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.

Higher sensitivity to hydrogen peroxide and light stress conditions of the microcystin producer Microcystis aeruginosa sp PCC7806 compared to non-producer strains.

The increasing incidence of cyanobacterial blooms with their associated production of cyanotoxins lead managers of aquatics systems to control their biomass to limit the health risk. Among the variety of existing treatment approaches, hydrogen peroxide (H2O2) shows increasing use but the effects of environmental parameters on its effectiveness are still not completely known. With the aim to assess the efficiency of H2O2 treatments in the control of cyanobacterial blooms and decrease toxic risk, we tested three Microcystis strains according to their ability to produce cyanotoxins (a microcystin-producing, non-microcystin-producing and mcyB-knockout mutant). Photochemical efficiency, percentage of living cells and microcystin cell content were compared under various hydrogen peroxide concentrations coupled with stress conditions encountered during the life cycle of cyanobacteria as darkness and high light. The microcystin-producing strain appeared the more sensitive to hydrogen peroxide treatment and to light condition, probably due to a lower rate of repair of Photo System II (PSII). We also highlighted various responses of PSII activity according to Microcystis strains which could partly explain the shift of dominant genotypes often occurring during a bloom event. Our results confirm the link between light and microcystin content and variations of microcystin contents appear as a consequence of photosynthetic activity. These findings could be of particular interest regarding water quality management, especially the use of H2O2 as a potential algaecide which seems to be more effective to use during periods of high light.

Dynamics of the prokaryotic and eukaryotic microbial community during a cyanobacterial bloom.

Toxic cyanobacterial blooms frequently develop in eutrophic freshwater bodies worldwide. Microcystis species produce microcystins (MCs) as a cyanotoxin. Certain bacteria that harbor the mlr gene cluster, especially mlrA, are capable of degrading MCs. However, MC-degrading bacteria may possess or lack mlr genes (mlr+ and mlr- genotypes, respectively). In this study, we investigated the genotype that predominantly contributes to biodegradation and cyanobacterial predator community structure with change in total MC concentration in an aquatic environment. The 2 genotypes coexisted but mlr+ predominated, as indicated by the negative correlation between mlrA gene copy abundance and total MC concentration. At the highest MC concentrations, predation pressure by Phyllopoda, Copepoda, and Monogononta (rotifers) was reduced; thus, MCs may be toxic to cyanobacterial predators. The results suggest that cooperation between MC-degrading bacteria and predators may reduce Microcystis abundance and MC concentration.

Cyanobacterial Akinete Distribution, Viability, and Cyanotoxin Records in Sediment Archives From the Northern Baltic Sea.

Cyanobacteria of the order Nostocales, including Baltic Sea bloom-forming taxa Nodularia spumigena, Aphanizomenon flosaquae, and Dolichospermum spp., produce resting stages, known as akinetes, under unfavorable conditions. These akinetes can persist in the sediment and germinate if favorable conditions return, simultaneously representing past blooms and possibly contributing to future bloom formation. The present study characterized cyanobacterial akinete survival, germination, and potential cyanotoxin production in brackish water sediment archives from coastal and open Gulf of Finland in order to understand recent bloom expansion, akinete persistence, and cyanobacteria life cycles in the northern Baltic Sea. Results showed that cyanobacterial akinetes can persist in and germinate from Northern Baltic Sea sediment up to >40 and >400 years old, at coastal and open-sea locations, respectively. Akinete abundance and viability decreased with age and depth of vertical sediment layers. The detection of potential microcystin and nodularin production from akinetes was minimal and restricted to the surface sediment layers. Phylogenetic analysis of culturable cyanobacteria from the coastal sediment core indicated that most strains likely belonged to the benthic genus Anabaena. Potentially planktonic species of Dolichospermum could only be revived from the near-surface layers of the sediment, corresponding to an estimated age of 1-3 years. Results of germination experiments supported the notion that akinetes do not play an equally significant role in the life cycles of all bloom-forming cyanobacteria in the Baltic Sea. Overall, there was minimal congruence between akinete abundance, cyanotoxin concentration, and the presence of cyanotoxin biosynthetic genes in either sediment core. Further research is recommended to accurately detect and quantify akinetes and cyanotoxin genes from brackish water sediment samples in order to further describe species-specific benthic archives of cyanobacteria.

