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• The toxicity of OlyA6/PlyB complex was assessed on the Mediterranean sea urchin. • OlyA6 was localized in the sea urchin egg containing sphingolipid membrane receptors. • High OlyA6/PlyB levels affected sea urchin fertilization and early development. • Delayed development and incorrect migration of primary mesenchyme cells occurred. • A slight environmental impact at high OlyA6/PlyB concentrations was found. The use of alternative solutions for pest management to replace pesticides in agriculture is of great interest. Proteinaceous complexes deriving from edible oyster mushrooms were recently proposed as environmentally friendly bioinsecticides. Such complexes, composed of ostreolysin A6 (OlyA6) and pleurotolysin B (PlyB), target invertebrate-specific membrane sphingolipids in insect's midgut, causing death through the formation of transmembrane pores. In this work, the potential impact of OlyA6/PlyB complexes was tested in the Mediterranean sea urchin Paracentrotus lividus, as an indicator of environmental quality. The ability of the fluorescently tagged OlyA6 to bind sea urchin gametes (sperm, eggs), the lipidome of sea urchin gametes, and the potential toxic effects and developmental anomalies caused by OlyA6/PlyB complexes on P. lividus early development (embryo, larvae) were investigated. The binding of the fluorescently tagged OlyA6 could be observed only in sea urchin eggs, which harbor OlyA6 sphingolipid membrane receptors, conversely to sperm. High protein concentrations affected sea urchin fertilization (>750 µg/L) and early development (> 375 µg/L in embryos; >100 µg/L in larvae), by causing toxicity and morphological anomalies in embryos and larvae. The main anomalies consisted in delayed embryos and incorrect migration of the primary mesenchyme cells that caused larval skeletal anomalies. The classification of these anomalies indicated a slight environmental impact of OlyA6/PlyB complexes at concentrations higher than 750 µg/L. Such impact should not persist in the marine environment, due to the reversible anomalies observed in sea urchin embryos and larvae that may promote defense strategies. However, before promoting the use of OlyA6/PlyB complexes as bio-pesticides at low concentrations, further studies on other marine coastal species are needed. [Display omitted] [ABSTRACT FROM AUTHOR]

The fate and toxicity of ingested marine microplastics (MPs) have been of major concern in aquatic ecotoxicology for the last decade. Although their ingestion by a wide range of marine organisms has been proven, the uptake of MPs within organs is not yet fully understood and relies on the ability of ingested microplastics to transfer from the gut to tissues beyond the digestive wall (i.e., translocation). The present study investigates the in vitro transfer of fluorescent high-density polyethylene particles of different sizes classes (1–5 µm; 10–29 µm; 38–45 µm) across the intestinal wall of the sea urchin Paracentrotus lividus using Ussing chambers. Small microplastics (1–5 µm) were proven to be able to cross the intestinal wall of P. lividus and reach the coelomic fluid, while larger microplastics (≥ 10 µm) were not observed to cross the intestinal wall. Results demonstrate a size-dependent passage of polyethylene microparticles across the intestinal walls of P. lividus for the first time, highlighting the suitability of Ussing chamber systems to study the transfer of MPs across the intestinal wall of animals. [ABSTRACT FROM AUTHOR]

• Transgenerational effects of OA were studied in P. lividus sperm and larvae. • Gametogenesis under OA resulted in increased sperm ATP content. • Slower decrease of swimming velocity was found in sperm from males kept at low pH. • Parental exposure to OA decreased larval survival but increased larval growth. • Parental pH affected offspring performances more than post-spawning pH. In marine organisms, differing degree of sensitivity to ocean acidification (OA) is expected for each life stage, and disturbance at one stage can carry over into the following stage or following generation. In this study we investigated phenotypic changes of sperm and larvae of the sea urchin Paracentrotus lividus in response to different pH conditions (8.0, 7.7, 7.4) experienced by the parents during gametogenesis. In sperm from two-months exposed males, sperm motility, velocity, ATP content, ATP consumption and respiration rate were evaluated at three pH values of the activating medium (8.0, 7.7 and 7.4). Moreover, larvae from each parental group were reared at pH 8.0 and 7.7 for 20 days and larval mortality and growth were then assessed. Sperm motility and respiration rate were not affected either by exposure of males to low pH or by the post-activation pH. Sperm velocity did not differ among post-activation pH values in all sperm groups, but it decreased slower in sperm developed under acidified conditions, suggesting the presence of positive carryover effect on sperm longevity. This positive carryover effect of exposure of males to low pH values was highlighted also for the sperm ATP content, which was higher in these groups of sperm. ATP consumption rate was affected by post-activation pH with higher values at pH 8.0 in sperm from males maintained at control condition and pH 7.7 while the energy consumption appeared to be differently modulated at different experimental conditions. A negative carry over effect of OA was observed on survival of larvae from parents acclimated at pH 7.4 and additive negative effects of both parental and larval exposure to low pH can be suggested. In all groups of larvae, decreased somatic growth was observed at low rearing pH, thus larvae from parents maintained at low pH did not show an increased capability to cope with OA. [ABSTRACT FROM AUTHOR]

