through a standard copper-catalyzed click chemistry reaction. The genera Planktothrix and Microcystis specifically incorporated unnatural amino acids such as N-propargyloxy-carbonyl-L-lysine or O-propargyl-L-tyrosine, resulting in modified AP or MC peptides carrying the incorporated alkyne moiety. The labeled cells were quantitatively differentiated from the unlabeled control cells. MCs and APs occurred intracellularly as distinct entities showing a cell-wide distribution but a lowered spatial overlap with natural autofluorescence. Using the immunofluorescence technique, colocalization with markers of individual organelles was utilized to relate the distribution of labeled MCs to cellular compartments, e.g., using RbcL and FtsZ (cytosol) and PsbA (thylakoids). The colocalization correlation coefficients calculated pairwise between organelles and autofluorescence were highly positive as opposed to the relatively low positive indices derived from labeled MCs. The lower correlation coefficients imply that only a portion of the labeled MC molecules were related spatially to the organelles in the cell.

Temperature Response of Planktonic Microbiota in Remote Alpine Lakes.

Alpine lakes are considered pristine freshwater ecosystems and sensitive to direct and indirect changes in water temperature as induced by climate change. The bacterial plankton constitutes a key component in the water column and bacterial metabolic activity has direct consequences for water quality. In order to understand bacterial response to global temperature rise in five alpine lakes located in the Austrian Alps (1700-2188 m a.S.L.) water temperature was compared within a decadal period. Depth-integrated samples were characterized in community composition by 16S rDNA deep-amplicon sequencing early [56 ± 16 (SD) days after ice break up] and later (88 ± 16 days) in the growing season. Within the 10 years period, temperature rise was observed through reduced ice cover duration and increased average water temperature. During the early growing season, the average water temperature recorded between circulation in spring until sampling date (WAS), and the day of autumn circulation, as well as chemical composition including dissolved organic carbon influenced bacterial community composition. In contrast, only nutrients (such as nitrate) were found influential later in the growing season. Metabolic theory of ecology (MTE) was applied to explain the dependence of taxonomic richness on WAS in mathematical terms. The calculated activation energy exceeded the frequently reported prediction emphasizing the role of WAS during early growing season. Accordingly, the relative abundance of predicted metabolism related genes increased with WAS. Thus, the dominant influence of temperature after ice break up could be explained by overall climate change effects, such as a more intense warming in spring and an overall higher amplitude of temperature variation.

Benthic Diatom Communities in an Alpine River Impacted by Waste Water Treatment Effluents as Revealed Using DNA Metabarcoding.

Freshwater ecosystems are continuously affected by anthropogenic pressure. One of the main sources of contamination comes from wastewater treatment plant (WWTP) effluents that contain wide range of micro- and macropollutants. Chemical composition, toxicity levels and impact of treated effluents (TEs) on the recipient aquatic ecosystems may strongly differ depending on the wastewater origin. Compared to urban TEs, hospital ones may contain more active pharmaceutical substances. Benthic diatoms are relevant ecological indicators because of their high species and ecological diversity and rapid response to human pressure. They are routinely used for water quality monitoring. However, there is a knowledge gap on diatom communities' development and behavior in treated wastewater in relation to prevailing micro- and macropollutants. In this study, we aim to (1) investigate the response of diatom communities to urban and hospital TEs, and (2) evaluate TEs effect on communities in the recipient river. Environmental biofilms were colonized in TEs and the recipient river up- and downstream from the WWTP output to study benthic diatoms using DNA metabarcoding combined with high-throughput sequencing (HTS). In parallel, concentrations of nutrients, pharmaceuticals and seasonal conditions were recorded. Diatom metabarcoding showed that benthic communities differed strongly in their diversity and structure depending on the habitat. TE sites were generally dominated by few genera with polysaprobic preferences belonging to the motile guild, while river sites favored diverse communities from oligotrophic and oligosaprobic groups. Seasonal changes were visible to lower extent. To categorize parameters important for diatom changes we performed redundancy analysis which suggested that communities within TE sites were associated to higher concentrations of beta-blockers and non-steroidal anti-inflammatory drugs in urban effluents vs. antibiotics and orthophosphate in hospital effluents. Furthermore, indicator species analysis showed that 27% of OTUs detected in river downstream communities were indicator for urban or hospital TE sites and were absent in the river upstream. Finally, biological diatom index (BDI) calculated to evaluate the ecological status of the recipient river suggested water quality decrease linked to the release of TEs. Thus, in-depth assessment of diatom community composition using DNA metabarcoding is proposed as a promising technique to highlight the disturbing effect of pollutants in Alpine rivers.

Microcystin Content in Phytoplankton and in Small Fish from Eutrophic Nyanza Gulf, Lake Victoria, Kenya.

