Effects of unicellular and colonial forms of toxic Microcystis aeruginosa from laboratory cultures and natural populations on tropical cladocerans

Three life-table experiments, two growth experiments and one feedinginhibition experiment, were performed to study the effects of the toxiccyanobacterium Microcystis aeruginosa on the cladoceransofa tropical lagoon (Jacarepaguá Lagoon, Rio de Janeiro, Brazil).Differentexperimental designs were used to estimate toxic effects of both field samplesand laboratory cultures of Microcystis aeruginosa oncladoceran life history parameters and juvenile growth rates. Effects ofnutritional deficiency could be distinguished from toxic effects in experimentswhere green algae in high carbon concentration were mixed withMicrocystis. Our results show that natural assemblages ofMicrocystis caused much less pronounced toxic effects thanlaboratory cultures and that unicellular forms were more toxic than colonialforms, even though both contained high concentrations of toxins. One possibleexplanation is that colonies were too large to be ingested by the smallMoina micrura and Ceriodaphniacornuta. Feeding inhibition by single cells and small colonies seemsto be another mechanism that contributes to the harmful effects ofMicrocystis on cladocerans, both in the laboratory and inthe field. Thus, caution is needed in extrapolating results from the laboratoryto the field. We did find, however, that toxic algae in natural seston caninhibit growth and reproduction of native cladocerans populations.     

Different tolerances and responses to low temperature and darkness between waterbloom forming cyanobacterium <Emphasis Type="Italic">Microcystis</Emphasis> and a green alga <Emphasis Type="Italic">Scenedesmus</Emphasis>

The dynamics of planktonic cyanobacteria in eutrophicated freshwaters play an important role in formation of annual summer blooms, yet overwintering mechanisms of these water bloom forming cyanobacteria remain unknown. The responses to darkness and low temperature of three strains (unicellular Microcystis aeruginosa FACHB-905, colonial M. aeruginosa FACHB-938, and a green alga Scenedesmus quadricauda FACHB-45) were investigated in the present study. After a 30-day incubation under darkness and low temperature, cell morphology, cell numbers, chlorophyll a, photosynthetic activity (ETR_max and I _k), and malodialdehyde (MDA) content exhibited significant changes in Scenedesmus. In contrast, Microcystis aeruginosa cells did not change markedly in morphology, chlorophyll a, photosynthetic activity, and MDA content. The stress caused by low temperature and darkness resulted in an increase of the antioxidative enzyme-catalase (CAT) in all three strains. When the three strains re-grew under routine cultivated condition subjected to darkness and low temperature, specific growth rate of Scenedesmus was lower than that of Microcystis. Flow cytometry (FCM) examination indicated that two distinct types of metabolic response to darkness and low temperature existed in the three strains. The results from the present study reveal that the cyanobacterium Microcystis, especially colonial Microcystis, has greater endurance and adaptation ability to the stress of darkness and low temperature than the green alga Scenedesmus. Handling editor: D. Hamilton     

Nutrient limitation of Microcystis aeruginosa in northern California Klamath River reservoirs

Nutrient limitations were investigated in Copco and Iron Gate Reservoirs, on the Klamath River in California, where blooms of the toxin-producing cyanobacterium Microcystis aeruginosa were first reported in 2005. Nutrient enrichment experiments conducted in situ in June and August, 2007 and 2008, determined responses in phytoplankton biomass, Microcystis abundance and microcystin concentration to additions of phosphorus and different forms of nitrogen (NH₄⁺, NO₃, and urea). Microcystis abundance was determined using quantitative PCR targeting the phycocyanin intergenic spacer cpcBA.Total phytoplankton biomass increased with additions of N both before and during Microcystis blooms, with no primary effects from P, suggesting overall N limitation for phytoplankton growth during the summer season. NH₄⁺ generally produced the greatest response in phytoplankton growth, while Microcystis abundance increased in response to all forms of N. Microcystis doubling time in the in situ experiments was 1.24–1.39 days when N was not limiting growth. The results from this study suggest availability of N during the summer is a key growth-limiting factor for the initiation and maintenance of toxic Microcystis blooms in Copco and Iron Gate Reservoirs in the Klamath River.     

