Effects of Cell-Bound Microcystins on Survival and Feeding of Daphnia spp.

The influence of cell-bound microcystins on the survival time and feeding rates of six Daphnia clones belonging to five common species was studied. To do this, the effects of the microcystin-producing Microcystis strain PCC7806 and its mutant, which has been genetically engineered to knock out microcystin synthesis, were compared. Additionally, the relationship between microcystin ingestion rate by the Daphnia clones and Daphnia survival time was analyzed. Microcystins ingested with Microcystis cells were poisonous to all Daphnia clones tested. The median survival time of the animals was closely correlated to their microcystin ingestion rate. It was therefore suggested that differences in survival among Daphnia clones were due to variations in microcystin intake rather than due to differences in susceptibility to the toxins. The correlation between median survival time and microcystin ingestion rate could be described by a reciprocal power function. Feeding experiments showed that, independent of the occurrence of microcystins, cells of wild-type PCC7806 and its mutant are able to inhibit the feeding activity of Daphnia. Both variants of PCC7806 were thus ingested at low rates. In summary, our findings strongly suggest that (i) sensitivity to the toxic effect of cell-bound microcystins is typical for Daphnia spp., (ii) Daphnia spp. and clones may have a comparable sensitivity to microcystins ingested with food particles, (iii) Daphnia spp. may be unable to distinguish between microcystin-producing and -lacking cells, and (iv) the strength of the toxic effect can be predicted from the microcystin ingestion rate of the animals.     

Role of Microcystins in Poisoning and Food Ingestion Inhibition of Daphnia galeata Caused by the Cyanobacterium Microcystis aeruginosa

The effects of microcystins on Daphnia galeata, a typical filter-feeding grazer in eutrophic lakes, were investigated. To do this, the microcystin-producing wild-type strain Microcystis aeruginosa PCC7806 was compared with a mcy⁻ PCC7806 mutant, which could not synthesize any variant of microcystin due to mutation of a microcystin synthetase gene. The wild-type strain was found to be poisonous to D. galeata, whereas the mcy⁻ mutant did not have any lethal effect on the animals. Both variants of PCC7806 were able to reduce the Daphnia ingestion rate. Our results suggest that microcystins are the most likely cause of the daphnid poisoning observed when wild-type strain PCC7806 is fed to the animals, but these toxins are not responsible for inhibition of the ingestion process.     

Immunogold localisation of microcystins in cryosectioned cells of Microcystis

Insights into the origins, function(s), and fates of cyanobacterial toxins may be obtained by an understanding of their location within cyanobacterial cells. Here, we have localised microcystins in laboratory cultures of Microcystis PCC 7806 and PCC 7820 by immunogold labelling. Cryosectioning was used for immunoelectron microscopy since microcystins were extracted during the ethanol-based dehydration steps routinely used for sample preparation. Microcystins were specifically localised in the nucleoplasm and were associated with all major inclusions of the microcystin-producing strains Microcystis PCC 7806 (MC(+)) and Microcystis PCC 7820, and labelling was preferentially associated with the thylakoids and around polyphosphate bodies. A mutant strain of Microcystis PCC 7806 (MC(-)) which does not produce microcystins was used as a control. Distribution of total gold label within each cell region or associated with inclusions indicated that most of the cells' microcystin pool was associated with the thylakoids (69%, PCC 7806 (MC(+)); 78%, PCC 7820), followed by the nucleoplasmic region (19%, PCC 7806 (MC(+)); 12%, PCC 7820). Cryosectioning is a useful technique since it reduces the extraction of microcystins during sample preparation for electron microscopy.     

Iron uptake by toxic and nontoxic strains of Microcystis aeruginosa

Iron uptake by microcystin-producing and non-microcystin-producing strains of Microcystis aeruginosa was investigated through short-term uptake assays. Although strain-specific differences were observed, the siderophore-independent Fe uptake kinetics were essentially similar (e.g., maximum uptake rates of 2.0 to 3.3 amol·cell(-1)·h(-1)) for the wild-type toxic strain PCC7806 and a genetically engineered mutant unable to produce microcystin.     