The human health risks posed by exposure to cyanobacterial toxins such as microcystin (MC) through water and fish consumption remain poorly described. During the last two decades, coastal regions of Lake Victoria such as Nyanza Gulf (Kisumu Bay) have shown severe signs of eutrophication with blooms formed by Microcystis producing MC. In this study, the spatial variability in MC concentration in Kisumu Bay was investigated which was mostly caused by Microcystis buoyancy and wind drifting. Small fish (<6 cm) mainly composed of Rastrineobola argentea were examined for MC content by means of biological methods such as ELISA and protein phosphatase inhibition assay (PPIA) and partly by chemical-analytical methods such as LC-MS/MS. Overall, the MC content in small fish was related to the MC content observed in the seston. When comparing the MC content in the seston in relation to dry weight with the MC content in small fish the latter was found three orders of magnitude decreased. On average, the ELISA-determined MC contents exceeded the PPIA-determined MC contents by a factor of 8.2 ± 0.5 (SE) while the MC contents as determined by LC-MS/MS were close to the detection limit. Using PPIA, the MC content varied from 25⁻109 (mean 62 ± 7) ng/g fish dry weight in Kisumu Bay vs. 14 ± 0.8 ng MC/g in the more open water of L. Victoria at Rusinga channel. Drying the fish under the sun showed little effect on MC content, although increased humidity might indirectly favor photocatalyzed MC degradation.

Single colony genetic analysis of epilithic stream algae of the genus Chamaesiphon spp.

In order to understand Chamaesiphon spp. evolution and ecological diversification, we investigated the phylogenetic differentiation of three morphospecies from field samples by means of single colony genetics. Individual colonies of three different morphospecies (C. starmachii, C. polonicus, C. geitleri,) were isolated from lotic gravel streams and their 16S rDNA nucleotide variability was analyzed. For a number of individual colonies, microscopical and ultrastructural analysis was also performed. A phylogenetic tree of all major lineages of the phylum of Cyanobacteria assigned all Chamaesiphon genotypes (1149-1176 bp) most closely with the family of Gomontiellaceae of the order Oscillatoriales. The sequences obtained from colonies assigned to C. starmachii (n = 21), C. polonicus (n = 9), and C. geitleri (n = 17) were found to reveal high average (3.5%) nucleotide diversity. No phylogenetic sub-branching in correspondence with morphology was observed suggesting that the three Chamaesiphon morphospecies did not represent monophyletic taxa. We could not attribute specific thylakoid ultrastructure to phylogenetic sub-branches; however, the observed parietally and loosely arranged thylakoids indicate that for the genus Chamaesiphon, the variability in thylakoid ultrastructure might have been underestimated. In summary, the high nucleotide diversity of the 16S rDNA gene implies phylogenetic diversity that corresponds little to morphological classification.

Evolution of Anabaenopeptin Peptide Structural Variability in the Cyanobacterium Planktothrix.

Cyanobacteria are frequently involved in the formation of harmful algal blooms wherein, apart from the toxic microcystins, other groups of bioactive peptides are abundant as well, such as anabaenopeptins (APs). The APs are synthesized nonribosomally as cyclic hexapeptides with various amino acids at the exocyclic position. We investigated the presence and recombination of the AP synthesis gene cluster (apnA-E) through comparing 125 strains of the bloom-forming cyanobacterium Planktothrix spp., which were isolated from numerous shallow and deep water habitats in the temperate and tropical climatic zone. Ten ecologically divergent strains were purified and genome sequenced to compare their entire apnA-E gene cluster. In order to quantify apn gene distribution patterns, all the strains were investigated by PCR amplification of 2 kbp portions of the entire apn gene cluster without interruption. Within the 11 strains assigned to P. pseudagardhii, P. mougeotii, or P. tepida (Lineage 3), neither apnA-E genes nor remnants were observed. Within the P. agardhii/P. rubescens strains from shallow waters (Lineage 1, 52 strains), strains both carrying and lacking apn genes occurred, while among the strains lacking the apnA-E genes, the presence of the 5'end flanking region indicated a gene cluster deletion. Among the strains of the more derived deep water ecotype (Lineage 2, 62 strains), apnA-E genes were always present. A high similarity of apn genes of the genus Planktothrix when compared with strains of the genus Microcystis suggested its horizontal gene transfer during the speciation of P. agardhii/P. rubescens. Genetic analysis of the first (A1-) domain of the apnA gene, encoding synthesis of the exocyclic position of the AP molecule, revealed four genotype groups that corresponded with substrate activation. Groups of genotypes were either related to Arginine only, the coproduction of Arginine and Tyrosine or Arginine and Lysine, or even the coproduction of Arginine, Tyrosine, and Lysine in the exocyclic position of the AP-molecule. The increased structural diversity resulted from the evolution of apnA A1 genotypes through a small number of positively selected point mutations that occurred repeatedly and independently from phylogenetic association.