• The consequences vary considerably depending on the number and concentration of trace elements. • The effects of 15 trace elements at the highest concentrations are wider arms and delayed development. • Monitoring the entire larval development appears to be an efficient method to predict the effects of low levels of trace element contamination. • A hormesis phenomenon was observed with a mixture of 7 trace elements (Co, Cr, Cu, Fe, Hg, Ni and Zn) at average concentrations. • The provenance of spawners is important and must be precised in ecotoxicological studies. Several experiments were performed using larvae of Paracentrotus lividus (Lamarck, 1816) in order to determine the consequences of different chronic contamination with mixtures of (i) fifteen trace elements from concentrations measured in the world ocean seawater, and (ii) seven trace elements from contamination resulting from mining. To predict the impact of increased marine pollution, higher concentrations were also used. These bioassays were conducted using spawners collected from Calvi (reference site, Corsica), and Albo (mining area, Corsica). The effects of trace elements have been studied on the entire larval development. The results show wider arms and delayed development as the number and concentration of trace elements increases. Therefore, the synergy between the different trace elements is of paramount importance with regard to the impact on organisms. Probably due to a hormesis phenomenon, larvae contaminated with seven trace elements at average concentrations developed more quickly. This work also highlighted the importance of the origin of spawners in ecotoxicological studies. To our knowledge, this is the first study to investigate the effects of such a broad combination of trace elements for chronic contamination on the entire larval stage of Paracentrotus lividus. [ABSTRACT FROM AUTHOR]

We examined egg fertilisation in purple sea urchin ( Paracentrotus lividus ) after sperm exposure to carbon-based nanomaterials, carbon black (CB) and graphene oxide (GO), from 0.0001 mg/L to 1.0 mg/L. Gastrula stage embryos were investigated for acetylcholinesterase and propionylcholinesterase activities, and their morphological characteristics. Plutei were analysed for morphological abnormalities, with emphasis on skeletal rod formation. Egg fertilisation was significantly affected by CB, at all concentrations tested. Loss of cell adhesion at the gastrula surface was observed in eggs fertilised with sperm treated with CB. However, concentration-dependant morphological anomalies were observed in the gastrulae and plutei formed after sperm exposure to either CB or GO. The activities of both cholinesterases decreased in the gastrulae, although not in a concentration-dependent manner. These effects appear to arise from physical interactions between these carbon-based nanomaterials and the sperm, whereby nanomaterials attached to the sperm surface interfere with fertilisation, which leads to disturbances in the signalling pathways of early embryonic development. Reduced cholinesterase activity in gastrulae from eggs fertilised with nanomaterial-treated sperm confirms involvement of the cholinergic system in early sea urchin development, including skeletogenesis. [ABSTRACT FROM AUTHOR]

Usage of bisphenol A (BPA) in production of polycarbonate plastics has resulted in global distribution of BPA in the environment. These high concentrations cause numerous negative effects to the aquatic biota, among which the most known is the induction of endocrine disruption. The focus of this research was to determine the effects of two experimentally determined concentrations of BPA (100 nM and 4 μM) on cellular detoxification mechanisms during the embryonic development (2-cell, pluteus) of the rocky sea urchin ( Paracentrotus lividus ), primarily the potential involvement of multidrug efflux transport in the BPA intercellular efflux. The results of transport assay, measurements of the intracellular BPA and gene expression surveys, for the first time indicate the importance of P-glycoprotein (P-gp/ABCB1) in defense against BPA. Cytotoxic effects of BPA, validated by the immunohistochemistry (IHC) and the transmission electron microscopy (TEM), induced the aberrant karyokinesis, and consequently, the impairment of embryo development through the first cell division and retardation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights: [•] Pb and Zn exposure resulted in disruption of Ca homeostasis. [•] Ca uptake rates and whole body Ca accumulation were severely inhibited by both metals. Ca2+ ATPase activity was significantly inhibited by Zn but not by Pb. [•] Pb exhibited marked bioaccumulation whereas Zn was well-regulated. [•] Both metals had little effect on growth, measured as larval dry weight, or on Na, K, or Mg concentrations. [•] Embryos exposed to Pb showed some capacity for recovery. [Copyright &y& Elsevier]