Population dynamics and production of cladoceran zooplankton in the highly eutrophic Lake Kasumigaura

The growth rate, birth rate, death rate and production of the cladocera of Lake Kasumigaura were studied. Standing crop of zooplankton seemed to be governed by predation rather than food. Maximum productivity of cladocerans was observed in late August and early September. There were differences in production between sampling stations. The highest production was recorded in the most eutrophic basin, where heavy water blooms of Microcystis aeruginosa occurred. Maximum secondary production coincided with maximum primary production, which was mainly due to M. aeruginosa. Cladocerans probably utilize decomposed or decomposing Microcystis cells and bacteria in summer. Estimates of annual production of cladocerans varied from 4.2 to 13.1 g dry wt · m^–3, and annual P:B ratios ranged from 36 to 108. The production of cladocerans in Takahamairi Bay was 2.7% of gross primary production.     

Morphological and physiological changes in Microcystis aeruginosa as a result of interactions with heterotrophic bacteria

1. To reveal the role of aquatic heterotrophic bacteria in the process of development of Microcystis blooms in natural waters, we cocultured unicellular Microcystis aeruginosa with a natural Microcystis‐associated heterotrophic bacterial community. 2. Unicellular M. aeruginosa at different initial cell densities aggregated into colonies in the presence of heterotrophic bacteria, while axenic Microcystis continued to grow as single cells. The specific growth rate, the chl a content, the maximum electron transport rate (ETR_max) and the synthesis and secretion of extracellular polysaccharide (EPS) were higher in non‐axenic M. aeruginosa than in axenic M. aeruginosa after cell aggregation, whereas axenic and non‐axenic M. aeruginosa displayed the same physiological characteristic before aggregation. 3. Heterotrophic bacterial community composition was analysed by PCR–denaturing gradient gel electrophoresis (PCR–DGGE) fingerprinting. The biomass of heterotrophic bacteria strongly increased in the coinoculated cultures, but the DGGE banding patterns in coinoculated cultures were distinctly dissimilar to those in control cultures with only heterotrophic bacteria. Sequencing of DGGE bands suggested that Porphyrobacter, Flavobacteriaceae and one uncultured bacterium could be specialist bacteria responsible for the aggregation of M. aeruginosa. 4. The production of EPS in non‐axenic M. aeruginosa created microenvironments that probably served to link both cyanobacterial cells and their associated bacterial cells into mutually beneficial colonies. Microcystis colony formation facilitates the maintenance of high biomass for a long time, and the growth of heterotrophic bacteria was enhanced by EPS secretion from M. aeruginosa. 5. The results from our study suggest that natural heterotrophic bacterial communities have a role in the development of Microcystis blooms in natural waters. The mechanisms behind the changes of the bacterial community and interaction between cyanobacteria and heterotrophic bacteria need further investigations.     

Growth interactions among blue-green (Anabaena Oscillarioides,Microcystis aeruginosa) and green (Chlorella sp.) algae

The growth interactions amongst the blue-green algal species Anabaena oscillarioides, Microcystis aeruginosa and the green alga, Chlorella sp. were studied both in mixed cultures and in filter cultures separated by a membrane filter in the two arms of an interaction U-tube. The role of nutrients especially phosphate upon the interaction has also been studied. Anabaena and Microcystis both inhibited the growth of Chlorella while Microcystis also inhibited the growth of Anabaena. The inhibitory effect of Microcystis was found to be dependent on high concentrations of the initial algal inocula and independent of the initial concentration of nutrients such as inorganic phosphate, indicating that the nature of the inhibition is probably due to the production of inhibitory extracellular products by Microcystis. On the other hand, the inhibitory effect of Anabaena on Chlorella is the consequence of nutrient competition with Anabaena competing more effectively for the available phosphate.     

