Fluorescence in situ hybridization of Microcystis strains producing microcystin using specific mRNA probes.

Cyanobacteria are ubiquitous micro-organisms that can produce toxic compounds, the cyanotoxins. The monitoring of such producers in the environment is of prime importance for human health. An attractive technology for such monitoring is fluorescence in situ hybridization (FISH), which allows the detection and enumeration of environmental micro-organisms. We present here the application of tyramide signal amplification fluorescence in situ hybridization (TSA-FISH) to the detection of microcystin-producing Microcystis strains. We used a 16S rRNA-specific probe, MICR3, to specifically label and observe by epifluorescence microscopy Microcystis aeruginosa strains. Using confocal laser scanning microscopy and a specific probe, MCYA, targeting the mcyA mRNA we have labelled M. aeruginosa PCC 7806, which produces microcystins. Microcystis aeruginosa PCC 7005 which does not produce microcystins is not labelled by this probe. Furthermore, we show here that this specific mRNA labelling in M. aeruginosa PCC 7806 is enhanced in cells illuminated for 1 h just after a dark period of cultivation of 24 h, conditions in which the mcyA gene is up regulated. The data presented here might be applicable to the monitoring of toxic Microcystis strains in the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Cyanobacteria producing toxic compounds (cyanotoxins) are present in the environment and in water bodies. Their presence poses a threat on human and animal health. It is thus important to detect, identify and enumerate these toxic Cyanobacteria. Using tyramide signal amplification fluorescence in situ hybridization (TSA-FISH) and specific probes, with confocal laser scanning microscopy, we have specifically detected Microcystis strains producing microcystin toxins. The data presented here might be applied to the monitoring of water bodies at early stages and all along the formation of Microcystis blooms.

Quantification and genetic diversity of total and microcystin-producing Microcystis during blooming season in Tai and Yang-cheng lakes, China.

AIMS: The aims of present study were to evaluate the abundances, genetic diversity of total and microcystin-producing Microcystis over temporal and spatial scales, and to investigate relationships among Microcystis and water parameters in Tai and Yang-cheng lakes. METHODS AND RESULTS: Abundances of total and microcystin-producing Microcystis varied across sampling periods and locations, which were assessed using qPCR with primers specific to Microcystis 16S rDNA and mcyA genes. The 16S rDNA from two lakes were relatively diverse. However, mcyA genes were rather conservative and were >97% identical to reference sequences. The highly positive correlations between mcyA and microcystin presence (r = 0·671 in Tai; r = 0·799 in Yang-cheng) suggested that mcyA can be used as a good biomarker for microcystin productions. CONCLUSION: The results demonstrated that Microcystis were genetically diverse between these conjunctive lakes; however, mcyA genes were relatively conservative in two lakes. Quantifying mcyA by qPCR was an efficient tool for monitoring toxic Microcystis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has improved our understanding of observable differences within and between each lake on spatial and temporal scales. And the discovery of new mcyA sequences in natural water enriched the understanding of phylogenetic diversity of Microcystis and toxin-production-related mcy gene.

Spatio-temporal variation of toxin-producing gene abundance in Microcystis aeruginosa from Poyang Lake.

Microcystis aeruginosa (M. aeruginosa) causes massive blooms in eutrophic freshwater and releases microcystin. Poyang Lake is the largest freshwater lake in China and has kept a mid-nutrient level in recent years. However, there is little research on microcystin production in Poyang Lake. In this study, water and sediment samples from ten sampling sites in Poyang Lake were collected from May to December in 2020, and from January to April in 2021 respectively. Microcystis genes (mcyA, mcyB, 16 s rDNA) were quantified by real-time fluorescence quantitative PCR analysis, and then the spatial and temporal variation of mcy genes, physicochemical factors, and bacterial population structure in the lake was analyzed. The relationship between the abundance of mcy genes and physicochemical factors in water column was also revealed. Results indicated that the microcystin-producing genes mcyA and mcyB showed significant differences in spatial and temporal levels as well, which is closely related to the physicochemical factors especially the water temperature (p < 0.05) and the nitrogen content (p < 0.05). The abundance of mcy genes in the sediment in December affected the abundance of mcy genes in the water column in the next year, while the toxic Microcystis would accumulate in the sediment. In addition to the toxic Microcystis, we also found a large number of non-toxic Microcystis in the water column and sediment, and the ratio of toxic to non-toxic species can also affect the toxicity production of M. aeruginosa. Overall, the results showed that M. aeruginosa toxin-producing genes in Poyang Lake distributed spatially and temporally which related to the physicochemical factors of Poyang Lake.

