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Widely appreciated in gastronomy, many harvested and farmed Crustacean species are a great economic resource in the fishery business, throughout the world. Factors like pollutants and/or improper management in on-board/on-shore practices may adversely affect their edible quality and shelf-life. Recent studies from applied scientific research have been supporting the fishing industry and trading companies, in order to improve post-harvest practices and offer a safe and competitive product of high quality. Here, we developed an innovative molecular approach to get basic information on crustacean quality by using the European lobster Nephrops norvegicus (Linnaeus, 1758). For the first time, we investigated muscle-exudates of thawed lobsters for the presence of nucleic acids and analysed mRNA targets selected at NCBI to test their potential use as biomarkers of quality in frozen-thawed seafood. By RT-PCRs and sequencing we validated small amplicons of Actin, Cytochrome oxidase subunit 1 (COI), Calpain M, Prophenoloxidase (ppo) and Crustin-like antimicrobial peptide. In comparative One Step RT-PCR and/or One Step RT-qPCR assays, we analysed their amplification profiles at 1, 4 and 7 days after thawing (a.t.), in lobsters maintained to checked temperatures from capture. Based on amplification efficiency (AE), inversely correlated to the time after thawing and loss of RNA targets integrity, we identified new highly sensitive, species specific early indicators of quality. Our results highlight the advantage of using amplicons from exudates in different food science applications including authenticity/traceability, and provide new insights for the development of scientific tools applicable to crustacean quality monitoring on industrial-scale.

Ginger (Zingiber officinale, fam: Zingiberaceae) is a plant whose rhizome is used by humans as a spice or anti-inflammatory and antioxidant. It is composed of many constituents, though the majority are gingerols and shogaols. Although the bioactive components have been found, the mechanisms of action of the single components still need to be determined. Ginger compounds may be toxic at high doses, and therefore its use as a food/drug requires better guidelines. This study focuses on one of the major ginger compounds, the [6]-gingerol, and the relationships between the dosage used and the effects on sea urchin Paracentrotus lividus. Toxicity tests with morphological and viability evaluations were done on embryos and adult immune cells. A dose-dependent toxic effect of [6]-gingerol was observed. It increased the number of abnormal morphologies and reduced the size of embryos up to 100% with the dose of 100 μM. Cell viability of immune cells gradually decreased over time, up to 40% with 50 μM after 6 h. The expression of genes coding for inflammatory (Pl-NF-kB, Pl-Tlr4, Pl-tbk1, Pl-jun) and stress responsive (Pl-grp78, Pl-XPB-ERCC3) proteins were measured in immune cells stimulated with lipopolysaccharide after [6]-gingerol pre-treament. The results suggested that low doses of [6]-gingerol (10 and 20 μM) might confer protection to sea urchin immune cells. Different doses of [6]-gingerol (10, 20, and 50 μM) tested on three different non-pathogenic bacteria did not show any antimicrobial effects. The results suggested that [6]-gingerol might be beneficial as a functional food.

Paracentrotus lividus is a Mediterranean and Eastern Atlantic sea urchin species, very sensitive to chemical and physical environmental changes and widely used in eco-toxicological studies. Here, we applied a high throughput screening approach on P. lividus embryos exposed to UVB radiation (UV), Cadmium Chloride (Cd) and their combination (Cd/UV), to deeply characterize the molecular responses adopted by embryos to cope with these stressors. in vitro eco-toxicological assays were performed by exposing embryos to Cd (10−4 M) soon after fertilization, to UV (200 and 400J/m2) at early stage of development, while in co-exposure experiments, Cd-exposed embryos were irradiated with UV at 200 J/m2. By NanoString nCounter technology, custom-made probes were developed and hybridized on total RNA extracted from exposed embryos at 51h after fertilization. By in silico analyses, we selected and retrieved at the NCBI nucleotide database a panel of P. lividus transcripts encoding for many regulatory and structural proteins that we ranked in categories, i.e., Apoptosis, Biomineralization, Defense, Development, Immunity, Signaling and Transcription Factors. The analysis of 127 transcripts highlighted the dysregulation of many genes, some specifically activated to cope with stress agents, others involved in the complex molecular network of genes that regulate embryo development. We revealed the downregulation of Biomineralization and Development genes and the upregulation of Defensive genes in Cd and Cd/UV embryos. Our approach, using sea urchin embryo as an in vivomodel, contributes to advance our knowledge about cellular responses to UV, Cd and their combination.

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.

