Psammechinus Miliaris Research Articles

Handling, Reproducing and Cryopreserving Five European Sea Urchins (Echinodermata, Klein, 1778) for Biodiversity Conservation Purposes.

In this work, five local sea urchin species found in European waters were studied. Four were regular species: Sphaerechinus granularis, Psammechinus miliaris, Echinus esculentus (Linnaeus, 1758) and the edible sea urchin Paracentrotus lividus; and one was an irregular species, Echinocardium cordatum. These five species of sea urchins have been studied regarding their fertility, toxicity of cryoprotecting agents, cryopreservation of different cell types and chilling injury. The baseline fertility is similar in P. lividus, P. miliaris and S. granularis. Nonetheless, the sperm:egg ratio, contact time and development of the fertilization envelope would need to be studied further on a case-by-case basis. Sperm can be maintained inactively in the gonad (4 °C), and oocytes also maintain quality in sea water (4 °C), even after 72 h. Sperm was cryopreserved for four species with some post-thaw intra specific variability, and embryo cryopreservation was only possible for S. granularis. Overall, this study provided a wider vision of the biology and reproduction of these species that will help us develop tools for their biodiversity conservation through cryopreservation.

Interspecific differences in oxidative DNA damage after hydrogen peroxide exposure of sea urchin coelomocytes.

Interspecific comparison of DNA damage can provide information on the relative vulnerability of marine organisms to toxicants that induce oxidative genotoxicity. Hydrogen peroxide (H2O2) is an oxidative toxicant that causes DNA strand breaks and nucleotide oxidation and is used in multiple industries including Atlantic salmon aquaculture to treat infestations of ectoparasitic sea lice. H2O2 (up to 100 mM) can be released into the water after sea lice treatment, with potential consequences of exposure in nontarget marine organisms. The objective of the current study was to measure and compare differences in levels of H2O2-induced oxidative DNA damage in coelomocytes from Scottish sea urchins Echinus esculentus, Paracentrotus lividus, and Psammechinus miliaris. Coelomocytes were exposed to H2O2 (0-50 mM) for 10 min, cell concentration and viability were quantified, and DNA damage was measured by the fast micromethod, an alkaline unwinding DNA method, and the modified fast micromethod with nucleotide-specific enzymes. Cell viability was >92% in all exposures and did not differ from controls. Psammechinus miliaris coelomocytes had the highest oxidative DNA damage with 0.07 ± 0.01, 0.08 ± 0.01, and 0.07 ± 0.01 strand scission factors (mean ± SD) after incubation with phosphate-buffered saline, formamidopyrimidine-DNA glycosylase, and endonuclease-III, respectively, at 50 mM H2O2. Exposures to 0.5 mM H2O2 (100-fold dilution from recommended lice treatment concentration) induced oxidative DNA damage in all three species of sea urchins, suggesting interspecific differences in vulnerabilities to DNA damage and/or DNA repair mechanisms. Understanding impacts of environmental genotoxicants requires understanding species-specific susceptibilities to DNA damage, which can impact long-term stability in sea urchin populations in proximity to aquaculture farms.

Elevated toxicity of resuspended mine tailings over time

Resuspension of disposed mine tailings is an important secondary source of heavy metal pollution in affected regions. UK copper mine tailings were continuously resuspended in seawater over 14 days to understand potential metal dissolution and associated ecotoxicological impacts. Aqueous concentrations of arsenic increased 859%, nickel 85%, manganese and cadmium over 40%, and vanadium and zinc over 20%. Green sea urchin (Psammechinus miliaris) gametes were exposed to 18-h (18hr) and 14-day (14d) resuspension elutriates. Fertilisation success dropped from 72% (18hr) to 21.06% (14d). Toxicity Index (TI) values (reduced fertilisation membrane quality and fertilisation success) increased from 1.6 (18hr) to 2.8 (14d). Embryo polyspermy frequencies increased from 17.58% (18hr) to 21.13% (14d). Higher TI values and polyspermy rates indicate impacts may go beyond standard bioassay endpoints. Current predictions of mine tailing impacts may be underestimated. This is important for ecosystem resilience when considering other anthropogenic stressors such as climate change. CapsuleDisturbance of metal contaminated sediments over prolonged periods results in ever increasing dissolution concentrations, and elevated toxicity of the water medium to biological receptors.