Strong effects of amoebae grazing on the biomass and genetic structure of a Microcystis bloom (Cyanobacteria)

Despite its importance for bloom toxicity, the factors determining the population structure of cyanobacterial blooms are poorly understood. Here, we report the results of a two‐year field survey of the population dynamics of Microcystis blooms in a small hypertrophic urban pond. Microscopic enumeration of Microcystis and its predators and parasites was combined with pigment and microcystin analysis and denaturing gradient gel electrophoresis of the ITS rDNA region to assess population dynamics and structure. Two main Microcystis morpho‐ and ITS types were revealed, corresponding to M. aeruginosa and M. viridis. In both years, high population densities of naked amoebae grazing on Microcystis coincided with rapid decreases in Microcystis biomass. In one year, there was a shift from heavily infested M. aeruginosa to the less‐infested M. viridis, allowing the bloom to rapidly recover. The preference of amoebae for M. aeruginosa was confirmed by grazing experiments, in which several amoeba strains were capable of grazing down a strain of M. aeruginosa, but not of M. viridis. Zooplankton and chytrid parasites appeared to be of minor importance for these strong and fast reductions in Microcystis biomass. These findings demonstrate a strong impact of small protozoan grazers on the biomass and genetic structure of Microcystis blooms.     

Molecular detection of potentially toxic cyanobacteria and their associated bacteria in lake water column and sediment

We investigated the molecular diversity of cyanobacteria and bacteria during a water bloom in a lake with a long history of toxic cyanobacterial blooms (Lake Kastoria, Greece). We also tested the hypothesis whether bloom-forming cyanobacteria are preserved in the lake’s sediment 2 years after the bloom. The dominant cyanobacteria during the bloom included the potentially toxin-producing Microcystis aeruginosa and several other Chroococcales forms closely related to the genus Microcystis. This suggests that the use of cyanobacterial-specific primers seems to be very informative in describing the cyanobacteria during the water blooms. The bacterial community showed high diversity, consisting mostly of singleton and doubleton phylotypes. The majority of the phylotypes were typical lake bacteria including some potential pathogens and toxin metabolising bacteria, suggesting that the dominant toxic cyanobacteria did not have any significant effect on the bacterial community structure. In the sediment, 2 years after the water bloom, no bloom-forming cyanobacteria were retrieved, suggesting that they cannot be preserved in the sediment. Similar to the water column, sediment bacterial diversity was also high, consisting mostly of yet-uncultured bacteria that are related to environments where organic matter degradation takes place.     

Interspecific protection against oxidative stress: green algae protect harmful cyanobacteria against hydrogen peroxide

Oceanographic studies have shown that heterotrophic bacteria can protect marine cyanobacteria against oxidative stress caused by hydrogen peroxide (H₂O₂). Could a similar interspecific protection play a role in freshwater ecosystems? In a series of laboratory experiments and two lake treatments, we demonstrate that freshwater cyanobacteria are sensitive to H₂O₂ but can be protected by less-sensitive species such as green algae. Our laboratory results show that green algae degrade H₂O₂ much faster than cyanobacteria. Consequently, the cyanobacterium Microcystis was able to survive at higher H₂O₂ concentrations in mixtures with the green alga Chlorella than in monoculture. Interestingly, even the lysate of destructed Chlorella was capable to protect Microcystis, indicating a two-component H₂O₂ degradation system in which Chlorella provided antioxidant enzymes and Microcystis the reductants. The level of interspecific protection provided to Microcystis depended on the density of Chlorella. These findings have implications for the mitigation of toxic cyanobacterial blooms, which threaten the water quality of many eutrophic lakes and reservoirs worldwide. In several lakes, H₂O₂ has been successfully applied to suppress cyanobacterial blooms. Our results demonstrate that high densities of green algae can interfere with these lake treatments, as they may rapidly degrade the added H₂O₂ and thereby protect the bloom-forming cyanobacteria.     

