Eco-friendly management of harmful cyanobacterial blooms in eutrophic lakes through vertical flow multi-soil-layering technology.

Eutrophication has led to the widespread occurrence of cyanobacterial blooms. Toxic cyanobacterial blooms with high concentrations of microcystins (MCs) have been identified in the Lalla Takerkoust reservoir in Morocco. The objective of this study was to evaluate the efficiency of the Multi-Soil-Layering (MSL) ecotechnology in removing natural cyanobacterial blooms from the lake. Two MSL pilots were used in rectangular glass tanks (60 × 10 × 70 cm). They consisted of permeable layers (PLs) made of pozzolan and a soil mixture layer (SML) containing local soil, ferrous metal, charcoal and sawdust. The main difference between the two systems was the type of local soil used: sandy soil for MSL1 and clayey soil for MSL2. Both MSL pilots effectively reduced cyanobacterial cell concentrations in the treated water to very low levels (0.09 and 0.001 cells/mL). MSL1 showed a gradual improvement in MC removal from 52 % to 99 %, while MSL2 started higher at 90 % but dropped to 54% before reaching 86%. Both MSL systems significantly reduced organic matter levels (97.2 % for MSL1 and 95.8 % for MSL2). Both MSLs were shown to be effective in removing cyanobacteria, MCs, and organic matter with comparable performance.

Risk assessment of cyanobacteria toxic metabolites on freshwater ecosystems applying molecular methods.

Cyanobacteria, ancient prokaryotes, interfere with ecosystem water quality through the production of cyanotoxins and bloom formation. Therefore, for water safety and public health reasons, the application of faster, sensitive, and specific tools on its risk assessment is demanded. Polymerase chain reaction (PCR) coupled with DNA sequencing can be a helpful tool for the presence and potential to cyanotoxicity. To achieve these, seven waterbodies located on the North and Center regions of Portugal were sampled for two monitoring periods (2017 and 2018). Thus, given the five risk levels proposed (none up to four cyanotoxins - mcyA, cyrC, anaC, sxtI - being detected per risk level), results showed that the great majority of the ecosystems analyzed on the presence of blooms and under climate change phenomenon (heat waves) had an elevated risk (up to four cyanotoxins being detected) corresponding to a situation of high potential of cyanotoxicity. In the opposite conditions (i.e., absence of blooms and heat waves), the risk was lowered to none or only one cyanotoxin being detected. Two ecosystems escaped this trend and demonstrated little to no alterations among risk levels from 1 year to another corresponding to a high potential of cyanotoxicity and cyanotoxins persistence in comparison to other studied ecosystems. Overall, the risk assessment undertaken suggests that other ecosystems ecological variables (physical, hydrological, or chemical) are interfering on the occurrence and persistence of cyanotoxins biosynthesis genes. Given the observed conditions (eutrophic status, bloom occurrence, and heat waves) of the analyzed ecosystems, cyanobacterial potential for toxicity seems to have increased, suggesting a need of the incorporation of other cyanotoxins apart of the regulated microcystins-LR on cyanotoxins surveillance programs of Portugal.

Statistical Inferences Applying Non-Parametric Data on Cyanobacterial Investigations: Contributions to Water Quality and New Trends under Global Changes on Portuguese Freshwater Ecosystems.