• Ecotoxicological integration provide the status of chemical bioavailability. • Comparability of bioassays batteries confirms a higher sensibility of sea urchin. • Choice of 2° battery species doesn't real influence the ecotoxicological status. • Multidisciplinary WOE model provides the marine environmental quality assessment. This study was conducted under the Italian Ministerial Decree D.M. 173/2016 which regulates the assessment of the Sediment Class Quality in Italy using ecotoxicological bioassay and chemical analysis (Weight-Of-Evidence model). The aim of this work was to evaluate the real classification obtained by the theoretically equivalent responses of nine different combinations of batteries based on six different species: Aliivibrio fischeri (inhibition of bioluminescence), Phaeodactylum tricornutum, Skeletonema costatum, Dunaliella tertiolecta (inhibition of algal growth), Paracentrotus lividus and Crassostrea gigas (embryotoxicity). Bioassays, in many cases, showed a non-bioavailability effect of the pollutants; these one highly revealed by the chemical analyses. Algal species showed responses very similar from each other. Otherwise, species used for embryotoxicity produced wide responses, consequently modifying the quality class of sediments and the handling management (i.e. landfill confinement or beach nourishment) allowed by the Law. [ABSTRACT FROM AUTHOR]

Abstract: Androgen metabolism (androstenedione and testosterone) has been assessed in the digestive tube and gonads of the echinoderm Paracentrotus lividus exposed to different concentrations of the biocide triphenyltin (TPT) in a semi-static water regime for 4 weeks. Key enzymatic activities involved in both synthesis and metabolism of androgens, namely 17β-hydroxysteroid dehydrogenases (17β-HSDs), 3β-HSDs, 5α-reductases, P450-aromatase, palmitoyl-CoA:testosterone acyltransferases (ATAT) and testosterone sulfotransferases (SULT), were investigated in digestive tube and/or gonads of control and TPT-exposed specimens in an attempt to see whether androgen metabolism was altered by exposure. In agreement with previous data for vertebrates, exposure to TPT led to a concentration dependent decrease of P450-aromatase that was statistically significant at the highest TPT concentration tested (225ng/L). Additionally, increased metabolism of testosterone to form dihydrotestosterone (DHT) and 5α-androstane-3β,17β-diol was observed, suggesting increased 5α-reductase activity in the gonads of TPT-exposed individuals. Interestingly, exposure to TPT induced testosterone conjugating activities in organisms exposed to medium (SULT) and high (ATAT and SULT) TPT concentrations. Despite the changes of androgen metabolizing enzymes, testosterone levels in gonads remained rather stable. In contrast, an increase in testosterone and a concomitant decrease in estradiol were observed in the coelomic fluid of TPT-exposed organisms. Overall, the data indicate the ability of TPT to modulate androgen metabolism and circulating steroid levels in P. lividus and suggest the existence of regulatory mechanisms to maintain stable endogenous levels of testosterone in gonads. This study also contributes to a better knowledge of echinoderm endocrinology. [Copyright &y& Elsevier]

The effects of humic acid (HA) on the toxicity of copper to sea urchin Paracentrotus lividus larvae were studied in chemically defined seawater. Square Wave Anodic Stripping Voltammetry (SWASV) was employed to study the complexation of copper in seawater medium. A simple complexation model assuming one ligand type and a 1:1 reaction stoichiometry successfully explained the inverse titration experiments. A conditional stability constant of 6.53±0.05 and a complexating capacity of 230±7 μmol Cu/g HA were obtained. Sea urchin bioassay tests with two endpoints, embryogenesis success and larval growth were carried out in order to study the toxicity of dissolved copper in both the presence and absence of HA. The toxicity data obtained fitted well into a logistic model, and the high sensitivity of both endpoints (EC50 were 41.1 μg Cu/l and 32.9 μg Cu/l, respectively) encourages their use for biomonitoring. The HA had a clearly protective effect, reducing the toxicity of Cu to the sea urchin larvae. The labile copper, rather than the total copper concentrations, explained the toxicity of the Cu–HA solutions, and the Cu–HA complexes appeared as non-toxic forms. These results are in agreement with the Free Ion Activity Model, because the labile Cu concentrations in this buffered and chemically defined medium covary with the free ion activity of the Cu, validating the model to naturally occurring HA in the marine environment. [Copyright &y& Elsevier]