A spontaneous mutant of microcystin biosynthesis: genetic characterization and effect on Daphnia

Microcystis aeruginosa strain MRC is unique in its' possession of the mcyA-J gene cluster, which encodes microcystin synthetase, but its' inability to produce microcystins. M. aeruginosa strain MRD is genetically identical to MRC at numerous genomic loci examined, but produces a variety of microcystins, mainly with the amino acid tyrosine in the molecule. Zooplankton studies with Daphnia galeata and D. pulicaria , using the mutant (MRC) and its' wild type (MRD), showed for the first time that microcystins other than microcystin-LR can be responsible for the poisoning of Daphnia by Microcystis . Regardless of microcystin content, both Daphnia exhibited significantly reduced ingestion rates when fed with either strain of M. aeruginosa compared with the green alga Scenedesmus acutus . A disruption of the molting process in both Daphnia spp. was noted when these species were fed with MRC cells. Such symptoms on Daphnia have not been previously reported for cyanobacteria and may point to a bioactive compound, other than microcystin, which inhibits the hardening of protein–chitin complexes in Daphnia .     

Functional analysis of PilT from the toxic cyanobacterium Microcystis aeruginosa PCC 7806

The evolution of the microcystin toxin gene cluster in phylogenetically distant cyanobacteria has been attributed to recombination, inactivation, and deletion events, although gene transfer may also be involved. Since the microcystin-producing Microcystis aeruginosa PCC 7806 is naturally transformable, we have initiated the characterization of its type IV pilus system, involved in DNA uptake in many bacteria, to provide a physiological focus for the influence of gene transfer in microcystin evolution. The type IV pilus genes pilA, pilB, pilC, and pilT were shown to be expressed in M. aeruginosa PCC 7806. The purified PilT protein yielded a maximal ATPase activity of 37.5 +/- 1.8 nmol P(i) min(-1) mg protein(-1), with a requirement for Mg(2+). Heterologous expression indicated that it could complement the pilT mutant of Pseudomonas aeruginosa, but not that of the cyanobacterium Synechocystis sp. strain PCC 6803, which was unexpected. Differences in two critical residues between the M. aeruginosa PCC 7806 PilT (7806 PilT) and the Synechocystis sp. strain PCC 6803 PilT proteins affected their theoretical structural models, which may explain the nonfunctionality of 7806 PilT in its cyanobacterial counterpart. Screening of the pilT gene in toxic and nontoxic strains of Microcystis was also performed.     

Evidence of the cost of the production of microcystins by Microcystis aeruginosa under differing light and nitrate environmental conditions

The cyanobacterium Microcystis aeruginosa is known to proliferate in freshwater ecosystems and to produce microcystins. It is now well established that much of the variability of bloom toxicity is due to differences in the relative proportions of microcystin-producing and non-microcystin-producing cells in cyanobacterial populations. In an attempt to elucidate changes in their relative proportions during cyanobacterial blooms, we compared the fitness of the microcystin-producing M. aeruginosa PCC 7806 strain (WT) to that of its non-microcystin-producing mutant (MT). We investigated the effects of two light intensities and of limiting and non-limiting nitrate concentrations on the growth of these strains in monoculture and co-culture experiments. We also monitored various physiological parameters, and microcystin production by the WT strain. In monoculture experiments, no significant difference was found between the growth rates or physiological characteristics of the two strains during the exponential growth phase. In contrast, the MT strain was found to dominate the WT strain in co-culture experiments under favorable growth conditions. Moreover, we also found an increase in the growth rate of the MT strain and in the cellular MC content of the WT strain. Our findings suggest that differences in the fitness of these two strains under optimum growth conditions were attributable to the cost to microcystin-producing cells of producing microcystins, and to the putative existence of cooperation processes involving direct interactions between these strains.     