Using stable isotope analysis to determine the effects of ocean acidification and warming on trophic interactions in a maerl bed community

AbstractOcean acidification and warming are likely to affect the structure and functioning of marine benthic communities. This study experimentally examined the effects of ocean acidification and warming on trophic interactions within a maerl bed community by using stable carbon and nitrogen isotope analysis. Two three‐month experiments were conducted in winter and summer seasons with four different combinations of pCO2 (ambient and elevated pCO2) and temperature (ambient and +3°C). Experimental assemblages were created in tanks held in the laboratory and were composed of calcareous (Lithothamnion corallioides) and fleshy algae (Rhodymenia ardissonei, Solieria chordalis, and Ulva sp.), gastropods (Gibbula magus and Jujubinus exasperatus), and sea urchins (Psammechinus miliaris). Our results showed higher seaweed availability for grazers in summer than winter. Therefore, grazers were able to adapt their diet seasonally. Increased pCO2 and temperature did not modify the trophic structure in winter, while shifts in the contribution of seaweed were found in summer. Combined acidification and warming increased the contribution of biofilm in gastropods diet in summer conditions. Psammechinus miliaris mostly consumed L. corallioides under ambient conditions, while the alga S. chordalis became the dominant food source under high pCO2 in summer. Predicted changes in pCO2 and temperature had complex effects on assemblage trophic structure. Direct effects of acidification and warming on seaweed metabolism may modify their abundance and biomass, affecting their availability for grazers. Climate change may also modify seaweeds' nutritive value and their palatability for grazers. The grazers we investigated were able to change their diet in response to changes in algal assemblages, an advantage given that warming and acidification alter the composition of algal communities.

Responses to environmentally relevant microplastics are species-specific with dietary habit as a potential sensitivity indicator

There is a lack of information on understanding how marine organisms respond to environmentally relevant microplastics (MP) which hampers decision making for waste management strategies. This study addresses this information gap by determining whether responses to MPs are species specific within a functional group. Benthic residing sea urchins, Psammechinus miliaris and Paracentrotus lividus were used as a case study. Psammechinus miliaris are strong omnivores with dietary intake including hard components (e.g. shell, tubeworms) and therefore likely to cope with the ingestion of MPs, while P. lividus are strong herbivores consuming softer dietary items (e.g. biofilms, algae) and therefore more likely sensitive. Responses to environmentally relevant MPs were conducted across two trials. Trial one determined the impact of short term (24 h) external exposure to storm-like sediment resuspension of MP concentrations (53 μm polyvinyl chloride (PVC) 25,000 MP L−1) compared to a control without MPs. No significant impacts were observed for both P. lividus and P. miliaris on metabolic rate or righting time, and urchins were able to remove MPs from the body surface using pedicellariae and cilia. Trial two determined the impact of medium term (2 months) ingestion of a diet laced with PVC MPs (59 μm) at an inclusion rate of 0.5% mass and a control diet (without MPs) on somatic growth and animal condition. The ingestion of MPs did not significantly impact P. miliaris but significantly reduced the alimentary index within P. lividus, indicating a compromised nutritional state. This study shows that responses to microplastics are species-specific and therefore cannot be generalized. Furthermore, feeding habit could act as a potential indicator for sensitivity to MP ingestion which will be important for impact assessments of plastic pollution and management strategies.

Effects of seawater Mg2+ /Ca2+ ratio and diet on the biomineralization and growth of sea urchins and the relevance of fossil echinoderms to paleoenvironmental reconstructions.

