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. 2000 Apr;5(2):113–120. doi: 10.1379/1466-1268(2000)005<0113:cabrte>2.0.co;2

Cellular and biochemical responses to environmental and experimentally induced stress in sea urchin coelomocytes

Valeria Matranga 1,3, Giuseppe Toia 1, Rosa Bonaventura 1, Werner EG Müller 2
PMCID: PMC312897  PMID: 11147962

Abstract

Coelomocytes are considered to be immune effectors of sea urchins. Subpopulations of coelomocytes can be purified from a total cell suspension. The proportion of each cell type can vary not only among species, but also between individuals of the same species, according to their size and physiological conditions. We tested the hypothesis that coelomocytes play a role in defense mechanisms activated by adverse external conditions. Total coelomocytes from control and stressed (temperature, pollution, and injuries) sea urchins were analyzed for their expression of the 70 kDa heat shock protein (hsp70), a well recognized stress marker. Further analysis was performed by separation of coelomocytes into subpopulations by step gradients. We demonstrated that sea urchin coelomocytes respond to temperature shock and to polluted seawater by the upregulation of hsp70. Among coelomocytes certain cells, known as red spherula cells, showed a great increase in number in animals collected from polluted seawaters or subjected to “accidental” injury. The present study confirms the immunological function of sea urchin coelomocytes, as indicated by the upregulation of the hsp70 molecular marker, and suggests that sea urchin coelomocytes can be utilized as sensitive bio-indicators of environmental stress.

INTRODUCTION

In recent years interest has grown in the use of aquatic invertebrates as tools for monitoring environmental hazards. Most of the literature comes from studies on lower marine invertebrates, such as mussels (Kurelec 1992). However, few investigations report the effects of pollution on adults of higher marine invertebrates (eg, Echinoderms). This is probably due to the fact that effector systems that can be used for monitoring pollution are not known. In contrast, the teratogenic effects of various chemical agents and drugs on the development of sea urchin embryos have been extensively studied (Kobayashi 1980; Ozretic et al 1985; Sconzo et al 1995; Morale et al 1998).

The coelomic cavity of the sea urchin contains cells, generically called coelomocytes, that have been studied for many decades. Due to their capability to respond to injuries, host invasion, and cytotoxic agents, coelomocytes are regarded as the immune effectors of the sea urchin. In fact, coelomocytes react to challenges with modifications in their motility, increased phagocytic and encapsulation activities, and release of cytotoxic factors (Matranga 1996). Coelomocytes can be separated into subpopulations whose functions are not yet understood. It is not yet clear whether diverse coelomocytes, which differ in size and shape, have different functions or whether they have a common precursor cell resembling the human blood cell lineages.

The aim of this study was first to find a biological indicator to be used as a stress marker and to characterize the response to induced or accidental stresses by the use of molecular markers. Second, to establish the use of sea urchin coelomocytes as a cell laboratory to test different environmental hazards. For this purpose, sea urchin coelomocytes were challenged by conditions known to be adverse for the whole organism, such as changes in ambient temperature of the seawater in which the animals were kept or exposure to pollutants and injury. In all cases, the expression of stress protein (hsp70) was analyzed by means of specific monoclonal antibodies on Western Blots of total homogenates. Variations in the ratio of coelomocyte subpopulations were analyzed by purification and identification of cell subpopulations.

MATERIALS AND METHODS

Sampling

Paracentrotus lividus to be used for experimentally induced stresses were collected from the seacoast of the Palermo gulf. Animals for monitoring pollution effects were collected in the Northern Adriatic Sea, along the coasts near Rovinj (Istria), and kept in tanks under constant aeration in circulating seawater. Animals collected from the uncontaminated Limski Canal (north of Rovinj) were used as unpolluted controls. Those collected near the lab water front (in front of Ruder Boskovic Marine Station) were considered to be polluted, which means under the direct influence of urban runoff and industrial waste from a nearby fish cannery (Bihari and Batel 1994; Müller et al 1998).