Improved detection of mcyA genes and their phylogenetic origins in harmful algal blooms.

Microcystins, a group of cyanotoxins produced by cyanobacterial strains, have become a significant microbial hazard to human and animal health due to increases in the frequency and intensity of cyanobacterial harmful algal blooms (CyanoHABs). Many studies have explored the correlation between microcystin concentrations and abundances of toxin-producing genes (e.g., mcyA genes) measured using quantitative PCR, and discrepancies between toxin concentrations and gene abundances are often observed. In this study, the results show that these discrepancies are at least partially due to primer sets that do not capture the phylogenetic diversity of naturally present toxin-producers. We designed three novel primer gene probes based on known mcyA genes to improve the detection and quantification of these genes in environmental samples. These primers were shown to improve the identification of mcyA genes compared to previously published primers in freshwater metagenomes, cyanobacterial isolates, and lake water samples. Unlike previously published primers, our primer sets could selectively amplify and resolve Microcystis, Anabaena, and Planktothrix mcyA genes. In lake water samples, abundance estimations of mcyA genes were found to correlate strongly with microcystin concentrations. Based on our results, these primers offer significant improvements over previously published probes to accurately identify and quantify mcyA genes in the environment. There is an increasing need to develop models based on microbial information and environmental factors to predict CyanoHABs, and improved primers will play an important role in aiding monitoring efforts to collect reliable and consistent data on toxicity risks.

Limited Microcystin, Anatoxin and Cylindrospermopsin Production by Cyanobacteria from Microbial Mats in Cold Deserts.

Toxic metabolites are produced by many cyanobacterial species. There are limited data on toxigenic benthic, mat-forming cyanobacteria, and information on toxic cyanobacteria from Central Asia is even more scarce. In the present study, we examined cyanobacterial diversity and community structure, the presence of genes involved in toxin production and the occurrence of cyanotoxins in cyanobacterial mats from small water bodies in a cold high-mountain desert of Eastern Pamir. Diversity was explored using amplicon-based sequencing targeting the V3-V4 region of the 16S rRNA gene, toxin potential using PCR-based methods (mcy, nda, ana, sxt), and toxins by enzyme-linked immunosorbent assays (ELISAs) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Molecular identification of cyanobacteria showed a high similarity of abundant taxa to Nostoc PCC-73102, Nostoc PCC-7524, Nodularia PCC-935 and Leptolyngbya CYN68. The PCRs revealed the presence of mcyE and/or ndaF genes in 11 samples and mcyD in six. The partial sequences of the mcyE gene showed high sequence similarity to Nostoc, Planktothrix and uncultured cyanobacteria. LC-MS/MS analysis identified six microcystin congeners in two samples and unknown peptides in one. These results suggest that, in this extreme environment, cyanobacteria do not commonly produce microcystins, anatoxins and cylindrospermopsins, despite the high diversity and widespread occurrence of potentially toxic taxa.

Recruitment of cyanobacteria by reverse transcription quantitative real-time PCR based on expression of Microcystis gene.