A mannan binding lectin is involved in cell-cell attachment in a toxic strain of Microcystis aeruginosa

Microcystin, a hepatotoxin that represents a serious health risk for humans and livestock, is produced by the bloom-forming cyanobacterium Microcystis aeruginosa in freshwater bodies worldwide. Here we describe the discovery of a lectin, microvirin (MVN), in M. aeruginosa PCC7806 that shares 33% identity with the potent anti-HIV protein cyanovirin-N from Nostoc ellipsosporum. Carbohydrate microarrays were employed to demonstrate the high specificity of the protein for high-mannose structures containing alpha(1-->2) linked mannose residues. Lectin binding analyses and phenotypic characterizations of MVN-deficient mutants suggest that MVN is involved in cell-cell recognition and cell-cell attachment of Microcystis. A binding partner of MVN was identified in the lipopolysaccharide fraction of M. aeruginosa PCC7806. MVN is differentially expressed in mutants lacking the hepatotoxin microcystin. Additionally, MVN-deficient mutants contain much lower amounts of microcystin than the wild-type cells. We discuss a possible functional correlation between microcystin and the lectin and possible implications on Microcystis morphotype formation. This study provides the first experimental evidence that microcystins may have an impact on Microcystis colony formation that is highly important for the competitive advantage of Microcystis over other phytoplankton species.     

Effects of light on the microcystin content of Microcystis strain PCC 7806

Many cyanobacteria produce microcystins, hepatotoxic cyclic heptapeptides that can affect animals and humans. The effects of photosynthetically active radiation (PAR) on microcystin production by Microcystis strain PCC 7806 were studied in continuous cultures. Microcystis strain PCC 7806 was grown under PAR intensities between 10 and 403 micro mol of photons m(-2) s(-1) on a light-dark rhythm of 12 h -12 h. The microcystin concentration per cell, per unit biovolume and protein, was estimated under steady-state and transient-state conditions and on a diurnal timescale. The cellular microcystin content varied between 34.5 and 81.4 fg cell(-1) and was significantly positively correlated with growth rate under PAR-limited growth but not under PAR-saturated growth. Microcystin production and PAR showed a significant positive correlation under PAR-limited growth and a significant negative correlation under PAR-saturated growth. The microcystin concentration, as a ratio with respect to biovolume and protein, correlated neither with growth rate nor with PAR. Adaptation of microcystin production to a higher irradiance during transient states lasted for 5 days. During the period of illumination at a PAR of 10 and 40 micro mol of photons m(-2) s(-1), the intracellular microcystin content increased to values 10 to 20% higher than those at the end of the dark period. Extracellular (dissolved) microcystin concentrations were 20 times higher at 40 micro mol of photons m(-2) s(-1) than at 10 micro mol of photons m(-2) s(-1) and did not change significantly during the light-dark cycles at both irradiances. In summary, our results showed a positive effect of PAR on microcystin production and content of Microcystis strain PCC 7806 up to the point where the maximum growth rate is reached, while at higher irradiances the microcystin production is inhibited.     

Quantitative real-time PCR for determination of microcystin synthetase e copy numbers for microcystis and anabaena in lakes

Cyanobacterial mass occurrences in freshwater lakes are generally formed by Anabaena, Microcystis, and Planktothrix, which may produce cyclic heptapeptide hepatotoxins, microcystins. Thus far, identification of the most potent microcystin producer in a lake has not been possible due to a lack of quantitative methods. The aim of this study was to identify the microcystin-producing genera and to determine the copy numbers of microcystin synthetase gene E (mcyE) in Lake Tuusulanj?rvi and Lake Hiidenvesi in Finland by quantitative real-time PCR. The microcystin concentrations and cyanobacterial cell densities of these lakes were also determined. The microcystin concentrations correlated positively with the sum of Microcystis and Anabaena mcyE copy numbers from both Lake Tuusulanj?rvi and Lake Hiidenvesi, indicating that mcyE gene copy numbers can be used as surrogates for hepatotoxic Microcystis and ANABAENA: The main microcystin producer in Lake Tuusulanj?rvi was Microcystis spp., since average Microcystis mcyE copy numbers were >30 times more abundant than those of ANABAENA: Lake Hiidenvesi seemed to contain both nontoxic and toxic Anabaena as well as toxic Microcystis strains. Identifying the most potent microcystin producer in a lake could be valuable for designing lake restoration strategies, among other uses.     

