Valeria Matranga

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

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.

Common strategies and technologies for the ecosafety assessment and design of nanomaterials entering the marine environment.

The widespread use of engineered nanomaterials (ENMs) in a variety of technologies and consumer products inevitably causes their release into aquatic environments and final deposition into the oceans. In addition, a growing number of ENM products are being developed specifically for marine applications, such as antifouling coatings and environmental remediation systems, thus increasing the need to address any potential risks for marine organisms and ecosystems. To safeguard the marine environment, major scientific gaps related to assessing and designing ecosafe ENMs need to be filled. In this Nano Focus, we examine key issues related to the state-of-the-art models and analytical tools being developed to understand ecological risks and to design safeguards for marine organisms.

Stress to cadmium monitored by metallothionein gene induction in Paracentrotus lividus embryos

Abstract We used sea urchin embryos as bioindicators to study the effects of exposure to sublethal cadmium concentrations on the expression of the metallothionein (MT) gene stress marker. For this purpose, the complete complementary deoxyribonucleic acid of the species Paracentrotus lividus (Pl) was cloned and sequenced. Northern blot analysis showed that basal levels of Pl-MT messenger ribonucleic acid, having an apparent size of 700 bases, are expressed in all developmental stages analyzed, from early cleavage to pluteus. However, when embryos were continuously cultured in sublethal CdCl2 concentrations and harvested at cleavage, swimming blastula, late gastrula, and pluteus stages (6, 12, 24, and 48 hours after fertilization, respectively), a time- and dose-dependent increase in the transcription levels of the Pl-MT gene was observed. Interestingly, although microscopical inspection revealed the occurrence of abnormalities only after 24 hours of exposure to the pollutant, Northern blot and reverse transcriptase–polymerase chain reaction analyses revealed significant increases in Pl-MT expression levels already after 12 and 6 hours of exposure, respectively. Therefore, this study confirms the validity of MT as marker of exposure and provides evidence that Pl-MT and sea urchin embryos can be a potentially valuable and sensitive model for testing in very short periods of time seawaters heavily contaminated with cadmium.

Coelomocytes and post-traumatic response in the common sea star Asterias rubens

Abstract Coelomocytes are recognized as the main cellular component of the echinoderm immune system. They are the first line of defense and their number and type can vary dramatically during infections or following injury. Sea stars have been used as a model system to study the regeneration process after autotomy or predation. In the present study we examined the cellular and biochemical responses of coelomocytes from the European sea star Asterias rubens to traumatic stress using immunochemical and biochemical approaches. In terms of trauma and post-traumatic stress period, here we consider the experimental arm amputation and the repair phase involved in the first 24 hours post-amputation, which mimicked a natural predation event. Four cell morphotypes were distinguishable in the coelomic fluid of both control and post-traumatic-stressed animals (phagocytes, amoebocytes, vibratile cells, hemocytes), but phagocytes were the major components, accounting for about 95% of the total population. Thus, the effects measured relate to the overall population of coelomocytes. A modest increase in the total number of freely circulating coelomocytes was observed 6 hours post-amputation. Interestingly, a monoclonal antibody (McAb) to a sea urchin embryo adhesion protein (toposome) cross-reacted with isolated sea star coelomocytes and stained the coelomic epithelium of control animals with an increase in trauma-stressed arms. In addition, coelomocytes from trauma-stressed animals showed a time-dependent increase in Hsp70 levels, as detected by both immunocytochemistry and immunoblotting within 24 hours after arm tip amputation, with a peak at 6 hours after amputation. Our findings indicate a clear role for coelomocytes and classic stress molecules in the post-traumatic stress associated with the early repair phase of regeneration.

Sea urchin coelomocytes as a novel cellular biosensor of environmental stress: a field study in the Tremiti Island Marine Protected Area, Southern Adriatic Sea, Italy

The aim of the present study was to investigate on the suitability of the sea urchin as a sentinel organism for the assessment of the macro-zoobenthos health state in bio-monitoring programmes. A field study was carried out during two oceanographic campaigns using immuno-competent cells, the coelomocytes, from sea urchins living in a marine protected area. In particular, coelomocytes subpopulations ratio and heat shock protein 70 (HSC70) levels were measured in specimens of Paracentrotus lividus (Lamark, 1816) collected in two sampling sites, namely Pianosa and Caprara Islands, both belonging to the Tremiti Island Marine Protected Area (MPA) in the Southern Adriatic Sea, Italy. By density gradients separation performed on board the Astrea boat, we found an evident increase in red amoebocytes, a subpopulation increasing upon stress, in those specimens collected around Pianosa (strictly protected area with no human activities allowed), unlike those collected around Caprara (low restrictions for human activities). Likewise, we found higher HSC70 protein levels in the low impacted site (Pianosa) by Western blots on total coelomocyte lysates. The apparent paradox could be explained by the presence in the Pianosa sampling area of contaminating remains from Second World War conventional ammunitions and a merchant boat wreck. Metal determination performed using sea urchin gonads by inductively coupled plasma atomic emission spectrometry (ICP-AES) revealed higher Fe and lower Zn levels around Pianosa with respect to Caprara, in accordance with the persistent contaminating metal sources, and thus calling for remediation measures. Taken all together, our results confirm the feasibility of using sea urchin coelomocytes as biosensors of environmental stress.

