Harmful algal blooms (HABs) are a phenomenon caused by the local proliferation of algae, with deleterious consequences, particularly in coastal waters throughout the world. In the ESTTAL project, we conduct a limited genomic study of expressed sequence tags (ESTs) for a variety of toxigenic taxa representing major eukaryotic microalgal groups, including dinoflagellates, raphidophytes, prymnesiophytes and diatoms, as well as prokaryotic cyanobacteria. Toxigenic strains of each taxonomic group will be grown under optimal conditions for toxin expression. After cloning of cDNA into plasmid vectors, approximately 10,000 clones from each species are randomly sequenced for ESTs. Our approach is to annotate the ESTs and attempt to identify genes associated with toxin production. DNA microarrays are developed for screening of toxigenic and non-toxigenic strains. In addition, the sequence data are analysed to identify other genes that may be involved in cell regulation or growth, cell cycle events, stress response and the induction of sexuality. Cultures are grown under various environmental conditions to investigate the effects of external forcing functions on gene expression linked to toxicity and growth. Successful completion of thie ESTTAL project will yield new information on microalgal and cyanobacterial genomic sequences for a diversity of taxa and will assist in the diagnosis of genes related to toxin biosynthesis and the formation of toxic blooms.
Recent technological advances in genomics and bioinformatics, and the acquisition of extensive sequence databases, now permit the analysis of genome-wide expression profiles from a diversity of species. Ideally, such analyses are based on sequence data for many genes obtained from genome projects on target organisms, but for the toxic microalgae and cyanobacteria such data are not currently available. Our main objective is to examine the genetic basis for toxin production and bloom formation from a limited genomic perspective. It can be considered an advantage of this approach that our study plans to be useful in the search for genes associated with stress responses, mating and induction of sexuality, and other critical metabolic processes. When interpreted with reference to the EST sequence data-base, the integration of genetic data with environmental forcing functions should contribute to a better understanding of the regulation of growth, leading to the phenomenon of bloom formation. Such knowledge can also contribute to the formulation of EU policy regarding the potential effects on HAB dynamics of the input of anthropogenic nutrients into coastal waters. The aim of this project is to generate a seminal sequence data-set on genes related to toxin expression and the interactions of genetic and environmental factors (light, salinity, temperature, nutrients) influencing growth and hence bloom formation for a diversity of prokaryotic and eukaryotic algal species. Species will be selected to represent a wide range of systematic groups contributing to HABs in European waters, including the cyanobacteria, dinoflagellates, raphidophytes, prymnesiophytes, and diatoms.
More detailed, measurable objectives are:
|*||Development of a selected EST database for target toxic algal species for future genomic analysis|
|*||Provision of comparative sequence data to examine the gene expression of particular metabolites, e.g. phycotoxins, stress proteins, and other bioactive compounds|
|*||Contribution to the understanding of genetic regulation of biochemical pathways leading to synthesis of phycotoxins|
|*||Contribution to the understanding of genetic regulation of growth and algal bloom formation in response to environmental stimuli|
|*||Construction of DNA microarrays to study gene expression in toxic algae under controlled physiological conditions|
ESTTAL has adopted a common approach for all the WPs to better facilitate the comparative studies among the different taxa. For this reason, the activities proposed in each WP are very similar and will proceed according to an established sequence of work steps for each partner in order to use the same methodology on diverse species. For the eukaryotic algae, e.g. Alexandrium minutum, Prymnesium parvum, Pseudo-nitzschia, and Fibrocapsa japonica a large number of EST sequences will be generated to achieve saturation for one single toxigenic strain. In other cases, such as the cyanobacterium Planktothrix, the strategy will be to compare strains within a species that produce different classes of toxins. The identification and annotation of the differentially expressed ESTs in prokaryotes and eukaryotes will be greatly facilitated by the availability of a number of cyanobacterial and eukaryotic algal whole genome sequences, including those for the cyanobacteria Nostoc, Anabaena, Trichodesmium, Cyanidioschyzon merolae and the diatom Thalassiosira pseudonana. The results from a number of other EST sequencing projects, e.g., on Phaeodactylum, Ostreococcus and Chlamydomonas, can also be accessed and compared.
