The Microcystis-microbiome interactions: origins of the colonial lifestyle.

Species of the Microcystis genus are the most common bloom-forming toxic cyanobacteria worldwide. They belong to a clade of unicellular cyanobacteria whose ability to reach high biomasses during blooms is linked to the formation of colonies. Colonial lifestyle provides several advantages under stressing conditions of light intensity, ultraviolet light, toxic substances and grazing. The progression from a single-celled organism to multicellularity in Microcystis has usually been interpreted as individual phenotypic responses of the cyanobacterial cells to the environment. Here, we synthesize current knowledge about Microcystis colonial lifestyle and its role in the organism ecology. We then briefly review the available information on Microcystis microbiome and propose that changes leading from single cells to colonies are the consequence of specific and tightly regulated signals between the cyanobacterium and its microbiome through a biofilm-like mechanism. The resulting colony is a multi-specific community of interdependent microorganisms.

Effect of hydrological modification on the potential toxicity of Microcystis aeruginosa complex in Salto Grande reservoir, Uruguay.

It is widely known that the environmental conditions caused by the construction of reservoirs favor the proliferation of toxic cyanobacteria and the formation of blooms due to the high residence time of the water, low turbidity, temperature regimes, among others. Microcystin-producing cyanobacteria such as those from the Microcystis aeruginosa complex (MAC) are the most frequently found organisms in reservoirs worldwide, being the role of the environment on microcystin production poorly understood. Here, we addressed the community dynamics and potential toxicity of MAC cyanobacteria in a subtropical reservoir (Salto Grande) located in the low Uruguay river. Samples were taken from five different sites (upstream, inside the reservoir and downstream) during contrasting seasons (summer and winter) to analyze: (i) the MAC community structure by amplicon sequencing of the phycocyanin gene spacer, (ii) the genotype diversity of microcystin-producing MAC by high resolution melting analysis of the mcyJ gene, and (iii) the abundance and mcy transcription activity of the microcystin-producing (toxic) fraction. We found that MAC diversity decreased from summer to winter but, despite the observed changes in MAC community structure, the abundance of toxic organisms and the transcription of mcy genes were always higher inside the reservoir, regardless of the season. Two different genotypes of toxic MAC were detected inside the reservoir, one associated with low water temperature (15 °C) and one thriving at high water temperature (31 °C). These findings indicate that the environmental conditions inside the reservoir reduce community diversity while promoting the proliferation of toxic genotypes that actively transcribe mcy genes, whose relative abundance will depend on the water temperature.

The Effect of Agglomeration on Arsenic Adsorption Using Iron Oxide Nanoparticles.

The presence of arsenic in groundwater and other drinking water sources presents a notable public health concern. Although the utilization of iron oxide nanomaterials as arsenic adsorbents has shown promising results in batch experiments, few have succeeded in using nanomaterials in filter setups. In this study, the performance of nanomaterials, supported on sand, was first compared for arsenic adsorption by conducting continuous flow experiments. Iron oxide nanoparticles (IONPs) were prepared with different synthetic methodologies to control the degree of agglomeration. IONPs were prepared by thermal decomposition or coprecipitation and compared with commercially available IONPs. Electron microscopy was used to characterize the degree of agglomeration of the pristine materials after deposition onto the sand. The column experiments showed that IONPs that presented less agglomeration and were well dispersed over the sand had a tendency to be released during water treatment. To overcome this implementation challenge, we proposed the use of clusters of iron oxide nanoparticles (cIONPs), synthesized by a solvothermal methodology, which was explored. An isotherm experiment was also conducted to determine the arsenic adsorption capacities of the iron oxide nanomaterials. cIONPs showed higher adsorption capacities (121.4 mg/g) than the other IONPs (11.1, 6.6, and 0.6 mg/g for thermal decomposition, coprecipitation, and commercially available IONPs, respectively), without the implementation issues presented by IONPs. Our results show that the use of clusters of nanoparticles of other compositions opens up the possibilities for multiple water remediation applications.

Genotyping and Multivariate Regression Trees Reveal Ecological Diversification within the Microcystis aeruginosa Complex along a Wide Environmental Gradient.

