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Abstract The analysis of sediment DNA has emerged as a promising alternative to classical paleolimnological proxies, which typically rely on hard fossils to reconstruct environmental change. In recent studies, the timing and causes of apparent increases in toxic cyanobacterial blooms in lakes around the world have been identified by high‐throughput sequencing of cyanobacterial 16S rRNA genes from decadal‐ and centennial‐scale sediment records. However, the reconstruction of ecological time series can be influenced by DNA degradation processes interfering with taxonomic resolution and quantification of target genes. To determine whether some cyanobacterial taxa degrade more rapidly than others, a one‐year incubation experiment was performed to estimate degradation rates in sediments under contrasting environmental conditions of temperature (4°C vs. 25°C) and sediment redox (oxic vs. anoxic). Using sediments collected from an oligotrophic lake, serum bottles were inoculated with either Microcystis aeruginosa, Synechococcus sp., or Trichormus variabilis. These cyanobacteria were chosen because they are widespread yet exhibit contrasting morphologies. The absolute concentrations of the target genes were measured over time through droplet digital PCR. The three taxa exhibited log‐linear declines with different rates of decay. Based on first‐order linear decay models, Microcystis exhibited faster decay under oxic conditions, whereas Synechococcus sp. and Trichormus were both temperature and anoxic dependent. Overall, cyanobacterial decay rates were lowest under 4°C anoxic conditions, corresponding to conditions in deep temperate lakes. Under 4°C, Synechococcus exhibited the lowest degradation rates followed by Trichormus, then Microcystis. These findings will be useful in the interpretation of population and community structure data based on sediment DNA and advances our current understanding of cyanobacterial preservation in sediment.

Over the past three decades, the Rideau River (Ontario, Canada) watershed has experienced a land-use change from rural and agricultural land towards urban land, and the introduction and expansion of invasive species. This study examined spatial and temporal patterns of periphyton biomass (chlorophyll-a and ash-free dry mass) collected from riffle zones in 1995 and 2019 along a 66-km stretch of the Rideau River. This study also examined long-term changes in water nutrient and chloride concentrations collected through the Provincial Water Quality Monitoring Network between 2000 and 2018. Declines in total nitrogen between 2000 and 2018 occurred in areas that have not experienced urbanization since the 2000s. In contrast, declines in total phosphorus and increases in chloride between 2000 and 2018 occurred at midstream and downstream sites that have undergone urbanization during the same time period. Conductivity also showed an increase with distance downstream similarly in both 1995 and 2019. Average total phosphorus and total Kjeldahl nitrogen concentrations were lower in 2019 compared to 1995 but did not increase with distance downstream as seen in many river systems, including the Rideau River, in earlier years. Periphyton biomass did not change along the length of the river between 1995 and 2019, despite the declines in nutrient concentrations. These findings highlight the persistence of riverine periphyton in a multi-use watershed experiencing ongoing anthropogenic changes.

Sediment DNA (sedDNA) analyses are rapidly emerging as powerful tools for the reconstruction of environmental and evolutionary change. While there are an increasing number of studies using molecular genetic approaches to track changes over time, few studies have compared the coherence between quantitative polymerase chain reaction (PCR) methods and metabarcoding techniques. Primer specificity, bioinformatic analyses, and PCR inhibitors in sediments could affect the quantitative data obtained from these approaches. We compared the performance of droplet digital polymerase chain reaction (ddPCR) and high-throughput sequencing (HTS) for the quantification of target genes of cyanobacteria in lake sediments and tested whether the two techniques similarly reveal expected patterns through time. Absolute concentrations of cyanobacterial 16S rRNA genes were compared between ddPCR and HTS using dated sediment cores collected from two experimental (Lake 227, fertilized since 1969 and Lake 223, acidified from 1976 to 1983) and two reference lakes (Lakes 224 and 442) in the Experimental Lakes Area (ELA), Canada. Relative abundances of Microcystis 16S rRNA (MICR) genes were also compared between the two methods. Moderate to strong positive correlations were found between the molecular approaches among all four cores but results from ddPCR were more consistent with the known history of lake manipulations. A 100-fold increase in ddPCR estimates of cyanobacterial gene abundance beginning in ~1968 occurred in Lake 227, in keeping with experimental addition of nutrients and increase in planktonic cyanobacteria. In contrast, no significant rise in cyanobacterial abundance associated with lake fertilization was observed with HTS. Relative abundances of Microcystis between the two techniques showed moderate to strong levels of coherence in top intervals of the sediment cores. Both ddPCR and HTS approaches are suitable for sedDNA analysis, but studies aiming to quantify absolute abundances from complex environments should consider using ddPCR due to its high tolerance to PCR inhibitors.