• A high throughput technology was applied on embryos exposed to UV, Cd and Cd/UV. • We investigated the molecular responses adopted by embryos to cope with these stressors. • The analysis of 127 transcripts highlighted the dysregulation of many genes. 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 vivo model, contributes to advance our knowledge about cellular responses to UV, Cd and their combination. [ABSTRACT FROM AUTHOR]

• Increased temperature enhanced development and arm length, Gd had a stunting effect. • Near-future temperature projections (+3 °C, 21 °C) reduced the negative effects of Gd. • Extreme warming (+6 °C, 24 °C) had negative synergistic effects. • Gd was the main driver for the induction of heat shock proteins expression. • Temperature, with or without Gd, induced the activation of autophagy and apoptosis. Gradual ocean warming and marine heatwaves represent major threats for marine organisms already facing other anthropogenic-derived hazards, such as chemical contamination in coastal areas. In this study, the combined effects of thermal stress and exposure to gadolinium (Gd), a metal used as a contrasting agent in medical imaging which enters the aquatic environment, were investigated in the embryos and larvae of the sea urchin Paracentrotus lividus. Embryos were exposed to six treatments of three temperatures (18 °C, 21 °C, 24 °C) and two Gd concentrations (control: 0 μM; treated: 20 μM). With respect to developmental progression, increased temperature accelerated development and achievement of the larval stage, while Gd-exposed embryos at the control temperature (18 °C) showed a general delay in development at 24 h post-fertilization (hpf), and a stunting effect and impaired skeleton growth at 48 hpf. Elevated temperatures at near-future projections (+3 °C, 21 °C) reduced the negative effects of Gd on development with a lower percentage of abnormality and improved skeleton growth. Combined extreme warming at present-day marine heatwave conditions (+6 °C, 24 °C) and Gd treatment resulted in a lower proportion of embryos reaching the advanced larval stages compared to the 21 °C + Gd. At the molecular level, western blot analysis showed that Gd was the main driver for the induction of heat shock protein (HSP60, HSP70) expression. At 48 hpf, temperature increase was the main driver for activation of additional cellular stress response strategies such as autophagy and apoptosis. Combined treatments showed the induction of HSP60 at 24 hpf and autophagic and apoptotic processes at 48 hpf. Treatments having low levels of HSPs expression showed high levels of apoptosis, and vice versa , clearly demonstrating the antagonistic effects of HSPs expression and apoptosis. Detection of fragmented DNA in apoptotic nuclei showed selective apoptosis, likely in extremely damaged cells. Our results indicate that the negative effects of Gd-exposure on P. lividus larval development and biomineralization will be mitigated by a near-future ocean warming, up to a thermotolerance threshold when negative synergistic effects were evident. Our data highlight the use of biomarkers as sensitive tools to detect environmental impacts as well as the need for a better understanding of the interactions between the multiple stressors faced by marine species in coastal environments. [ABSTRACT FROM AUTHOR]

• In binary exposures, other metals inhibited bioaccumulation of Zn and Cd. • Inhibitory effects on bioaccumulation were not reflected in unidirectional metal influx rates • Low Ni levels, alone and in binary mixtures, delayed development Metal accumulation, disturbance of Ca2+ homeostasis, and occurrence of abnormalities are well-established consequences of single metal exposure during early development stages of sea urchins. However, the effects caused by low concentrations of metals and metal mixtures need to be better understood in marine invertebrates. Therefore, the present study investigated the effects of environmentally relevant concentrations of Zn (9 μg/L), Cd (30 μg/L) and Ni (5 μg/L) in single and binary exposures (Zn + Cd, Cd + Ni and Ni + Zn) to the early life stages of the purple sea urchin Strongylocentrotus purpuratus. Endpoints checked in all treatments after 48-h exposure were unidirectional metal influx rates, bioaccumulation, and Ca2+ influx rates. Additionally, the presence of abnormal larvae and developmental delay was evaluated at 24 h, 48 h and 72 h of exposure. Unidirectional influx rates of all three metals were significantly higher than control background rates in all single exposures and binary mixtures, and were generally not different between them. Net accumulation (body burden) of both Zn and Cd increased significantly as a result of their respective single exposures, while Ni accumulation decreased considerably. When Zn or Cd were presented in binary exposures with other metals, the net accumulations of Zn or Cd were reduced relative to single exposures to these metals, whereas this did not occur for Ni accumulation. Thus, bioaccumulation proved to be a better metric than influx rate measurements to analyze metal competition at a whole organism level at these low metal concentrations. Short-term Ca2+ influx also did not appear to be a sensitive metric, as the measured rates did not vary among all single and binary exposures, with the exception of a lower rate in Ni + Zn binary exposure. A critical aspect observed was the relationship between bioaccumulation versus influx measurements, which proved positive for Cd, but negative for Zn and Ni, demonstrating possible capacities for both Zn and Ni regulation by sea urchin larvae. Increases in larval abnormalities relative to controls occurred only after binary mixtures, starting at 48 h exposure and maintained until 72 h. However, delay of the sea urchin development by the presence of gastrula stage at 72 h exposure occurred in Zn and Ni single exposures and all metal mixtures, with very high abnormal development when Ni was present. [ABSTRACT FROM AUTHOR]

