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Sediment pollution is a major issue in coastal areas, potentially endangering human health and the marine environments. We investigated the short-term sublethal effects of sediments contaminated with polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) on the sea urchin Paracentrotus lividus for two months. Spiking occurred at concentrations below threshold limit values permitted by the law (TLVPAHs = 900 µg/L, TLVPCBs = 8 µg/L, Legislative Italian Decree 173/2016). A multi-endpoint approach was adopted, considering both adults (mortality, bioaccumulation and gonadal index) and embryos (embryotoxicity, genotoxicity and de novo transcriptome assembly). The slight concentrations of PAHs and PCBs added to the mesocosms were observed to readily compartmentalize in adults, resulting below the detection limits just one week after their addition. Reconstructed sediment and seawater, as negative controls, did not affect sea urchins. PAH- and PCB-spiked mesocosms were observed to impair P. lividus at various endpoints, including bioaccumulation and embryo development (mainly PAHs) and genotoxicity (PAHs and PCBs). In particular, genotoxicity tests revealed that PAHs and PCBs affected the development of P. lividus embryos deriving from exposed adults. Negative effects were also detected by generating a de novo transcriptome assembly and its annotation, as well as by real-time qPCR performed to identify genes differentially expressed in adults exposed to the two contaminants. The effects on sea urchins (both adults and embryos) at background concentrations of PAHs and PCBs below TLV suggest a need for further investigations on the impact of slight concentrations of such contaminants on marine biota.

Physiological effects of algal metabolites is a key step for the isolation of interesting bioactive compounds. Invertebrate grazers may be fed on live diatoms or dried, pelletized, and added to compound feeds. Any method may reveal some shortcomings, due to the leaking of wound-activated compounds in the water prior to ingestion. For this reason, encapsulation may represent an important step of bioassay-guided fractionation, because it may assure timely preservation of the active compounds. Here we test the effects of the inclusion in alginate (biocompatible and non-toxic delivery system) matrices to produce beads containing two benthic diatoms for sea urchin Paracentrotus lividus feeding. In particular, we compared the effects of a diatom whose influence on P. lividus was known (Nanofrustulum shiloi) and those of a diatom suspected to be harmful to marine invertebrates, because it is often present in blooms (Striatella unipunctata). Dried N. shiloi and S. unipunctata were offered for one month after encapsulation in alginate hydrogel beads and the larvae produced by sea urchins were checked for viability and malformations. The results indicated that N. shiloi, already known for its toxigenic effects on sea urchin larvae, fully conserved its activity after inclusion in alginate beads. On the whole, benthic diatoms affected the embryogenesis of P. lividus, altering the expression of several genes involved in stress response, development, skeletogenesis and detoxification processes. Interactomic analysis suggested that both diatoms activated a similar stress response pathway, through the up-regulation of hsp60, hsp70, NF-κB, 14-3-3 ε and MDR1 genes. This research also demonstrates that the inclusion in alginate beads may represent a feasible technique to isolate diatom-derived bioactive compounds.

Due to the continuous development, production and consumption of nanoparticles (NPs), their release, fate and effects in marine coastal environment can represent a major concern. The aim of this study was to evaluate the toxicity of ZnO nanoparticles (ZnO NPs) and compare it to bulk ZnSO4 on three macroinvertebrates: the isopod Cymodoce truncata (i.e. used for the first time in ecotoxicology), the amphipod Gammarus aequicauda and the sea urchin Paracentrotus lividus. This study showed concentration- and time-dependent relationships for all biological models for both ZnO NPs and ZnSO4. Both Zn forms elicited high toxicity to G. aequicauda and C. truncata juveniles, but ZnO NPs induced comparable responses to both species (96h-LC50 = 0.30 and 0.37 mg/L for G. aequicauda and C. truncata, respectively; p > 0.05), while differences were found after ZnSO4 exposure (96h-LC50 = 0.28 and 0.63 mg/L, respectively; p < 0.05). ZnO NPs generated sub-lethal effects on P. lividus embryos (72h-EC50 = 0.04 (0.03, 0.05) mg/L), not significantly different from ZnSO4 ones (72h-EC50 = 0.06 (0.05, 0.07) mg/L). Effects of ZnO NPs were similar to existing literature data for other testing species. C. truncata can be considered as a promising new biological model in (nano)ecotoxicology. Graphical abstract