[D-Leu1]MC-LY (1) ([M + H]+ m/z 1044.5673, Δ 2.0 ppm), a new microcystin, was isolated from Microcystis aeruginosa strain CPCC-464. The compound was characterized by 1H and 13C NMR spectroscopy, liquid chromatography−high resolution tandem mass spectrometry (LC−HRMS/MS) and UV spectroscopy. A calibration reference material was produced after quantitation by 1H NMR spectroscopy and LC with chemiluminescence nitrogen detection. The potency of 1 in a protein phosphatase 2A inhibition assay was essentially the same as for MC-LR (2). Related microcystins, [D-Leu1]MC-LR (3) ([M + H]+ m/z 1037.6041, Δ 1.0 ppm), [D-Leu1]MC-M(O)R (6) ([M + H]+ m/z 1071.5565, Δ 2.0 ppm) and [D-Leu1]MC-MR (7) ([M + H]+ m/z 1055.5617, Δ 2.2 ppm), were also identified in culture extracts, along with traces of [D-Leu1]MC-M(O2)R (8) ([M + H]+ m/z 1087.5510, Δ 1.6 ppm), by a combination of chemical derivatization and LC−HRMS/MS experiments. The relative abundances of 1, 3, 6, 7 and 8 in a freshly extracted culture in the positive ionization mode LC−HRMS were ca. 84, 100, 3.0, 11 and 0.05, respectively. These and other results indicate that [D-Leu1]-containing MCs may be more common in cyanobacterial blooms than is generally appreciated but are easily overlooked with standard targeted LC−MS/MS screening methods.

Cyanobacterial blooms increasingly impair inland waters, with the potential for a concurrent increase in cyanotoxins that have been linked to animal and human mortalities. Microcystins (MCs) are among the most commonly detected cyanotoxins, but little is known about the distribution of different MC congeners despite large differences in their biomagnification, persistence, and toxicity. Using raw-water intake data from sites around the Great Lakes basin, we applied multivariate canonical analyses and regression tree analyses to identify how different congeners (MC-LA, -LR, -RR, and -YR) varied with changes in meteorological and nutrient conditions over time (10 years) and space (longitude range: 77°2′60 to 94°29′23 W). We found that MC-LR was associated with strong winds, warm temperatures, and nutrient-rich conditions, whereas the equally toxic yet less commonly studied MC-LA tended to dominate under intermediate winds, wetter, and nutrient-poor conditions. A global synthesis of lake data in the peer-reviewed literature showed that the composition of MC congeners differs among regions, with MC-LA more commonly reported in North America than Europe. Global patterns of MC congeners tended to vary with lake nutrient conditions and lake morphometry. Ultimately, knowledge of the environmental factors leading to the formation of different MC congeners in freshwaters is necessary to assess the duration and degree of toxin exposure under future global change.

The suitability of pressurized liquid extraction (PLE) of cyanotoxins from cells was investigated. The stability of cyanotoxins (MCYST-RR, MCYST-LR, and anatoxin-a) was evaluated at nine combinations of pressure and temperature (7, 10, and 14 MPa and 60C, 80C and 100C) using 75% (v/v) methanol in water (MeOH) as solvent. Additional experiments investigated the stability of cyanotoxins when water was used as solvent (at a pressure of 14 MPa and a temperature of 40C, 50C, 60C, 80C, or 100C). Results using 75% MeOH showed that the MCYST-RR and MCYST-LR were stable under the tested pressures up to 80C. At 100C MCYST recovery decreased by 10% to 17%. When water was used as the solvent, no differences in recovery were observed for MCYST-LR, whereas for MCYST-RR, maximum recovery was obtained at 60C, and degradation occurred at 100C. In contrast, anatoxin-a was labile under all experimental conditions; the best recoveries (ca. 50%) were obtained at 60C at the three pressures using 75% MeOH. However, only 17%23% recovery was obtained with water extraction at all temperatures. The extraction of MCYST-LR and variants from cells (Microcystis aeruginosa, UTCC299) was studied using two solvents, 75% MeOH and 100% water, at 14 MPa and 60C and 100C. PLE extracts were compared with extracts obtained with 75% MeOH and ultrasonication. Complete extraction was achieved in both solvents in one 5-min cycle (at 100C). Although lower recovery was obtained using PLE (79%105%), shorter extraction time and automation are advantageous over ultrasonication. 2005 Wiley Periodicals, Inc. Environ Toxicol 20: 390396, 2005. [ABSTRACT FROM AUTHOR]