Simultaneous Microcystis algicidal and microcystin synthesis inhibition by a red pigment prodigiosin.

Microcystis blooms and their secondary metabolites microcystins (MCs) occurred all over the world, which have damaged aquatic ecosystems and threatened public health. Techniques to reduce the Microcystis blooms and MCs are urgently needed. This study aimed to investigate the algicidal and inhibitory mechanisms of a red pigment prodigiosin (PG) against the growth and MC-producing abilities of Microcystis aeruginosa (M. aeruginosa). The numbers of Microcystis cells were counted under microscope. The expression of microcystin synthase B gene (mcyB) and concentrations of MCs were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme linked immunosorbent assay (ELISA) methods, respectively. The inhibitory effects of PG against M. aeruginosa strain FACHB 905 with 50% algicidal concentration (LC50) at 120 h was 0.12 µg/mL. When M. aeruginosa cells exposed to 0.08 µg/mL, 0.16 µg/mL, 0.32 µg/mL PG, the expression of mcyB of M. aeruginosa was down-regulated 4.36, 8.16 and 18.51 times lower than that of the control at 120 h. The concentrations of total MC (TMC) also were 1.66, 1.72 and 5.75 times lower than that of the control at 120 h. PG had high algicidal effects against M. aeruginosa, with the activities of superoxide dismutase (SOD) initially increased and then decreased after 72 h, the contents of malondialdehyde (MDA) increase, the expression of mcyB gene down-regulation, and MCs synthesis inhibition. This study was first to report the PG can simultaneously lyse Microcystis cells, down-regulate of mcyB expression and inhibit MCs production effectively probably due to oxidative stress, which indicated PG poses a great potential for regulating Microcystis blooms and MCs pollution in the environment.

Spatial-temporal survey of Microcystis oligopeptide chemotypes in reservoirs with dissimilar waterbody features and their relation to genetic variation.

The ability of cyanobacteria to produce toxins and other secondary metabolites is patchily distributed in natural populations, enabling the use of cellular oligopeptide compositions as markers to classify strains into ecologically-relevant chemotypical subpopulations. The composition and spatiotemporal distribution of Microcystis chemotypes within and among waterbodies was studied at different time scales by analyzing (i) Microcystis strains isolated between 1998 and 2007 from different Spanish reservoirs and (ii) individual Microcystis aeruginosa colonies collected from pelagic and littoral habitats in Valmayor reservoir (Spain) during a bloom. No agreement between chemotypes and both morphotypes and genotypes (based on cpcBA-IGS, 16S-23S rRNA ITS and mcyB genes) was found, suggesting that oligopeptide profiles in individual strains evolve independently across morphospecies and phylogenetic genotypes, and that the diversity of microcystin variants produced cannot be explained by mcyB gene variations alone. The presence of identical chemotypes in spatially-distant reservoirs with dissimilar trophic state, lithology or depth indicate that waterbody characteristics and geographical boundaries weakly affect chemotype composition and distribution. At smaller spatiotemporal scales (i.e. during bloom), M. aeruginosa populations showed high number of chemotypes, as well as marked differences in chemotype composition and relative abundance among the littoral and pelagic habitats. This indicates that the factors influencing chemotype composition, relative abundance and dynamics operate at short spatial and temporal scales, and supports emerging hypotheses about interactions with antagonistic microorganisms as possible drivers for widespread chemical polymorphisms in cyanobacteria.

Detection of microcystin-producing cyanobacteria in water samples using loop-mediated isothermal amplification targeting mcyB gene.