Addressing the ecological and evolutionary processes underlying biodiversity patterns is essential to identify the mechanisms shaping community structure and function. In bacteria, the formation of new ecologically distinct populations (ecotypes) is proposed as one of the main drivers of diversification. New ecotypes arise when mutations in key functional genes or acquisition of new metabolic pathways by horizontal gene transfer allow the population to exploit new resources, permitting their coexistence with the parental population. We previously reported the presence of microcystin-producing organisms of the Microcystis aeruginosa complex (toxic MAC) through an 800-km environmental gradient ranging from freshwater to estuarine-marine waters in South America. We hypothesize that the success of toxic MAC in such a gradient is due to the existence of very closely related populations that are ecologically distinct (ecotypes), each specialized to a specific arrangement of environmental variables. Here, we analyzed toxic MAC genetic diversity through quantitative PCR (qPCR) and high-resolution melting analysis (HRMA) of a functional gene (mcyJ, microcystin synthetase cluster). We explored the variability of the mcyJ gene along the environmental gradient by multivariate classification and regression trees (mCART). Six groups of mcyJ genotypes were distinguished and associated with different combinations of water temperature, conductivity, and turbidity. We propose that each mcyJ variant associated with a defined environmental condition is an ecotype (or species) whose relative abundances vary according to their fitness in the local environment. This mechanism would explain the success of toxic MAC in such a wide array of environmental conditions. IMPORTANCE Organisms of the Microcystis aeruginosa complex form harmful algal blooms (HABs) in nutrient-rich water bodies worldwide. MAC HABs are difficult to manage owing to the production of potent toxins (microcystins) that resist water treatment. In addition, the role of microcystins in the ecology of MAC organisms is still elusive, meaning that the environmental conditions driving the toxicity of the bloom are not clear. Furthermore, the lack of coherence between morphology-based and genomic-based species classification makes it difficult to draw sound conclusions about when and where each member species of the MAC will dominate the bloom. Here, we propose that the diversification process and success of toxic MAC in a wide range of water bodies involves the generation of ecotypes, each specialized in a particular niche, whose relative abundance varies according to its fitness in the local environment. This knowledge can improve the generation of accurate prediction models of MAC growth and toxicity, helping to prevent human and animal intoxication.

Rapid freshwater discharge on the coastal ocean as a mean of long distance spreading of an unprecedented toxic cyanobacteria bloom.

Cyanobacterial toxic blooms are a worldwide problem. The Río de la Plata (RdlP) basin makes up about one fourth of South America areal surface, second only to the Amazonian. Intensive agro-industrial land use and the construction of dams have led to generalized eutrophication of main tributaries and increased the intensity and duration of cyanobacteria blooms. Here we analyse the evolution of an exceptional bloom at the low RdlP basin and Atlantic coast during the summer of 2019. A large array of biological, genetic, meteorological, oceanographic and satellite data is combined to discuss the driving mechanisms. The bloom covered the whole stripe of the RdlP estuary and the Uruguayan Atlantic coasts (around 500 km) for approximately 4 months. It was caused by the Microcystis aeruginosa complex (MAC), which produces hepatotoxins (microcystin). Extreme precipitation in the upstream regions of Uruguay and Negro rivers' basins caused high water flows and discharges. The evolution of meteorological and oceanographic conditions as well as the similarity of organisms' traits in the affected area suggest that the bloom originated in eutrophic reservoirs at the lower RdlP basin, Salto Grande in the Uruguay river, and Negro river reservoirs. High temperatures and weak Eastern winds prompted the rapid dispersion of the bloom over the freshwater plume along the RdlP northern and Atlantic coasts. The long-distance rapid drift allowed active MAC organisms to inoculate freshwater bodies from the Atlantic basin, impacting environments relevant for biodiversity conservation. Climate projections for the RdlP basin suggest an increase in precipitation and river water flux, which, in conjunction with agriculture intensification and dams' construction, might turn this extraordinary event into an ordinary situation.

Morphology captures toxicity in Microcystis aeruginosa complex: Evidence from a wide environmental gradient✰.