Cyanobacteria are a bloom-forming ancient group of photosynthetic prokaryotes. A rise in temperature is a major contributor to its massive proliferation, namely on freshwater ecosystems, with social and economic impacts. Thus, reliable and cost-effective tools can permit the fast surveillance and assessment of temperature effects on potentially toxic cyanobacteria distribution and impacts. The occurrence of three potentially toxic cyanobacteria species was assessed on seven sampling points across three sampling years. Moreover, the association between the occurrence of those cyanobacteria species with climate change events was addressed. Here, we combined molecular and statistical methods to study the impacts of temperature on the occurrence of three globally occurring cyanotoxin-producing cyanobacteria species-Microcystis aeruginosa (microcystins), Raphidiopsis raciborskii (cylindrospermopsins and saxitoxins) and Planktothrix agardhii (microcystins and saxitoxins). Samples were collected on seven European temperate freshwater systems located on the North and Centre regions of Portugal, across three distinct sampling years with distinct ranges of air temperature. Data support that M. aeruginosa is still a common inhabitant of Portuguese freshwater ecosystems and a new trend was found on R. raciborskii recent invasion and establishment on the colder north ecosystems of Portugal. Additionally, the highest frequency of detection of both cyanobacteria was associated with warmer years. P. agardhii also revealed a new trend, being reported for the first time on North and Centre Regions of Portugal, however with no statistical relation with air temperature, demonstrating a higher ecological fitness. Distinct profiles of the statistical analysis on the three tested cyanobacteria species contribute to deepen the studies on other species as well as of our analyzed species on a global level. This assessment may help to anticipate possible repercussions on water quality and public health due to most probable alterations on cyanotoxins profile given the ecological fitness established among air temperature and PCR detection of potentially toxic cyanobacteria.

Preliminary evidence on the presence of cyanobacteria and cyanotoxins from culture enrichments followed by PCR analysis: new perspectives from Africa (Mali) and South Pacific (Fiji) countries.

Cyanobacteria are a group of microorganisms that can be found in a diverse range of biogeographical areas and produce potent and damaging cyanotoxins, which reveal importance for continuous studies and surveillance efforts. In this study, we analyzed worldwide two-month culture-enriched water samples collected from 12 distinct countries (Costa Rica, Cuba, Fiji, France, Indonesia, Mali, Portugal, South Africa, Spain, Thailand, USA, Vietnam) including two undisclosed areas (Fiji and Mali). We performed a PCR-based molecular multi-step scheme that consisted in the detection of the main cyanobacterial species, genera, and cyanotoxins biosynthesis genes. Results from this study indicate that Microcystis aeruginosa followed by Planktothrix agardhii were the most prevalent species of all the 12 countries analyzed. Cylindrospermospis raciborskii was detected in Costa Rica, while P. agardhii was detected in Fiji and South Africa. M. aeruginosa was detected in Fiji and Mali. Regarding the main cyanotoxins biosynthesis genes, a cyrC gene fragment (cylindrospermopsins) was amplified in the African continent (South Africa), while anaC (anatoxin-a) was detected in two distinct locations, Mali and Vietnam. Saxitoxins biosynthesis gene was also detected in Fiji and Vietnam. Microcystins biosynthesis gene (mcyA) was co-detected with anatoxin-a biosynthesis gene in Mali and with saxitoxins biosynthesis gene (sxtI) in Portugal. This study therefore constitutes a major contribution to the global biogeography of cyanobacteria and its cyanotoxins and recommends continuous vigilance of toxic cyanobacteria particularly in the more undisclosed areas of the world. The PCR analysis data obtained in our 2-month culture-enriched water samples supports molecular methods as a preliminary tool in the environmental surveillance of cyanobacteria and cyanotoxins in undisclosed locations, particularly since the several positive amplifications detected may indicate that though samples were collected under non-bloom conditions, if environmental conditions change in the ecosystem, there is a risk that bloom-forming species may arose along with their detected cyanotoxicity.

Cyanotoxins Occurrence in Portugal: A New Report on Their Recent Multiplication.

