Centre for Applied Gensensorik (CAG), UFT, University of Bremen
Universität Bremen, FB2-UFT, Biotechnology and Molecular Genetics
Leobener Strasse
D-28359 Bremen
Germany
Prof Dr Dietmar Blohm, Dr Marc Kochzius

Universidad de Oviedo
C/ Julian Claveria, s/n.
E-33006 Oviedo
Spain
Dr Eva Garcia Vazquez

InfoConsult Gesellschaft fuer Informationstechnik mbH
Universitaetsallee 17
D-28359 Bremen
Germany
Uwe Kaufmann

Prokaria EHF
Gylfaflöt 5
IS-112 Reykjavik
Iceland
Dr Jakob K. Kristjansson

Institute of Marine Biology of Crete
P.O. Box 2214
GR-71003 Iraklio
Greece
Dr Andonis Magoulas

Stiftung Alfred Wegener Institut fuer Polar- und Meeresforschung
Am Handelshafen 12(Building A-126)
D-27570 Bremerhaven
Germany
Dr Linda Medlin

Ecole Pratique des Hautes Etudes, Université de Perpignan (CNRS)
F-66860 Perpignan Cedex
France
Dr Serge Planes

Exiqon A/S
Bygstubben 9
DK-2950 Vedbaek
Denmark
Dr Sakari Kauppinen

Interdepartment Centre of Research in Environmental Sciences, Università di Bologna
via Sant'Alberto, 163
I-48100 Ravenna
Italy
Dr Fausto Tinti

Fisheries Genetics Laboratory, College of Fisheries and Aquaculture, Mustafa Kernal University
Faculty of Fisheries and Aquaculture
Fish Genetics Laboratory
TR-31040 Antakya, Hatay
Turkey
Dr Cemal Turan

Barlow BA and Tzotzos GT (1995) Biotechnology. In: Heywood VH (ed) Global biodiversity assessment. UNEP/Cambridge University Press; pp 671-710
Bell JL, Grassle JP (1998) A DNA probe for identification of larvae of the commercial surfclam (Spisula solidissima) Molecular Marine Biology and Biotechnology 7(2): 127-137
Bakan G and Büyükgüngör H (2000) The Black Sea. Marine Pollution Bulletin 41(1 6): 24-43
Bianchi CN and Morri C (2000) Marine biodiversity of the Mediterranean Sea: Situation, problems and prospects for future research. Marine Pollution Bulletin 40(5): 367-376
Blohm D, Guiseppi-Elie A (2001) New developments in microarray technology. Current Opinion in Biotechnology 12:41-47
Bucklin A, Guarnieri M, Hill RS, Bentley AM, Kaartvedt S (1999) Taxonomic and systematic assessment of planktonic copepods using mitochondrial COI sequence variation and competitive, species-specific PCR. Hydrobiologia 401: 239-254
Cognetti G, Lardicci C, Abbiati M, Castelli A (2000) The Adriatic Sea and the Tyrrhenian Sea. In: Sheppard CRC (ed) Seas at the millennium: An Environmental evaluation. Volume I, Pergamon, Amsterdam, pp 267-284
Delbaere B (2002) An inventory of biodiversity indicators in Europe. Final draft, European Centre for Nature Conservation
Ducrotoy J-P, Elliott M, De Jonge VN (2000) The North Sea. Marine Pollution Bulletin 41(1-6): 5-23
EU (2001) A sustainable Europe for a better world: A European Union strategy for sustainable development (Commission’s proposal to the Gothenburg European Council). COM(2001)264 final
Féral J-P (2002) How useful are the genetic markers in attempts to understand and manage marine biodiversity? Journal of Experimental Marine Biology and Ecology 268: 121-145
Groombridge B and Jenkins MD (2002) World atlas of biodiversity. University of California Press, Berkeley
Kappel K, von Westernhagen H, Blohm D (2003) Microarry-based identification of eggs and larvae from fish species common in the North Sea. Abstract, Dechema Chip-Technology Meeting 24.-25. February 2003, Frankfurt, Germany
Kerkmann K, Medlin L (in press) Microarrays: the flurorescent wave of the future, Nova Hedwigia
Kiesling TL, Wilkinson E, Rabalais J, Ortner PB, McCabe MM, Fell JW (2002) Rapd identification of adult an naupliar stages of copepods using DNA hybridisation methodology. Marine Biotechnology 4: 30-39
Niemeyer CM and Blohm D (1999) DNA microarrays. Angewandte Chemie International Edition 38: 2865-2869
Palumbi S (1996) Nucleic acids II: The polymerase chain reaction. In: Hillis DM, Moritz C, Mable BK (eds) Molecular systematics; Sinauer Associates, Inc., Sunderland; pp 205-247
Peplies J, Glöckner FO, Amann R (2003) Optimization strategies for DNA microarray-based detection of bacteria with 16S rRNA-targeting oligonucleotide probes. Applied and Environmental Microbiology 69(3): 1397-1407
Rocha-Olivares A (1998) Multiplex haplotype-specific PCR: a new approach for spieces identification of the early life stages of rockfishes of the species-rich genus Sebastes Cuvier. Journal of Experimental Marine Biology and Ecology 231: 279-290
Rosel PE, Kocher TD (2002) DNA-based identification of larval cod in stomach contents of predatory fishes. Journal of Experimental Marine Biology and Ecology 267: 75-88
Stork NE (1997) Measuring global biodiversity and its decline. In: Reaka-Kudla ML, Wilson DE, Wilson EO (eds) Biodiversity II, Joseph Henry Press, Washington D.C., pp 41-68
Sweijd NA, Bowie RCK, Evans BS, Lopata AL (2000) Molecular genetics and the management and conservation of marine organisms. Hydrobiologia 420: 153-164
Zaitsev Y and Mamaev V (1997) Marine Biological Diversity in the Black Sea. A study of change and decline. United Nations Publications, New York

