A Novel Wide-Range Freshwater Cyanophage MinS1 Infecting the Harmful Cyanobacterium Microcystis aeruginosa.

Microcystis aeruginosa, as one of the major players in algal bloom, produces microcystins, which are strongly hepatotoxic, endangering human health and damaging the ecological environment. Biological control of the overgrowth of Microcystis with cyanophage has been proposed to be a promising solution for algal bloom. In this study, a novel strain of Microcystis cyanophage, MinS1, was isolated. MinS1 contains an icosahedral head approximately 54 nm in diameter and a 260 nm-long non-contractile tail. The phage genome consists of a linear, double-stranded 49,966 bp DNA molecule, which shares very low homology with known phages in the NCBI database (only 1% of the genome showed weak homology with known phages when analyzed by megablast). The phage contains 75 ORFs, of which 23 ORFs were predicted to code for proteins of known function, 39 ORFs were predicted to code for proteins of unknown function, and 13 ORFs showed no similarity to any protein sequences. Transmission electron microscopy and phylogenetic analysis showed that MinS1 belongs to the family Siphoviridae. Various experiments confirmed that the phage could infect several different orders of cyanobacteria, including Chroococcales, Nostocales, Oscillatoriales, Hormogonales, and Synechococcales, indicating that it has a very broad host range. In addition, MinS1 has no known antibiotic tolerance genes, virulence genes, and tRNAs, and it is tolerant to temperature, pH, UV, and salinity, suggesting that MinS1 has good potential for application as a biological control agent against cyanobacterial blooms. This study expands the diversity and knowledge of cyanophages, and it provides useful information for the development of novel prevention and control measures against cyanobacterial blooms.

Analytical Technique Optimization on the Detection of ß-cyclocitral in Microcystis Species.

ß-Cyclocitral, specifically produced by Microcystis, is one of the volatile organic compounds (VOCs) derived from cyanobacteria and has a lytic activity. It is postulated that ß-cyclocitral is a key compound for regulating the occurrence of cyanobacteria and related microorganisms in an aquatic environment. ß-Cyclocitral is sensitively detected when a high density of the cells is achieved from late summer to autumn. Moreover, it is expected to be involved in changes in the species composition of cyanobacteria in a lake. Although several analysis methods for ß-cyclocitral have already been reported, ß-cyclocitral could be detected using only solid phase micro-extraction (SPME), whereas it could not be found at all using the solvent extraction method in a previous study. In this study, we investigated why ß-cyclocitral was detected using only SPME GC/MS. Particularly, three operations in SPME, i.e., extraction temperature, sample stirring rate, and the effect of salt, were examined for the production of ß-cyclocitral. Among these, heating (60 °C) was critical for the ß-cyclocitral formation. Furthermore, acidification with a 1-h storage was more effective than heating when comparing the obtained amounts. The present results indicated that ß-cyclocitral did not exist as the intact form in cells, because it was formed by heating or acidification of the resulting intermediates during the analysis by SPME. The obtained results would be helpful to understand the formation and role of ß-cyclocitral in an aquatic environment.

Field cyanobacterial blooms producing retinoid compounds cause teratogenicity in zebrafish embryos.

Cyanobacteria routinely release potentially harmful bioactive compounds into the aquatic environment. Several recent studies suggested a potential link between the teratogenicity of effects caused by cyanobacteria and production of retinoids. To investigate this relationship, we analysed the teratogenicity of field-collected cyanobacterial bloom samples by means of an in vivo zebrafish embryo test, an in vitro reporter gene bioassay and by the chemical analysis of retinoids. Extracts of biomass from cyanobacterial blooms with the dominance of Microcystis aeruginosa and Aphanizomenon klebahnii were collected from water bodies in the Czech Republic and showed significant retinoid-like activity in vitro, as well as high degrees of teratogenicity in vivo. Chemical analysis was then used to identify a set of retinoids in ng per gram of dry weight concentration range. Subsequent fractionation and bioassay-based characterization identified two fractions with significant in vitro retinoid-like activity. Moreover, in most of the retinoids eluted from these fractions, teratogenicity with malformations typical for retinoid signalling disruption was observed in zebrafish embryos after exposure to the total extracts and these in vitro effective fractions. The zebrafish embryo test proved to be a sensitive toxicity indicator of the biomass extracts, as the teratogenic effects occurred at even lower concentrations than those expected from the activity detected in vitro. In fact, teratogenicity with retinoid-like activity was detected at concentrations that are commonly found in biomasses and even in bulk water surrounding cyanobacterial blooms. Overall, these results provide evidence of a link between retinoid-like activity, teratogenicity and the retinoids produced by cyanobacterial water blooms in the surrounding environment.