Historical reports show that in Portugal, cyanotoxins reports were mainly in the Center (cylindrospermopsins) and South (cylindrospermopsins, saxitoxins) regions of the country apart from the well distributed microcystins. Therefore, in our study, seven freshwater ecosystems located in the North and Center Regions of Portugal were screened between April and September of 2017 for the main cyanotoxins (microcystins, cylindrospermopsins, anatoxin-a, and saxitoxins) by a two methods approach that combined the application of molecular (PCR) and immunological (ELISA) assays. Results from our survey reveal that both methods revealed the presence of all main cyanotoxins. ELISA results showed that 48% of the samples were above (1.6-18.8 µg/L) the guideline value established for microcystins (1 µg/L), while in the remaining cyanotoxins, 33% of the samples were above (1.1-6.8 µg/L) the guideline value established for anatoxin-a (1 µg/L). Further, for saxitoxins, only one sample gave a value above (4.3 µg/L) the guideline (3 µg/L) and this corresponded to a North Region ecosystem. In the cytotoxin cylindrospermopsins, none of the samples were above the guideline established value (1 µg/L). This study will improve the risk assessment strategy in Portugal, as well as advance water quality and water management.

New Insights into the Role of Epithelial⁻Mesenchymal Transition during Aging.

Epithelial⁻mesenchymal transition (EMT) is a cellular process by which differentiated epithelial cells undergo a phenotypic conversion to a mesenchymal nature. The EMT has been increasingly recognized as an essential process for tissue fibrogenesis during disease and normal aging. Higher levels of EMT proteins in aged tissues support the involvement of EMT as a possible cause and/or consequence of the aging process. Here, we will highlight the existing understanding of EMT supporting the phenotypical alterations that occur during normal aging or pathogenesis, covering the impact of EMT deregulation in tissue homeostasis and stem cell function.

Cyanobacterial diversity held in microbial biological resource centers as a biotechnological asset: the case study of the newly established LEGE culture collection.

Cyanobacteria are a well-known source of bioproducts which renders culturable strains a valuable resource for biotechnology purposes. We describe here the establishment of a cyanobacterial culture collection (CC) and present the first version of the strain catalog and its online database (http://lege.ciimar.up.pt/). The LEGE CC holds 386 strains, mainly collected in coastal (48%), estuarine (11%), and fresh (34%) water bodies, for the most part from Portugal (84%). By following the most recent taxonomic classification, LEGE CC strains were classified into at least 46 genera from six orders (41% belong to the Synechococcales), several of them are unique among the phylogenetic diversity of the cyanobacteria. For all strains, primary data were obtained and secondary data were surveyed and reviewed, which can be reached through the strain sheets either in the catalog or in the online database. An overview on the notable biodiversity of LEGE CC strains is showcased, including a searchable phylogenetic tree and images for all strains. With this work, 80% of the LEGE CC strains have now their 16S rRNA gene sequences deposited in GenBank. Also, based in primary data, it is demonstrated that several LEGE CC strains are a promising source of extracellular polymeric substances (EPS). Through a review of previously published data, it is exposed that LEGE CC strains have the potential or actual capacity to produce a variety of biotechnologically interesting compounds, including common cyanotoxins or unprecedented bioactive molecules. Phylogenetic diversity of LEGE CC strains does not entirely reflect chemodiversity. Further bioprospecting should, therefore, account for strain specificity of the valuable cyanobacterial holdings of LEGE CC.

First occurrence of cylindrospermopsin in Portugal: a contribution to its continuous global dispersal.

Cylindrospermopsin (CYN) was found to occur in Portugal for the first time. In this study CYN values varied from a minimum of 1.4 µg L-1 to a maximum of 12 µg L-1 detected through HPLC technique and confirmed by LC-MS method. Amplification of the cyrC gene was done and was confirmed to be from the genera Aphanizomenon. This study is therefore an important contribution to the knowledge on the dispersal and biogeography of CYN.

Genetic characterization of Microcystis aeruginosa isolates from Portuguese freshwater systems.