Compared to terrestrial ecosystems very little is known about marine biodiversity and changes in species richness and ecosystem function. This is mainly due to sampling difficulties and problems in taxonomy. Many species have not yet been described and there are few scientific specialists for several groups of marine organisms, including phytoplankton, invertebrates, as well as the eggs and larvae of fishes. Even though the status of marine biodiversity is alarming, scientific expertise in biodiversity is currently being lost (Bianchi and Morri 2000). Therefore, the necessary monitoring of marine biodiversity is hampered, because taxonomic expertise for the identification of sampled organisms is lacking. DNA chips, the new DNA-based identification technology, can facilitate a reliable and efficient processing of samples in biodiversity monitoring and ecosystem science.

DNA-based identification methods are a powerful tool for the identification of marine organisms (Barlow and Tzotzos 1995, Féral 2000) and are able to differentiate species, subspecies and variants of organisms with unprecedented accuracy. However, they are not yet widely used in marine biodiversity and ecosystem science. There are successful reports about DNA-based identification of marine animals, such as eggs, larvae and adults of fishes (Rocha-Olivares 1998, Sweijd et al. 2000, Ward 2000), zooplankton (Bucklin et al. 1999), invertebrates larvae (Bell and Grassle 1998), and prey in gut contents analysis (Rosel and Kocher 2002). Nevertheless, all these methods, which are based on PCR amplification followed by gel electrophoresis or sequencing, are specific for one or a few species. Additionally they are very laborious and only marginally useful for routine analysis.

DNA chips however have the potential to identify hundreds of species at once, enhancing the state-of-the-art in the field of DNA-based identification methods. Such chips are glass microscope slides with spots of probe oligonucleotides that are complementary to a specific target DNA sequence. The targets hybridise with the capture oligonucleotides on the chip and this is detected by a fluorescent label which was attached to the target during PCR.

To develop DNA chips for the identification of marine organisms a combined biological and technical approach has been initiated: The biological material will be sampled and supplied by marine biologists experienced in the identification of these organisms.

The next step is the sequencing of suitable molecular markers for probe design. The following technical part consists mainly of constructing gene probe libraries and determining their specificity experimentally. For each organism to be recognised by DNA chip methods a characteristic set of specific molecular probes defined by specific nucleic acid sequences is needed. These actvities will be carried out by biotech research centres in connection with companies engaged in DNA chip technology.

The application of DNA chips for the identification of marine organisms is a very new and innovative field of research, just starting to be developed. DNA hybridisation assays for the identification of zooplankton (Kiesling et al. 2002) and prey in gut content analysis of fishes (Rosel and Kocher 2002) are reported, but currently there are virtually no applications of DNA chips for the identification of marine animals and phytoplankton published. Exceptions are pilot studies on fishes (Kappel et al. 2003) in the laboratory of the coordinator, and on phytoplankton by consortium members (Kerkmann and Medlin, in press).

Sustainable development is a fundamental goal of the European Union, implemented during a process of conferences (Rio de Janeiro 1992), treaties (Amsterdam 1997), and EU council decisions (Helsinki 1999, Göteborg 2001). Loss of biodiversity was emphasised as one of the main threats to sustainable development during the Göteborg European Council (EU 2001). This underlines the importance of biodiversity and ecosystem science to facilitate the realisation of sustainable development. Therefore, the following priorities for action have been identified among others (EU 2001):

(1) Halt of biodiversity loss (by 2010)
(2) Improvement of fisheries management to ensure healthy marine ecosystems
(3) Establishment of a biodiversity indicator system (by 2003)

A basis for the implementation of these priorities for action in the marine environment is a cost effective, reliable and efficient technology for the identification of marine organisms. DNA chips are a powerful and innovative technology which facilitates surveying and monitoring of marine organisms, necessary to track changes in biodiversity and ecosystem functioning. Especially for a biodiversity indicator system which aims to answer policy questions about the threat status and trends of Europe’s biodiversity (Delbaere 2002), the identification of species is a fundamental task.

Global biodiversity is estimated to 14 million species, but currently only 12.5% of these species have been described. Known marine biodiversity reaches around 250.000 species and 315 animal species are considered as threatened. This seems to be a rather low number, but is due to the fact that only mammals and birds have been comprehensively assessed (Groombridge and Jenkins 2002). Estimates of global extinction rates are between 2% and 30% per decade (Stork 1997).