Effects of different concentrations of Microcystis aeruginosa on the intestinal microbiota and immunity of zebrafish (Danio rerio).

Microcystis aeruginosa is a primary species of toxin-producing cyanobacteria. This study explored the effects of short-term exposure (96 h) to M. aeruginosa on the intestinal microflora variation and immune function of zebrafish. After exposure to different cell concentrations of M. aeruginosa, marked histological variation was observed in the intestine, such as goblet cells proliferation and intestinal desquamation. In addition high-concentration M. aeruginosa treatment (initial concentration: 1.59 × 105 cells mL-1) induced a significant increase in cytokine levels compared with other groups. Low-concentration M. aeruginosa treatment (initial concentration: 0.88 × 105 cells mL-1) promoted the transcription of inflammatory genes, while high-concentration treatment restrained the transcription of these genes. Moreover, M. aeruginosa exposure also changed the intestinal microbial diversity. At the phylum level, bacteria belonging to Proteobacteria were the most abundant in all groups, and Gammaproteobacteria were the dominant bacteria with major changes. Pathogenic microorganisms such as Shewanella, Plesiomonas, Halomonas, Pseudomonas, and Lactobacillus increased greatly after treatment with different cell concentrations of M. aeruginosa. This study indicates that M. aeruginosa induces an increase in zebrafish goblet cells and enhances the inflammatory response, which may produce detrimental effects in zebrafish, resulting in a greater proportion of pathogenic bacteria and intestinal injury. The results of this study will help improve the understanding of the effects of M. aeruginosa on the intestines of aquatic organisms.

Nitrogen limitation, toxin synthesis potential, and toxicity of cyanobacterial populations in Lake Okeechobee and the St. Lucie River Estuary, Florida, during the 2016 state of emergency event.

Lake Okeechobee, FL, USA, has been subjected to intensifying cyanobacterial blooms that can spread to the adjacent St. Lucie River and Estuary via natural and anthropogenically-induced flooding events. In July 2016, a large, toxic cyanobacterial bloom occurred in Lake Okeechobee and throughout the St. Lucie River and Estuary, leading Florida to declare a state of emergency. This study reports on measurements and nutrient amendment experiments performed in this freshwater-estuarine ecosystem (salinity 0-25 PSU) during and after the bloom. In July, all sites along the bloom exhibited dissolved inorganic nitrogen-to-phosphorus ratios < 6, while Microcystis dominated (> 95%) phytoplankton inventories from the lake to the central part of the estuary. Chlorophyll a and microcystin concentrations peaked (100 and 34 µg L-1, respectively) within Lake Okeechobee and decreased eastwards. Metagenomic analyses indicated that genes associated with the production of microcystin (mcyE) and the algal neurotoxin saxitoxin (sxtA) originated from Microcystis and multiple diazotrophic genera, respectively. There were highly significant correlations between levels of total nitrogen, microcystin, and microcystin synthesis gene abundance across all surveyed sites (p < 0.001), suggesting high levels of nitrogen supported the production of microcystin during this event. Consistent with this, experiments performed with low salinity water from the St. Lucie River during the event indicated that algal biomass was nitrogen-limited. In the fall, densities of Microcystis and concentrations of microcystin were significantly lower, green algae co-dominated with cyanobacteria, and multiple algal groups displayed nitrogen-limitation. These results indicate that monitoring and regulatory strategies in Lake Okeechobee and the St. Lucie River and Estuary should consider managing loads of nitrogen to control future algal and microcystin-producing cyanobacterial blooms.