The steps of all ESTTAL work packages can be summarised as follows:
|1.||Grow toxic eukaryotic algal species representing major planktonic clades under a wide range of growth conditions. Grow cyanobacterial strains containing multiple toxin types and expressing different toxicity|
|2.||Assay and analyse cell extracts to confirm toxin expression|
|3.||Extract total RNA and reverse transcribe one part into cDNA|
|4.||Clone cDNA into plasmid vectors and randomly sequence ~10,000 clones from each species, group sequences into contigs (= ESTs)|
|5.||Carefully annotate ESTs and identify genes potentially involved in toxin synthesis or growth regulation leading to bloom formation|
|6.||Create DNA microarrays for each species from a subset of all ESTs|
|7.||Hybridize DNA microarrays with RNA of the cultures for expression analysis of potential toxin- or bloom-related genes|
Toxic strains of the eukaryotic species (Alexandrium minutum, Fibrocapsa japonica, Prymnesium parvum, and Pseudo-nitzschia multistriata) are grown in replicate batch cultures on defined enriched natural seawater growth medium under various conditions to determine their growth characteristics. Conditions include environmental factors known to favour maximal growth leading to bloom formation and the expression of toxicity. In addition, alternative growth conditions are chosen to establish the upper and lower threshold levels for growth tolerance, including manipulation of light, temperature, salinity, and nutrients such as nitrate and phosphate concentrations. Approximately 10 different culture treatments are tested as environmental factors are known to have a great influence on algal growth rates as well as on cell toxin quota and expressed toxicity. Therefore, both the growth kinetics and the expressed toxin levels throughout the culture cycle for each species is followed closely. With the possible exception of Pseudo-nitzschia, for which maximal domoic acid production tends to occur upon entry into stationary growth phase under nutrient-stressed conditions, cultures are harvested for RNA extraction and toxin analysis in the late exponential growth phase, to maximize biomass yield and cell toxin quota and to limit the effect of other leaked or excreted metabolites that may be produced by cell lysis.The cell toxicity and toxin content will be monitored by bioassays and/or by chemical analytical methods. We have preferentially selected a plasmid vector over a phage system because the outcome, i.e., plasmid preparations from bacteria, is more suitable for subsequent sequencing. By using plasmids as templates for sequencing rather than ?-phages, we expect to achieve a higher percentage of successful sequencing reactions and, more importantly, a longer reading length. Sequences will be assembled into contigs and then compared to sequences in the databanks. The outcome of this comparison will be carefully analysed by manual methods. Subsequently, a workshop will be held to bring together all results and to identify and collect all genes resulting from the ESTs. At the annotation workshop, we will collaboratively choose the ESTs of interest for the microarrays. We will explicitly select a basic similar set of genes for all species, supplemented by specific genes for individual species. The outcome will be species-specific arrays containing a large number of genes covering all areas of metabolism
|Work package No||Work package title||Partners involved (in bold lead party)|
|WP 1||ESTs from Alexandrium minutum & Prymnesium parvum||Alfred Wegener Institut; MPI for Chemical Ecology; Fritz Lipmann Institute for Age Research|
|WP 2||ESTs from Pseudo-nitzschia||Stazione Zoologica Anton Dohrn; MPI for Chemical Ecology; Fritz Lipmann Institute for Age Research|
|WP 3||ESTs from Fibrocapsa japonica||Ecole Normale Superieure, Department of Biology; MPI for Chemical Ecology; Fritz Lipmann Institute for Age Research|
|WP 4||ESTs from Planktothrix rubescens||School of Biological Sciences University of Bristol; MPI for Chemical Ecology; Fritz Lipmann Institute for Age Research|
In summary, the proposed project will provide a data-set that will allow a better understanding of formation of HABs and the molecular basis of toxin production. Knowing the sequences of toxin genes will allow for the recognition of these genes, or their transcripts, in the environment. Understanding the factors influencing toxicity will ultimately assist in prediction of the magnitude and environmental consequences of specific HABs and therefore constitutes an important element of an early warning system.
