Culture-Independent Study of the Late-Stage of a Bloom of the Toxic Dinoflagellate Ostreopsis cf. ovata: Preliminary Findings Suggest Genetic Differences at the Sub-Species Level and Allow ITS2 Structure Characterization.

Available genomic data for the toxic, bloom-forming, benthic Ostreopsis spp. are traditionally obtained from isolates rather than from individuals originally present in environmental samples. Samples from the final phase of the first reported Ostreopsis bloom in European North Atlantic waters (Algarve, south coast of Portugal) were studied and characterized, using a culture-independent approach. In the first instance, a microscopy-based analysis revealed the intricate complexity of the samples. Then, we evaluated the adequacy of commonly used molecular tools (i.e., primers and nuclear ribosomal markers) for the study of Ostreopsis diversity in natural samples. A PCR-based methodology previously developed to identify/detect common Ostreopsis species was tested, including one new combination of existing PCR primers. Two sets of environmental rRNA sequences were obtained, one of them (1052 bp) with the newly tested primer set. These latter sequences encompass both the ITS1-5.8S-ITS2 region and the D1/D2 domain of the LSU rRNA gene, leading us to an accurate identification of ITS2. In turn, this allowed us to predict and show for the first time the ITS2 secondary structure of Ostreopsis. With 92 bp in length and a two-helix structure, the ITS2 of this genus revealed to be unique among the dinoflagellates. Both the PCR approach as the phylogenetic analyses allowed to place the Ostreopsis cells observed in the samples within the O. cf. ovata phylospecies' complex, discarding the presence of O. cf. siamensis. The (phylo)genetic results point out a certain level of nucleotide sequence divergence, but were inconclusive in relation to a possible geographic origin of the O. cf. ovata population from the Algarve's bloom.

Microbial community changes elicited by exposure to cyanobacterial allelochemicals.

An increasing body of evidence points out that allelopathy may be an important process shaping microbial communities in aquatic ecosystems. Cyanobacteria have well-documented allelopathic properties, mainly derived from the evaluation of the activity of allelopathic extracts or pure compounds towards monocultures of selected target microorganisms. Consequently, little is known regarding the community dynamics of microorganisms associated with allelopathic interactions. In this laboratory-based study, a Microcystis spp.-dominated microbial community from a freshwater lake was exposed, for 15 days, to exudates from the cyanobacterium Oscillatoria sp. strain LEGE 05292 in laboratory conditions. This cyanobacterium is known to produce the allelochemicals portoamides, which were among the exuded compounds. The community composition was followed (by means of polymerase chain reaction followed by denaturing gradient gel electrophoresis and microscopic analyses) and compared to that of a non-exposed situation. Following exposure, clear differences in the community structure were observed, in particular for cyanobacteria and unicellular eukaryotic taxa. Interestingly, distinct Microcystis genotypes present in the community were differentially impacted by the exposure, highlighting the fine-scale dynamics elicited by the exudates. These results support a role for cyanobacterial allelochemicals in the structuring of aquatic microbial communities.

Seasonal dynamics of Microcystis spp. and their toxigenicity as assessed by qPCR in a temperate reservoir.

Blooms of toxic cyanobacteria are becoming increasingly frequent, mainly due to water quality degradation. This work applied qPCR as a tool for early warning of microcystin(MC)-producer cyanobacteria and risk assessment of water supplies. Specific marker genes for cyanobacteria, Microcystis and MC-producing Microcystis, were quantified to determine the genotypic composition of the natural Microcystis population. Correlations between limnological parameters, pH, water temperature, dissolved oxygen and conductivity and MC concentrations as well as Microcystis abundance were assessed. A negative significant correlation was observed between toxic (with mcy genes) to non-toxic (without mcy genes) genotypes ratio and the overall Microcystis density. The highest proportions of toxic Microcystis genotypes were found 4-6 weeks before and 8-10 weeks after the peak of the bloom, with the lowest being observed at its peak. These results suggest positive selection of non-toxic genotypes under favorable environmental growth conditions. Significant positive correlations could be found between quantity of toxic genotypes and MC concentration, suggesting that the method applied can be useful to predict potential MC toxicity risk. No significant correlation was found between the limnological parameters measured and MC concentrations or toxic genotypes proportions indicating that other abiotic and biotic factors should be governing MC production and toxic genotypes dynamics. The qPCR method here applied is useful to rapidly estimate the potential toxicity of environmental samples and so, it may contribute to the more efficient management of water use in eutrophic systems.