Microcystis bloom containing microcystin-LR induces type 2 diabetes mellitus.

Epidemiological data from Lake Taihu showed significantly higher incidences of type 2 diabetes mellitus (T2DM) than in other areas of China. This may be related to the occurrence of a Microcystis bloom in Lake Taihu in the summer and autumn every year. The objective of this study is to investigate whether the contaminated water from the Microcystis bloom and the derivative pollutant microcystin-LR (MC-LR) can explain the higher incidences of T2DM. Healthy male mice were fed with water from different regions of Lake Taihu, and were either acutely or chronically exposed to MC-LR through oral administration or intraperitoneal injection. Serum lipid profiles were determined, and the effects on T2DM-related gene expression and insulin receptor signaling pathway were investigated. Intraperitoneal glucose tolerance (IPGTT) and insulin resistance (IRT) tests were implemented, and the functions of pancreatic islet and ß-cell were also evaluated. The results showed that both water sampled from the region with a Microcysis bloom and those containing MC-LR altered the serum glucide and lipid profiles in mice after exposure. The exposure to a Microcysis bloom water affected the expression T2DM-related genes: up-regulated the mRNA levels of FASn, ACACA, G6pc, LPL, and Insig2, and down-regulated the mRNA level of PEPCK and Gsk-3ß. Both acute and chronic exposure of MC-LR, even at very low concentrations (1 µg/L), impaired the insulin receptor signalling pathway and induced hyperinsulinemia and insulin resistance in mice. In this study, the most important intracellular target of MC-LR was found to be hetapocellular mitochondria. Thus, exposure to Microcystis bloom water containing microcystin-LR can induce the incidence of T2DM, by impairing the function of mitochondria by microcystin-LR. The study suggests a review of the risk assessment concerning 1 µg/L MC-LR as the reference dose in surface water.

Proteomic evidences for microcystin-RR-induced toxicological alterations in mice liver.

This study deals with the isolation and purification of an important variant of microcystins namely microcystin-RR (MCYST-RR) from Microcystis aeruginosa and reports its effects on mice liver protein profile and cellular functions. Protein profiling by 2-dimensional gel electrophoresis revealed changes in the number and accumulation of protein spots in liver of mice treated with different concentrations of MCYST-RR. Untreated (control) mice liver showed 368 protein spots while the number was 355, 348 and 332 in liver of mice treated with 200, 300 and 400 µg kg body wt-1 of MCYST-RR respectively. Altogether 102, 97, and 92 spots were differentially up-accumulated and 93, 91, and 87 spots were down- accumulated respectively with the treatment of 200, 300, 400 µg kg body wt-1. Eighteen differentially accumulated proteins present in all the four conditions were identified by MALDI-TOF MS. Of these eighteen proteins, 12 appeared to be involved in apoptosis/toxicological manifestations. Pathway analysis by Reactome and PANTHER database also mapped the identified proteins to programmed cell death/apoptosis clade. That MCYST-RR induces apoptosis in liver tissues was also confirmed by DNA fragmentation assay. Results of this study elucidate the proteomic basis for the hepatotoxicity of MCYST-RR which is otherwise poorly understood till date.

Reproductive and endocrine-disrupting toxicity of Microcystis aeruginosa in female zebrafish.