In this study, a SYBR Green quantitative real-time PCR method was established and applied. Relative expression of the synthetic genes from Microcystis gas vesicles (gvpC), algal toxin genes (mcyA), and polysaccharides (espL) from water and sediments of Meiliang Bay and from the center of Lake Taihu were tested from January to June, 2017. Indoor Microcystis aeruginosa was used as the control group. The kit for total RNA extraction in Microcystis was optimized. Results showed that the optimized kit extracted high-concentrations and high-quality total RNA from Microcystis. The extraction purity and concentration were significantly higher than those extracted by the original kit. The transcription level of gvpC increased gradually until a peak was reached in March. However, expression of gvpC decreased continuously at the proliferating and floating stages of Cyanobacterial biomass. The maximum level of expression of gvpC in April in comparison to expression of mcyA in March occurred first. We found that the SYBR Green qRT-PCR method, which is characterized by high specificity, repeatability, is rapid, and can be used for quantitative detection of expression of gvpC, mcyA, and espL. The recruitment of cyanobacteria is the process in which cyanobacteria in the sediment began to regain their activity, started to grow and migrated to the water column.

Benthic Archives Reveal Recurrence and Dominance of Toxigenic Cyanobacteria in a Eutrophic Lake over the Last 220 Years.

Akinetes are resistant cells which have the ability to persist in sediment for several decades. We have investigated the temporal distribution of akinetes of two species, Dolichospermum macrosporum and Dolichospermum flos-aquae, in a sediment core sampled in Lake Aydat (France), which covers 220 years. The upper part, from 1907 to 2016, the number of akinetes fluctuated but stayed at high concentrations, especially for D. macrosporum in surface sediment (with the maximal value close to 6.105 akinetes g DW-1 of sediment), suggesting a recurrence of blooms of this species which was probably closely related to anthropic eutrophication since the 1960s. Before 1907, the abundance of akinetes of both species was very low, suggesting only a modest presence of these cyanobacteria. In addition, the percentage of intact akinetes was different for each species, suggesting different ecological processes in the water column. This percentage also decreased with depth, revealing a reduction in germination potential over time. In addition, biosynthetic genes of anatoxin-a (anaC) and microcystin (mcyA) were detected. First results show a high occurrence of mcyA all down the core. In contrast, anaC gene was mostly detected in the surface sediment (since the 1980s), revealing a potentially more recent occurrence of this cyanotoxin in Lake Aydat which may be associated with the recurrence of blooms of D. macrosporum and thus with anthropic activities.

Detection of a Planktothrix agardhii Bloom in Portuguese Marine Coastal Waters.

Cyanobacteria blooms are frequent in freshwaters and are responsible for water quality deterioration and human intoxication. Although, not a new phenomenon, concern exists on the increasing persistence, scale, and toxicity of these blooms. There is evidence, in recent years, of the transfer of these toxins from inland to marine waters through freshwater outflow. However, the true impact of these blooms in marine habitats has been overlooked. In the present work, we describe the detection of Planktothrix agardhii, which is a common microcystin producer, in the Portuguese marine coastal waters nearby a river outfall in an area used for shellfish harvesting and recreational activities. P. agardhii was first observed in November of 2016 in seawater samples that are in the scope of the national shellfish monitoring system. This occurrence was followed closely between November and December of 2016 by a weekly sampling of mussels and water from the sea pier and adjacent river mouth with salinity ranging from 35 to 3. High cell densities were found in the water from both sea pier and river outfall, reaching concentrations of 4,960,608 cells·L-1 and 6810.3 × 106 cells·L-1 respectively. Cultures were also established with success from the environment and microplate salinity growth assays showed that the isolates grew at salinity 10. HPLC-PDA analysis of total microcystin content in mussel tissue, water biomass, and P. agardhii cultures did not retrieve a positive result. In addition, microcystin related genes were not detected in the water nor cultures. So, the P. agardhii present in the environment was probably a non-toxic strain. This is, to our knowledge, the first report on a P. agardhii bloom reaching the sea and points to the relevance to also monitoring freshwater harmful phytoplankton and related toxins in seafood harvesting and recreational coastal areas, particularly under the influence of river plumes.

Distribution and Habitat Specificity of Potentially-Toxic Microcystis across Climate, Land, and Water Use Gradients.

