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The effects of ocean acidification, a major anthropogenic impact on marine life, have been mainly investigated in laboratory/mesocosm experiments. We used the CO2 vents at Ischia as a natural laboratory to study the long-term effects of ocean acidification on the sea urchin Paracentrotus lividus population resident in low-pH (7.8 ± 0.2) compared to that at two control sites (pH 8.02 ± 0.00; 8.02 ± 0.01). The novelty of the present study is the analysis of the sea urchin immune cells, the sentinels of environmental stress responses, by a wide-ranging approach, including cell morphology, biochemistry and proteomics. Immune cell proteomics showed that 311 proteins were differentially expressed in urchins across sites with a general shift towards antioxidant processes in the vent urchins. The vent urchin immune cells showed higher levels of total antioxidant capacity, up-regulation of phagosome and microsomal proteins, enzymes of ammonium metabolism, amino-acid degradation, and modulation of carbon metabolism proteins. Lipid-hydroperoxides and nitric oxide levels were not different in urchins from the different sites. No differences in the coelomic fluid pH, immune cell composition, animal respiration, nitrogen excretion and skeletal mineralogy were observed. Our results reveal the phenotypic plasticity of the immune system of sea urchins adapted to life at vent site, under conditions commensurate with near-future ocean acidification projections.

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

The vast category of microplastics in the marine environment, encompassing among other aspects their persistence, degradation and impact on biota, has become an important topic of research. in spite of environmental health concerns, much work has yet to be done on understanding the potential roles of polymer sources, composition and particle sizes in causing adverse effects which have already been observed in a number of biota. the present study was aimed at adding to current knowledge by verifying if, and to what extent, embryogenesis in the sea urchin species Paracentrotus lividus is adversely affected by polystyrene and polymethylmethacrylate virgin microparticles over a size range 1-230 mu m and at concentrations of 0.1-10 mg L-1. Developing embryos which came in contact with the microplastics only after fertilisation did not display a significant increase of developmental defects. Unlike embryo exposures, when P. lividus sperm were exposed to the microplastics or their leachates, modest, yet significant effects were observed, both in terms of decreased fertilisation rate and increase of transmissible damage to offspring. Further, it was noted that larvae more readily ingested polymethylmethacrylate than polystyrene microparticles after 3 days which may represent a route for enhancing the toxicity of the former compared to the latter. Overall, these findings provide evidence for lesser sensitivity of P. lividus early life stages to microplastics compared to other urchins such as Sphaerechinus granularis. in turn, the more robust response of P. lividus highlights the importance of choosing an appropriate test species with the highest sensitivity when investigating mildly harmful materials., Croatian Science Foundation [IP-2018-01-5351]; Environment and Climate Change Canada, This work has been supported by the Croatian Science Foundation under grant number IP-2018-01-5351, and Environment and Climate Change Canada. the authors are grateful to Dr. Ozge Kozgus Guldu for DLS measurements.

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.

Marine pollution due to disused industrial activities is a major threat to ecosystems and human health, for example through the effects of re-suspension of toxic substances that are present in contaminated sediments. Here, we examined the effects of different re-suspension patterns of polluted sediments from the site of national interest Bagnoli-Coroglio, on the immune system of the sea urchin Paracentrotus lividus. An indoor experiment was set up exposing sea urchins for 34 days to such sediments and evaluating the effects of two patterns of water turbulence, mimicking natural storms at sea. One group of animals experienced an "aggregated" pattern of turbulence, consisting in two events, each lasting 2 days, separated by only 3 calm days, while a second group experienced two events of turbulence separated by 17 calm days (spaced pattern). At different times from the beginning of the experiment, coelomic fluid was collected from the animals and immune cells were examined for cell count and morphology, oxidative stress variables, and expression of genes involved in metal detoxification, stress response and inflammation. Our results highlighted that the aggregated pattern of turbulence was more noxious for sea urchins. Indeed, their immune system was altered, over the exposure time, as indicated by the increase of red amoebocytes number. Moreover, despite of an increase of the antioxidant power, animals from this group displayed a very significant ROS over-production at the end of the experiment. Conversely, animals in the spaced condition activated a different immune response, mainly having phagocytes as actors, and were able to partially recover from the received stress at the end of the experiment. No changes in the expression of genes related to antioxidant and anti-inflammatory responses were observed in both groups. By contrast, a down-regulation of various metallothioneins (4, 6, 7 and 8) in the group subjected to aggregated pattern was observed, while metallothionein 8 was up-regulated in the animals from the group exposed to the spaced pattern of turbulence. This work provides the first evidence of how sea urchins can respond to different re-suspension patterns of polluted sediments by modulating their immune system functions. The present data are relevant in relation to the possible environmental restoration of the study site, whose priorities include the assessment of the effects of marine pollution on local organisms, among which P. lividus represents a key benthic species.

