Rosa Bonaventura

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.

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.

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.

Titanium dioxide nanoparticles stimulate sea urchin immune cell phagocytic activity involving TLR/p38 MAPK-mediated signalling pathway.

Titanium dioxide nanoparticles (TiO2NPs) are one of the most widespread-engineered particles in use for drug delivery, cosmetics, and electronics. However, TiO2NP safety is still an open issue, even for ethical reasons. In this work, we investigated the sea urchin Paracentrotus lividus immune cell model as a proxy to humans, to elucidate a potential pathway that can be involved in the persistent TiO2NP-immune cell interaction in vivo. Morphology, phagocytic ability, changes in activation/inactivation of a few mitogen-activated protein kinases (p38 MAPK, ERK), variations of other key proteins triggering immune response (Toll-like receptor 4-like, Heat shock protein 70, Interleukin-6) and modifications in the expression of related immune response genes were investigated. Our findings indicate that TiO2NPs influence the signal transduction downstream targets of p38 MAPK without eliciting an inflammatory response or other harmful effects on biological functions. We strongly recommend sea urchin immune cells as a new powerful model for nano-safety/nano-toxicity investigations without the ethical normative issue.

Echinoderms as Blueprints for Biocalcification: Regulation of Skeletogenic Genes and Matrices

Echinoderms have an extensive endoskeleton composed of magnesian calcite, a form of calcium carbonate that contains small amounts of magnesium carbonate and occluded matrix proteins. Adult sea urchins have several calcified structures, including test, teeth, and spines, composed of numerous ossicles which form a three-dimensional meshwork of mineral trabeculae, the stereom. The biomineral development begins in 24-hour-old embryos within the primary mesenchyme cells (PMCs), the only cells producing a set of necessary matrix proteins. The deposition of the biomineral occurs in a privileged extracellular space produced by the fused filopodial processes of the PMCs. We showed for the first time that signals from ectoderm cells overlying PMCs play an important role in the regulation of biomineralization-related genes. It is believed that growth factors are produced by ectoderm cells and released into the blastocoel where they interact with cognate receptor tyrosine kinases restricted to PMCs, which activate signaling cascades regulating the expression of biomineralization-related genes. We demonstrated the implication of a TGF-beta family factor by a perturbation model in which skeleton elongation was indirectly blocked by monoclonal antibodies to an extracellular matrix (ECM) protein located on the apical surface of ectoderm. Thus, it was inferred that interfering with the binding of the ECM ligand, a member of the discoidin family, to its cell surface receptor, a βC integrin, disrupts the ectodermal cell signaling cascade, resulting in reduced or aberrant skeletons. During the last few years, we analyzed the expression of biomineralization-related genes in other examples of experimentally induced skeleton malformations, produced by the exposure to toxic metals, such as Cd and Mn or ionizing radiations, such as UV-B and X-rays. Besides the obvious toxicological implication, since the mis-expression of spicule matrix genes paralleled skeleton defects, we believe that by means of these studies we can dissect the molecular steps taking place and possibly understand the physiological events regulating embryonic biomineralization.

Combined Effects of Cadmium and UVB Radiation on Sea Urchin Embryos: Skeleton Impairment Parallels p38 MAPK Activation and Stress Genes Overexpression.

Human and natural activities release many pollutants in the marine environment. The mixture of pollutants can affect many organisms concurrently. We used Paracentrotus lividus as a model to analyze the effects on signal transduction pathways and stress gene expression in embryos exposed continuously to double stress, i.e., cadmium (Cd) from fertilization and UVB at cleavage (Cd/UVB-embryos). By microscopical inspection, we evaluated embryonic morphology after 72 h of development. Tissue-specific markers were used to assess mesoderm differentiation by immunofluorescence. We analyzed p38MAPK, ERK1/2, and JNK activation by Western blot and mRNA profiles of Pl-MT, Pl-14-3-3epsilon, and Pl-jun genes by real-time quantitative polymerase chain reaction (qPCR) and the localization of their transcripts by whole mount in situ hybridization (WMISH). We found that the Cd/UVB combined exposure induced morphological malformations in 76% of pluteus embryos, mainly affecting the development of the skeleton, including the normal branching of skeletal roads. In Cd/UVB-embryos, p38MAPK was activated 1 h after UVB exposure and a remarkable overexpression of the Pl-MT, Pl-14.3.3epsilon, and Pl-jun genes 24 h after UVB exposure. Pl-MT and Pl-14.3.3epsilon mRNAs were misexpressed as they were localized in a position different from that observed in wild-type embryos, i.e., the intestine. On the contrary, Pl-jun mRNA has remained localized in the skeletogenic cells despite their displacement in exposed embryos. In conclusion, Cd/UVB exposure affected skeletal patterning producing alternative morphologies in which p38MAPK activation and Pl-MT, Pl-14.3.3epsilon, and Pl-jun gene overexpression seem linked to a protective role against the stress response induced by Cd/UVB.

The newly characterized Pl-jun is specifically expressed in skeletogenic cells of the Paracentrotus lividus sea urchin embryo.