Microcystin toxin-producing cyanobacteria are known to have harmful effects on humans and animals. We have developed a loop-mediated isothermal amplification (LAMP)-based detection method by targeting the microcystin synthetase B gene (mcyB), the gene responsible for the production of microcystin. The sensitivity of the method was found to be 1 fg per reaction, and it was 1000-fold higher than the conventional PCR. The LAMP method was able to amplify the target gene with a minimum amount of dNTP (0.4 mM), which further reduces the cost of reaction. The improved LAMP assay could detect the presence of the toxin-producing cyanobacteria in water samples within 2 h of time, which demonstrates the rapidness of the method. Freshwater samples were screened using the developed LAMP, and seven water samples collected from lakes and a bird sanctuary tested positive for mcyB gene harboring cyanobacteria, and negative in all other drinking waters. Hence, the developed LAMP could be a possible alternative to the existing molecular methods for screening for microcystin in environmental samples with greater sensitivity.

Influence of cyanobacteria, mixotrophic flagellates, and virioplankton size fraction on transcription of microcystin synthesis genes in the toxic cyanobacterium Microcystis aeruginosa.

Toxic cyanobacteria such as Microcystis aeruginosa are a worldwide concern in freshwater reservoirs. Problems associated with their mass occurrence are predicted to increase in the future due to global warming. The hepatotoxic secondary metabolite microcystin is of particular concern in this context. This study aimed to determine whether co-occurring microorganisms influence the expression of microcystin biosynthesis genes. To this end, we performed cocultivation experiments and measured mcyB and mcyD transcripts in M. aeruginosa using RT-qPCR. We utilized representatives from three different plankton groups: the picocyanobacterium Synechococcus elongatus, the unicellular flagellate grazer Ochromonas danica, and virioplankton from two different lakes. The presence of S. elongatus significantly increased mcyB and mcyD transcription in M. aeruginosa. Cocultivation with the mixotrophic chrysophyte O. danica did not increase the transcription of mcyB and mcyD; in fact, mcyD transcripts decreased significantly. The virioplankton size fraction of environmental water samples induced a significant increase in mcyB and mcyD transcription when obtained from lakes with cyanobacterial blooms. Our results show that co-occurring microorganisms influence the expression of microcystin biosynthesis genes in M. aeruginosa.

Temporal Dynamics of the Microbial Community Composition with a Focus on Toxic Cyanobacteria and Toxin Presence during Harmful Algal Blooms in Two South German Lakes.

Bacterioplankton plays an essential role in aquatic ecosystems, and cyanobacteria are an influential part of the microbiome in many water bodies. In freshwaters used for recreational activities or drinking water, toxic cyanobacteria cause concerns due to the risk of intoxication with cyanotoxins, such as microcystins. In this study, we aimed to unmask relationships between toxicity, cyanobacterial community composition, and environmental factors. At the same time, we assessed the correlation of a genetic marker with microcystin concentration and aimed to identify the main microcystin producer. We used Illumina MiSeq sequencing to study the bacterioplankton in two recreational lakes in South Germany. We quantified a microcystin biosynthesis gene (mcyB) using qPCR and linked this information with microcystin concentration to assess toxicity. Microcystin biosynthesis gene (mcyE)-clone libraries were used to determine the origin of microcystin biosynthesis genes. Bloom toxicity did not alter the bacterial community composition, which was highly dynamic at the lowest taxonomic level for some phyla such as Cyanobacteria. At the OTU level, we found distinctly different degrees of temporal variation between major bacteria phyla. Cyanobacteria and Bacteroidetes showed drastic temporal changes in their community compositions, while the composition of Actinobacteria remained rather stable in both lakes. The bacterial community composition of Alpha- and Beta-proteobacteria remained stable over time in Lake Klostersee, but it showed temporal variations in Lake Bergknappweiher. The presence of potential microcystin degraders and potential algicidal bacteria amongst prevalent Bacteroidetes and Alphaproteobacteria implied a role of those co-occurring heterotrophic bacteria in cyanobacterial bloom dynamics. Comparison of both lakes studied revealed a large shared microbiome, which was shaped toward the lake specific community composition by environmental factors. Microcystin variants detected were microcystin-LR, -RR, and -YR. The maximum microcystin concentrations measured was 6.7 µg/L, a value still acceptable for recreational waters but not drinking water. Microcystin concentration correlated positively with total phosphorus and mcyB copy number. We identified low abundant Microcystis sp. as the only microcystin producer in both lakes. Therefore, risk assessment efforts need to take into account the fact that non-dominant species may cause toxicity of the blooms observed.