Understanding the regulation of toxin genes, as well as those responsible for stress response, growth and other metabolic processes, will allow for improved prediction of conditions under which toxin synthesis and growth may be enhanced or suppressed, and therefore enable better forecasting of HABs and the magnitude of their toxic impact. Although it is beyond the scope of this project, it is not unreasonable to expect that genomic studies will eventually assist in the development of mitigation strategies. For the present and near future, information on sequence and regulation information on toxin genes will enable monitoring their expression in natural plankton communities.
Several thousand genes will be isolated that are likely not involved in toxin biosynthesis from the targeted microalgal and cyanobacterial phytoplankton groups. Studying the expression of these genes is critical to the general understanding of ecophysiology and metabolism of these organisms, e.g. their impact on bloom formation. The sequences of these genes will be made available to the scientific community and thus will help marine biologists and genomics researchers from a variety of disciplines, including those involved in phylogeny, systematics, taxonomy, gene expression and biodiversity research. The sequence data will also add to the data-set generated by the EU Network of Excellence on Marine Genomics.
Standardization The ESTTAL comparative approach to sequence data-base analysis will allow for considerable standardization in methodologies. In the global HAB content, this proposal represents the first coordinated attempt to link the production of toxins among diverse phycotoxigenic groups of algae and cyanobacteria by means of EST sequencing and to study gene expression of these target analytes by both molecular and chemical-analytical techniques. Rather than scatter the efforts of diverse research groups we have chosen to unite the specialists in genomics, bioinformatics, sequencing and gene expression, microarray development, organismal biology and toxin detection, whereby each partner provides the leading role for the project in a particular area of expertise. This will assist in standardizing methodologies and ensure a minimum of operator-introduced variability in the respective data sets. The development of novel microarrays for toxic algae is fundamentally dependent upon the strict standardization of protocols. Since there are very few studies on EST expression in microalgae, much less for toxic species, there is scope for significant new advances in molecular technology.
Monitoring The EU expansion, coupled with increasing globalization of trade in seafood, will require the development of new monitoring technologies and harmonization of seafood safety regulations to ensure public health of the consumers. On the other side, the global increase and dependence upon aquaculture production in areas that may be subjected to episodic outbreaks of toxicity due to Harmful Algal Blooms can have a dramatic effect upon social and economic development in producing countries, most importantly in developing economies. Therefore the implementation of appropriate HAB bloom and toxin monitoring strategies is required. Even a modest improvement in understanding the causative factors of toxin production and diagnostic methods with field applications to be developed within ESTTAL can help to minimize and mitigate these deleterious consequences.
Policy Monitoring toxic algae in European waters is subject to the EU Directive, 91/1491/CEE. At present, this is only achieved using more traditional methods such as microscopic counts and bioassays. No bloom prediction has been implemented because the basis of bloom formation is poorly understood and ecosystem models are not yet available. Furthermore, in most cases, no distinction is made between toxic and non-toxic strains of a given species, leading to the possibility of false alarms where a non-toxic strain of a potentially toxic species is detected. In the future, through the identification of toxin related genes, we will be able to detect these genes and their transcripts in environmental samples by deploying microarray and microchip technology. Knowledge of potential effects of increasing bloom occurrence and toxin production relative to anthropogenic input will inform EU policies regarding coastal regulations and monitoring.
Dissemination and Exploitation ESTTAL endeavours to disseminate the key results by addressing the issues in laymans language. In this regard, ESTTAL members maintain close links with the communication departments of their respective institutes. Access to the EST data-base is restricted to the consortium participants under password protection until such time as the SSC deems it appropriate to widen access the this information. Limited sharing of the data-base may be granted to members of other research consortia if this is viewed by the SSC to be mutually beneficial. The consortium expects to provide all the sequence data to public scientific archives (e.g. GENBANK) upon completion of the tasks and eventual publication