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Design and testing of dna microarrays to monitor microbial diversity with adequate biodiversity indexes, using cyanobacteria in freshwater as a model system

Deliverables

Diversity software is currently being developed which will allow diversity indices to be calculated from molecular style data for microbes. In the future this software will be marketed and sold by PISCES Conservation Ltd to other similar projects.
The database of European planktic cyanobacteria (natural morphospecies) (called CYATAXO) can serve to scientists as well as to workers in laboratories of water company and environmental research to help to identify samples from reservoirs and other water bodies. Particular importance for work with toxic strains.The Database (in English) consists of: Taxonomic names + authors; synonymics; phenotype description; dimensions; original and further drawings and microphotographs; ultrastructural characters; ecological and ecophysiological remarks and list of strains available (with a reference strain; if available). Data about variability in nature and in cultures as much as possible.
In addition to the numerous tests on representative strains that were carried out for the validation; the performance of the DNA micro-arrays has been tested on 20 environmental samples from European lakes. This gave an evaluation of how well they can be used as a water-monitoring tool. For this use as a monitoring tool; there is obviously a long learning curve to master the technology.
Physical (e.g. temperature) and chemical (e.g nutrients) are measured during the 2-year-study in lake Tuusulanjärvi. A cross-analysis between these variables and cyanobacterial biomass and community structure will be performed in order to reveal possible causal relationships between cyanobacterial populations and the environmental status of the eutrophic lake.The results serve as an important information for environmental authorities in explaining the formation of cyanobacterial blooms in lakes.
Data of cyanobacterial populations in the MIDI-CHIP sampling sites (Esch-sur-Sûre reservoir in Luxembourg; Bacino Bubano in Italy and Tuusulanjärvi in Finland) will be compared and related to the environmental characteristics on each site. The data will be analysed in oder to pinpoint probable common and distinctive parameters for the freshwater bodies. Diversity indices developed in the project will be used.
We have established a robust procedure based on microarray technology able to detect potentially toxic cyanobacteria. This procedure relies on previously known art and properly defined molecular probes which sequences constitute the main result in terms of intellectual property that could be protected (patented).
We have established a robust procedure based on the microarray technology able to detect cyanobacterial diversity. This procedure relies on previously known detection method (Ligation Detection Assay) and properly defined molecular probes which sequences constitute the main result in term of intellectual property that could be protected (patented)
The cyanobacterial biocoenosis of the selected lakes and basins has been characterised and their two years dynamics has been followed. The techniques used include counting; microscopic observation; isolation of the cyanobacterial morphotypes and molecular methods; thus allowing the comparison of the traditional and the molecular methods. The molecular approach included several different techniques performed either on total cyanobacterial DNA extracted from natural samples and on pure cyanobacterial strains isolated from the studied waterbodies. High-resolution electrophoretic approaches (DGGE and TGGE) were used to obtain a fingerprint of the cyanobacterial community and to determine the partial sequence of the 16S rRNA gene from members of the community. Cloning of the same gene was used to complete the characterisation. The correlation of the cyanobacterial diversity with limnological and ecological characteristics of the lakes (from WP1) can yield factors; which allow for the sustainable management of the water resources. Having considered different types of waterbodies located in 3 countries; allow observing ecological and geographical variations.
A database has been developed specifically to hold data from the MIDI CHIP project. The database holds sampling data; morphological data of fresh; fixed and cultured organisms; and molecular data (cloning; DGGE; TGGE and strain sequences). The database is centered on the sample name and all other information relates back to the original sample from which it came. There is also information on all the partners and contacts involved in the project.
Cyanobacteria are responsible for the production of hepatotoxins (microcystins) and neurotoxins (anatoxin-a and homoanatoxin-a); which are poisoning animals and causing problems for recreational and drinking water. Therefore; toxicity monitoring is required. Two biochemical tests; PPIA and ELISA; have been used as a first screening of toxins. Then; a full analytical investigation using HPLC-UV/DAD (for microcystins) and HPLC-UV/DAD and HPLC-UV/FL (for neurotoxins) has been performed. These results include the data concerning the toxicity; quantity and quality of cyanotoxins in MIDI-CHIPs field samples and strains samples. The data will be put in the context of environmental diagnostics and blooms management.
The cyanobacterial genus Microcystis is delimited according to genetic criteria (molecular sequencing by 16S rRNA); but the subgeneric classification is unclear; and the existence of traditional morphospecies is doubtful. However; this genus forms heavy water blooms in eutrophic waters in Europe; numerous populations produce toxins; and the orientation in the natural subgeneric diversity is important. The review contains therefore the descriptions and characteristocs of the main morphospecies recognized in European waters with their phenotype diacritical markers.
An effort to bring into laboratory culture as many members of the cyanobacterial community of the selected European water bodies as possible has been carried out. To widen the number of different cyanobacterial types brought to cultivation; several different enrichment and isolation media and also different isolation techniques were used. As a result; hundreds of cyanobacterial strains are now available for future use after two year's sampling. The diversity of the cyanobacteria present in nature is well represented in the collection of the strains obtained in culture. These strains are of a fundamental value for further characterisation of the freshwater cyanobacterial community for the research on cyanotoxins and for the assessment of cyanobacterial taxonomy and biodiversity. The cyanobacterial strains isolated are a valuable resource. They can be used to study biodiversity and taxonomy of planktic cyanobacteria; sources of bioactive compounds that they may produce and sources of gene sequences to be used in micro-arrays; or other molecular methods (primer design and testing for PCR; real time PCR etc.) to monitor water quality.
Many problems persist with the microscopical identification of cyanobacteria in natural samples. Through the comparison of natural material and isolated strains; through the study of their morphology and the combination with the molecular data; the existing taxonomic system and identification keys will be improved by the people in charge of the monitoring of the cyanobacteria developpement in European waterbodies.
Microphotographs of the cyanobacterial forms observed in the natural samples of the three lakes as well as of the isolated strains are taken and put in a common database; together with same information on identification. This database will constitute an important tool for water managers for the help in identification of cyanobacteria developing in their lakes.

Exploitable results

Cyanobacterial blooms are a more and more frequent phenomenon in surface waters, and are a concern for human and animal health. Indeed, certain taxa may produce potent hepatotoxins and neurotoxins. A DNA chip was designed by 4 partners of the MIDI-CHIP project (http://www.cip.ulg.ac.be/midichip) and patented (PCT/FI2004/000310 PCT notification WO2004/104211) by Licentia Ltd. The purpose is to use the chip for an extensive, automated monitoring of surface waters to detect toxic and non-toxic cyanobacteria.

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