Background The immune system of echinoderm sea urchins is characterised by a high degree of complexity that is not completely understood. The Mediterranean sea urchin Paracentrotus lividus coelomocytes mediate immune responses through phagocytosis, encapsulation of non-self particles, and production of diffusible factors including antimicrobial molecules. Details of these processes, and molecular pathways driving these mechanisms, are still to be fully elucidated. Principal findings In the present study we treated the sea urchin P. lividus with the bacterial lipopolysaccharide (LPS) and collected coelomocytes at different time-points (1, 3, 6 and 24 hours). We have shown, using label-free quantitative mass spectrometry, how LPS is able to modulate the coelomocyte proteome and to effect cellular pathways, such as endocytosis and phagocytosis, as soon as the immunomodulating agent is injected. The present study has also shown that treatment can modulate various cellular processes such as cytoskeleton reorganisation, and stress and energetic homeostasis. Conclusions Our data demonstrates, through mass spectrometry and the following functional annotation bioinformatics analysis, how the bacterial wall constituent is sufficient to set off an immune response inducing cytoskeleton reorganisation, the appearance of clusters of heat shock proteins (Hsp) and histone proteins and the activation of the endocytic and phagocytic pathways. Data are available via ProteomeXchange with identifier PXD008439.

Oxygenated derivatives of fatty acids, collectively called oxylipins, are a highly diverse family of lipoxygenase (LOX) products well described in planktonic diatoms. Here we report the first investigation of these molecules in four benthic diatoms, Cylindrotheca closterium, Nanofrustulum shiloi, Cocconeis scutellum, and Diploneis sp. isolated from the leaves of the seagrass Posidonia oceanica from the Gulf of Naples. Analysis by hyphenated MS techniques revealed that C. closterium, N. shiloi, and C. scutellum produce several polyunsaturated aldehydes (PUAs) and linear oxygenated fatty acids (LOFAs) related to the products of LOX pathways in planktonic species. Diploneis sp. also produced other unidentified fatty acid derivatives that are not related to LOX metabolism. The levels and composition of oxylipins in the benthic species match their negative effects on the reproductive success in the sea urchin Paracentrotus lividus. In agreement with this correlation, the most toxic species N. shiloi revealed the same LOX pathways of Skeletonema marinoi and Thalassiosira rotula, two bloom-forming planktonic diatoms that affect copepod reproduction. Overall, our data highlight for the first time a major role of oxylipins, namely LOFAs, as info-chemicals for benthic diatoms, and open new perspectives in the study of the structuring of benthic communities.

The major yolk protein toposome plays crucial roles during gametogenesis and development of sea urchins. We previously found that nitration of toposome increases in the gonads of a Paracentrotus lividus population living in a marine protected area affected by toxic blooms of Ostreospsis cf. ovata, compared to control populations. This modification is associated with ovatoxin accumulation, high levels of nitric oxide in the gonads, and a remarkable impairment of progeny development. However, nothing is known about the environmental-mediated-regulation of the structure and biological function of toposome. Here, we characterize through wide-ranging biochemical and structural analyses the nitrated toposome of sea urchins exposed to the bloom, and subsequently detoxified. The increased number of nitrated tyrosines in toposome of sea urchins collected during algal bloom induced structural changes and improvement of the Ca2+-binding affinity of the protein. After 3 months’ detoxification, ovatoxin was undetectable, and the number of nitric oxide-modified tyrosines was reduced. However, the nitration of specific residues was irreversible and occurred also in embryos treated with metals, used as a proxy of environmental pollutants. The structural and functional changes of toposome caused by nitration under adverse environmental conditions may be related to the defective development of sea urchins’ progeny.