It has been argued that skeletal Mg/Ca ratio in echinoderms is mostly governed by Mg2+ and Ca2+ concentrations in the ambient seawater. Accordingly, well-preserved fossil echinoderms were used to reconstruct Phanerozoic seawater Mg2+ /Ca2+ ratio. However, Mg/Ca ratio in echinoderm skeleton can be affected by a number of environmental and physiological factors, the effects of which are still poorly understood. Notably, experimental data supporting the applicability of echinoderms in paleoenvironmental reconstructions remain limited. Here, we investigated the effect of ambient Mg2+ /Ca2+ seawater ratio and diet on skeletal Mg/Ca ratio and growth rate in two echinoid species (Psammechinus miliaris and Prionocidaris baculosa). Sea urchins were tagged with manganese and then cultured in different Mg2+ /Ca2+ conditions to simulate fluctuations in the Mg2+ /Ca2+ seawater ratios in the Phanerozoic. Simultaneously, they were fed on a diet containing different amounts of magnesium. Our results show that the skeletal Mg/Ca ratio in both species varied not only between ossicle types but also between different types of stereom within a single ossicle. Importantly, the skeletal Mg/Ca ratio in both species decreased proportionally with decreasing seawater Mg2+ /Ca2+ ratio. However, sea urchins feeding on Mg-enriched diet produced a skeleton with a higher Mg/Ca ratio. We also found that although incubation in lower ambient Mg2+ /Ca2+ ratio did not affect echinoid respiration rates, it led to a decrease or inhibition of their growth. Overall, these results demonstrate that although skeletal Mg/Ca ratios in echinoderms can be largely determined by seawater chemistry, the type of diet may also influence skeletal geochemistry, which imposes constraints on the application of fossil echinoderms as a reliable proxy. The accuracy of paleoseawater Mg2+ /Ca2+ calculations is further limited by the fact that Mg partition coefficients vary significantly at different scales (between species, specimens feeding on different types of food, different ossicle types, and stereom types within a single ossicle).

Multiple New Paralytic Shellfish Toxin Vectors in Offshore North Sea Benthos, a Deep Secret Exposed.

In early 2018, a large easterly storm hit the East Anglian coast of the UK, colloquially known as the ‘Beast from the East’, which also resulted in mass strandings of benthic organisms. There were subsequent instances of dogs consuming such organisms, leading to illness and, in some cases, fatalities. Epidemiological investigations identified paralytic shellfish toxins (PSTs) as the cause, with toxins present in a range of species and concentrations exceeding 14,000 µg STX eq./kg in the sunstar Crossaster papposus. This study sought to better elucidate the geographic spread of any toxicity and identify any key organisms of concern. During the summers of 2018 and 2019, various species of benthic invertebrates were collected from demersal trawl surveys conducted across a variety of locations in the North Sea. An analysis of the benthic epifauna using two independent PST testing methods identified a ‘hot spot’ of toxic organisms in the Southern Bight, with a mean toxicity of 449 µg STX eq./kg. PSTs were quantified in sea chervil (Alcyonidium diaphanum), the first known detection in the phylum bryozoan, as well as eleven other new vectors (>50 µg STX eq./kg), namely the opisthobranch Scaphander lignarius, the starfish Anseropoda placenta, Asterias rubens, Luidia ciliaris, Astropecten irregularis and Stichastrella rosea, the brittlestar Ophiura ophiura, the crustaceans Atelecyclus rotundatus and Munida rugosa, the sea mouse Aphrodita aculeata, and the sea urchin Psammechinus miliaris. The two species that showed consistently high PST concentrations were C. papposus and A. diaphanum. Two toxic profiles were identified, with one dominated by dcSTX (decarbamoylsaxitoxin) associated with the majority of samples across the whole sampling region. The second profile occurred only in North-Eastern England and consisted of mostly STX (Saxitoxin) and GTX2 (gonyautoxin 2). Consequently, this study highlights widespread and variable levels of PSTs in the marine benthos, together with the first evidence for toxicity in a large number of new species. These findings highlight impacts to ‘One Health’, with the unexpected sources of toxins potentially creating risks to animal, human and environmental health, with further work required to assess the severity and geographical/temporal extent of these impacts.