Seasonal variations of Microcystis populations in sediments of Lake Biwa, Japan

Seasonal variations of colony numbers of Microcystis aeruginosa(Kütz.) Kütz. and M. wesenbergii(Komárek) Komárek in N. V. Kondrat. in sediments of Lake Biwa were investigated over a period of 1 year. At two stations located in the shallow South Basin of Lake Biwa (ca. 4 m water depth), the colony number of Microcystisfluctuated seasonally. The number had a tendency to gradually decrease from winter to early summer, while it increased through mid-summer and autumn. Since the Microcystispopulation in sediment was rather small, intensive growth and accumulation in the water column should be important for the formation of Microcystisblooms in Lake Biwa. Microcystiscolonies in the sediment samples after June were observed to be floating in a counting chamber under a microscope. The observation suggests that the recruitment of Microcystis colonies into the water column mostly occurs in early summer. The number of Microcystiscolonies in the deep North Basin of Lake Biwa (70 – 90 m water depth) was larger than in the South Basin. Because the seasonal variation of colony numbers was not observed in the North Basin, and Microcystiscells do not have gas vesicles, these colonies will not return into the water column. The colonies isolated from the sediment of the North Basin were able to grow in cultured conditions, in the same way as those from the sediment of the South Basin. Therefore, Microcystiscolonies may survive for a long time under stable conditions of low temperature (ca. 8 °C) and darkness, in the sediment of the deep North Basin, accumulating gradually each year.     

Toxic Blue-green Algae Water Blooms Found in some Lakes in the German Democratic Republic

From 1977 to 1979 plankton samples were taken from 6 lakes in the German Democratic Republic (GDR) during water blooms and examined for their toxicity to homothermal animals. Microcystis aeruginosa, Aphanizomenon flos-aquae, Anabaena spiroides, and Oscillatoria redekei were dominant in the samples. With the exception of Oscillatoria redekei the algae tested had toxic effects on mice after intraperitoneal injection. The rate of survival of the test animals was particularly low when the algae were disintegrated by ultrasound or freeze-drying prior to injection, this indicating the endogenic character of the toxins. Water blooms of Microcystis aeruginosa taken from Lake Pehlitzsee (Eberswalde District) showed the highest toxicity with an LD₃₀ as high as 45 and 43.7 mg/kg, respectively. Injection of the lyophilized cells of Aphanizomenon flos-aquae brought about the same symptoms in the test animals as in the case of Microcystis, but the LD₃₀ was 200 mg/kg.     

Inhibition of phytoplankton species by co-culture with a fungus

Harmful algal blooms are a serious worldwide environmental problem. Algicidal microorganisms, especially bacteria, have attracted extensive attention as possible agents for inhibiting water blooms. Such capabilities, however, are rarely reported for fungi. In this work, a fungal strain, Trichaptum abietinum 1302BG, is studied for its capability to inhibit four phytoplankton species: Microcystis aeruginosa FACH-918, Microcystis flos-aquae FACH-1028, Oocystis borgei FACH-1108, and Microcystis aeruginosa PCC 7806. The results show that the chlorophyll-a from these phytoplankton species co-cultured with Trichaptum abietinum 1302BG decreases rapidly 24 h after inoculation, and phytoplankton cells almost completely disappear 48 h after incubation. These results suggest that the fungus strain has great potential for the degradation of phytoplankton species. To the best of our knowledge, this is the first report of a white-rot fungus that can inhibit harmful algae blooms species.     

Photochemical responses of phytoplankton to rapid increasing‐temperature process

Phytoplankton is sensitive to rapidly increasing temperature in spring. However, studies on the effect of temperature on phytoplankton have mainly focused on constant temperatures. It is necessary to clarify the determining parameters of phytoplankton shifts during temperature increases, as temperatures are predicted to fluctuate more intensively and frequently in the future. In the study, we analyzed the responses of photosynthetic properties and growth in a cyanobacterium (Microcystis aeruginosa) and a green alga (Chlorella pyrenoidosa), the dominant species in Taihu, to rapid increasing‐temperature process in the laboratory and in the field. The results show that gradually increasing temperature inhibited photosynthesis and the growth of C. pyrenoidosa and had almost no effect on M. aeruginosa. Elevated increasing temperature range also had more significant effects on the photosynthetic properties and growth rates of C. pyrenoidosa than those of M. aeruginosa in the laboratory and in the field. All of these results suggest that the photosynthetic performance of M. aeruginosa is more suitable to gradually increasing temperature and relatively strong temperature variations than that of C. pyrenoidosa, which might partially contribute to Microcystis excluding Chlorella competitively in aquatic ecosystem. Our findings point out the possible importance of the rapid and dramatic increasing‐temperature process to the formation of cyanobacterial blooms.     