Toxic cyanobacterial blooms are a growing threat to freshwater bodies worldwide. In order for a toxic bloom to occur, a population of cells with the genetic capacity to produce toxins must be present together with the appropriate environmental conditions. In this study, we investigated the distribution patterns and phylogeny of potentially-toxic Microcystis (indicated by the presence and/or phylogeny of the mcyD and mcyA genes). Samples were collected from the water column of almost 60 water bodies across widely differing gradients of environmental conditions and land use in Israel. Potentially, toxic populations were common but not ubiquitous, detected in ~65% of the studied sites. Local environmental factors, including phosphorus and ammonia concentrations and pH, as well as regional conditions such as the distance from built areas and nature reserves, were correlated with the distribution of the mcyD gene. A specific phylogenetic clade of Microcystis, defined using the sequence of the mcyA gene, was preferentially associated with aquaculture facilities but not irrigation reservoirs. Our results reveal important environmental, geospatial, and land use parameters affecting the geographic distribution of toxinogenic Microcystis, suggesting non-random dispersal of these globally abundant toxic cyanobacteria.

Evaluating the influence of light intensity in mcyA gene expression and microcystin production in toxic strains of Planktothrix agardhii and Microcystis aeruginosa.

Cyanobacteria are phytoplanktonic organisms widely occurring in freshwaters, being frequently associated with the production of toxins, namely microcystins (MCs). MCs are produced non-ribosomally by a multienzyme complex (mcy genes). It has been reported that environmental factors, such as light intensity, can influence toxin production. The aim of this study was to assess the influence of light intensity in the transcription of the mcyA gene and corresponding production of microcystins in toxic isolates of Planktothrix agardhii, where little is known, and compare them to Microcystis aeruginosa. For that purpose, cultures were exposed to three different light intensities (4, 20 and 30 µmol photons m(-2) s(-1)) for 18 days at 20 ± 1 °C. The growth was followed daily using absorbance readings. Samples were collected at each growth stage for cell counting, microcystins quantification and RNA extraction. The level of transcripts was quantified by RT-qPCR and the relative expression determined using 16S rDNA, gltA and rpoC1 as reference genes. The most stable reference genes in M. aeruginosa were rpoC1 and gltA, whereas in P. agardhii were 16S rDNA and gltA. There was a correspondence between the growth rate and light intensity in M. aeruginosa and P. agardhii. The growth rates for both species were lower at 4 and higher at 30 µmol photons m(-2) s(-1). Microcystin concentration per cell was similar between light intensities in M. aeruginosa and over time, while in P. agardhii it was higher in the stationary phase at 4 µmol photons m(-2) s(-1). There were differences in the expression of mcyA between the two species. In M. aeruginosa, the highest levels of expression occurred at 4 µmol photons m(-2) s(-1) in the adaptation phase, whereas for P. agardhii it was at 4µmol photons m(-2) s(-1) in the exponential growth phase. Our results indicate that the light intensities tested had distinct influences on the growth, microcystin production and mcyA expression levels, presenting considerable differences in M. aeruginosa and P. agardhii.

Temporal variations in microcystin-producing cells and microcystin concentrations in two fresh water ponds.

The relationship between microcystin production, microcystin-producing cyanobacteria, including Microcystis spp., and various biological and physicochemical parameters in Sankuldhara and Lakshmikund, situated in the same geographical area was studied over a period of 1.5 years. Seasonal variation in cyanobacterial 16S rRNA, Microcystis spp. 16S rRNA, mcyA and mcyB genes were quantitatively determined by real-time PCR. Microcystis was the dominant microcystin producer in both study sites constituting 67% and 97% of the total microcystin-producing cyanobacteria at Sankuldhara and Lakshmikund, respectively. Microcystin concentrations were 2.19-39.60 µg/L and 15.22-128.14 µg/L at Sankuldhara and Lakshmikund, respectively, as determined by LC-MS. Principal component analysis revealed a strong positive correlation between microcystin concentration and the copy number of mcyA and mcyB, chlorophyll a and cyanobacterial biomass at both sites. The higher microcystin concentrations in Lakshmikund pond were attributed to the high copy number of mcy genes present coupled with the pond's eutrophication status, as indicated by high total algal biomass, high chlorophyll a content, high nutrient load and low DO. Therefore, a significant difference in microcystin concentrations, correlating with these various biological and physicochemical parameters, confirms the importance of local environmental variables in the overall regulation of microcystins production.