Biotic factors in induced defence revisited: cell aggregate formation in the toxic cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> PCC 7806 is triggered by spent <Emphasis Type="Italic">Daphnia</Emphasis> medium and disrupted cells

Bioassays with the toxic cyanobacterium Microcystis aeruginosa PCC 7806, its non-toxic mutant ΔmcyB, and Daphnia magna as grazer were used to evaluate biotic factors in induced defence, in particular cyanobacterial and grazer-released info-chemicals. Three main questions were addressed in this study: Does Daphnia grazing lead to a loss of cyanobaterial biomass? Is the survival time of Daphnia shorter in a culture of the toxic cyanobacterium? Does direct grazing or the presence of spent Daphnia medium or a high number of disrupted toxic Microcystis cells in the assays lead to an increase in the cellular microcystin content in the remaining intact cells? The biovolume (growth) as well as size and abundance of Microcystis aggregates were determined by particle analysis, while the survival time of Daphnia individuals was recorded by daily observation and counting, with the relative concentration of cell-bound microcystin-LR, was measured by HPLC analysis. Compared to some recent studies in the field of induced defence, in this study, evidence was found for a direct grazing effect, i.e. the loss of biovolume in the toxic culture. In addition, Daphnia magna ingested more non-toxic than toxic cells, and survived longer with non-toxic cells. In terms of increased cell-bound toxin concentration as a means of defence reported in some studies, a higher cell-bound microcystin-LR content was not measured in this study in any of the treatments (P > 0.05). Under low light conditions with impaired growth of Microcystis, and the presence of a high number of particles with less than 1-μm diameter (possibly heterotrophic bacteria), Daphnia medium was associated with a strong reduction in cell-bound toxin concentration (P < 0.05). This study showed no increased cell aggregation under direct grazing (P > 0.05), but increased aggregation with spent Daphnia medium under high light conditions (P < 0.05). Further, the addition of cell-free extract from disrupted toxic Microcystis cells strongly increased the aggregation of the intact cells under low light (P < 0.05). These findings are discussed with the possible role of microcystin and other infochemicals in the expression of proteins and morphology changes in Microcystis.     

The abundance of microcystin-producing genotypes correlates positively with colony size in Microcystis sp. and determines its microcystin net production in Lake Wannsee

The working hypotheses tested on a natural population of Microcystis sp. in Lake Wannsee (Berlin, Germany) were that (i) the varying abundance of microcystin-producing genotypes versus non-microcystin-producing genotypes is a key factor for microcystin net production and (ii) the occurrence of a gene for microcystin net production is related to colony morphology, particularly colony size. To test these hypotheses, samples were fractionated by colony size with a sieving procedure during the summer of 2000. Each colony size class was analyzed for cell numbers, the proportion of microcystin-producing genotypes, and microcystin concentrations. The smallest size class of Microcystis colonies (<50 microm) showed the lowest proportion of microcystin-producing genotypes, the highest proportion of non-microcystin-producing cells, and the lowest microcystin cell quotas (sum of microcystins RR, YR, LR, and WR). In contrast, the larger size classes of Microcystis colonies (>100 microm) showed the highest proportion of microcystin-producing genotypes, the lowest proportion of non-microcystin-producing cells, and the highest microcystin cell quotas. The microcystin net production rate was nearly one to one positively related to the population growth rate for the larger colony size classes (>100 microm); however, no relationship could be found for the smaller size classes. It was concluded that the variations found in microcystin net production between colony size classes are chiefly due to differences in genotype composition and that the microcystin net production in the lake is mainly influenced by the abundance of the larger (>100- microm) microcystin-producing colonies.     