Coelomocyte separation in Na-metrizoate gradients

Sea urchins were bled individually through a cut in the peristomal membrane. Usually between 1 and 2 × 106 cells per mL are contained in the coelomic fluid collected from sea urchins, and a volume of 5–10 mL total, depending on the size of the sea urchin. The fluid, approximately 10 mL, containing the total coelomocyte population was poured on 30-mL ice-cold 20 mM Tris -0.5 M NaCl -30 mM Ethylenediaminetetraacetic acid pH 7.5 (ISO-EDTA). Cells were centrifuged at 50× g and the pellet was resuspended in 1 mL ISO-EDTA. The suspension was loaded on a 5 mL Metrizoic Acid (MA)-ISO-EDTA, step gradient (MA: 1 mL each of 37.5%, 25%, 18.75%, 15%, and 12.5%). The gradients were centrifuged at 50× g for 15’ with a swing-out rotor as previously described (Gerardi et al 1990).

Sea urchin coelomocyte stress induction and hsp70 expression analysis

Individuals of sea urchin P lividus were temperature stressed in the laboratory and the expression of hsp70 was determined by immunological methods. Induction of heat or cold stress was performed by placing urchins for 2 hours in warm water at 35°C or cold water at 4°C respectively, followed by 1 hour recovery at 16°C. For time course experiments, sea urchins were exposed to a cold temperature for variable periods of time followed by 1 hour recovery at 16°C.

The coelomic fluid from stressed or control sea urchins was collected by a cut in the peristomal membrane and immediately poured on 30 mL ice-cold 20 mM Tris-0.5 M NaCl- 30 mM Ethylenediaminetetracetic acid pH 7.5 (ISO-EDTA). No significant differences in total number of cells were found between control and stressed sea urchins. The total coelomocyte population from either control or stressed urchins constituted an experimental point. Coelomocytes from different urchins were never pooled. The number of animals used and the individual variations are presented in the results section.

Cells were centrifuged at 50× g for 5’, the supernatant discarded, and the pellet lysed in 500 μL of a dilution 1:10 of ISO-EDTA, supplemented with a cocktail of protease inhibitors: 1 μL MIX (Antipain 1 mg/mL, Leupeptin 1 mg/mL, Benzamidine 500 mM), 1 μL aprotinin (1 mg/mL), 1 μL pepstatin (1 mg/mL), 0.25 μL PMSF (200 mM). The lysates were centrifuged at 12000 rpm for 10’, the supernatant was recovered, and the protein content was determined by the Lowry method.

SDS-PAGE and Western blot

Coelomocytes lysates, (20 μg), were separated on 6% SDS-PAGE under reducing conditions according to Laemmli (1970). Molecular weight markers were β-galactosidase (116 kDa), phosphorylase b (97kDa), and bovine serum albumin (67kDa) from BIORAD (Hercules, CA, USA). The SDS-polyacrylamide minigels were transferred to nitrocellulose paper as reported by Towbin et al (1979). Western blots were performed using an anti-bovine brain 70 kDa heat shock protein monoclonal antibody (hsp70 McAb) commercially available (SIGMA Chemical Company, H-5147, St Louis, MO, USA) diluted 1 to 1000. The second antibody was an anti-mouse conjugated with peroxidase from SIGMA, diluted 1 to 2000. 4-chloro-1-naphtol was used for detection of bands, and reactions were terminated by addition of distilled water. In order to compare band intensities, immunoblots were scanned with a Bio-Rad imaging densitometer (Model GS-670) equipped with an analysis program automatic integrator (Molecular Analyst).

RESULTS

Sea urchin coelomocytes respond to temperature shock by hsp70 protein overexpression