Management of dredged materials disposal is regulated by several environmental normative requirements, and it is often supported by the integration of chemical data with ecotoxicological characterization. The reliability of a bioassay to assess the potential toxicity of dredged sediments requires the selection of quality criteria that should be based on simple analytical methods and easily understandable hazard for politicians and environmental managers. The sea urchin embryo-toxicity bioassay is considered an essential component for evaluating the quality of sediments in harbour areas but its use, when based exclusively on the observation of normal vs. abnormal embryos, may alter the interpretation of the results, overestimating the risk assessment. To improve the reliability of this assay in establishing a causative relationship between quality of sediments and sea urchin embryonic development, here we developed and validated three Integrative Toxicity Indexes (ITI 2.0, ITI 3.0, ITI 4.0), modifying the already-known ITI (here ITI 1.0). Based on this aim, we used Taranto harbour as a model pilot-study to compare results to those obtained from standard criteria. Among the tested indexes, the ITI 4.0, discriminating strictly developmental delay and morphological defects from fertilized egg to gastrula stage, resulted in the most promising.

The sea urchin embryo develops a well-defined biomineralized endoskeleton, synthesized exclusively by the skeletogenic cells, supported by ectodermal cues for the correct skeleton patterning. The biomineralization process is tightly regulated via a hierarchical order of gene expression, including transcription and growth factors, biomineralization proteins. Recently, the role of kinases and intracellular signaling pathways in sea urchin skeletogenesis has been addressed, although the downstream components still remain unknown. In this study, we investigated the role of phosphatidylinositide 3-kinase (PI3K)-mediated signaling pathway in Paracentrotus lividus, to identify its genes/proteins targets. The effects of LY294002 (LY), a PI3K-specific inhibitor, were evaluated at morphological and molecular levels. Treatment with 40 μM LY from the blastula stage completely blocked skeleton deposition, which was reversed by wash out experiments. Besides, LY caused a slight delay in the tripartite gut development. Despite the skeleton absence, a few skeleton-specific proteins/mRNAs were regularly expressed and localized in LY-treated embryos, as shown for MSP130 and SM50 by immunofluorescence and in situ hybridization experiments. QPCR analyses showed that LY differently affected the expression of genes coding for other biomineralization proteins, transcription and growth factors. SM30 and carbonic anhydrase expression was severely downregulated, while almost all the transcription factors analyzed were upregulated. Based on the present results and in silico analyses, we propose an “interactomic” model simulating PI3K connections in P. lividus embryos. Our findings define a novel regulatory step in the embryonic skeletogenesis, and provide valuable molecular data for further studies on the role of PI3K signaling in invertebrate biomineralization.

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.

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-14-3-3e; 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.

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.

Gadolinium (Gd), a metal of the lanthanide series used as contrast agent for magnetic resonance imaging, is released into the aquatic environment. We investigated the effects of Gd on the development of four sea urchin species: two from Europe, Paracentrotus lividus and Arbacia lixula, and two from Australia, Heliocidaris tuberculata and Centrostephanus rodgersii. Exposure to Gd from fertilization resulted in inhibition or alteration of skeleton growth in the plutei. The similar morphological response to Gd in the four species indicates a similar mechanism underlying abnormal skeletogenesis. Sensitivity to Gd greatly varied, with the EC50 ranging from 56 nM to 132 μM across the four species. These different sensitivities highlight the importance of testing toxicity in several species for risk assessment. The strong negative effects of Gd on calcification in plutei, together with the plethora of marine species that have calcifying larvae, indicates that Gd pollution is urgent issue that needs to be addressed.

Submersible sea-cages represent an alternative method to standard surface-based cages, and could help solve several production problems that exist in surface-based farming, such as heavy storms, algal and jellyfish blooms and attacks by predators. In this study, we investigated growth, haematological, biochemical and immunological parameters of European sea bass ( Dicentrarchus labrax ) farmed in surface and submersible cages, to compare the physiological status under different rearing conditions. In addition, we evaluated the use of erythrocyte HSP70 levels as a biomarker of fish welfare. The study was conducted in two submerged and two surface cages filled with D. labrax . No significant differences were observed in the growth rate and the relative condition factor between the two groups. Significantly higher levels of serum cortisol, haematocrit plasma glucose and total plasma proteins were measured in surface-based cages. Conversely, fish in submerged cages showed higher haemolytic activity and lysozyme levels in both mucus and kidney than surface reared sea bass. Results obtained in this study indicate that submergence could be a favorable condition for sea bass farming, suggesting mariculture in submerged net cages as a promising system that allows producers to minimize stress (included that from predation by fish-eating birds) and therefore favor animal welfare.