Role of toxic and bioactive secondary metabolites in colonization and bloom formation by filamentous cyanobacteria Planktothrix.

Bloom-forming cyanobacteria Planktothrix agardhii and P. rubescens are regularly involved in the occurrence of cyanotoxin in lakes and reservoirs. Besides microcystins (MCs), which inhibit eukaryotic protein phosphatase 1 and 2A, several families of bioactive peptides are produced, thereby resulting in impressive secondary metabolite structural diversity. This review will focus on the current knowledge of the phylogeny, morphology, and ecophysiological adaptations of Planktothrix as well as the toxins and bioactive peptides produced. The relatively well studied ecophysiological adaptations (buoyancy, shade tolerance, nutrient storage capacity) can partly explain the invasiveness of this group of cyanobacteria that bloom within short periods (weeks to months). The more recent elucidation of the genetic basis of toxin and bioactive peptide synthesis paved the way for investigating its regulation both in the laboratory using cell cultures as well as under field conditions. The high frequency of several toxin and bioactive peptide synthesis genes observed within P. agardhii and P. rubescens, but not for other Planktothrix species (e.g. P. pseudagardhii), suggests a potential functional linkage between bioactive peptide production and the colonization potential and possible dominance in habitats. It is hypothesized that, through toxin and bioactive peptide production, Planktothrix act as a niche constructor at the ecosystem scale, possibly resulting in an even higher ability to monopolize resources, positive feedback loops, and resilience under stable environmental conditions. Thus, refocusing harmful algal bloom management by integrating ecological and phylogenetic factors acting on toxin and bioactive peptide synthesis gene distribution and concentrations could increase the predictability of the risks originating from Planktothrix blooms.

Emergence of nontoxic mutants as revealed by single filament analysis in bloom-forming cyanobacteria of the genus Planktothrix.

BACKGROUND: Bloom-forming cyanobacteria cause toxic algae outbreaks in lakes and reservoirs. We aimed to explore and quantify mutation events occurring within the large mcy gene cluster (55 kbp) encoding microcystin (MC) biosynthesis that inactivate MC net production. For this purpose we developed a workflow to detect mutations in situ occurring anywhere within the large mcy gene cluster as amplified from one single filament of the red-pigmented cyanobacterium Planktothrix rubescens. From five lakes of the Alps eight hundred Planktothrix filaments were isolated and each individual filament was analyzed for mutations affecting the mcy genes. RESULTS: Mutations inactivating MC synthesis were either through an insertion element ISPlr1 or the partial deletion of mcy genes. Neutral mutations not affecting MC biosynthesis occurred within two intergenic spacer regions, either through the insertion of a Holliday-junction resolvase RusA or ISPlr1. Altogether, the insertions affected a few mcy genes only and their location was correlated with regions similar to repetitive extragenic palindromic DNA sequences (REPs). Taking all of the filaments together, the mutations leading to the inactivation of MC synthesis were more rare (0.5-6.9%), when compared with the neutral mutations (7.5-20.6%). On a spatial-temporal scale the ratio of MC synthesis-inactivating vs. neutral mutations was variable, e.g., the filament abundance carrying partial deletion of mcyD (5.2-19.4%) and/or mcyHA (0-7.3%) exceeded the abundance of neutral mutations. CONCLUSIONS: It is concluded that insertion events occurring within the Planktothrix mcy gene cluster are predictable due to their correlation with REPs. The frequency of occurrence of the REPs within the mcy gene cluster of Planktothrix relates to the rather common mutation of mcy genes in Planktothrix. Spatial-temporal variable conditions may favor the emergence of partial mcy deletion mutants in Planktothrix, in particular a higher proportion of genotypes resulting in inactivation of MC synthesis might be caused by increased ISPlr1 activity.

Global solutions to regional problems: Collecting global expertise to address the problem of harmful cyanobacterial blooms. A Lake Erie case study.

In early August 2014, the municipality of Toledo, OH (USA) issued a 'do not drink' advisory on their water supply directly affecting over 400,000 residential customers and hundreds of businesses (Wilson, 2014). This order was attributable to levels of microcystin, a potent liver toxin, which rose to 2.5µgL-1 in finished drinking water. The Toledo crisis afforded an opportunity to bring together scientists from around the world to share ideas regarding factors that contribute to bloom formation and toxigenicity, bloom and toxin detection as well as prevention and remediation of bloom events. These discussions took place at an NSF- and NOAA-sponsored workshop at Bowling Green State University on April 13 and 14, 2015. In all, more than 100 attendees from six countries and 15 US states gathered together to share their perspectives. The purpose of this review is to present the consensus summary of these issues that emerged from discussions at the Workshop. As additional reports in this special issue provide detailed reviews on many major CHAB species, this paper focuses on the general themes common to all blooms, such as bloom detection, modeling, nutrient loading, and strategies to reduce nutrients.