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Contenu archivé le 2022-12-23

Surveillance of Pollution in the Mediterranean Sea : Marine Organims as Ubiquitous Markers - Novel Approach

Objectif

- To study and characterise the stress proteins and the MFO system of four common Mediterranean organisms (the seabream Sparus aurata, the colonial sea squirt Botryllus schlosseri, the siliceous sponge Geodia cydonium, and mytilid mussels) under defined field and laboratory conditions;
- To study the effects of pollutants on cell cultures raised from tunicates and sponges;
- To develop an array of probes (oligonucleotides and antibodies) for fast and simple detection of stress protein levels;
- To establish a combined bioassay method for surveying different levels and sources of pollution in the Mediterranean Sea.
As examples for the progress of the research activities the results from the cloning studies, using the sponge Geodia cydonium, are reported here.
The following biomarkers have been cloned :

- Heat Shock Protein-70 (HSP70)
The biomarker concept involves the use of biochemical, cellular and physiological parameters as screening tools in environmental surveillance. Stress proteins, such as heat shock proteins [HSPs], fulfil many of the requirements for being ideal candidates in a biomarker strategy for environmental monitoring. Sponges [Porifera] are one of the major phyla found in the marine hard-substrate benthos, both with respect to the number of species and biomass. However, only recently have genes from sponges been cloned. Here we describe the isolation of the cDNA encoding a heat shock protein of Mr 70 kDa [HSP70]. The cDNA HSP70, from the marine sponge G. cydonium has a length of 2.3 kb and encodes an aa sequence of Mr 72,579. The sponge HSP70 displays characteristic features of the HSP70 family. The HSP70 protein is induced by natural stressors including changes in temperature as well as pH as demonstrated by Western blot analysis. No response was observed after treating the samples with hypotonic- or hypertonic conditions. Our results provide the first molecular evidence that HSP70 of sponges is a useful biomarker.

- GDP-dissociation inhibitor
We have isolated the Rab GDP-dissociation inhibitor [GDI], which has previously been shown to be a key element in the intracellular traffic system. The 1,521 bp long cDNA, encoding sponge GDI, has been isolated and analysed. The deduced aa sequence [Mr 50,215] displays high homology to vertebrate, invertebrate and also yeast GDIs. Northern blot analysis revealed that the 1.6 kb sponge transcript that hybridised with the sponge GDI is downregulated under temperature stress; in contrast, the transcription of the sponge HSP70 is strongly enhanced. Due to the high sequence-conservation of the GDIs one can assume that sponge GDI probes can be used as functional biomarkers for temperature stress, especially when applied simultaneously with the highly conserved HSP70 probes.

- DnaJ
In preparation.

- Application of biomarkers
The marine sponge Suberites domuncula was used to identify early markers for thermal stress. Cubes from sponges have been kept for 30 min at 31ºC, a temperature that is 10ºC higher than the ambient temperature. After this treatment the sponge cubes were maintained again at 21ºC. To demonstrate that the animals reacted to the elevated temperature, the expression of heat shock protein [HSP] was determined. Using an antibody rose against HSP70 it was found by Western blotting that the animals specifically express an 45 kDa polypeptide after heat treatment. It was shown that already after 10 min of heat treatment the steady-state concentration of trehalose drops by 40% from a basis level of 13 nmol/mg protein. The activity of the trehalose-degrading enzyme, trehalase, remained unchanged. As second immediate early biomarkers for thermal stress, the enzyme activity of glutathione S-transferase [GST] as well as glutathione can be used. After a 5-min period the activity of GST decreased from 25 nmol/min x mg to 15 nmol/min x mg. Similarly, the level of glutathione [GSH] dropped from 22 nmol/mg to 12 nmol/mg after a 15 to 20 min exposure time. The original state of the biomarkers, trehalose, GST and GSH, was reached again after a recovery period of less than 180 min. The steady-state level for polyphosphates did not change during heat treatment. These data show that in S. domuncula the concentration level of trehalose and the activity of GST are biomarkers for immediate early response towards heat stress.

Follow-up

- The results that will be obtained from the examined species will be evaluated to find the common proteins and the specific ones that are synthesised as a response to different stressors. Putting all the data together, it will be tried to establish a common quantitative index that will indicate the relative stress of an examined organism.
- The studied areas
- Haifa Bay
Adjacent to one of the major industrial areas of Israel, and a considerable number of industries discharge their effluents into the bay either directly or through the Kishon river. It is also an area that is used as a commercial fishing ground;
- The Northern Adriatic (Rovinj)
A highly polluted area including zones heavily polluted and others that are less affected by human activities. The gradient of pollution at the marine sites near Rovinj ranges from unimpacted sites through gradually more impacted sites, and areas heavily impacted by the wastes of a local cannery;
- The Aegean coast (Izmir Bay)
With a total length of 2,800 km, Turkey's longest coastline. Wastewater discharges into the Aegean occur at a total of fifteen points along it's Turkish coastline, including seven rivers, six tourist enterprises, other sources of household wastes of various sizes, and one industrial zone. The total pollution load of these sources is equivalent to that of a population of around 10 million.

- The selected organisms
- Botryllus schlosseri (Pallas) is a cosmopolitan encrusting colonial tunicate (Protochordata);
- The siliceous sponge Geodia cydonium (Jameson) is a common cosmopolitan organism;
- The mytilid mussels [Mytilus galloprovincialis, Mytilaster minimus] are cosmopolitan bivalves;
- The gilthead seabream (Sparus aurata; Sparidae) is an economically important marine fish.
All four organisms were found to be useful as model organisms for biochemical monitoring of environmental pollutants.

- Characterisation of stress proteins
The study will be done simultaneously on all four animals collected from clean, relatively non-polluted areas (laboratory studies) and on animals collected from polluted or semi polluted areas for evaluation of the levels of different heat shock proteins (hsps) in their tissues (field studies). Additionally, laboratory studies on hsp synthesis will be performed in vitro on the Botryllus cell line recently developed. In the laboratory studies different stressors and combinations of different stressors will be used such as heat shock, heavy metals, water soluble fractions of oil, sedimentation, detergents, carcinogens, different drugs, aerial exposure, mechanical damages, polychlorinated biphenyls and more. In these experiments, the study of stress proteins will be conducted through three routes: 1. metabolic labelling, 2. hsp-expression using specific antibodies as probes, 3. measurement of hsp-mRNA level using cDNA probes, to obtain the rate of expression of the hsp-gene on the level of transcription/post-transcription.

- Study on MFO systems in fish and mussels
This study will be done simultaneously on the gilthead seabream and the mussel M. galloprovincialis. The methods being used are partly similar to those used in the section of the hsp (such as using of monoclonal antibodies, immunoprecipitation, cloning of MFO genes, determination of the MFO mRNA expression rates by Northern blotting) and will not be repeated here.

- Establishing bioassays with tissue cultures
Cell cultures derived from tunicate blood cells and sponge tissues will be used. While permanent cell lines from tunicates are already established in the groups, until now only short term cultures are available from sponges. Permanent lines will be obtained from G. cydonium following the successful procedures applied previously with B. schlosseri.

Thème(s)

Data not available

Appel à propositions

Data not available

Régime de financement

CSC - Cost-sharing contracts

Coordinateur

JOHANNES GUTENBERG UNIVERSITAET MAINZ
Contribution de l’UE
Aucune donnée
Adresse
Duesbergweg 6
55099 MAINZ
Allemagne

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Participants (3)