European Sea Urchin Somatic and Gonadal Responses to Differing Stocking Densities and Seawater Flow Rates: A Case Study for Experimental Design Considerations

Sea urchins are often used as model species in anthropogenic studies, and their aquaculture productions have expanded in recent years, yet basic rearing techniques applied in these studies vary and have received little investigation. This study assessed the effect of seawater flow rates and stocking densities on the somatic and physiological characteristics of European sea urchin species Paracentrotus lividus and Psammechinus miliaris. Two experiments were carried out for each species: one using low, medium, and high seawater flow rates (∼0.2, 0.8, and 1.60 L min–1, respectively) and another using low, medium, and high stocking densities [∼38, 76, and 115 ind. m–2 or 0.8, 1.6, and 2.4 kg m–2 (wet mass), respectively]. Within the flow rate experiment, P. lividus showed no significant treatment effects. By contrast, P. miliaris reared in low seawater flow rates had significantly lower relative spine lengths and gonad indices than conspecifics reared under higher flow rates (medium and high). Within the stocking density experiment, P. miliaris reared under low stocking densities demonstrated significantly larger somatic growth (test diameter and whole animal wet mass) relative to those stocked at high densities but were similar to those stocked at medium densities. For both species, gonad indices were largest within low densities compared with those within higher stocking densities (high only for P. lividus, and medium and high for P. miliaris). This study suggests that careful consideration for general holding conditions, and comparison across anthropogenic studies is required, especially across different species. Furthermore, this information could help improve the production effort of European sea urchin species while achieving high marketable attributes.

Molecular mechanisms underpinning transgenerational plasticity in the green sea urchin Psammechinus miliaris

The pre-conditioning of adult marine invertebrates to altered conditions, such as low pH, can significantly impact offspring outcomes, a process which is often referred to as transgenerational plasticity (TGP). This study describes for the first time, the gene expression profiles associated with TGP in the green sea urchin Psammechinus miliaris and evaluates the transcriptional contribution to larval resilience. RNA-Seq was used to determine how the expression profiles of larvae spawned into low pH from pre-acclimated adults differed to those of larvae produced from adults cultured under ambient pH. The main findings demonstrated that adult conditioning to low pH critically pre-loads the embryonic transcriptional pool with antioxidants to prepare the larvae for the “new” conditions. In addition, the classic cellular stress response, measured via the production of heat shock proteins (the heat shock response (HSR)), was separately evaluated. None of the early stage larvae either spawned in low pH (produced from both ambient and pre-acclimated adults) or subjected to a separate heat shock experiment were able to activate the full HSR as measured in adults, but the capacity to mount an HSR increased as development proceeded. This compromised ability clearly contributes to the vulnerability of early stage larvae to acute environmental challenge.

Benthic biodiversity on old platforms, young wind farms, and rocky reefs

Abstract The introduction of artificial hard substrates in an area dominated by a sandy seabed increases habitat available to epifouling organisms. To investigate this, samples were taken on old offshore oil and gas platforms, and data were compared with data of a young wind farm and a natural reef. Depth, sampling date, abundance of Mytilus edulis, Psammechinus miliaris, Metridium dianthus, and the presence of Tubulariidae and substrate (rock or steel) all correlated with species richness. Multivariate analysis showed a large overlap in communities on steel and rock and between the wind farm and platforms. The community changed over a gradient from deep rocks to shallow steel substrate, but no strong community differentiation was observed. Deep steel was more similar to natural rocks than shallow steel. When an artificial reef is intended to be colonized by communities similar to those on a natural reef, its structure should resemble a natural reef as much as possible.