An extracellular glycoprotein is implicated in cell-cell contacts in the toxic cyanobacterium Microcystis aeruginosa PCC 7806

Microcystins are the most common cyanobacterial toxins found in freshwater lakes and reservoirs throughout the world. They are frequently produced by the unicellular, colonial cyanobacterium Microcystis aeruginosa; however, the role of the peptide for the producing organism is poorly understood. Differences in the cellular aggregation of M. aeruginosa PCC 7806 and a microcystin-deficient Delta mcyB mutant guided the discovery of a surface-exposed protein that shows increased abundance in PCC 7806 mutants deficient in microcystin production compared to the abundance of this protein in the wild type. Mass spectrometric and immunoblot analyses revealed that the protein, designated microcystin-related protein C (MrpC), is posttranslationally glycosylated, suggesting that it may be a potential target of a putative O-glycosyltransferase of the SPINDLY family encoded downstream of the mrpC gene. Immunofluorescence microscopy detected MrpC at the cell surface, suggesting an involvement of the protein in cellular interactions in strain PCC 7806. Further analyses of field samples of Microcystis demonstrated a strain-specific occurrence of MrpC possibly associated with distinct Microcystis colony types. Our results support the implication of microcystin in the colony specificity of and colony formation by Microcystis.     

藻类连续培养体系的构建与优化及其在产毒和无毒微囊藻竞争中的应用

利用发酵罐加装外置环形光源构建藻类连续培养系统, 以产毒微囊藻PCC 7806及其无毒突变株PCC 7806 mcyB–为培养材料, 通过对补料时间、接种密度和稀释率参数的优化, 获得最优培养条件, 并应用于产毒与无毒微囊藻的竞争实验中。通过优化得到连续培养的最优培养条件: 补料时间为第4天, 起始接种密度为4×106 cells/mL, 稀释率为0.15/d。在连续培养下, 光照为35 μmol/(m2·s)时, 以1﹕1的起始比例接种产毒与无毒微囊藻, 二者间的竞争会达到平衡, 并以无毒微囊藻占据优势, 且两者以不同的优势度长时间维持不变。在此基础上, 开展了不同光强对产毒与无毒微囊藻竞争影响的实验, 结果表明, 光强为35和80 μmol/(m2·s)时, 无毒株在连续培养中占据优势; 而光强为5和15 μmol/(m2·s)时, 无毒和产毒微囊藻维持起始接种比例不变。研究通过优化连续培养条件为室内藻类竞争实验提供了更为适宜的培养模式。     

Release of heptapeptide toxin (microcystin) during the decomposition process of Microcystis aeruginosa.

The decomposition process of toxic blue-green alga (cyanobacteria), Microcystis aeruginosa, under dark and aerobic condition was investigated in relation to the change of the amounts of heptapeptide toxins (microcystins YR and LR) by two experiments: one with Microcystis cells and the other with two purified microcystins. In the experiment with Microcystis cells, an increase of heterotrophic bacteria observed from the beginning of the experiment, was followed by decomposition of the algal cells and the subsequent release of microcystins into the filtrate fraction. The amounts of the toxins initially present in the cells were quantitatively detected in the filtrate fraction on the 35th day. The decomposition of microcystin YR began on the 42nd day. The decomposition rate of the two toxins was different. The decomposition rate of purified microcystins YR and LR, compared in distilled water and culture medium, respectively, indicated clearly that microcystin YR was more labile to decomposition than microcystin LR in the culture medium. At the end of the experiment (45th day) microcystin YR decreased to 58.6%, while 86.2% of microcystin LR remained.     