Microcystins production and antibacterial activity of cyanobacterial strains of Synechocystis, Synechococcus and Romeria from water and coral reef organisms (Brazil) / Producción de microcistina y actividad antibacteriana de cepas cianobacterianas de Synechocystis, Synechococcus y Romeria aisladas de agua y organismos de arrecifes de coral del litoral brasileño

AbstractCyanobacteria are widely distributed in terrestrial, freshwater and marine environments, and over the past decades have been recognized as a powerful source of bioactive compounds. In this study, some cyanobacterial strains were isolated from samples of seawater, brackish water and tissue of reef benthic invertebrates (zoanthid Protopalythoa variabilis, the sponges Cynachrella sp. and Haliclona sp., the coral Siderastrea stellata, and ascidians), collected at the states of Paraíba and Rio Grande do Norte (Northeast of Brazil), during the period between July 2010 and February 2014. After standard isolation methods, the cultivation of the strains was carried out in acclimatized culture chamber (25 °C) under constant aeration, for 15 days at 12-hour photoperiod, using Conway and BG11 media made with filtered seawater. The cyanobacterial cells were analysed for the microcystin production by the ELISA technique and their ethanolic and methanolic extracts for the antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa by the agar well diffusion method. The detection of the mcyB gene, one of the genes related to the microcystin synthesis, was done by the Polymerase Chain Reaction (PCR) technique. The majority of the eighteen cyanobacterial strains belonged to Synechococcaceae Family. The genera of Synechocystis, Synechococcus and Romeria were represented by ten, six and two strains, respectively. The production of microcystins was observed in five strains belonging to the genus Synechocystis. The presence of mcyB gene was detected in 12 strains of cyanobacteria: Synechocystis (three strains), Synechococcus (six strains) and Romeria (two strains). Only one strain (Synechocystis aquatilis) showed both the microcystin production and the mcyB gene presence. The antibacterial activity was observed for one strain of Romeria gracilis, one strain of Synechocystis aquatilis and two strains of Synechococcus sp. The ethanolic extracts of R. gracilis strain and two Synechococcus spp. strains inhibited the growth of P. aeruginosa. Among methanolic extracts of cyanobacteria, only one strain of S. aquatilis showed activity against S. aureus, and one R. gracilis strain against P. aeruginosa. Some cyanobacterial strains studied were positive for the microcystin production and antibacterial activity against pathogenic bacteria S. aureus and P. aeuruginosa, and may be further explored for additional biotechnological applications. Rev. Biol. Trop. 65 (3): 890-899. Epub 2017 September 01.