Microcystis aeruginosa, a primary species in cyanobacterial blooms, is ubiquitously distributed in water. Microcystins (MCs) purified from M. aeruginosa can exert reproductive toxicity in fish. However, the effects of M. aeruginosa at environmentally relevant levels on the reproductive and endocrine systems of zebrafish are still unknown. The present study investigated the reproductive and endocrine-disrupting toxicity of M. aeruginosa on female zebrafish (Danio rerio) by short-term exposure (96 h). After exposure, marked histological lesions in the liver or gonads, such as nuclear pyknosis and deformation, were observed, and the fertilization rate and hatchability of eggs spawned from treated females were both significantly lower than they were in females in the control group, suggesting the possibility of transgenerational effects of M. aeruginosa exposure. Moreover, M. aeruginosa exposure decreased the concentration of 17ß-estradiol (E2) and testosterone (T) in female zebrafish. Interestingly, the vtg1 transcriptional level significantly decreased in the liver, whereas plasma vitellogenin (VTG) protein levels increased. The present findings indicate that M. aeruginosa could modulate endocrine function by disrupting transcription of hypothalamic-pituitary-gonadal-liver (HPGL) axis-related genes, and impair the reproductive capacity of female zebrafish, suggesting that M. aeruginosa causes potential adverse effects on fish reproduction in Microcystis bloom-contaminated aquatic environments.

Predicting blooms of toxic cyanobacteria in eutrophic lakes with diverse cyanobacterial communities.

We investigated possibility of predicting whether blooms, if they occur, would be formed of microcystin-producing cyanobacteria. DGGE analysis of 16S-ITS and mcyA genes revealed that only Planktothrix and Microcystis possessed mcy-genes and Planktothrix was the main microcystin producer. qPCR analysis revealed that the proportion of cells with mcy-genes in Planktothrix populations was almost 100%. Microcystin concentration correlated with the number of potentially toxic and total Planktothrix cells and the proportion of Planktothrix within all cyanobacteria, but not with the proportion of cells with mcy-genes in total Planktothrix. The share of Microcystis cells with mcy-genes was low and variable in time. Neither the number of mcy-possessing cells, nor the proportion of these cells in total Microcystis, correlated with the concentration of microcystins. This suggests that it is possible to predict whether the bloom in the Masurian Lakes will be toxic based on Planktothrix occurrence. Two species of toxin producing Planktothrix, P. agardhii and P. rubescens, were identified by phylogenetic analysis of 16S-ITS. Based on morphological and ecological features, the toxic Planktothrix was identified as P. agardhii. However, the very high proportion of cells with mcy-genes suggests P. rubescens. Our study reveals the need of universal primers for mcyA genes from environment.

Behaviors of Microcystis aeruginosa cells during floc storage in drinking water treatment process.

This is the first study to systematically investigate the different behaviors of Microcystis aeruginosa in the sludges formed by AlCl3, FeCl3, and polymeric aluminium ferric chloride (PAFC) coagulants during storage. Results show that the viability of Microcystis aeruginosa in PAFC sludge was stronger than that of cells in either AlCl3 or FeCl3 sludge after the same storage time, while the cells' viability in the latter two systems stayed at almost the same level. In AlCl3 and FeCl3 sludges high concentrations of Al and Fe were toxic to Microcystis aeruginosa, whereas in PAFC sludge low levels of Al showed little toxic effect on Microcystis aeruginosa growth and moderate amounts of Fe were beneficial to growth. The lysis of Microcystis aeruginosa in AlCl3 sludge was more serious than that in PAFC sludge, for the same storage time. Although the cell viability in FeCl3 sludge was low (similar to AlCl3 sludge), the Microcystis aeruginosa cells remained basically intact after 10 d storage (similar to PAFC sludge). The maintenance of cellular integrity in FeCl3 sludge might be due to the large floc size and high density, which had a protective effect for Microcystis aeruginosa.

Electronic tongue for microcystin screening in waters.

The potentiometric E-tongue system was employed for water toxicity estimation in terms of cyanobacterial microcystin toxins (MCs) detection. The data obtained from E-tongue were correlated to the MCs content detected by the standard chromatographic technique UHPLC-DAD (Ultra High Performance Liquid Chromatography with Diode Array Detector), as far as by the colorimetric enzymatic approach. The prediction of MCs released by toxic Microcystis aeruginosa strains was possible with Root Mean Squared Error of Validation (RMSEV) lower or very close to 1µg/L, the provisional guideline value of WHO for MCs content in potable waters. The application of E-tongue system opens up a new perspective offset for fast and inexpensive analysis in the field of environmental monitoring, offering also the possibility to distinguish toxin producing and non-toxic M. aeruginosa strains present in potable water.