Molecular Response of the Bloom-Forming Cyanobacterium, Microcystis aeruginosa, to Phosphorus Limitation

Cyanobacteria blooms caused by species such as Microcystis have become commonplace in many freshwater ecosystems. Although phosphorus (P) typically limits the growth of freshwater phytoplankton populations, little is known regarding the molecular response of Microcystis to variation in P concentrations and sources. For this study, we examined genes involved in P acquisition in Microcystis including two high-affinity phosphate-binding proteins (pstS and sphX) and a putative alkaline phosphatase (phoX). Sequence analyses among ten clones of Microcystis aeruginosa and one clone of Microcystis wesenbergii indicates that these genes are present and conserved within the species, but perhaps not the genus, as phoX was not identified in M. wesenbergii. Experiments with clones of M. aeruginosa indicated that expression of these three genes was strongly upregulated (50- to 400-fold) under low inorganic P conditions and that the expression of phoX was correlated with alkaline phosphatase activity (p < 0.005). In contrast, cultures grown exclusively on high levels of organic phosphorus sources (adenosine 5′-monophosphate, β-glycerol phosphate, and d-glucose-6-phosphate) or under nitrogen-limited conditions displayed neither high levels of gene expression nor alkaline phosphatase activity. Since Microcystis dominates phytoplankton assemblages in summer when levels of inorganic P (P_i) are often low and/or dominate lakes with low P_i and high organic P, our findings suggest this cyanobacterium may rely on pstS, sphX, and phoX to efficiently transport P_i and exploit organic sources of P to form blooms.     

Effects of Calcium Levels on Colonial Aggregation and Buoyancy of <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis>

Colonial aggregation of Microcystis plays a key role in bloom formation. Limited studies have been reported about effects of environmental factors on the aggregation of Microcystis. Calcium is an important chemical element in water system. In this study, we investigated the effects of a low- (0.015 g l⁻¹) and a high-concentration of calcium (0.100 g l⁻¹) on the aggregation and buoyancy of a colonial strain M. aeruginosa XW01. Results show that compared to the low concentration of calcium, the high-calcium condition results in bigger colonial size, higher level of buoyancy and increased production of extracellular polysaccharides (EPS) of M. aeruginosa XW01. Increased production of EPS induced by the high-calcium concentration should contribute to the colonial aggregation and buoyancy of M. aeruginosa XW01. These results suggest that an increase in calcium concentration may be beneficial for Microcystis blooms occurring in a soft water lake.     

The Influence of Toxic and Filamentous Blue-green Algae on Feeding and Population Growth of the Rotifer Brachionus rubens

The effect of toxic and of filamentous blue-green algae on feeding and population growth of the rotifer Brachionus rubens was investigated in laboratory experiments. A toxic strain of Microcystis aeruginosa was ingested, but rotifers cultured with Microcystis died faster than nonfed controls. The rigid filaments of Cylindrospermopsis raciborskii were not ingested, reduced the ingestion of simultaneously offered algae, and depressed population growth rates. The soft filaments of Anabaena flos-aquae were ingested at a moderate rate, did not reduce ingestion of other algae, and were used as an additional food in population growth experiments.     

PCR primers for selective detection of intra-species variations in the bloom-forming cyanobacterium,Microcystis