Cyanobacteria are microorganisms that pose a serious threat to the aquatic waterways through the production of dense blooms under eutrophic conditions and the release of toxic secondary metabolites-cyanotoxins. Within cyanobacteria, the colonial planktonic Microcystis aeruginosa is widely distributed in both fresh and brackish aquatic environments throughout the world being frequently observed in the Portuguese water systems. Apart from the well-established distribution of M. aeruginosa in Portugal, knowledge of its genetic diversity and population structure is unknown. Therefore, in this study twenty-seven strains were obtained from the North, Centre and South regions of Portugal and were subjected to extensive phylogenetic analyses using simultaneously four distinct genetic markers (16S rRNA, 16S-23S ITS, DNA gyrase subunit ß and cell division protein (ftsZ)) encompassing in total 2834 bp. With this work we characterized the phylogenetic relationship among the Portuguese strains, with the southern strains showing higher genetic structure relatively to the North and Centre strains. A total of fifteen genotypes were determined for M. aeruginosa in Portuguese water systems revealing a high genetic diversity. This is also the first study to report geographic variation on the population structure of the Portuguese M. aeruginosa.

Phylogeny and biogeography of the invasive cyanobacterium Cylindrospermopsis raciborskii.

Cylindrospermopsis raciborskii is a toxic cyanobacterium with an invasive nature. The species is found in all the main continents but its origin and dispersal routes on a worldwide perspective remain yet mostly unknown. In this study, 27 isolates of C. raciborskii gathered worldwide have been used for an in-deep phylogenetic analyses with a concatenated system of three genetic markers (16 rRNA, 16S-23S ITS larger subunit, and RNA polymerase rpoC1) comprehending 3,188 bp. Our results provide support for an origin of C. raciborskii in the American continent. Dispersal routes included afterward a spread into the African continent and then Asia and Australia, being Europe the last continent to be colonized by this species. Our phylogenetic inferences suggest that C. raciborskii seem to have a well-defined dispersal behavior with a well-established population structure around the world.

Methods to detect cyanobacteria and their toxins in the environment.

Cyanobacteria blooms are since early times a cause for environmental concern because of their negative impact through the release of odors, water discoloration, and more dangerously through the release of toxic compounds (i.e. the cyanotoxins) that can affect both human and animal welfare. Surveillance of the aquatic ecosystems is therefore obligatory, and methods to achieve such require a prompt answer not only regarding the species that are producing the blooms but also the cyanotoxins that are being produced and/or released. Moreover, besides this well-known source of possible intoxication, it has been demonstrated the existence of several other potential routes of exposure, either for humans or other biota such as through food additives and in terrestrial environments (in plants, lichens, biological soil crusts) and the recognition of their harmful impact on less studied ecosystems (e.g. coral reefs). Nowadays, the most frequent approaches to detect toxic cyanobacteria and/or their toxins are the chemical-, biochemical-, and molecular-based methods. Above their particular characteristics and possible applications, they all bring to the environmental monitoring several aspects that are needed to be discussed and scrutinized. The end outcome of this review will be to provide newer insights and recommendations regarding the methods needed to apply in an environmental risk assessment program. Therefore, a current state of the knowledge concerning the three methodological approaches will be presented, while highlighting positive and negative aspects of each of those methods within the purpose of monitoring or studying cyanobacteria and their toxins in the environment.

African origin and europe-mediated global dispersal of the cyanobacterium Microcystis aeruginosa.

Microcystis aeruginosa is a bloom-forming cyanobacteria, which currently has a cosmopolitan distribution. Since M. aeruginosa can produce toxic compounds across all continents that it inhabits, it is of major public health relevance to assess its origin and dispersal. Thus, we conducted a worldwide study using 29 isolates representative of all the main continents, and used a concatenated genetic system for phylogenetic analyses consisting of four genetic markers (spanning ca. 3,485 bp). Our results support an early origin of M. aeruginosa in the African continent, with a subsequent dispersal to establish a second genetic pool in the European continent, from where M. aeruginosa then colonized the remaining continental regions. Our findings indicate that the European population has a cosmopolitan distribution, and is genetically closer to populations from Africa and North America. Our study also highlights the utility of using a concatenated dataset for phylogenetic inferences in cyanobacteria.

Phylogeny and biogeography of cyanobacteria and their produced toxins.

