MIDI-CHIP Co-ordinator Dr. Annick Wilmotte CIP - Institute of Chemistry B6 University of Liège B-4000 Liège, Belgium Tel: + 32 4 366 38 56 Fax: + 32 4 366 33 64 Design and testing of DNA microarrays to monitor microbial diversity with adequate biodiversity indexes, using cyanobacteria in freshwater as a model system [email protected] LUXEMBOURG Centre de Recherche Public Gabriel Lippmann: Project Managers and Participating Organisations Consiglio Nazionale delle Ricerche Instituto di Tecnologie Biomediche (CNR-ITB) Via Fratelli Cervi 93 I-20090 Segrate (MI) Italy Contact: Dr. Gianluca De Bellis [email protected] Finnish Environment Institute (SYKE) Mechelininkatu 34A PO Box 140 FIN-00251 Helsinki Finland Contact: Dr. Pirjo Kuuppo [email protected] Azienda Multiservizi Imolese - AMI SpA Via Casalegno, 1 I-40026 Imola Italy Contact: Dr. Giovanni Tedioli [email protected] Centre de Recherche Public - Gabriel Lippmann Cellule de Recherche en Environnement et Biotechnologies 162a, avenue de la Faïencerie L-1511 Luxembourg Grand Duché de Luxembourg Contact: Dr. Lucien Hoffmann [email protected] Department of Applied Chemistry and Microbiology - University of Helsinki P.O. Box 56, Biocenter Viikki FIN-00014 Helsinki University Finland Contact: Prof. Kaarina Sivonen [email protected] Consiglio Nazionale delle Ricerche Istituto per lo Studio degli Ecosistemi Sezione di Firenze (CNR-ISE) Piazzale delle Cascine 28 I-50144 Firenze Italy Contact: Dr. Stefano Ventura [email protected] PISCES Conservation Ltd IRC House, The Square Pennington, Lymington Hants SO41 8GN United Kingdom Contact: Dr. Peter Henderson [email protected] Department of Botany University of South Bohemia Branišovská 31 CZ-37005 Ceské Budejovice Czech Republic Contact: Prof. Jirí Komárek [email protected] Web-Site: http://www.itba.mi.cnr.it/midi-chip Science Officer: Dr Martin Sharman Research DG (Environment) Biodiversity and Global Change EC, Rue de la Loi 200 (SDME 4-75) B-1049 Brussels, Belgium Fax: +32 2 296 05 88 Phone: +32 2 295 97 98 [email protected] Sampling, Isolation, Enumeration and Microscopic Identification Esch-sur-Sûre Lake (Luxembourg) Esch-sur-Sûre Lake FINLAND UNITED KINGDOM University of Helsinki: Molecular Analysis and Isolation PISCES Conservation Ltd: Finnish Environment Institute Software and Databases (FEI): Sampling and Enumeration BELGIUM Tuusulanjärvi Lake University of Liège (ULG): Molecular Analysis CZECH REPUBLIC University of South Bohemia: Microscopic Identification ITALY Consiglio Nazionale delle Ricerce (CNR-ITB): Microarray Design Consiglio Nazionale delle Ricerce (CNR-ISE): Isolation and Molecular Analysis Azienda Multiservizi Imolese AMI SpA: Sampling Bubano Basin Cyanobacterial bloom (Esch-sur-Sûre Lake) In order to be protected biodiversity it must be measured. For cyanobacteria, the traditional morphological measures of biodiversity are unsatisfactory. A new approach utilises molecular markers (like SSU rRNA) to define taxa on the basis of phylogeny. In freshwater ecosystems huge populations of cyanobacteria can occur. These blooms may release toxins that make the water poisonous. A second part of the study is to compare the temporal dynamics of biodiversity in both natural and disturbed lakes. This comparative analysis requires the use of ecological indexes to summarise the diversity of the samples and allow inferences about the ecosystem to be made. Duration: 1st April 2000 to 31th July 2003 Project funded by the European Commission Research Directorate-General Contract N° EVK2 EVK2--CT1999 CT1999--00026 To advance knowledge of microbial biodiversity - improving established techniques - test innovative DNA microarrays - develop software to calculate diversity indices Molecular biodiversity sampling Sampling From three lakes, combined water column samples are taken bewteen 0 to 2 meters. Chemical analysis is undertaken and a Lugol fixed sample prepared for cyanobacteria counting. Further, for morphological studies to describe the cyanobacterial taxa present and to create isolate cultures, formalin fixed and natural samples are collected with a 10 µm mesh plankton net. The objectives are to measure the molecular biodiversity of cyanobacteria using the rDNA operon and to follow cyanobacterial population dynamics for 2 years in lakes in Finland, Luxembourg, and Italy. Alternative molecular markers will be tested on isolated cyanobacterial strains and one or two of these markers selected to measure genotypic diversity in samples from the participating countries. An in situ hybridization sample is prepared by mixing a concentrated sample with ethanol. Volumes from 100 to 500ml are filtered on 0.45µm pore size membranes, put in lysis buffer and immediately frozen at –20°C. The frozen samples are sent to partners working on molecular analysis for DNA extraction. morphological biodiversity sequences in situ hybridization molecular biodiversity oligonucleotides DNA microarrays DNA microarray The microarray will be tested on natural samples. After testing, new sets of oligonucleotides including interesting regions for selected markers and polymorphic regions for the new rRNA sequences will be selected and included in the final version of the microarray. biodiversity indices Morphological biodiversity The objectives are to carry out and improve identification and quantification of cyanobacterial morphotypes. Such techniques are routinely used by water companies and environmental agencies in Europe. It is cheap and quite fast, but plagued by problems linked to morphological variability and the skill levels and experience of the observer. Incorrect identifications are probably frequent and cause ecological, biogeographical and toxicological confusion. This study is a unique opportunity for a phenotypic and genotypic study of cyanobacterial diversity, to critically assess the complementarity of techniques. Biodiversity indices Computer software has been developed to analyse data collected by a variety of bacteriological and molecular biological techniques to calculate microbial diversity. Suitable diversity indices have been identified for cyanobacteria in freshwater lakes. A database has been built to hold information on sample collection, environmental conditions and the results of morphological and molecular analyses.