International concern over environmental nuclear contamination of salt water fisheries and coastal resources has attracted the interests of ecologists, marine biologists and stakeholders. There are not many studies on the effects of X-rays, a component of radionuclides emissions, on embryonic development and gene expression. The sea urchin embryo is emerging as a useful model system for environmental and eco-toxicological studies. Here, we describe how X-rays affect development and gene expression in embryos of the Mediterranean sea urchin Paracentrotus lividus. Cleavage embryos were exposed to doses from 0.1 to 5 Gy, using an Ag source of X radiation. We found a dose-dependent increase in developmental delays and severe morphological defects in embryos microscopically inspected at two endpoints, 24 and 48 h after irradiation. By analogy with classical toxicity tests parameters we defined the No Observed Effect Dose at 0.1 Gy, the Lowest Observed Effect Dose at 0.5 Gy and ED50 at 1.0 Gy. Major perturbations concerned primitive intestine and skeleton differentiation and development: X-rays exposed embryos had both no gut and arms or poorly and abnormally developed ones. We found a dose-dependent reduction in the mRNA levels of two skeleton-specific genes, Pl-SM30 (spicule matrix 30) and Pl-msp130 (matrix spicule protein 130), as measured by semi-quantitative RT-PCR and whole mount in situ hybridization, respectively. These findings indicate the sea urchin embryo as a sensible bioindicator of X-radiation and propose its use as an alternative model, emphasizing the need for further investigation aimed to protect ecosystem health.

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.

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.

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. Database Nucleotide sequence data have been deposited in the EMBL databases under the accession number: HE817756.

The increase of UVB radiation reaching marine environment has harmful effects on living organisms. Paracentrotus lividus sea urchin embryos living in shallow water are exposed to radiations, providing a good model for studying the molecular mechanisms activated upon UV stress. Here, we report the modulated time- and dose-dependent expression of six genes, known to be involved in stress response, in embryos exposed at cleavage to 400 and 800 J/m2 UVB, and collected at early (morula) and later (gastrula) stages. We analyzed their mRNA levels by QPCR and found that Pl-14-3-3 showed a dose-dependent induction, both early and late in development; Pl-c-jun was up-regulated proportionally to the UVB dose at early stages and only at 800 J/m2 UVB at later stages; Pl-XPB-ERCC3, Pl-MT and Pl-NF-kB were highly up-regulated later in development at the high dose, with the exception of Pl-XPB-ERCC3 whose mRNA levels were high also at the lower dose; Pl-FOXO expression was not affected by UVB radiation. We believe that the identification of UVB-responsive genes in irradiated sea urchin embryos, reported for the first time in this study, will be helpful for the understanding of the involved molecular pathways. The correlation between the impaired morphogenesis, affecting endo-mesoderm differentiation, and gene modulations described herewith is also discussed.

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.

We used Paracentrotus lividus sea urchin embryos, a well-established model in developmental biology and ecotoxicology, for investigation on stress/anti-apoptotic protein expression elicited in response to harmful ionizing radiation, such as X-rays. We evaluated the acute effects of a high-dose exposure (5 Gy) on P. lividus analyzing by Western blotting the accumulation levels of HSP60, HSP70, BAG3 and a putative p63 at 24 and 48 h after irradiation. We found an increase in the HSP70, BAG3, and p63 protein levels only 48 h after irradiation, whereas no HSP60 increase was detected either at 24 or 48 h. Levels of the mRNA coding for HSP70 and p63 were also investigated by relative RT-PCR and were found to increase 24 h after irradiation, returning to their initial levels at 48 h. Results demonstrate the presence of an adaptive regulatory mechanism operating at the transcriptional level at 24 h, followed by a translational activation at 48 h post-irradiation. In conclusion, our findings confirm the sea urchin embryo as a sensible bioindicator of cell damage and we propose this model for studies on the protective pathways activated in response to X-rays. The novel result of the involvement of BAG3 and p63 in the response to X-rays, never tested so far in any other embryonic system, opens the way for their use as biomarkers of X-ray hazards.

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.