Reversal in competitive dominance of a toxic versus non-toxic cyanobacterium in response to rising CO2

Climate change scenarios predict a doubling of the atmospheric CO₂ concentration by the end of this century. Yet, how rising CO₂ will affect the species composition of aquatic microbial communities is still largely an open question. In this study, we develop a resource competition model to investigate competition for dissolved inorganic carbon in dense algal blooms. The model predicts how dynamic changes in carbon chemistry, pH and light conditions during bloom development feed back on competing phytoplankton species. We test the model predictions in chemostat experiments with monocultures and mixtures of a toxic and non-toxic strain of the freshwater cyanobacterium Microcystis aeruginosa. The toxic strain was able to reduce dissolved CO₂ to lower concentrations than the non-toxic strain, and became dominant in competition at low CO₂ levels. Conversely, the non-toxic strain could grow at lower light levels, and became dominant in competition at high CO₂ levels but low light availability. The model captured the observed reversal in competitive dominance, and was quantitatively in good agreement with the results of the competition experiments. To assess whether microcystins might have a role in this reversal of competitive dominance, we performed further competition experiments with the wild-type strain M. aeruginosa PCC 7806 and its mcyB mutant impaired in microcystin production. The microcystin-producing wild type had a strong selective advantage at low CO₂ levels but not at high CO₂ levels. Our results thus demonstrate both in theory and experiment that rising CO₂ levels can alter the community composition and toxicity of harmful algal blooms.     

蓝藻毒素的研究概况

综述了近20年来有关藻毒素检测技术、脱毒降毒方法、微囊藻毒素（MC）与环境因子关系、MC对水生生物的影响、合成机理、Microcystis aeruginosa PCC7806专题及其他相关研究的进展。国内在此领域上的研究还比较薄弱,且更多集中在宏观层面上。     

The involvement of α-proteobacteria Phenylobacterium in maintaining the dominance of toxic Microcystis blooms in Lake Taihu,China

Lake Taihu in China has suffered serious harmful cyanobacterial blooms for decades. The algal blooms threaten the ecological sustainability, drinking water safety, and human health. Although the roles of abiotic factors (such as water temperature and nutrient loading) in promoting Microcystis blooms have been well studied, the importance of biotic factors (e.g. bacterial community) in promoting and meditating Microcystis blooms remains unclear. In this study, we investigated the ecological dynamics of bacterial community, the ratio of toxic Microcystis, as well as microcystin in Lake Taihu. High-throughput 16S rRNA sequencing and principal component analysis (PCA) revealed that the bacteria community compositions (BCCs) clustered into three groups, the partitioning of which corresponded to that of groups according to the toxic profiles (the ratio of toxic Microcystis to total Microcystis, and the microcystin concentrations) of the samples. Further Spearman's correlation network showed that the α-proteobacteria Phenylobacterium strongly positively correlated with the toxic profiles. Subsequent laboratory chemostats experiments demonstrated that three Phenylobacterium strains promoted the dominance of the toxic Microcystis aeruginosa PCC7806 when co-culturing with the non-toxic PCC7806 mcyB⁻ mutant. Taken together, our data suggested that the α-proteobacteria Phenylobacterium may play a vital role in the maintenance of toxic Microcystis dominance in Lake Taihu.     

Recombination,cryptic clades and neutral molecular divergence of the microcystin synthetase (<Emphasis Type="Italic">mcy</Emphasis>) genes of toxic cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis>

Background  

The water-bloom-forming cyanobacterium Microcystis aeruginosa is a known producer of various kinds of toxic and bioactive chemicals. Of these, hepatotoxic cyclic heptapeptides microcystins have been studied most intensively due to increasing concerns for human health risks and environmental damage. More than 70 variants of microcystins are known, and a single microcystin synthetase (mcy) gene cluster consisting of 10 genes (mcyA to mcyJ) has been identified to be responsible for the production of all known variants of microcystins. Our previous multilocus sequence typing (MLST) analysis of the seven housekeeping genes indicated that microcystin-producing strains of M. aeruginosa are classified into two phylogenetic groups.