ResumenLas cianobacterias se encuentran ampliamente distribuidas en ecosistemas terrestres, de agua dulce y marinos, y en las últimas décadas han sido reconocidas como una poderosa fuente de compuestos bioactivos. En este estudio, las cepas de cianobacterias fueron aisladas a partir de agua de mar, agua salobre y muestras de tejidos de invertebrados bentónicos de arrecifes (zoanthid Protopalythoa variabilis, las esponjas Cynachrella sp. y Haliclona sp., el coral Siderastrea stellata y ascidias) recogidas en los estados de Paraíba y Rio Grande do Norte, en el noreste de Brasil, en el período comprendido entre julio 2010 y febrero 2014. La mayoría de las dieciocho cepas de cianobacterias pertenecían a la Familia Synechococcaceae. Los géneros: Synechocystis, Synechococcus y Romeria estuvieron representados por diez, seis y dos cepas, respectivamente. Las cepas fueron analizadas para la producción de microcistina por ELISA y para la actividad antibacteriana contra Staphylococcus aureus y Pseudomonas aeruginosa por el método de difusión en agar. La detección del gen mcyB, uno de los genes relacionados con la síntesis de microcistina, se realizó mediante la técnica de reacción en cadena de la polimerasa (PCR). El cultivo de las cepas se realizó en cámara de cultivo aclimatada (25 ° C) bajo aireación constante durante 15 días con un fotoperíodo de 12 horas utilizando los medios Conway y BG11 elaborados con agua de mar filtrada. Se observó la producción de microcistina en cinco cepas pertenecientes al género Synechocystis. La presencia del gen mcyB fue detectada en doce cepas de cianobactérias: Synechocystis (tres cepas), Synechococcus (seis cepas) y Romeria (dos cepas). Sólo una cepa (Synechocystis aquatilis) mostró tanto la producción de microcistina como la presencia del gen mcyB. Se observó la actividad antibacteriana de una cepa de Romeria gracilis, de una cepa de Synechocystis aquatilis y dos cepas de Synechococcus sp. Los extractos etanólicos de las cepas de R. gracilis y Synechococcus sp. inhibieron el crecimiento de P. aeruginosa. Entre los extractos metanólicos de cianobacterias solamente S. aquatilis mostró actividad contra S. aureus y R. gracilis contra P. aeruginosa. Varias cepas de cianobacterias estudiadas en este trabajo fueron positivas para la producción de microcistina y actividad antibacteriana frente a bacterias patógenas de S. aureus y P. aeuruginosa, y pueden ser explotadas para aplicaciones biotecnológicas.

Influence of temperature, mixing, and addition of microcystin-LR on microcystin gene expression in Microcystis aeruginosa.

Cyanobacteria, such as the toxin producer Microcystis aeruginosa, are predicted to be favored by global warming both directly, through elevated water temperatures, and indirectly, through factors such as prolonged stratification of waterbodies. M. aeruginosa is able to produce the hepatotoxin microcystin, which causes great concern in freshwater management worldwide. However, little is known about the expression of microcystin synthesis genes in response to climate change-related factors. In this study, a new RT-qPCR assay employing four reference genes (GAPDH, gltA, rpoC1, and rpoD) was developed to assess the expression of two target genes (the microcystin synthesis genes mcyB and mcyD). This assay was used to investigate changes in mcyB and mcyD expression in response to selected environmental factors associated with global warming. A 10°C rise in temperature significantly increased mcyB expression, but not mcyD expression. Neither mixing nor the addition of microcystin-LR (10 µg L-1 or 60 µg L-1 ) significantly altered mcyB and mcyD expression. The expression levels of mcyB and mcyD were correlated but not identical.

Microcystins do not necessarily lower the sensitivity of Microcystis aeruginosa to tannic acid.

Different phytoplankton strains have been shown to possess varying sensitivities towards macrophyte allelochemicals, yet the reasons for this are largely unknown. To test whether microcystin (MC) is responsible for strain-specific sensitivities of Microcystis aeruginosa to macrophyte allelochemicals, we compared the sensitivity of 12 MC- and non-MC-producing M. aeruginosa strains, including an MC-deficient mutant and its wild type, to the polyphenolic allelochemical tannic acid (TA). Non-MC-producing strains showed a significantly higher sensitivity to TA than MC-producing strains, both in Chlorophyll a concentrations and quantum yields of photosystem II. In contrast, an MC-deficient mutant displayed a higher fitness against TA compared to its wild type. These results suggest that the resistance of M. aeruginosa to polyphenolic allelochemicals is not primarily related to MCs per se, but to other yet unknown protective mechanisms related to MCs.

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.