Members of the 14-3-3 protein family are involved in many important cellular events, including stress response, survival and apoptosis. Genes of the 14-3-3 family are conserved from plants to humans, and some members are responsive to UV radiation. Here, we report the isolation of the complete cDNA encoding the 14-3-3 epsilon isoform from Paracentrotus lividus sea urchin embryos, referred to as Pl14-3-3ε, and the phylogenetic relationship with other homologues described in different phyla. Pl14-3-3ε mRNA levels were measured by QPCR during development and found to increase from the mesenchyme blastula to the prism stage. In response to UV-B (312 nm) exposure, early stage embryos collected 2 h later showed a 2.3-fold (at 400 J/m(2)) and a 2.7-fold (at 800 J/m(2)) increase in Pl14-3-3ε transcript levels compared with controls. The spatial expression of Pl14-3-3ε mRNA, detected by whole mount in situ hybridization in both control and UV-B exposed embryos, harvested at late developmental stages, showed transcripts to be located in the archenteron of gastrula stage and widely distributed in all germ layers, respectively. The Pl14-3-3ε mRNA delocalization parallels the failure in archenteron elongation observed morphologically, as well as the lack of specific endoderm markers, investigated by indirect immuno-fluorescence on whole mount embryos. Results confirm the involvement of 14-3-3ε in the stress response elicited by UV-B and demonstrate, for the first time, its contribution at the transcriptional level in the sea urchin embryo.

Sea urchin embryos and larvae represent suitable model systems on where to investigate the effects of heavy metals on development and cell viability. Here, we tested the toxic effects of low (10(-12 )M), medium (10(-9 )M), and high (10(-6 )M) cadmium chloride concentrations, mimicking unpolluted, moderately and highly polluted seawaters, respectively, on Paracentrotus lividus sea urchins offspring. Larvae were continuously treated from fertilization and inspected at time intervals comprised between 10 and 30 days of development. Delays and/or morphological abnormalities were firstly evident in larvae treated for 15 days with high cadmium (10(-6 )M) and for 25 days with medium cadmium (10(-9 )M). Major defects consisted in the reduction and lack of arms and skeleton elongation. No obvious differences with respect to controls were observed in embryos/larvae exposed to low cadmium (10(-12) M), even after 30 days of exposure. Using in situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay (TUNEL) assay on larvae whole mounts, we detected apoptosis after 10 days of treatment with 10(-6) and 10(-9) M CdCl(2,) when no morphological abnormalities were recognizable yet. Supernumerary apoptotic cells were found in arm buds, ciliary bands, and apex. In conclusion, echinoderm embryos and larvae represent candidates of choice for the study of stress and defense mechanisms activated by cadmium exposure.

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.

The depletion of the stratospheric ozone layer and the resulting increase in hazardous ultraviolet-B (UV-B) radiation reaching the Earth are of major concern not only for terrestrial but also for aquatic organisms. UV-B is able to penetrate clear water to ecologically significant depths. This chapter deals with the effects of UV radiation on DNA integrity in marine benthic organisms, in particular sea urchins in comparison to other marine invertebrates (sponges and corals). These animals cannot escape the damaging effects of UV-B radiation and may be additionally exposed to pollution from natural or anthropogenic sources. Besides eggs and larvae that lack a protective epidermal layer and are particularly prone to the damaging effects of UV radiation, coelomocytes from the sea urchin Paracentrotus lividus were used as a “cellular sensor” to analyse the effects on DNA caused by UV-B, heavy metals (cadmium), and their combined actions. From our data we conclude that sea urchin coelomocytes as well as cells from other marine invertebrates are useful bioindicators of UV-B and heavy metal stress, responding to these stressors with different extents of DNA damage.

Ozone depletion results in an increased flux of biologically damaging radiations reaching the earth. Although ultraviolet (UV) penetration is attenuated by the seawater, harmful effects can be still observed at low depths where sea urchin embryos are living. We have used Paracentrotus lividus embryos to study the impacts of UV radiation on their development. Blastula cultures were exposed to different doses of UVB (312 nm) radiations and the resulting endpoint effects were evaluated in terms of embryonic morphological abnormalities, variations in specific gene expression, and changes in the levels of stress proteins. We found that embryos were moderately sensitive to 50 J/m2 UVB radiation; an increase in the number of developmentally delayed and malformed embryos was detected when increasing doses, up to 1000 J/m2, were used. Major developmental defects, observed 24 and 48 h after exposure, consisted in the failure of skeleton elongation and patterning. Accordingly, we found a reduction in the number of primary mesenchyme cells that expressed Pl-SM30, a gene coding for one of the specific matrix proteins of the skeleton. The morphological effects observed 1, 24, and 48 h after exposure were correlated with a dose-dependent increase in the level and in the activation of two recognized stress markers, namely hsp70 and p38 MAPk, respectively, consistent with their role in mediating cellular response to stress and suggesting a function in embryo survival.