Blooms of the Microcystis aeruginosa complex (MAC) consist of mixtures of toxin-producing and non-toxin-producing populations, but the environmental conditions that determine their relative abundance and shift are not clear. Morphological traits reflect the responses of MAC organisms to environmental changes, thus they could be useful to improve the predictability of the abundance of both toxic and nontoxic populations. In this work, the response of MAC toxic populations to environmental conditions and their relationship with morphology (size of organisms) were investigated in different water bodies (reservoir, river, and estuary) covering wide salinity (0-33) and temperature (10-36 °C) gradients. Sub-surface water samples were collected and divided into 4 size classes (mesh size 〈20 µm, 20-60 µm, 60-150 µm and〉 150 µm) and three toxicity proxies were assessed (mcyE gene and transcripts copy numbers and microcystin concentration) for each size-class. For all the size-classes, the logarithm of the number of mcyE gene copies per sample was proportional to the logarithm of the corresponding biovolume fraction, showing that MAC biovolume is a good indicator of toxicity potential. When toxicity was analyzed through mcyE transcript abundance and microcystin concentration, the largest size fraction (>150 µm) showed the highest toxicity values of both proxies. Nevertheless, mcyE transcription and toxin production per cell were higher in the colonies retained in the 60 to 150 µm size fractions, followed by single cells (<20 µm). At the reservoir, where environmental variability is low, the total abundance of mcyE gene copies was significantly explained by MAC biovolume, regardless of the environmental conditions. However, when data from the reservoir to the estuary were modeled, biovolume and temperature (with a minor contribution of salinity and wind intensity) were selected in the best models. According to these results, the size distribution of MAC biovolume appears as a good predictor of active toxin production, being the colonies in the 60-150 µm size fraction good indicators of higher toxicity. These results can be used to predict MAC toxicity based on the size structure of the community.

A multilevel trait-based approach to the ecological performance of Microcystis aeruginosa complex from headwaters to the ocean.

The Microcystis aeruginosa complex (MAC) clusters cosmopolitan and conspicuous harmful bloom-forming cyanobacteria able to produce cyanotoxins. It is hypothesized that low temperatures and brackish salinities are the main barriers to MAC proliferation. Here, patterns at multiple levels of organization irrespective of taxonomic identity (i.e. a trait-based approach) were analyzed. MAC responses from the intracellular (e.g. respiratory activity) to the ecosystem level (e.g. blooms) were evaluated in wide environmental gradients. Experimental results on buoyancy and respiratory activity in response to increased salinity (0-35) and a literature review of maximum growth rates under different temperatures and salinities were combined with field sampling from headwaters (800km upstream) to the marine end of the Rio de la Plata estuary (Uruguay-South America). Salinity and temperature were the major variables affecting MAC responses. Experimentally, freshwater MAC cells remained active for 24h in brackish waters (salinity=15) while colonies increased their flotation velocity. At the population level, maximum growth rate decreased with salinity and presented a unimodal exponential response with temperature, showing an optimum at 27.5°C and a rapid decrease thereafter. At the community and ecosystem levels, MAC occurred from fresh to marine waters (salinity 30) with a sustained relative increase of large mucilaginous colonies biovolume with respect to individual cells. Similarly, total biomass and, specific and morphological richness decreased with salinity while blooms were only detected in freshwater both at high (33°C) and low (11°C) temperatures. In brackish waters, large mucilaginous colonies presented advantages under osmotic restrictive conditions. These traits values have also been associated with higher toxicity potential. This suggest salinity or low temperatures would not represent effective barriers for the survival and transport of potentially toxic MAC under likely near future scenarios of increasing human impacts (i.e. eutrophication, dam construction and climate change).

Dynamics of toxic genotypes of Microcystis aeruginosa complex (MAC) through a wide freshwater to marine environmental gradient.

Bloom-forming species belonging to Microcystis aeruginosa complex (MAC) are the most commonly reported worldwide. MAC blooms are composed by toxic and non-toxic genotypes and the environmental conditions favouring the dominance of toxic genotypes are still a matter of debate among the scientific community. In this study, we evaluated the distribution of toxic MAC genotypes along a seasonal cycle and over an environmental gradient spanning 800km, from a eutrophic freshwater reservoir in Río Uruguay to marine water in the outer limit of Río de la Plata. Abundance of four mcy genes, mcyB, mcyD, mcyE and mcyJ was determined by qPCR and used as a proxy of abundance of toxic MAC genotypes. All the mcy genes were detected through the seasonal cycle at all sampling sites, being systematically higher in the freshwater reservoir and decreasing towards the marine site. The highest toxic genotype abundance was found during the austral summer months. According to generalized linear regressions and random forest models, temperature and conductivity were the most relevant explanatory variables. This suggests that although toxic MAC genotypes grow optimally in freshwater, they are also able to tolerate the high-salinity and low temperature conditions found in estuarine and marine waters. This ability to resist harsh conditions impose a health risk and a management challenge. To our knowledge, this is the first report addressing several mcy genes in a broad gradient that includes a wide array of different environmental conditions.