Optimising stocking density for the commercial cultivation of sea urchin larvae

Increased pressure on wild stocks of sea urchins had led to a requirement for aquaculture based production. However, effective and efficient methodologies still remain under development. The effects of stocking density on Psammechinus miliaris and Paracentrotus lividus were investigated in order to evaluate optimum stocking densities for large scale production. Larvae were reared at stocking densities of 1, 2, 3 and 4 larvae mL−1 and the effects on survival, development, abnormality and morphology were recorded. Additional cultures were maintained at a high density of 3 larvae mL−1 and then displaced to a lower density of 1 larvae mL−1 part way through the larval life cycle (‘displacement treatment’; day 13), to evaluate whether negative effects of high stocking densities could be mitigated. Responses from each species differed. P. miliaris demonstrated the highest growth at 1 larvae mL−1, resulting in larger larval and rudiment sizes by the end of the experiment (day 16). Rearing at 2 larvae mL−1 also demonstrated good growth performance, but only up to day 12. Higher densities of 3 and 4 larvae mL−1 did not affect survival or development, but significantly negatively impacted growth. There was no significant impact on survival, development, and morphology at any of the tested stocking densities for P. lividus. However, of note is that P. lividus reared at a high density of 4 larvae mL−1 had 25% lower survival than controls by the end of the experimental period (day 16). Displacement (larvae transferred from 3 to 1 larvae mL−1 on day 13) was effective for both P. miliaris and P. lividus with survival and rudiment sizes similar to larvae stocked continuously at low densities of 1 larvae mL−1. Although, P. lividus generally performed well at high densities, this demonstrates that displacement approaches could be possible for this species if required. However, of note is that displaced P. lividus had 30% lower survival than controls by the end of the experimental period (day 16). Therefore, this cultivation approach may be a generally viable option for large scale cultivation of these species. This study highlights that species responses can be different when reared at differing stocking densities highlighting a need to expand this approach to a wider range of marketable species. It also demonstrates that more efficient means of production (e.g. displacing larval densities part way through the production process) might be possible for some species (e.g. P. miliaris).

Echinochrome A Release by Red Spherule Cells Is an Iron-Withholding Strategy of Sea Urchin Innate Immunity

Cellular immune defences in sea urchins are shared amongst the coelomocytes - a heterogeneous population of cells residing in the coelomic fluid (blood equivalent) and tissues. The most iconic coelomocyte morphotype is the red spherule cell (or amebocyte), so named due to the abundance of cytoplasmic vesicles containing the naphthoquinone pigment echinochrome A. Despite their identification over a century ago, and evidence of antiseptic properties, little progress has been made in characterising the immunocompetence of these cells. Upon exposure of red spherule cells from sea urchins, i.e., Paracentrotus lividus and Psammechinus miliaris, to microbial ligands, intact microbes, and damage signals, we observed cellular degranulation and increased detection of cell-free echinochrome in the coelomic fluid ex vivo. Treatment of the cells with ionomycin, a calcium-specific ionophore, confirmed that an increase in intracellular levels of Ca²⁺ is a trigger of echinochrome release. Incubating Gram-positive/negative bacteria as well as yeast with lysates of red spherule cells led to significant reductions in colony-forming units. Such antimicrobial properties were counteracted by the addition of ferric iron (Fe³⁺), suggesting that echinochrome acts as a primitive iron chelator in echinoid biological defences.

Assessing the impact of diclofenac, ibuprofen and sildenafil citrate (Viagra®) on the fertilisation biology of broadcast spawning marine invertebrates

Exposure to synthetic chemicals is a key environmental challenge faced by aquatic organisms. The time and dose effects of the pharmaceuticals diclofenac, ibuprofen, and sildenafil citrate on sperm motility and successful fertilisation are studied using the echinoderms, Asterias rubens and Psammechinus miliaris, and the polychaete worm Arenicola marina, all important components of the marine benthos. Motility was reduced for all species when exposed to diclofenac concentrations ≥0.1 μg/L. Exposure to ≥1.0 μg/L of ibuprofen affected only P. miliaris gametes and fertilisation success of A. marina. A. rubens and P. miliaris sperm increased in both percentage motility and swimming velocity when exposed to sildenafil citrate at concentrations ≥18 and ≥ 50 ng/L, respectively. Pre-incubation of sperm with sildenafil citrate significantly increased fertilisation success in A. rubens and P. miliaris but not in A. marina. Pre-incubated A. rubens oocytes fertilised successfully in ibuprofen. According to EU Directive 93/67/EEC, diclofenac is classified as a very toxic substance to gametes of A. rubens, P. miliaris, and A. marina (EC50 = 100–1000 μg/L) while ibuprofen is classified as very toxic to gametes of P. miliaris but non-toxic to gametes of A. marina (EC50 > 10,000 μg/L). The present study indicates that diclofenac exposure may have negative impacts on invertebrate reproductive success, whereas ibuprofen potentially may compromise P. miliaris reproduction. This study provides a valuable insight into the mechanisms that allow marine invertebrates to survive and reproduce in contaminated and changing habitats.