Cyanotoxins are a group of compounds produced by cyanobacteria that can have severe physiological effects on other organisms, including humans. The potential allelopathic effects of Microcystis aeruginosa, a toxic cyanobacterium, on the duckweed plant, Lemna gibba L., were examined using three experimental methods: (1) a series of toxicity bioassays, (2) evaluation of toxin production by M. aeruginosa in the direct and indirect presence of L. gibba, and (3) inhibition of oxygen evolution in photosynthesis. The results showed that, first, there were no clear dose-dependent effects of the microcystin-LR standard or the toxic M. aeruginosa culture filtrate on any of the end points measured in the toxicity bioassays (plant and frond number, dry weight, growth rate, chlorophyll content; one-way ANOVA, p > 0.05). In those cases in which an EC50 value could be obtained, chlorophyll a was the most sensitive end point, as it had the lowest EC50 value (14.47 g/L microcystin-LR) of all the end points. Second, the presence of L. gibba did not result in higher microcystin-LR production in the toxic M. aeruginosa culture. And, last, oxygen evolution was not affected in isolated chloroplasts exposed directly to microcystin-LR. Therefore, microcystins from the toxic cyanobacterium Microcystis aeruginosa do not appear to have an allelopathic effect on the common aquatic macrophyte Lemna gibba. 2005 Wiley Periodicals, Inc. Environ Toxicol 20: 6773, 2005. [ABSTRACT FROM AUTHOR]

The eutrophication of freshwaters is a global health concern as lakes with excess nutrients are often subject to toxic cyanobacterial blooms. Although phosphorus is considered the main element regulating cyanobacterial biomass, nitrogen (N) concentration and more specifically the availability of different N forms may influence the overall toxicity of blooms. In this study of three eutrophic lakes prone to cyanobacterial blooms, we examined the effects of nitrogen species and concentrations and other environmental factors in influencing cyanobacterial community structure, microcystin (MC) concentrations and MC congener composition. The identification of specific MC congeners was of particular interest as they vary widely in toxicity. Different nitrogen forms appeared to influence cyanobacterial community structure leading to corresponding effects on MC concentrations and composition. Total MC concentrations across the lakes were largely explained by a combination of abiotic factors: dissolved organic nitrogen, water temperature and ammonium, but Microcystis spp. biomass was overall the best predictor of MC concentrations. Environmental factors did not appear to affect MC congener composition directly but there were significant associations between specific MC congeners and particular species. Based on redundancy analyses (RDA), the relative biomass of Microcystis aeruginosa was associated with MC-RR, M. wesenbergii with MC-LA and Aphanizomenon flos-aquae with MC-YR. The latter two species are not generally considered capable of MC production. Total nitrogen, water temperature, ammonium and dissolved organic nitrogen influenced the cyanobacterial community structure, which in turn resulted in differences in the dominant MC congener and the overall toxicity.

Anatoxin-a (ANTX) is a neurotoxin produced by several freshwater cyanobacteria and implicated in lethal poisonings of domesticated animals and wildlife. The factors leading to its production in nature and in culture are not well understood. Resource availability may influence its cellular production as suggested by the carbon-nutrient hypothesis, which links the amount of secondary metabolites produced by plants or microbes to the relative abundance of nutrients. We tested the effects of nitrogen supply on ANTX production and release in a toxic strain of the cyanobacterium Aphanizomenon issatschenkoi (Nostocales). We hypothesized that nitrogen deficiency might constrain the production of ANTX. However, the total concentration and more significantly the cellular content of anatoxin-a peaked (max. 146 µg/L and 1683 µg•g-1 dry weight) at intermediate levels of nitrogen supply when N-deficiency was evident based on phycocyanin to chlorophyll a and carbon to nitrogen ratios. The results suggest that the cellular production of anatoxin-a may be stimulated by moderate nutrient stress as described recently for another cyanotoxin (microcystin).

The importance of nitrogen (N) versus phosphorus (P) in explaining total cyanobacterial biovolume, the biovolume of specific cyanobacterial taxa, and the incidence of cyanotoxins was determined for 102 north German lakes, using methods to separate the effects of joint variation in N and P concentration from those of differential variation in N versus P. While the positive relationship between total cyanobacteria biovolume and P concentration disappeared at high P concentrations, cyanobacteria biovolume increased continually with N concentration, indicating potential N limitation in highly P enriched lakes. The biovolumes of all cyanobacterial taxa were higher in lakes with above average joint NP concentrations, although the relative biovolumes of some Nostocales were higher in less enriched lakes. Taxa were found to have diverse responses to differential N versus P concentration, and the differences between taxa were not consistent with the hypothesis that potentially N(2)-fixing Nostocales taxa would be favoured in low N relative to P conditions. In particular Aphanizomenon gracile and the subtropical invasive species Cylindrospermopsis raciborskii often reached their highest biovolumes in lakes with high nitrogen relative to phosphorus concentration. Concentrations of all cyanotoxin groups increased with increasing TP and TN, congruent with the biovolumes of their likely producers. Microcystin concentration was strongly correlated with the biovolume of Planktothrix agardhii but concentrations of anatoxin, cylindrospermopsin and paralytic shellfish poison were not strongly related to any individual taxa. Cyanobacteria should not be treated as a single group when considering the potential effects of changes in nutrient loading on phytoplankton community structure and neither should the N(2)-fixing Nostocales. This is of particular importance when considering the occurrence of cyanotoxins, as the two most abundant potentially toxin producing Nostocales in our study were found in lakes with high N relative to P enrichment.