Biodiversity and ecosystems of European Seas are under anthropogenic induced pressure, such as pollution, eutrophication, coastal construction, and fishery overexploitation (Bakan and Büyükgüngör 2000, Bianchi and Morri 2000, Ducrotoy et al. 2000).

Biodiversity and ecosystem functioning in the North Sea is under threat, because 185 million people live in its catchment area, exposing it to pollution from industrial and agricultural activities. Commercial fishing activity is another severe anthropogenic disturbance, removing 30-40% of the biomass of exploited fish species, and altering the ecosystem functioning by exhaustive trawling (Ducrotoy et al. 2000). To facilitate biodiversity and ecosystem science, the "Fish & Chips" project aims to develop a "Fish Chip", a "Invertebrate Chip" as well as a "Phytoplankton Chip" for the North Sea and adjacent areas, such as Baltic Sea and North-Eastern Atlantic.

The situation in the Mediterranean Sea is quite similar and most of its marine habitats are in danger, due to heavy demographic, urban, and industrial pressure. For a better knowledge of the role and patterns of biodiversity as well as ecosystem functioning, it is essential to start monitoring at a the pan-Mediterranean scale (Bianchi and Morri 2000). Because of its hydrographical and hydrobiological features the Adriatic Sea is a peculiar basin within the Mediterranean Sea. The human population affecting the area reaches up to 30 million and results in heavy industrial, urban, and agricultural pollution load.

Overfishing, e.g. of bivalves, causes severe damage to benthic communities (Cognetti et al. 2000) which leads to changes in the trophic interactions, and hence ecosystem functioning. To address such environmental problems on a whole Mediterranean scale, the "Fish & Chips" consortium includes partners from all EU countries bordering the Mediterranean Sea and Turkey. The goal is the development of a "Fish Chip" and an "Invertebrate Chip". These DNA chips can facilitate the identification of fish eggs and larvae, zooplankton, as well as benthic organisms, e.g. in gut contents analyses of demersal fishes and monitoring of biondicators.

The Black Sea environment is heavily impacted by water pollution from 17 countries, because almost one third of continental Europe drains into it. This has lead to widespread alterations in biodiversity and ecosystem functioning in recent times, e.g. quantitative and qualitative changes in benthic invertebrate and fish communities (Bakan and Büyükgüngör 2000). The Black Sea is considered as the most damaged sea on the planet. It is one of the most isolated seas in the world and experiences the introduction of exotic species with severe effects on the ecosystem (Zaitsev and Mamaev 1997). The Black Sea fauna which is shared with the Mediterranean will be covered by the "Fish Chip" and "Invertebrate Chip".

The aim of the "Fish & Chips" project is the development of DNA chips for the identification of marine organisms in European Seas as a cost effective, reliable and efficient technology in marine biodiversity and ecosystem science.

Many marine organisms, such as (1) eggs and larvae of fishes and invertebrates, (2) zoo- and phytoplankton, and (3) benthic invertebrates, are difficult to identify by morphological characters, and for many groups the skills of specialised taxonomists are needed. The classical microscopy methods are extremely time consuming and require a high degree of taxonomic expertise. Consequently, the basic step of identifying such organisms is a major bottleneck in marine biodiversity and ecosystem science.

Currently DNA chips are mainly a research tool for gene expression analysis (Blohm and Guiseppi-Elie 2001, Niemeyer and Blohm 1999) and are not yet widely applied for the identification of organisms, except in a few studies on bacteria (e.g. Peplies et al. 2003, Small et al. 2001) and viruses (e.g. Wang et al. 2002). The "Fish & Chips" project aims to demonstrate that DNA chips can be a new innovative tool for the identification of marine animals and phytoplankton:

The fish chips will comprise capture oligonucleotides for key species of fishes of the North Sea, Baltic Sea, North-Eastern Atlantic, Mediterranean and Black Sea. This chip will facilitate ecosystem research in terms of ichthyoplankton community studies, as well as dispersal of fish eggs and larvae.

The phytoplankton chip will focus mainly on unicellular algae from the North Sea. This DNA chip will enable monitoring of biodiversity, especially for pico- and nanoplankton species which lack morphological features for identification. The detection of harmful algae blooms s also an important application.

The fish and phytoplankton chips can capitalise on samples and sequence information already obtained by consortium members and from other projects.

The invertebrate chip will focus on important prey species of demersal fishes and harpacticoid copepods in the Mediterranean and North Sea that are difficult to identify by morphological characters. Prey organisms of demersal fishes have to be identified by classical methods in gut contents analysis before the species for genetic identification can be selected.

This new innovative tool will enhance ecosystem research on energy flow and food web structure, because it will facilitate identification of disintegrated prey in gut contents analysis. It will further strengthen regular monitoring of bioindicator organisms in marine ecological research by increasing both accuracy of identification and frequency of monitoring.

A further result of the DNA chip development is an assessment of the genetic diversity of the studied species. These data can

(a) reveal phylogenetic relationships
(b) indicate taxonomic boundaries as well as cryptic species, and
(c) deliver insight into phylogeographic patterns