• Zinc-sunscreens interfere with sea urchin skeletal formation and axial development. • Zinc-sunscreens are internalized by sea urchin embryos in a dose-dependent manner. • Exposure to sunscreens reduces sea urchin multidrug-resistant transporter activity. The growing popularity of physical sunscreens will lead to an increased release of ingredients from zinc oxide (ZnO) sunscreens into marine environments. Though zinc (Zn) is a necessary micronutrient in the ocean, greater than natural Zn concentrations may be released into marine environments by use of sunscreens. The extent of the consequences of this addition of Zn to the ocean are not fully understood. We investigated the effects of materials released by ZnO- sunscreens on the development of California purple sea urchin, Strongylocentrotus purpuratus. Embryos incubated in various concentrations of Zn (0.01, 0.05, 0.1, 0.5, and 1 mg/L), the sources of which included zinc-containing compounds: ZnO and zinc sulfate (ZnSO 4); and ZnO sunscreens: All Good, Badger, and Raw Elements brands. Based on EC 50 values, ZnO-containing sunscreens were slightly, but not significantly, more toxic than ZnO and ZnSO 4 , suggesting that sunscreens may release additional unknown materials that are detrimental to sea urchin embryo development. All concentrations of Zn-exposure resulted in significant malformations (skeletal abnormality, stage arrest, axis determination disruption), which were identified using light and fluorescence confocal microscopy. The concentration of Zn2+ internalized by the developing embryos correlated positively with the concentration of Zn in seawater. Additionally, exposure to both ZnO sunscreens and ZnO and ZnSO 4 at 1 mg/L Zn, significantly increased calcein-AM (CAM) accumulation, indicating decreased multidrug resistant (MDR) transporter activity. This is one of the first studies documenting ZnO-containing sunscreens release high concentrations of Zn that are internalized by and have detrimental effects on aquatic organisms. [ABSTRACT FROM AUTHOR]

• Cd and Zn exposure affect antioxidant system in early life stage of S. purpuratus. • Cd is more potent than Zn in exerting sublethal effects on oxidative stress parameters. • SOD activity and lipid peroxidation are the most sensitive biomarkers. • Sublethal effects of mixtures are less than additive in embryos of purple sea urchin. Oxidative stress parameters were evaluated during the first 72 h of embryonic development of purple sea urchin Strongylocentrotus purpuratus continuously exposed to control conditions, to cadmium alone (Cd, 30 μg/L), to zinc alone (Zn, 9 μg/L) or to a Cd (28 μg/L) plus Zn (9 μg/L) mixture. These sublethal concentrations represent ∼ 10% of the acute EC50. Bioaccumulation, antioxidant capacity against peroxyl radicals (ACAP), total glutathione (GSH) level, glutathione-S-transferase (GST), glucose-6-phosphate dehydrogenase (G6PDH) and superoxide dismutase (SOD) activity, and lipid peroxidation (LPO) were analyzed at 24 h (blastula), 48 h (gastrula), and 72 h (pluteus) stages of development. Zinc (an essential metal) was well-regulated, whereas Cd (non-essential) bioaccumulated and whole-body [Cd] increased from blastula to pluteus stage in sea urchin larvae. In controls, ACAP progressively declined from 24 h to 72 h, while LPO reciprocally increased, but other parameters did not change. Cd alone was more potent than Zn alone as a pro-oxidant, with the major effects being decreases in SOD activity and parallel increases in LPO throughout development; GST activity also increased at 24 h. Zn alone caused only biphasic disturbances of ACAP. In all cases, the simultaneous presence of the other metal prevented the effects, and there was no instance where the oxidative stress response in the presence of the Cd/Zn mixture was greater than in the presence of either Cd or Zn alone. Therefore the sublethal effects of joint exposures were always less than additive or even protective, in agreement with classical toxicity data. Furthermore, our results indicate that SOD and Zn can play important roles in protecting sea urchin embryos against Cd-induced lipid peroxidation. [ABSTRACT FROM AUTHOR]