Co-occurrence of microcystin and anatoxin-a in the freshwater lake Aydat (France): Analytical and molecular approaches during a three-year survey.

Cyanobacterial mass occurrence is becoming a growing concern worldwide. They notably pose a threat to water users when cyanotoxins are produced. The aim of this study was to evaluate the occurrence and the dynamics of two cyanotoxins: microcystin (MC) and anatoxin-a (ANTX-a), and of two of the genes responsible for their production (respectively mcyA and anaC) during three consecutive bloom periods (2011, 2012 and 2013) in Lake Aydat (Auvergne, France). MC was detected at all sampling dates, but its concentration showed strong inter- and intra-annual variations. MC content did not correlate with cyanobacterial abundance, nor with any genera taken individually, but it significantly correlated with mcyA gene abundance (R2=0.51; p=0.042). MC content and mcyA gene abundance were maximal when cyanobacterial abundance was low, either at the onset of the bloom or during a trough of biomass. The LC-MS/MS analysis showed the presence of ANTX-a in the 2011 samples. To our knowledge, this is the first report of the presence of this neurotoxin in a French lake. The presence of ANTX-a corresponded to the only year for which Anabaena did not dominate the cyanobacterial community alone, and several cyanobacterial genera were present, including notably Aphanizomenon. anaC gene detection by PCR was not coherent with ANTX-a presence, both gene and toxin were never found for a same sample. This implies that molecular tools to study genes responsible for the production of anatoxin-a are still imperfect and the development of new primers is needed. This study also highlights the need for better monitoring practices that would not necessarily focus only on the peak of cyanobacterial abundance and that would take cyanotoxins other than MC into account.

Identificação do gene mcyA em florações naturais de Radiocystis fernandoi, em um tributário do reservatório de Rosana, Brasil / Identification of the mcyA gene in natural blooms of Radiocystis fernandoi from a tributary of the Rosana reservoir, Brazil

As cianobactérias são conhecidamente produtoras de toxinas. Dentro de uma mesma espécie, podemos encontrar variedades tóxicas e não-tóxicas, impossíveis de diferenciação apenas pela morfologia. A principal toxina produzida pelas cianobactérias é a microcistina. Esta proteína é biossintetizada por um grupo de genes conhecidos como mcy. A detecção destes genes a partir de PCR permite a distinção das variedades tóxicas e não-tóxicas. Desse modo, o objetivo desse trabalho foi investigar a ocorrência de florações produtoras de toxinas em um rio tributário do reservatório de Rosana, via amplificação do gene mcyA por PCR. Foram coletadas duas amostras de água da subsuperfície. As duas amostras coletadas no rio do Corvo foram dominadas pela espécie Radiocystis fernandoi e apresentaram resultados positivos para a presença do gene mcyA, confirmando o potencial tóxico dessa espécie. Os resultados representam alerta sobre a qualidade da água do rio do Corvo. A técnica PCR foi eficiente para a rápida detecção de cianobactérias produtoras de toxinas, inclusive podendo ser utilizada antes mesmo do agravamento das condições ambientais pela produção de toxinas, além de apresentar baixo custo.

Cyanobacterias are known as toxin producers. Within the same species, toxic and non-toxic varieties can be found and it is impossible to differentiate them only by morphology. The most important toxin produced by cyanobacteria is microcystin. This protein is synthesized by a cluster of genes known as mcy. The detection of these genes by PCR allows the differentiation of the producing toxin strain from the non-producing toxin strain. Thus, the goal of this work was to investigate the occurrence of toxigenic blooms of cyanobacteria in the Corvo River through PCR amplification of mcyA gene. For this, two samples of blooms of cyanobacteria were collected in Corvo River. Both samples were dominated by Radiocystis fernandoi and presented positive results for the presence of the mcyA gene, which may confirm the potential toxigenicity for that species. These results are an alert about water quality in the Corvo River. Here we demonstrate that amplification of the mcyA gene by PCR is a fast, cheap and efficient method for the detection of toxin- producing cyanobacteria.