Phylogeny is an evolutionary reconstruction of the past relationships of DNA or protein sequences and it can further be used as a tool to assess population structuring, genetic diversity and biogeographic patterns. In the microbial world, the concept that everything is everywhere is widely accepted. However, it is much debated whether microbes are easily dispersed globally or whether they, like many macro-organisms, have historical biogeographies. Biogeography can be defined as the science that documents the spatial and temporal distribution of a given taxa in the environment at local, regional and continental scales. Speciation, extinction and dispersal are proposed to explain the generation of biogeographic patterns. Cyanobacteria are a diverse group of microorganisms that inhabit a wide range of ecological niches and are well known for their toxic secondary metabolite production. Knowledge of the evolution and dispersal of these microorganisms is still limited, and further research to understand such topics is imperative. Here, we provide a compilation of the most relevant information regarding these issues to better understand the present state of the art as a platform for future studies, and we highlight examples of both phylogenetic and biogeographic studies in non-symbiotic cyanobacteria and cyanotoxins.

Phylogeny of microcystins: evidence of a biogeographical trend?

Microcystins, the most prevalent cyanotoxins occurring worldwide, were first recorded in the species Microcystis aeruginosa. Its production has been reported in all continents; thus, we propose a comprehensive phylogenetic study to characterize M. aeruginosa microcystin-producing strains and establish whether or not the species has an historic biogeography. To accomplish this, we compared phylogenetically the nucleotide sequences of three genes of the mcy gene cluster (mcyA, mcyD and mcyG) from toxin producing M. aeruginosa strains across all the five continents. The obtained results provided valuable insight on the biogeography of M. aeruginosa produced microcystins: (i) the Asian strains showed to be distinct from the other continental groups indicating a genetically unique population and (ii) Asian strains were more related to European and North American strains. Moreover, the evidence of positive selection was determined in all the three mcy genes indicating that some functionality yet to be determined could be under selection for these genes.

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.

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.

Genetic variability of the invasive cyanobacteria Cylindrospermopsis raciborskii from Bir M'cherga reservoir (Tunisia).

Cylindrospermopsis raciborskii is an invasive freshwater cyanobacteria of tropical origin, also found in temperate regions. Due to its known ability to produce potent toxins, such as cylindrospermopsin and the paralytic shellfish poisoning, this species is of major concern from a water quality perspective. This study presents a genetic characterization of four C. raciborskii strains isolated from the Bir M'cherga Tunisian reservoir. The toxicity assessment was investigated via molecular biology tools, which suggested that all the isolated strains were not producing cylindrospermopsin, saxitoxin, or microcystin. This result was further confirmed by HPLC and MALDI-TOF analyses. However, we report for the first time in C. raciborskii the presence of mcyA and mcyE, two segments of the microcystin synthetase mcy cluster. All the strains were identified taxonomically based on the 16S rRNA sequences, and their phylogenetic relationships were assessed using the rpoC1 region. Tunisian strains formed a distinct clade separated from the other African strains.

Molecular and phylogenetic characterization of potentially toxic cyanobacteria in Tunisian freshwaters.

This study presents a genetic characterization of 27 potentially toxic cyanobacterial strains isolated from seven reservoirs located in the north and centre of Tunisia. These strains belonged mainly to Microcystis aeruginosa, Cylindrospermopsis raciborskii and Planktothrix agardhii species. Their toxicological potential was evaluated by molecular biology tools, which showed that none of the isolated strains carried segments of the gene cluster responsible for the production of cylindrospermopsin and saxitoxin. The majority of Microcystis isolates were able to synthesize microcystin, since they presented the six characteristic segments of the microcystin synthetase mcy cluster (mcyA, -B, -C, -D, -E and -G). This was further confirmed by MALDI-TOF analysis that showed the presence of eight microcystin variants, including microcystin-LR. The taxonomic identification of the strains was assessed based on the variability of the 16S rRNA gene sequences. Furthermore, the 16S-23S rRNA ITS sequences of Microcystis isolates and rpoC1 sequences of Cylindrospermopsis strains were also used in the phylogenetic analysis.