Effects of nitrate reduction on the eutrophication of an urban man-made lake (Palácio de Cristal, Porto, Portugal).

The monitoring of phytoplankton is an important component of water quality assessment in artificial and natural lakes. The evolution of eutrophication in an urban man-made lake (Palácio de Cristal, Porto, Portugal) was followed after remediation measures were applied. Nutrients as well as the phytoplankton community were monitored and the results compared with those of a previous study. Sampling was done in four sites of the lake so as to assess the efficiency of NO3 removal by the phytoplankton. The data of this study showed a reduction in NO3 levels in the underground source water, which caused a reduction in the phytoplankton abundance. The nitrate removal by the phytoplankton in the lake was on average 51% and was much lower compared with previous studies of the same lake. The dominant cyanobacteria species were Pseudanabaena spp., probably due to the low water residence time. Monitoring of potentially toxic cyanobacteria, by molecular methods based on the use of toxin gene primers, will be an efficient early warning method. This will allow the application of corrective management measures before an intense toxic bloom occurs.

Application of real-time PCR in the assessment of the toxic cyanobacterium Cylindrospermopsis raciborskii abundance and toxicological potential.

Cyanobacteria are prokaryotic photosynthetic microorganisms that pose a serious threat to aquatic environments because they are able to form blooms under eutrophic conditions and produce toxins. Cylindrospermopsis raciborskii is a planktonic heterocystous filamentous cyanobacterium initially assigned to the tropics but currently being found in more temperate regions such as Portugal, the southernmost record for this species in Europe. Cylindrospermopsin originally isolated from C. raciborskii is a cytotoxic alkaloid that affects the liver, kidney, and other organs. It has a great environmental impact associated with cattle mortality and human morbidity. Aiming in monitoring this cyanobacterium and its related toxin, a shallow pond located in the littoral center of Portugal, Vela Lake, used for agriculture and recreational purposes was monitored for a 2-year period. To accomplish this, we used the real-time PCR methodology in field samples to quantify the variation of specific genetic markers with primers previously described characterizing total cyanobacteria (16S rRNA), C. raciborskii (rpoC1), and cylindrospermopsin synthetase gene (pks). The results report the high abundance of both cyanobacteria and C. raciborskii in Vela Lake, with C. raciborskii representing 0.4% to 58% of the total cyanobacteria population. Cylindrospermopsin synthetase gene was detected in one of the samples. We believe that with the approach developed in this study, it will be possible to monitor C. raciborskii population dynamics and seasonal variation, as well as the potential toxin production in other aquatic environments.

Microcystins and cyanobacteria trends in a 14 year monitoring of a temperate eutrophic reservoir (Aguieira, Portugal).

A monitoring program of cyanobacteria and cyanotoxins in the framework of the surveillance of the Water Treatment Plant efficiency of the municipality of Santa Comba Dão (Portugal) was conducted from 1994 until 2007. With these data we aimed to answer the question, are MCs produced evenly over the years in a single water body? Samples were taken by the local health authorities in the site of the Water treatment plant and analyzed for total phytoplankton, cyanobacteria and the hepatotoxic cyanotoxins microcystins (MCs). Apart from 1999 and 2000, cyanobacteria represented a high proportion of total phytoplankton, attaining during several months of the year more than 90% of total phytoplankton density. A total of 24 cyanobacteria species were identified and Microcystis aeruginosa, Anabaena flos-aquae and Aphanizomenon flos-aquae were the main potentially toxic cyanobacteria species present throughout the period. MC concentration varied between 0.3 µg MC-LR eq l⁻¹ in October 98 and 87.0 µg MC-LR eq l⁻¹ in September 2001. The evolution of the average cell quota does not reveal any especial trend, although in 2001 the quota was the highest but not significantly different from the other years. The threshold limit of 5000 cells per ml of Microcystis aeruginosa should be taken into consideration in monitoring programs because the 10,000 cells per ml proposed by several other national programs might not be enough to prevent human health risks. Due to high annual variation in MC cell quota, monitoring programs of cyanobacteria and MC should be extended in time, since short term studies do not provide us the data needed for a safe management of a water body used for human purposes.