The response to temperature stress has been fully analyzed and described in sea urchin embryos of the species P. lividus (Giudice 1989) and the genes involved have been identified (Sconzo et al 1992). However, no studies on the presence of constitutive or inducible hsp70 protein in adult sea urchin cells have been reported. To test whether sea urchin coelomocytes respond to changes in water temperature by activating expression of hsp70, adult individuals were kept at 16°C, the control temperature (CT), with subsequent 35°C heat stress (HS) or 4°C cold stress (CS) for 4 hours. After a 1 hour recovery at 16°C, coelomocytes were gently collected at 4°C to prevent handling stress as much as possible, lysed as previously described, and equal amounts of protein were loaded on SDS-PAGE and analyzed for the expression of hsp70 by immunoblotting. The McAb used reacts specifically against hsp70 of bovine, human, rat, rabbit, chicken, guinea pigs, Drosophila, and human fibroblast cells. Moreover, it has been shown to react to heat shock proteins of other invertebrate cells (Koziol et al 1997). As shown in Figure 1A, which depicts a representative experiment, an homogenate from total coelomocytes obtained from sea urchins maintained at 16°C (CT) contains an immunoreactive faint band which migrates with an estimated molecular weight of 70 kDa, indicating that coelomocytes express very low levels of hsp70 as a constitutive component. A darker hsp70 band is observed in coelomocytes from heat stressed or cold stressed sea urchins, indicating an induction of the hsp70 stress marker. In order to quantify and compare band intensities, nitrocellulose membranes were scanned using an imaging densitometer equipped with an analysis program. Values obtained, reported in arbitrary units, are shown in Figure 1B. A 2-fold increase in the expression of hsp70 is found in heat-stressed coelomocytes as compared to controls. The induction of hsp70 was much higher in cells obtained from cold-stressed sea urchins kept at 4°C, where a 5-fold increase over controls was found.

Fig. 1.

Fig. 1.

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Sea urchin coelomocytes respond to temperature stress by activation of hsp70. (A) Western blot analysis of hsp70 expression in coelomocytes from sea urchin exposed to temperature stress in the laboratory. Coelomocytes obtained from control sea urchin reared at 16°C (CT), or sea urchin kept for 4 hours at 35°C (HS) or 4°C (CS) were run on SDS-PAGE, electroblotted and probed with anti-hsp70 antibody. On the left is the position reached by molecular weight markers. (B) Quantification of hsp70 expression. The histogram shows the results obtained by densitometric scanning of the filter shown in (A)

Apparently a basal level of hsp70 expression is found in control urchins, even when coelomocytes are prepared at 0°C immediately following their arrival in the laboratory (not shown). The possibility of harvesting coelomocytes immediately following collection (on the boat) would possibly allow the measurement of true basal levels of hsp70, but would require equipment not currently available.

It should be noted that eukaryotic cells contain a multigene family that encodes several related 70kDa proteins that differ in their intracellular location and regulation. These include the consitutive (or cognate) hsp73 and the stress-inducible hsp72. Since the antibody is actually recognizing both molecular species, it should be considered that what appears as a single band may actually correspond to several distinct proteins coded by distinct genes with distinct regulations.

Accumulation of hsp70 in coelomocytes which have been stressed for different time periods

It was of interest to determine if there was a minimum time required for the activation of hsp70 expression and if the amount of protein produced increased with increasing time of exposure to stress conditions. As a higher response was obtained under cold stress activation, we decided to use this condition for time course experiments on hsp70 expression. Groups of 4 individuals were kept at 4°C for 30’, 60’ and 240’, followed by a 1-hour recovery period at 16°C. Coelomocytes were then collected at 4°C, with care taken to prevent handling stress as much as possible, lysed and then equal amounts of proteins were analyzed for the expression of hsp70 by immunoblotting. In Figure 2, results of a representative experiment are shown, with the corresponding quantification of results shown in Figure 2B. After 30 minutes of stress induction the response is modest, with an estimated 1.25-fold increase in the amount of hsp70 protein over that of the control temperature sample. A 2.41-fold increase is observed in coelomocytes from sea urchins that underwent 60 minutes of cold stress, which corresponds to the maximal hsp70 expression observed. Two hundred and forty minutes of cold stress did not result in a further increase in levels of hsp70 expression, as a 2.37-fold increase over that observed in control temperature urchins was observed. No increases in expression were found for periods between 60 and 240 minutes (not shown). Therefore, more than 30 minutes or up to an hour of cold stress is required to provoke an expression of the hsp70 over control values. Again, as shown in Figure 1, hsp 70 seems to be constitutively expressed in coelomocytes from urchins that were not given cold stress. This finding reflects variability among individuals, as expected. A comparison of results from several experiments is shown in Figure 3A, in order to account for the variability among experiments. In this case, as it was not possible to average the absolute arbitrary figures obtained from scanning of WB, the increase relative to control nonstressed coelomocytes is given.

Fig. 2.

Fig. 2.