Manganese Interferes with Calcium, Perturbs ERK Signaling, and Produces Embryos with No Skeleton

Manganese (Mn) has been associated with embryo toxicity as it impairs differentiation of neural and skeletogenic cells in vertebrates. Nevertheless, information on the mechanisms operating at the cellular level remains scant. We took advantage of an amenable embryonic model to investigate the effects of Mn in biomineral formation. Sea urchin (Paracentrotus lividus) embryos were exposed to Mn from fertilization, harvested at different developmental stages, and analyzed for their content in calcium (Ca), expression of skeletogenic genes, localization of germ layer markers, and activation of the extracellular signal-regulated kinase (ERK). By optical and immunofluorescence microscopy, we found that Mn exposure produced embryos with no skeleton, by preventing the deposition of the triradiate calcitic spicules usually produced only by specialized mesoderm cells. On the contrary, ectoderm and endoderm differentiation was not impaired. Endogenous Ca content in whole embryos and its localization in Golgi regions of skeletogenic cells was strongly reduced, as measured by atomic absorption spectrometry and in vivo calcein labeling. Spicule-lacking embryos showed persistent ERK activation by immunocytochemistry and immunoblotting, contrary to the physiological oscillations observed in normal embryos. The expression of the skeletogenic genes, Pl-msp130 and Pl-sm30, was also differentially affected if compared with controls. Here, we showed for the first time the ability of Mn to interfere with Ca uptake and internalization into skeletogenic cells and demonstrate that Ca content regulates ERK activation/inactivation during sea urchin embryo morphogenesis. The use of Mn-exposed sea urchin embryos as a new model to study signaling pathways occurring during skeletogenesis will provide new insights into the mechanisms involved in Mn embryo toxicity and underlie the role of calcium in the biomineralization process in vertebrates.

Monitoring chemical and physical stress using sea urchin immune cells.

Coelomocytes are the cells freely circulating in the body fluid contained in echinoderm coelom and constitute the defence system, which, in response to injuries, host invasion, and adverse conditions, is capable of chemotaxis, phagocytosis, and production of cytotoxic metabolites. Red and colourless amoebocytes, petaloid and philopodial phagocytes, and vibratile cells are the cell types that, in different proportions, constitute the mixed coelomocyte cell population found in sea urchins. Advances in cellular and molecular biology have made it possible to identify a number of specific proteins expressed in coelomocytes under resting conditions or when activated by experimentally induced stress. Only recently, coelomocytes have been used for pollution studies with the aim of introducing a new biosensor for detection of stress at both cellular and molecular levels, as sentinel of sea health. In this chapter, we briefly review the important features of these valuable cells and describe studies on their use in the laboratory and in the field for the assessment of chemical and physical pollution of the sea.

Evidence of a precursor-product relationship between vitellogenin and toposome, a glycoprotein complex mediating cell adhesion

Toposome, a large and oligomeric glycoprotein complex isolated from mesenchyme-blastula embryos, was defined as a cell-adhesion molecule expressing positional information specificities during sea urchin embryogenesis. This report describes the biochemical and functional characterization of the toposome precursor from sea urchin coelomic fluids of both male and female organisms. The molecule is isolated in the form of a 22S particle which has an apparent molecular mass of 200 kDa. An intermediate form is present in yolk granules of unfertilized eggs with a molecular mass of 180 kDa. The 200 kDa and 180 kDa polypeptides are defined as toposome precursors by Western blot and immunoprecipitation analyses using polyclonal and monoclonal toposome-specific antibodies. Comparison of the 200 kDa polypeptide and mesenchyme-blastula toposome by partial-proteolysis peptide-mapping shows that they are related in a precursor-product relationship. A morphogenetic cell-aggregation assay shows that toposome precursors promote cell adhesion of dissociated blastula cells, suggesting that processing is not required for the cell-adhesion function. The studies reported here present the first evidence that cell adhesion molecules first appear in the form of a 200 kDa polypeptide, previously named vitellogenin, and to which only a function as major-yolk-protein precursor has been ascribed.

The multixenobiotic resistance mechanism in the marine sponge Suberites domuncula: its potential applicability for the evaluation of environmental pollution by toxic compounds

Experiments were carried out with the marine sponge Suberites domuncula to determine whether sponges may express-like mammalian tumor cells-a multidrug-like transporter system. The results demonstrate that sponge cells possess such a protective system termed multixenobiotic resistance (MXR) pump or P-glycoprotein-like pump. The protein was identified by antisera for the mammalian P170 multidrug resistance protein as a 130 kDa molecule. Binding studies were performed with 3H-vincristine (3H-VCR) and membrane vesicles; this process is ATP-dependent and inhibited by verapamil, which is known to reverse the multidrug-resistance phenotype in mammalian systems. Accumulation experiments were performed to demonstrate that the uptake of 3H-VCR is time-dependent, and increases at elevated extracellular levels of 3H-VCR. Application of the dyeing technique with calcein-AM, a suitable functional assay for multidrug transporter systems in mammal cells, also revealed the existence of the MXR pump in S. domuncula plasma membranes. These data demonstrate that S. domuncula is provided with a multidrug-like transporter, the MXR pump, which might function as a protection system for sponges in polluted environments.

Sea urchin immune cells as sentinels of environmental stress

Echinoderms, an ancient and very successful phylum of marine invertebrates, play a central role in the maintenance of ecosystem integrity and are constantly exposed to environmental pressure, including: predation, changes in temperature and pH, hypoxia, pathogens, UV radiation, metals, toxicants, and emerging pollutants like nanomaterials. The annotation of the sea urchin genome, so closely related to humans and other vertebrate genomes, revealed an unusually complex immune system, which may be the basis for why sea urchins can adapt to different marine environments and survive even in hazardous conditions. In this review, we give a brief overview of the morphological features and recognized functions of echinoderm immune cells with a focus on studies correlating stress and immunity in the sea urchin. Immune cells from adult Paracentrotus lividus, which have been introduced in the last fifteen years as sentinels of environmental stress, are valid tools to uncover basic molecular and regulatory mechanisms of immune responses, supporting their use in immunological research. Here we summarize laboratory and field studies that reveal the amenability of sea urchin immune cells for toxicological testing.