Growing evidence suggests that the transcription factors belonging to the Jun family are involved in many important cellular events, such as the control of bone development in mammalians. We have characterized, for the first time, a member of the Jun family from embryos of the sea urchin Paracentrotus lividus. The Pl‐jun protein sequence includes all the functional domains characteristic of members of the Jun family (i.e. the basic leucine zipper, the basic DNA‐binding and the c‐Jun N‐terminal kinase docking‐like domains), which are evolutionarily conserved. Moreover, all the key serine and threonine residues, which are phosphorylation targets for different kinases necessary for jun activation, appear to be well preserved. A model of the monomeric protein provides a simulation of the three‐dimensional structure and shows the potential sites for dimerization and DNA binding. Pl‐jun mRNA is expressed in the unfertilized egg and throughout sea urchin embryo development. As the development proceeds, Pl‐jun mRNA becomes exclusively expressed in the skeletogenic cells. Intriguingly, these cells contain significant amounts of the phosphorylated active protein entirely localized into their nuclei. These findings strengthen our hypothesis that suggests an active role for Pl‐jun in skeletogenic cells, thus indicating that this transcription factor is a novel component of the gene regulatory networks controlling skeletogenesis.

Response to metals treatment of Fra1, a member of the AP-1 transcription factor family, in P. lividus sea urchin embryos

Lithium (Li), Nickel (Ni), and Zinc (Zn) are metals normally present in the seawater, although they can have adverse effects on the marine ecosystem at high concentrations by interfering with many biological processes. These metals are toxic for sea urchin embryos, affecting their morphology and developmental pathways. In particular, they perturb differently the correct organization of the embryonic axes (animal-vegetal, dorso-ventral): Li is a vegetalizing agent and Ni disrupts the dorso-ventral axis, while Zn has an animalizing effect. To deeply address the response of Paracentrotus lividus embryos to these metals, we studied the expression profiling of Pl-Fra transcription factor (TF), relating it to Pl-jun, a potential partner for AP-1 complex formation, and to Pl-MT, known to be an AP-1 target and to have a protective role against heavy metals. The AP-1 TFs are found throughout the animal kingdom and are involved in many cellular events, i.e. cell proliferation and differentiation, immune and stress responses, cancer growth. Here, we isolated the complete Pl-Fra cDNA and showed that Pl-Fra transcript, already present in the unfertilized eggs, was newly synthesized from the blastula stage, while its spatial distribution was mainly observed in skeletogenic cells, similarly to Pl-jun. Interestingly, Pl-Fra expression was induced by the different metals and the induction kinetics revealed its persistent expression during treatments. Moreover, its temporal and spatial behavior in response to the three metals was comparable to that of Pl-jun and Pl-MT. The understanding of AP-1 functions in invertebrates may provide new knowledge about the mechanisms of response to metal injuries, as well as it might lead to acknowledge the TFs as new type of biomarkers for the evaluation of hazards in polluted environment.

Nickel toxicity in P. lividus embryos: Dose dependent effects and gene expression analysis

Many industrial activities release Nickel (Ni) in the environment with harmful effects for terrestrial and marine organisms. Despite many studies on the mechanisms of Ni toxicity are available, the understanding about its toxic effects on marine organisms is more limited. We used Paracentrotus lividus as a model to analyze the effects on the stress pathways in embryos continuously exposed to different Ni doses, ranging from 0.03 to 0.5 mM. We deeply examined the altered embryonic morphologies at 24 and 48 h after Ni exposure. Some different phenotypes have been classified, showing alterations at the expenses of the dorso-ventral axis as well as the skeleton and/or the pigment cells. At the lowest dose used, Ni mainly induced a multi-spicule phenotype observed at 24 h after treatment. On the contrary, at the highest dose of Ni (0.5 mM), 90% of embryos showed no skeleton and no pigment cells. Therefore, we focused on this dose to study protein and gene expression patterns at 24 and 48 h after exposure. Among the proteins analyzed, i.e. p38MAPK, Grp78 and Mn-SOD, only p38MAPK was induced by Ni treatment. Moreover, we analyzed the mRNA profiles of a pool of genes that are involved in stress response and in development mechanisms, i.e. the transcription factors Pl-NFkB and Pl-FOXO; a marker of DNA repair, Pl-XPB/ERCC3; a mitogen-activated protein kinase (MAPK), Pl-p38; an ER stress gene, Pl-grp78; an adapter protein, Pl-14-3-3ε; two markers of pigment cells, Pl-PKS1 and Pl-gcm. The spatial expression of mesenchymal marker genes has been evaluated in Ni-treated embryos at both 24 and 48 h after exposure. Our results indicated that Ni acts at several levels in P. lividus sea urchin, by affecting embryo development, influencing the embryonic immune response and activating stress response pathways to counteract the suffered injury and to promote embryos surviving.

The 70-kDa Heat-shock Protein as a Potential Biomarker of Quality of the Parapenaeus longirostris Shrimp Flesh

ABSTRACT The deep-water rose shrimp, Parapenaeus longirostris, is a target species of the Mediterranean fisheries mostly caught by trawlers offshore, processed, and frozen on board. The effects of thawing on shrimp muscle exudate collected at 0, 1, 2, 3 days after thawing were investigated. In total, 70-kDa heat-shock protein (Hsp70), alpha (α)-enolase, and manganese superoxide dismutase (Mn-SOD) were selected as metabolic and stress-related proteins and analyzed by immunoblotting on exudates. Data were compared for the amount of exudates collected and the pH values. Among the investigated proteins, only the Hsp70 levels showed a decrease related to the post-thawing period and correlated with both the significant increase of the exudate amount and the pH values. These data strongly suggest the potential use of Hsp70 as an early predictive biomarker for quality of the P. longirostris shrimp after thawing.