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Time course of cold stress and analysis of hsp70 expression. (A) Coelomocytes lysates from control sea urchin (0), or sea urchin kept at 4°C for 30 minutes, 60 minutes and 240 minutes were run on SDS-PAGE, electroblotted, and probed with anti-hsp70 antibody. Indicated on the left is the position reached by molecular weight markers (B) Quantification of hsp70 expression. The histogram shows the results obtained by densitometric scanning of the filter shown in (A)

Fig. 3.

Fig. 3.

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Variabilities and different coelomocyte cell types contribution to hsp70 expression. (A) Levels of hsp70 are expressed as fold increase in the expression of hsp70 referred to the basal level of control coelomocytes in 4 different experiments, where sea urchins were exposed to cold stress for 30 and 60 minutes. (B) Total coelomocytes obtained from 7 different unstressed sea urchins and 7 different cold stressed sea urchins were run on SDS-PAGE, electroblotted and probed with anti-hsp70 antibody. (C) Total coelomocytes obtained from control sea urchin (c), or band A (a) and band F (f) purified cell types from a cold stressed sea urchin (2 hours stress) were run on SDS-PAGE, electroblotted and probed with anti-hsp70 antibody

To obtain information on the spontaneous variability in the basal levels of hsp70 among individuals, the same amount of protein from 7 unstressed individuals and 7 cold stressed individuals were loaded on a gel, electroblotted, and probed with the anti-hsp70 antibody. Results, shown in Figure 3B, demonstrate variability both in the basal and stress levels of hsp70, as detected by means of an antibody that could recognize different proteins of the same family.

As previously shown, coelomocytes can be separated by metrizoic acid step gradients into a number of subpopulations which have been named on the basis of their morphological features: amoebocytes, colorless spherula cells, and colored spherula cells (Gerardi et al 1990; Cervello et al 1994; Matranga 1996). Among the cells of the last 2 phenotypes, differences occur in terms of cell size, whereas, the morphology of the cells differs only in the accumulation of echinochrome pigments which can be yellow or red. To assess if red spherula cells could account entirely for the overexpression of hsp70, single subpopulations were separated by metrizoic acid gradient from total coelomocytes of a cold stressed sea urchin and lysed. Equal amounts of proteins were separated on SDS-PAGE and electroblotted with anti-hsp70 antibody. Results are given in Figure 3C where lysates from band A (a) and band F (f) and unstressed control from total coelomocytes (c) are shown. It was not possible to obtain enough material to load on a gel from other bands, which contained principally colorless spherula cells (bands B-E). As observed in Figure 3C similar amounts of hsp70 are present in band A and F lysates, indicating that at least 2 different cell types contribute to the overexpression of hsp70.

Polluted seawater increases the proportion of a subpopulation of red spherula cells

To investigate the ability of coelomocytes to respond to environmental stress, sea urchins were sampled from unpolluted and polluted seawaters along the coast near Rovinj, Croatia. Some tracts of this coast have previously been designated as polluted areas as a result of being under the direct influence of urban runoff and industrial waste from a nearby fish cannery, and others as nonpolluted (Muller et al 1998). Coelomocytes from unpolluted and polluted seawater were separated by density gradients and the content of each cell population was examined. Figure 4 is a schematic drawing of results obtained in several samplings, representing 100% of the pattern of subpopulation separations from unpolluted urchins (10 out of 10) and 82% of the pattern of subpopulation separations from polluted urchins (9 out of 11). A single experiment is shown in Figure 5A-D. We found that, when separated into subpopulations, coelomocytes obtained from sea urchins living in polluted seawater showed an intense red colored band starting at the interface between 37% and 25% of metrizoic acid step gradient (Fig 5A right), designated as band F (see Fig 4). Conversely, red spherula cells were present in lower amounts and did not band at the F position in control cells obtained from animals collected in unpolluted sea waters (Fig 5A, left, Fig 4). Cell counts indicated that there was no significant difference between the total number of coelomocytes from sea urchins from unpolluted and polluted environments.

Fig. 4.

Fig. 4.

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Schematic drawing of coelomocytes obtained from sea urchins found in unpolluted and polluted seawater separated by metrizoic acid step gradients into subpopulations. Number of animals showing the represented patterns are indicated below each gradient

Fig. 5.

Fig. 5.