Members of the cyanobacterial genus Microcystis commonly form blooms in eutrophic freshwater systems, and some produce cyclic heptapeptide hepatotoxins called microcystins, thereby often causing serious water management problems. Microcystis species were unified into the single Microcystis aeruginosa classification based on 16S rRNA gene sequences and DNA–DNA re-association experiments; however, the morphological features of the organisms differ in different culturing conditions. Here, we describe a new real-time quantitative PCR (qPCR) method of determining Microcystis intradiversity using the SYBR Green I assay. We analyzed 71 Microcystis 16S-23S rDNA internal transcribed spacer region (16S-23S ITS) sequences, designed three group-specific PCR primers that successfully selected a morphologically M. wesenbergii-like non-toxic group (Group-3), and differentiated between M. viridis-like toxic group (Group-4) and M. aeruginosa-like Group-1 organisms including toxic and non-toxic Microcystis strains. The primers covered 76% of the Microcystis 16S-23S ITS regions from all over the world (six continents) included in GenBank. We constructed a mixed culture with representative Microcystis strains from each group, and estimated their cell densities by qPCR over 7 weeks. Group-1 and Group-3 grew exponentially for 4 weeks; however, the growth of Group-4 declined after 2 weeks, revealing different growth properties for the Microcystis groups in the mixed culture. Finally, we applied this method to natural Microcystis blooms at four freshwater sites, and found the dominance of Group-1 in three blooms and of Group-3 in one bloom, thereby showing the geographically uneven distribution of Microcystis genotypes. The developed qPCR technique targeting the 16S-23S ITS region is both rapid and simple and is useful for selective quantification of group variations among sympatric Microcystis genotypes, such as in mixed cultures and the natural environment.     

Effect of nutrient availability on the C, N, and P elemental ratios in the cyanobacterium Microcystis aeruginosa

To clarify whether nutrients limit the growth of Microcystis aeruginosa (Kütz.) Kütz during the growing season in Lake Yogo, we examined the cellular ratios of carbon (C), nitrogen (N), and phosphorus (P) in the populations of M. aeruginosa from August to December 2001. We also measured cellular C, N, and P ratios of M. aeruginosa under batch culture conditions. The cellular levels of N and P of M. aeruginosa in natural population changed more than twofold. The atomic N: C ratio of natural populations of Microcystis fluctuated from 0.11 to 0.26. The atomic P: C ratio fluctuated from 0.0080 to 0.024. The N: C, P: C, and N: P ratios of exponentially growing M. aeruginosa in N-and P-rich medium were 0.19, 0.013, and 15 on average. The growth of M. aeruginosa was suppressed below the N: C ratio of 0.13 under the N-free condition and below the P: C ratio of 0.0026 in the P-free condition. In the natural population, the N: C ratio was low on August 1-2 (0.11) and the P: C ratio was low (less than 0.011) until September. The Microcystis population on August 1-2 was N limited, judging from the results of the culture experiment. In other periods, the population seemed to be supplied with a sufficient amount of N. Although the P: C ratio was low (approximately 0.01) during August and September, it was several times larger than the value of the reduction of growth rate that occurred in culture. P limitation did not occur during the study period. N became more of a limiting factor than P for the formation of blooms of Microcystis. No blooms were observed in August and September, in spite of the increase of cellular levels of N. The formation of Microcystis blooms in Lake Yogo seems to be affected by artificial manipulations such as pumping from Lake Biwa and outflow.     

The effect of temperature on growth characteristics and competitions of <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and <Emphasis Type="Italic">Oscillatoria mougeotii</Emphasis> in a shallow,eutrophic lake simulator system

Blue-green algal blooms formed by Microcystis and Oscillatoria often occur in shallow eutrophic lakes, such as Lake Taihu (China) and Lake Kasumigaura (Japan). Growth characteristics and competitions between Microcystis aeruginosa and Oscillatoria mougeotii were investigated using lake simulator systems (microcosms) at various temperatures. Oscillatoria was the superior competitor, which suppressed Microcystis, when temperature was <20°C, whereas the opposite phenomenon occurred at 30°C. Oscillatoria had a long exponential phase (20 day) and a low growth rate of 0.22 day⁻¹ and 0.20 day⁻¹ at 15°C and 20°C, respectively, whereas Microcystis had a shorter exponential phase (2–3 days) at 30°C and a higher growth rate (0.86 day⁻¹). Interactions between the algae were stronger and more complex in the lake simulator system than flask systems. Algal growth in the lake simulator system was susceptible to light attenuation and pH change, and algae biomasses were lower than those in flasks. The outcome of competition between Microcystis and Oscillatoria at different temperatures agrees with field observations of algal communities in Lake Taihu, indicating that temperature is a significant factor affecting competition between Microcystis and Oscillatoria in shallow, eutrophic lakes.