Molecular Cloning and Expression Determination of p38 MAPK from the Liver and Kidney of Silver Carp.

The sequence of p38MAPK in silver carp (Hm-p38a) was cloned and sequenced. Additionally, the acute toxicity of crude microcystins (MCs) on silver carp and induction expression of Hm-p38a by MCs exposure were also determined in this study. The results reveal that the length of Hm-p38a is 2418 bp and it contains a 1086 bp open reading frame. Hm-p38a could encode 361 amino acids. Sequence analysis indicates that Hm-p38a contains the conserved structures of Thr-Gly-Tyr motif and substrate binding site Ala-Thr-Arg-Trp, and it is highly conserved in fish. Phylogenetic analysis reveals that Hm-p38a is more closely related to fish than mammals and it belongs to p38α subfamily. Moreover, Hm-p38a in silver carp is constitutively expressed in all examined tissues. In addition, our results indicate that MCs exposure significantly promotes the transcription of Hm-p38a in fish liver or kidney, suggesting that mitogen-activated protein kinase should also be the signal pathway of MCs hepatotoxicity in fish.

Potassium sensitivity differs among strains of the harmful cyanobacterium Microcystis and correlates with the presence of salt tolerance genes.

Microcystis aeruginosa is a ubiquitous harmful cyanobacterium that causes problems in eutrophic lakes. Potassium ion (K(+)) addition is one of the suggested methods to combat harmful cyanobacterial blooms. To investigate the effectiveness of this method, we compared the potassium ion sensitivity of four Microcystis strains. Microcystis strains PCC 7005 and NIES-843 were very susceptible to potassium ion concentrations of ∼ 12 mmol L(-1), whereas strain PCC 7806 and its non-toxic mutant PCC 7806 ΔmcyB were not affected by added potassium ions. The origin of the strain appears to be of importance. Strain PCC 7806 originates from brackish water and possesses genes for the synthesis of the compatible solute sucrose, the water channel protein gene aqpZ and the sodium influx gene nhaS2, whereas strains PCC 7005 and NIES-843 have a freshwater origin and lack these genes. We conclude that potassium ion addition will not be a successful mitigation strategy in brackish waters, but may temporarily suppress Microcystis blooms in freshwater lakes. However, in the long run other Microcystis strains or other cyanobacteria with a higher salt tolerance will likely take over. In addition, our results also have implications for the potassium ion concentrations of mineral media used in laboratory studies with cyanobacteria.

Microcystin mcyA and mcyE Gene Abundances Are Not Appropriate Indicators of Microcystin Concentrations in Lakes.

Cyanobacterial harmful algal blooms (cyanoHABs) are a primary source of water quality degradation in eutrophic lakes. The occurrence of cyanoHABs is ubiquitous and expected to increase with current climate and land use change scenarios. However, it is currently unknown what environmental parameters are important for indicating the presence of cyanoHAB toxins making them difficult to predict or even monitor on time-scales relevant to protecting public health. Using qPCR, we aimed to quantify genes within the microcystin operon (mcy) to determine which cyanobacterial taxa, and what percentage of the total cyanobacterial community, were responsible for microcystin production in four eutrophic lakes. We targeted Microcystis-16S, mcyA, and Microcystis, Planktothrix, and Anabaena-specific mcyE genes. We also measured microcystins and several biological, chemical, and physical parameters--such as temperature, lake stability, nutrients, pigments and cyanobacterial community composition (CCC)--to search for possible correlations to gene copy abundance and MC production. All four lakes contained Microcystis-mcyE genes and high percentages of toxic Microcystis, suggesting Microcystis was the dominant microcystin producer. However, all genes were highly variable temporally, and in few cases, correlated with increased temperature and nutrients as the summer progressed. Interestingly, toxin gene abundances (and biomass indicators) were anti-correlated with microcystin in all lakes except the largest lake, Lake Mendota. Similarly, gene abundance and microcystins differentially correlated to CCC in all lakes. Thus, we conclude that the presence of microcystin genes are not a useful tool for eliciting an ecological role for toxins in the environment, nor are microcystin genes (e.g. DNA) a good indicator of toxins in the environment.