Marine organisms are highly sensitive to many environmental stresses, and consequently, the analysis of their bio-molecular responses to different stress agents is very important for the understanding of putative repair mechanisms. Sea urchin embryos represent a simple though significant model system to test how specific stress can simultaneously affect development and protein expression. Here, we used Paracentrotus lividus sea urchin embryos to study the effects of time-dependent continuous exposure to subacute/sublethal cadmium concentrations. We found that, between 15 and 24 h of exposure, the synthesis of a specific set of stress proteins (90, 72–70, 56, 28, and 25 kDa) was induced, with an increase in the rate of synthesis of 72–70 kDa (hsps), 56 kDa (hsp), and 25 kDa, which was dependent on the lengths of treatment. Recovery experiments in which cadmium was removed showed that while stress proteins continued to be synthesized, embryo development was resumed only after short lengths of exposure.

Coelomocytes are the immune effectors of the sea urchin and have shown to respond to environmental and experimental challenge by the activation of stress markers. We extended our in vivo studies to in vitro short term cultures of sea urchin coelomocytes by analysing their response to temperature being stress, acid pH and heavy metals, using the hsp70 protein as a stress marker. We found that the in vitro time course of temperature stress recapitulates results obtained in vivo where the highest overexpression was observed after 1 hour. Coelomocytes overexpress hsp70 in a time-dependent manner when cultured for 1 to 6 hr at pH 4.7 +/- 0.2 in isotonic buffer, supplemented with EDTA as anticoagulant. A peak in the level of hsp70 expression was observed at 2 hr of culture, corresponding to a 10-fold increase over the levels of control coelomocytes cultured at pH 7.3 +/- 0.2. The effect of different concentrations of CdCl2 in the culture over a period of 4 hr was also tested. We found that CdCl2 greatly increases the hsp70 expression, with 10(-3) M the dose at which the highest overexpression is observed.

Paracentrotus lividus embryos at the early pluteus stage undergo spontaneous apoptosis. Using a TUNEL (TdT-mediated dUTP Nick-End Labelling) assay on whole mount embryos, we showed that there was a different distribution of the apoptotic cells in different optical sections. Not more than 20% of cells in plutei were spontaneously apoptotic, as confirmed by the counts of dissociated ectoderm and intestine cells. Observation of larva stages closer to metamorphosis confirmed that apoptosis is a physiological event for the development of the adult. In particular, larvae at different developmental stages showed apoptotic cells in the oral and aboral arms, intestine, ciliary band and both apical and oral ganglia. Moreover, we found that the number of apoptotic cells decreased in later larva stages, possibly because in the organism approaching metamorphosis, a smaller number of cells needs to be eliminated. Furthermore, combined phorbol ester (TPA) and heat shock treatment enhanced apoptosis by increasing the number of cells involved in the phenomenon.

A number of studies aimed to assess the health of aquatic ecosystems have focused on the dangerous effects of ultraviolet B (UV-B) radiation on marine organisms, especially those living in shallow waters such as sea urchin embryos and their larvae. We studied the effects of UV-B radiation on Paracentrotus lividus embryos at early stages of development, in laboratory experiments. Embryos at the 16-cell stage were exposed to different doses of UV-B, ranging from 10 to 800 J/m2, and the resulting morphological abnormalities were observed. In addition, putative variations in the expression of the heat shock proteins 70 (hsp70) stress marker, at the protein and mRNA levels, were evaluated. The first effective dose was 150 J/m2, producing a significant number of abnormal embryos, scored 24 h after exposure. This number increased up to 100% when the two highest doses were used (400 and 800 J/m2). Besides delays in embryonic development, the most abnormal morphology (abnormal blastula) consisted of blastula-like embryos, with cells unusually filling the blastocoelic cavity. Abnormal blastulae were not able to continue development as they were found 48 h after exposure. Some embryos with less severe abnormalities showed tri-radiate spicule rudiments, but had major defects in gut elongation. A corresponding dose-dependent increase in the hsp70 protein levels was observed in embryos exposed to different UV-B doses. Conversely, slight, if any variations were detected in the hsp70 mRNA levels for any of the doses tested, claiming for a post-transcriptional regulation of UV-B-induced stress response.

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.