Application of ancient DNA to the reconstruction of past microbial assemblages and for the detection of toxic cyanobacteria in subtropical freshwater ecosystems.

Ancient DNA (aDNA) analysis of lake sediments is a promising tool for detecting shifts in past microbial assemblages in response to changing environmental conditions. We examined sediment core samples from subtropical, freshwater Laguna Blanca (Uruguay), which has been severely affected by cultural eutrophication since 1960 and where cyanobacterial blooms, particularly those of the saxitoxin-producer Cylindrospermopsis raciborskii, have been reported since the 1990s. Samples corresponding to ~1846, 1852, 2000 and 2007 AD were selected to perform denaturing gradient gel electrophoresis (DGGE) analysis of the 16S-23S rRNA intergenic transcribed spacer (ribosomal ITS) to compare their prokaryotic assemblage composition. Each stratum showed different ITS patterns, but the composition of 21st century samples was clearly different than those of mid-19th century. This compositional change was correlated with shifts in sediment organic matter and chlorophyll a content, which were significantly higher in recent samples. The presence of saxitoxin-producing cyanobacteria was addressed by quantitative real-time PCR of the sxtU gene involved in toxin biosynthesis. This gene was present only in recent samples, for which clone libraries and ITS sequencing indicated the presence of Cyanobacteria. Phylogenetic analyses identified C. raciborskii only in the 2000 sample, shortly after several years when blooms were recorded in the lake. These data suggest the utility of aDNA for the reconstruction of microbial assemblage shifts in subtropical lakes, at least on centennial scales. The application of aDNA analysis to genes involved in cyanotoxin synthesis extends the applicability of molecular techniques in palaeolimnological studies to include key microbial community characteristics of great scientific and social interest.

Size-dependent impacts of silver nanoparticles on the lifespan, fertility, growth, and locomotion of Caenorhabditis elegans.

The increased bioavailability of nanoparticles engineered for good dispersion in water may have biological and environmental impacts. To examine this issue, the authors assessed the biological effects in nematodes as they relate to exposure to silver nanoparticles (AgNPs) of different sizes at low (1 mg/L Ag), medium (10 mg/L Ag), and high concentrations (100 mg/L Ag). Over multiple generations, the authors found that the smallest particle, at 2 nm, had a notable impact on nematode fertility. In contrast, the largest particle, at 10 nm, significantly reduced the lifespan of parent nematodes (P0 ) by 28.8% and over the span of 3 generations (F1 -F3). In addition, a computer vision system automatically measured the adverse effects in body length and motility, which were not size-dependent.

Toxicity of quantum dots and cadmium salt to Caenorhabditis elegans after multigenerational exposure.

To fully understand the biological and environmental impacts of nanomaterials requires studies that address both sublethal end points and multigenerational effects. Here, we use a nematode to examine these issues as they relate to exposure to two different types of quantum dots, core (CdSe) and core-shell (CdSe/ZnS), and to compare the effect to those observed after cadmium salt exposures. The strong fluorescence of the core-shell QDs allowed for the direct visualization of the materials in the digestive track within a few hours of exposure. Multiple end points, including both developmental and locomotive, were examined at QD exposures of low (10 mg/L Cd), medium (50 mg/L Cd), and high concentrations (100 mg/L Cd). While the core-shell QDs showed no effect on fitness (lifespan, fertility, growth, and three parameters of motility behavior), the core QDs caused acute effects similar to those found for cadmium salts, suggesting that biological effects may be attributed to cadmium leaching from the more soluble QDs. Over multiple generations, we commonly found that for lower life-cycle exposures to core QDs the parents response was generally a poor predictor of the effects on progeny. At the highest concentrations, however, biological effects found for the first generation were commonly similar in magnitude to those found in future generations.