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Sea urchins respond to environmental stress by increasing their red spherula cells coelomocyte subpopulation and overexpressing hsp70. (A) Separation of total coelomocytes obtained from sea urchins reared in unpolluted (left) and polluted (right) seawater. (B) Coelomocytes obtained from sea urchin from unpolluted (left) or polluted (right) seawater were run on SDS-PAGE, electroblotted and probed with anti-hsp70 antibody. (C) Amoebocytes from control shown in A (left); Bar = 50 μm. (D) red spherula cells from polluted sample shown in A (right). (E) Separation of total coelomocytes obtained from control (left) and injured (right) sea urchin

We noticed that animals collected from unpolluted seawater and kept in tanks for at least 2 days exhibited relatively high amounts of red spherula cells, similar to the amounts observed in polluted sea urchins (not shown). For this reason laboratory induced stress tests were performed only on freshly collected sea urchins in order to avoid spontaneous stress responses, possibly with consequent hsp70 activation (see below). Conversely, when sea urchins from polluted seawater were maintained in tanks for at least 2 days, they had a lower amount of red spherula cells than those analyzed immediately after collection (not shown). The morphology of amoebocytes, which banded at the top of gradients and constituted from 90% to 95% of the total cell population, is shown in Fig 5C. The morphology of red spherula cells, collected from band F of the gradient shown in Fig 5A right, is shown in Fig 5D. A homogenous cell population of intensely red pigmented cells can be observed. In 1969, Johnson named this morphotype on the basis of color (Johnson 1969); however, this classification is still valid for invertebrate immunologists working with the sea urchin model system (Smith et al 1992). Red spherula cells have been reported to be the cell type that responds to injury by degranulation of their Echinochrome pigment, a bactericidal agent (Service and Wardlaw 1984). To assess if hsp70 expression was also induced by environmental stress, immunoblotting analysis of total coelomocyte lysates from sea urchins living in unpolluted (5A, left) and polluted (5A, right) seawater was performed using anti-hsp70 antibody. An increase in the expression of hsp70 was observed in coelomocytes from sea urchins found in polluted seawater (5B, right), as compared to control (5B, left). When quantified by volumetric analysis a 1.9-fold increase over the control was calculated.

Sea urchin coelomocytes respond to injury by an increase in the number of red spherula cells

Sea urchins respond to pathogens or to mechanical injuries by activating defense mechanisms which include: (1) expression of humoral molecules and (2) proliferation of different cell types circulating in the body cavity (Matranga 1996). In recent years the response mediated by the activation of a specific gene, a homologue of the mammalian profilin gene (Smith and Davidson 1992) has been demonstrated. However, in the previous studies coelomocytes were always taken as a homogeneous cell population without separating them into subpopulations with possible different functions. This is partly due to the technical difficulty of separating cells from single individuals with very low amounts of material available. By chance, we obtained a few individuals from the seacoast around Sicily, which showed lesions of different types on their bodies, with a consequent lack of spines in 10% to 20% of their bodies and areas of scar tissue (not shown). We called these lesions generic injuries, as we do not know if their cause was accidental caused by encounters with rocks or predators, or pathological due to bacterial or other microbial infections. We decided to compare cell populations obtained from intact and injured urchins with the aim of comparing these results to those we had obtained with urchins from polluted seawater. Total coelomocytes were collected and fractionated by metrizoic-acid gradients as described. When injured sea urchins were analyzed as to their coelomocyte subpopulations, a very high amount of red spherula cells was observed (Fig 5E right), as compared to those in control sea urchins (Fig 5E left). These red spherula cells induced in response to injury are a homogeneous population whose morphology is similar to that of red spherula cells observed in polluted urchins (Fig 5D).

DISCUSSION

In this report we describe the capability of sea urchin coelomocytes to respond to experimentally induced stress caused by shifts in temperature, by the activation of hsp70 expression, and by an increase in the number of red spherula cells. Measured responses to cold stress were more dramatic than those observed for heat stress, using 4°C and 35°C, respectively. We do not have an explanation for this observation; however, the simplest argument would be that the P lividus species of sea urchins, which lives around Sicily, is accustomed to warm sea water, and experiences cold water as a more severe stress. A logical prediction would be that the opposite is true for species living in cold waters, for example those living in the North Sea.