First report on the occurrence of microcystins in planktonic cyanobacteria from Central Mexico.

Although toxic cyanobacteria are commonly described worldwide, only one recent paper on the toxicity of cyanobacteria in Mexico has been published reporting the occurrence of cylindrospermopsin and saxitoxins. Microcystins are the most commonly studied cyanotoxins in many countries and those that may cause the most dramatic problems in terms of human health. In this paper, we studied the occurrence of potentially toxic cyanobacteria in different ecosystems in Central Mexico. Samples were collected in natural lakes (Zumpango, Laguna Atotonilco and Cienega Chica), reservoirs (Los Angeles and Valle de Bravo), man-made channels (Cuemanco, Tlameleca) and urban lakes (Chapultepec). A multi-technique approach was applied by the use of molecular, immunological and chemical techniques. Cyanobacteria were found in all the sites, ranging from 1.6 x 10(3) cells/mL in Tlameleca to 7.5 x 10(6) cells/mL in Chapultepec Grande, representing between 67 and 100% of the total phytoplankton density. The concentration of total microcystins varied between 4.9 and 78.0 microg MC-LR eq/L. The results revealed the occurrence of MC-LR in all the sites analyzed by MALDI-TOF, and MC-FR, MC-RR and MC-H(4)YR in two sites. Most of the studied sites are used either as a source of drinking water, as a recreational area, or for agriculture irrigation purposes, so the risk for human health may be high if not properly monitored. This is the first report on the MC profiles and concentrations in blooms collected in Mexico.

Comparison of sensitivity of grasses (Lolium perenne L. and Festuca rubra L.) and lettuce (Lactuca sativa L.) exposed to water contaminated with microcystins.

The effects of aqueous extracts from Microcysts aeruginosa strains (both microcystin-producers and non-microcystin producers) on germination and root growth were investigated for three economically important plant species: Festuca rubra L., Lolium perenne L., and Lactuca sativa L. There was a clear inhibition of root growth for L. sativa exposed to strains containing microcystins (5.9-56.4 microg L(-1)). The strain that produced the most pronounced effects contained the lowest concentration of microcystin suggesting that other cellular compounds may also affect growth.

Molecular techniques for the early warning of toxic cyanobacteria blooms in freshwater lakes and rivers.

The aim of this work was to test the efficacy of molecular techniques for detecting toxigenic cyanobacteria in environmental water samples collected from freshwater lakes, rivers and reservoirs in Portugal. Of 26 environmental samples tested, 21 were found to contain Microcystis using a genus-specific polymerase chain reaction (PCR). Another primer pair was applied to the same DNA template to test for the presence of microcystin synthetase genes. This primer pair resulted in the formation of a PCR product in 15 of the samples containing Microcystis and one sample that did not give a positive result in the Microcystis genus-specific PCR. A restriction assay using the enzyme EcoRV was then applied to show that in most cases, the gene fragment was from toxigenic strains of Microcystis and, in one above-mentioned case, from a microcystin-producing strain of Planktothrix. All environmental samples were examined microscopically to confirm the presence of cyanobacteria species. Samples were also tested for the presence of microcystins using the ELISA plate assay. There was good agreement between the results obtained with molecular techniques and those obtained from microscopy and chemical methods. The PCR techniques applied in this paper were found to be useful, particularly when the concentration of the target organism was very low compared with other organisms. This technique can be used to detect inocula for cyanobacterial populations and therefore provide a useful tool for assessing under which conditions particular species can grow into bloom populations.