Oxylipins such as polyunsaturated aldehydes (PUAs) and hydroxyacids (HEPEs) are signaling molecules derived from the oxidation of polyunsaturated fatty acids. They are common in diatoms that constitute a major group of microalgae in freshwater and oceanic ecosystems. Although HEPEs represent the most common oxylipins produced by diatoms, little information is available on their effects on marine invertebrates, and most of the information has been obtained by testing individual HEPEs. Our previous studies reported that four hydroxyacids, i.e., 5-, 9-, 11-, and 15-HEPE, were able to induce malformations and a marked developmental delay in sea urchin Paracentrotus lividus embryos, which had not been reported for other oxylipins. Here, we tested a mixture of 5-, 9-, 11-, and 15-HEPE at different concentrations for the first time. The results showed that mixtures of HEPEs have synergistic effects that are much more severe compared to those of individual HEPEs: The HEPE mixtures induced malformations in sea urchin embryos at lower concentrations. Increasing HEPE mixture concentrations induced a marked increase in the number of delayed embryos, until all embryos were delayed at the highest concentration tested. At the molecular level, the HEPE mixtures induced variations in the expression of 50 genes involved in different functional processes, mainly down-regulating these genes at the earliest stages of embryonic development. These findings are ecologically significant, considering that during diatom blooms, sea urchins could accumulate HEPEs in concentrations comparable to those tested in the present study.

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With the aim to obtain new antimicrobials against important pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa, we focused on antimicrobial peptides (AMPs) from Echinoderms. An example of such peptides is Paracentrin 1 (SP1), a chemically synthesised peptide fragment of a sea urchin thymosin. In the present paper, we report on the biological activity of a Paracentrin 1 derivative obtained by recombination. The recombinant paracentrin RP1, in comparison to the synthetic SP1, is 22 amino acids longer and it was considerably more active against the planktonic forms of S. aureus ATCC 25923 and P. aeruginosa ATCC 15442 at concentrations of 50 &micro, g/mL. Moreover, it was able to inhibit biofilm formation of staphylococcal and P. aeruginosa strains at concentrations equal to 5.0 and 10.7 &micro, g/mL, respectively. Molecular dynamics (MD) simulations allowed to rationalise the results of the experimental investigations, providing atomistic insights on the binding of RP1 toward models of mammalian and bacterial cell membranes. Overall, the results obtained point out that RP1 shows a remarkable preference for bacterial membranes, in excellent agreement with the antibacterial activity, highlighting the promising potential of using the tested peptide as a template for the development of novel antimicrobial agents.

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.

Increasing concentrations of contaminants, often resulting from anthropogenic activities, have been reported to occur in the marine environment and affect marine organisms. Among these, the metal ions cadmium and manganese have been shown to induce developmental delay and abnormalities, mainly reflecting skeleton elongation perturbation, in the sea urchin Paracentrotus lividus, an established model for toxicological studies. Here, we provide evidence that the physiological messenger nitric oxide (NO), formed by l-arginine oxidation by NO synthase (NOS), mediates the stress response induced by cadmium and manganese in sea urchins. When NO levels were lowered by inhibiting NOS, the proportion of abnormal plutei increased. Quantitative expression of a panel of 19 genes involved in stress response, skeletogenesis, detoxification and multidrug efflux processes was followed at different developmental stages and under different conditions: metals alone, metals in the presence of NOS inhibitor, NO donor and NOS inhibitor alone. These data allowed the identification of different classes of genes whose metal-induced transcriptional expression was directly or indirectly mediated by NO. These results open new perspectives on the role of NO as a sensor of different stress agents in sea urchin developing embryos. © 2014 Elsevier B.V.