Fast and direct extraction of cell-associated hepatotoxins from toxic cyanobacteria.

Microcystins are an important group of toxins produced by cyanobacteria of different genera. An increasing number of water contaminations by this class of toxins have been reported that are susceptible to generate important public health problems. We designed an efficient method for extracting these toxins on-site for a rapid testing of potentially contaminated water. The extraction parameters have been optimized using Microcystis aeruginosa and the technique successfully applied to different laboratory cultures and field samples. The procedure employs a simple and stable reagent mix of propanol, Tween 20, and trifluoroacetic acid. It is directly active on crude cell suspensions without any pre-treatment. Extraction yields measured by immunological quantification were at least equal to the best values obtained with the most commonly used laboratory techniques. An additional simple concentration/extraction step is also described that allows measurements on samples too dilute for direct detection by immunochromatographic techniques.

Genome-wide transcription profiles reveal genotype-dependent responses of biological pathways and gene-families in Daphnia exposed to single and mixed stressors.

The present study investigated the possibilities and limitations of implementing a genome-wide transcription-based approach that takes into account genetic and environmental variation to better understand the response of natural populations to stressors. When exposing two different Daphnia pulex genotypes (a cadmium-sensitive and a cadmium-tolerant one) to cadmium, the toxic cyanobacteria Microcystis aeruginosa, and their mixture, we found that observations at the transcriptomic level do not always explain observations at a higher level (growth, reproduction). For example, although cadmium elicited an adverse effect at the organismal level, almost no genes were differentially expressed after cadmium exposure. In addition, we identified oxidative stress and polyunsaturated fatty acid metabolism-related pathways, as well as trypsin and neurexin IV gene-families as candidates for the underlying causes of genotypic differences in tolerance to Microcystis. Furthermore, the whole-genome transcriptomic data of a stressor mixture allowed a better understanding of mixture responses by evaluating interactions between two stressors at the gene-expression level against the independent action baseline model. This approach has indicated that ubiquinone pathway and the MAPK serine-threonine protein kinase and collagens gene-families were enriched with genes showing an interactive effect in expression response to exposure to the mixture of the stressors, while transcription and translation-related pathways and gene-families were mostly related with genotypic differences in interactive responses to this mixture. Collectively, our results indicate that the methods we employed may improve further characterization of the possibilities and limitations of transcriptomics approaches in the adverse outcome pathway framework and in predictions of multistressor effects on natural populations.

Identification of pathways, gene networks, and paralogous gene families in Daphnia pulex responding to exposure to the toxic cyanobacterium Microcystis aeruginosa.

Although cyanobacteria produce a wide range of natural toxins that impact aquatic organisms, food webs, and water quality, the mechanisms of toxicity are still insufficiently understood. Here, we implemented a whole-genome expression microarray to identify pathways, gene networks, and paralogous gene families responsive to Microcystis stress in Daphnia pulex . Therefore, neonates of a sensitive isolate were given a diet contaminated with Microcystis to contrast with those given a control diet for 16 days. The microarray revealed 2247 differentially expressed (DE) genes (7.6% of the array) in response to Microcystis , of which 17% are lineage-specific (i.e., these genes have no detectable homology to any other gene in currently available databases) and 49% are gene duplicates (paralogues). We identified four pathways/gene networks and eight paralogous gene families affected by Microcystis . Differential regulation of the ribosome, including three paralogous gene families encoding 40S, 60S, and mitochondrial ribosomal proteins, suggests an impact of Microcystis on protein synthesis of D. pulex . In addition, differential regulation of the oxidative phosphorylation pathway (including the NADH:ubquinone oxidoreductase gene family) and the trypsin paralogous gene family (a major component of the digestive system in D. pulex ) could explain why fitness is reduced based on energy budget considerations.