Recently, the activation of murine peritoneal macrophages induced by cold water stress has been shown to be mediated by endogeneous production of Substance P (Zhu et al 1996). We do not know whether a similar mechanism is operative in sea urchin coelomocytes, but it is an attractive hypothesis to be tested.

The highest hsp70 induction observed occurs within 1 hour of stimulus. The question remains as to how this can be achieved in such a short time.

This is the first report describing the use of cells obtained from living adult sea urchins as indicators of pollution, as previous studies have utilized whole sea urchin embryos and larvae as indicators of cytotoxicity induced by several chemicals (Sconzo et al 1995). The method used for the purification and analysis of coelomocytes subpopulation is relatively easy and the presence of high amounts of red spherula cells is a clear indicator of pollution or stress. This is in agreement with earlier reports describing that in sea cucumbers (Smith 1983) and sand dollars (Smith and Smith 1985), the coelomic fluids of stressed individuals was red, probably due to degranulation and lysis of the red spherula cells. Particularly in the sand dollar Mellita quinquesperforata, it has been suggested that the red pigment is protective and histamine release after contact with foreign proteins has been measured (Smith and Smith 1985).

On the other hand, extracellular pigments in terrestrial bacteria may play a photoprotective role in absorbing UV-A radiation (Garcia-Pichel et al 1992).

In conclusion, the development of an embryo is not needed to test seawater pollution, but adult cells can be used as biomarkers. Therefore, red spherula cells have to be regarded as the primary cell population to be affected by stress such as pollution and hot or cold seawater. It would be interesting to know the origins of the greater number of red spherula cells. We can hypothesize that other cell types present in the coelomic fluid are converted to red spherula cells, perhaps confirming old theories that colorless spherula cells give rise to red spherula cells. Alternatively a mitogenic signal is causing very rapid cell duplication of a preexisting subset of red spherula cells, justifying the production of trophic factors by coelomocytes, such as inflammatory cytokines IL-1 (Beck and Habicht 1986), TNF (Beck et al 1989) and IL-6 (Beck and Habicht 1996). Another possibility is that the so-called hematopoietic areas present in the adult urchin (axial organ) would promptly release red spherula cells. Reports favor all possibilities, and these issues remain to be further investigated.

Our results agree with the notion that coelomocytes are the progenitors of vertebrate immune effectors, considering the Echinoderm phylum in the direct evolutionary line leading to the vertebrates (Smith and Davidson 1992). This concept has been documented by the finding that the SpCoel1 gene, homologous to the mammalian profilin gene (Goldschmidt-Clermont et al 1991), a protein present in platelets which acts as transductor to the cytoskeletal organization system, is activated in stimulated coelomocytes (Smith et al 1992). Furthermore, recent studies have shown that coelomocytes respond to injury by the activation of a series of genes related to the immune response (Smith et al 1996). Vertebrate homologues of complement components B and C3 have been cloned and sequenced in sea urchin, and transcripts are specifically expressed in LPS-activated coelomocytes (Al-Sharif et al 1998; Smith et al 1998).

Furthermore, the finding that biochemical mechanisms underlying multixenobiotic resistence of marine invertebrates, mussels (Kurelec 1992), and sponges (Muller et al 1996) is similar to the mechanism of multidrug resistence found in vertebrate tumor cells, opens the door to the use of sea urchin coelomocytes as cellular models with which to investigate diseases that target vertebrate blood cells. Sea urchin coelomocytes may also be a useful model to study effects of toxic compounds on the elevation of molecular markers known to be involved in immune and degenerative responses.

Acknowledgments

Many thanks to the staff of the Marine Laboratory of Rovinj, Croatia, for their hospitality and support during part of the work. The authors wish to thank Dr M. Cervello and Prof G. Sconzo for pioneering experiments and helpful suggestions on experimental procedures. This work was partially supported by EU Med-Campus Programme Med-Env-Stress Contract N° C034: “Monitoring of environmental stress using modern techniques”. G. Toia and R. Bonaventura were participants in the Med-Campus Course. We also thank the CNR Research and Training Programme for Third Mediterranean Countries to VM titled: “Monitoring of sea coasts pollution by biological indicators and biomolecular probes” for partial support of the project.

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