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.

Microplastics are defined as plastic fragments

Marine organisms are simultaneously exposed to numerous pollutants, among which metals probably represent the most abundant in marine environments. In order to evaluate the effects of metal exposure at molecular level in reproductive tissues, we profiled the sea urchin transcriptional response after non-lethal exposures using pathway-focused mRNA expression analyses. Herein, we show that exposures to relatively high concentrations of both essential and toxic metals hugely affected the gonadic expression of several genes involved in stress-response, detoxification, transcriptional and post-transcriptional regulation, without significant changes in gonadosomatic indices. Even though treatments did not result in reproductive tissues visible alterations, metal exposures negatively affected the main mechanisms of stress-response, detoxification and survival of adult P. lividus. Additionally, transcriptional changes observed in P. lividus gonads may cause altered gametogenesis and maintenance of heritable aberrant epigenetic effects. This study leads to the conclusion that exposures to metals, as usually occurs in polluted coastal areas, may affect sea urchin gametogenesis, thus supporting the hypothesis that parental exposure to environmental stressors affects the phenotype of the offspring.

Pharmaceuticals are ubiquitously detected in the marine environment at the ng–μg/L range. Given their biological activity, these compounds are known to induce detrimental effects on biota at relatively low exposure levels; however, whether they affect early life stages of marine species is still unclear. In this study, a set of bioassays was performed to assess the effects of propranolol (PROP), 17-α ethinylestradiol (EE2), and gemfibrozil (GEM) on gamete fertilization and embryonic development of mussels (Mytilus galloprovincialis) and sea urchins (Paracentrotus lividus), and on the survival of seabream (Sparus aurata) larvae. Treatments of PROP (500, 5000, 50,000 ng/L), EE2 (5, 50, 500 ng/L), and GEM (50, 500, 5000 ng/L) were selected to encompass levels comparable or superior to environmental concentrations. Obtained data were tested for dose-response curve fitting and the lowest EC10/LC10 used to calculate risk quotients (RQs) based on the MEC/PNEC. No alteration was induced by PROP on the mussel gamete fertilization, while inhibitory effects were observed at environmental levels of EE2 (500 ng/L) and GEM (5000 ng/L). Fertilization was significantly reduced in sea urchin at all PROP and EE2 dosages. The 48-h exposure to all pharmaceuticals induced the onset of morphological abnormalities in either mussel or sea urchin embryos. Alterations were generally observed at environmentally relevant dosages, except for PROP in mussels, in which alterations occurred only at 50,000 ng/L. A decreased survival of seabream larvae was recorded after 96-h exposure to PROP (all treatments), EE2 (50–500 ng/L), and GEM (500 ng/L). A median RQ > 1 was obtained for all pharmaceuticals, assigning a high risk to their occurrence in marine environments. Overall, results showed that current levels of contamination by pharmaceuticals can impact early stages of marine species, which represent critical junctures in the resilience of coastal ecosystems.

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.

The health of the sea urchin Paracentrotus lividus, a key species in the Mediterranean Sea, is menaced by several pressures in coastal environments. Here, we aimed at assessing the reproductive ability of apparently healthy P. lividus population in a marine protected area affected by toxic blooms of Ostreospsis cf. ovata. Wide-ranging analyses were performed in animals collected prior to and during the bloom, as well as at several times thereafter, during the reproductive season. Adults showed a low fertilization rate, along with high nitric oxide (NO) levels in the gonads and the nitration of the major yolk protein toposome, which is an important player in sea urchin development. Serious developmental anomalies were observed in the progeny, which persist several months after the bloom. NO levels were high in the different developmental stages, which also showed variations in the transcription of several genes that were found to be directly or indirectly modulated by NO. These results highlight subtle but important reproductive flaws transmitted from the female gonads to the offspring with the NO involvement. Despite a recovery along time after the bloom, insidious damages can be envisaged in the local sea urchin population, with possible reverberation on the whole benthic system.