Effect of Microcystis aeruginosa on the rotifer Brachionus calyciflorus at different temperatures.

Rotifers are one of the smallest metazoans. They serve as a model organism for ecotoxicological studies. More than 60% of the lakes in China are increasingly eutrophic and they are susceptible to blooms of Microcystis aeruginosa. We investigated the effects of M. aeruginosa on the survival and reproduction of Brachionus calyciflorus using the life table method at different temperatures. The findings showed that concentration of M. aeruginosa significantly affected the intrinsic rate of increase (r(m)), net reproductive rate (R(0)), average lifespan (L) and offspring number (p < 0.05). Temperature also significantly affected the generation time (T), average lifespan (L) and offspring number (p < 0.05). Moreover, the interaction between temperature and concentration had statistically significant effects on offspring number (p < 0.05). M. aeruginosa suppressed the survival and reproduction of B. calyciflorus, particularly at a concentration of 10(6) cells/mL. The r(m) values of the rotifers exposed at 10(6) cells/mL decreased more than 200% compared with those of the control group. However, at a lower concentration, 10(4) cells/mL, M. aeruginosa may supply appropriate nourishment to rotifers. In addition, at concentrations of 10(5) and 10(6) cells/mL, the inhibition of rotifers by M. aeruginosa heightened with increasing temperature.

High-frequency monitoring of the genetic diversity and the potential toxicity of a Microcystis aeruginosa bloom in a French shallow lake.

During cyanobacterial blooms, processes influencing the population dynamics of blooming species remain partially unexplained. To provide new information, we performed a high-frequency monitoring ­ every 2 days at six sampling points ­ of a Microcystis aeruginosa population blooming in a shallow lake. At each sampling date, there was no spatial heterogeneity in the ITS genotypic composition of the population and in the proportion of potentially microcystin- producing (mcyB+) cells, whereas high variations were recorded in cell abundances. In contrast, when looking at the temporal evolution of these parameters, the ITS genotypic composition of the population and in a lesser extent the percentage of mcyB+ cells displayed high variations during the growth phase of the bloom, but not during the plateau phase or the subsequent decline. This suggests that during the development of the bloom, there was no directional selection leading to the dominance of a restricted number of genotypes and that a balancing selection process permitted the maintenance of a high genetic diversity in the Microcystis population. Finally, no relationship was found between these variations occurring in the Microcystis population and those recorded for several environmental parameters, suggesting that many factors and processes interacting together might be involved in these variations.

Genotoxicity in Oreochromis niloticus (Cichlidae) induced by Microcystis spp bloom extract containing microcystins.

Studies of genotoxicity in fish caused by cyanobacterial extracts containing microcystins (MCs) can be useful in determining their carcinogenic risk due to a genotoxic mechanism. An extract of cyanobacterial Microcystis ssp, containing MC-LR and -LA from a bloom collected in a eutrophic lake, showed genotoxicity to Oreochromis niloticus. DNA damage (comet assay) was significantly induced in peripheral erythrocytes with both tested concentrations of 6.90 µg kg(-1) bw and 13.80 µg kg(-1) bw through intraperitoneal injection (ip). There was no micronucleus induction after ip injection at concentrations of 6.90 µg kg(-1) bw and 13.80 µg kg(-1) bw. Body exposure resulted in micronucleus induction and DNA damage only at the highest tested concentrations of 103.72 µg L(-1). Thus, comet assay and ip injection revealed the highest levels of the genotoxicity of MCs. Apoptosis-necrosis test carried out at concentrations of 6.90 µg kg(-1) bw and 13.80 µg kg(-1) bw revealed that at low concentrations more apoptosis than necrosis occurred. At higher concentrations more necrosis than apoptosis occurred.