Tissue and spine regeneration in the temperate sea urchin Psammechinus miliaris

The tissue regenerative capabilities of echinoderms are well known at the morphogenic level, yet significant knowledge gaps remain concerning the molecular control of these processes. This pilot study assayed two pharmacological agents (vincristine sulphate and DAPT) injected directly into the body cavity of specimens of the temperate echinoid Psammechinus miliaris which had had their spines and tube feet deliberately amputated. Vincristine sulphate, which is a generalised mitotic inhibitor, was used as a positive control, whereas DAPT is a y-secretase inhibitor known to block sea urchin embryo development and suppress echinoid regeneration by interfering with Notch signalling pathways. Significant differences in regeneration rate became apparent in both treatments 29 days post amputation with both inhibitors slowing regeneration of tube feet (0.6 μg/g vincristine sulphate by 44.4% relative to controls; 4 μg/g DAPT by 55.6% relative to controls) and spines (0.6 μg/g vincristine sulphate by 53.3% relative to controls; 4 μg/g DAPT by 66.7% relative to controls). Vincristine sulphate was more clearly dose-dependent than DAPT. This initial inhibition-based approach allows inferences to be made concerning possible molecular pathways controlling regeneration within P. miliaris and adds further support to the hypothesis that Notch signalling plays a major role in regulating regeneration in echinoids.

Enemy release an unlikely explanation for the invasive potential of the brown alga Sargassum muticum: experimental results, literature review and meta-analysis

The enemy release hypothesis predicts that non-indigenous species (NIS) are released from natural enemies (e.g. grazers etc.) in their new environment, thus facilitating invasion. Sargassum muticum is a conspicuous and successful invasive brown alga and several studies have investigated whether native herbivores feed less on S. muticum relative to native algae within the invaded range. Some of these studies have concluded that S. muticum is avoided by native herbivores, but this conclusion may be premature as the results are rather inconclusive and seem to depend partly on the type of algae included in the comparison. We conducted therefore a series of feeding experiments to test if Danish S. muticum is grazed less upon than a range of native algae by the sea urchin Psammechinus miliaris and complemented the experiments with a meta-analysis based on published data. In no-choice trials, P. miliaris consumed S. muticum faster than it consumed slow-growing Fucus vesiculosus and Halidrys siliquosa, whereas Saccharina latissima, Ceramium virgatum and Ulva intestinalis were grazed upon at higher or the same rates as S. muticum. More or less identical patterns were seen in the two- and multiple-choice experiments, where S. muticum was generally consumed faster than F. vesiculosus and H. siliquosa, but slower than S. latissima and the two most fast-growing algal species (U. intestinalis and C. virgatum). We screened the literature for comparable data and found 26 experiments with 27 species of algae and 14 species of invertebrate grazers. Meta-analysis on these data showed the same overall trend as observed in our experiments; S. muticum is generally consumed at the same rate or faster than other thick, leathery and canopy-forming algae, which are assumed to constitute the major competitors to S. muticum, but slower than more fast-growing sub-canopy species and more opportunistic algae. We question therefore that enemy release can explain the invasion success of S. muticum.

Sound production and associated behaviours of benthic invertebrates from a coastal habitat in the north-east Atlantic

Maerl beds are among the most endangered habitats in coastal temperate waters and a priority for conservation. Passive acoustics is a potential non-intrusive approach for surveying this fragile ecosystem with minor disturbances. Invertebrate sounds can be major contributors to natural coastal soundscapes but are not well studied. We conducted controlled tank-based experiments to identify sound-producing invertebrates inhabiting north-east Atlantic maerl beds and to characterise their sounds in terms of frequency features and source levels. We also determined which sound types are able to be detected above natural maerl ambient noise and suitable for in situ monitoring. Tank recording sessions of 20 abundant and potentially soniferous invertebrates from maerl beds revealed eight soniferous species and 15 different sound types. Two new sound-producing families were identified, Calyptraeidae and Majidae. Six sound types had properties consistent with detectability and identification for in situ acoustic studies: the feeding sound of sea urchins Echinus esculentus, Paracentrotus lividus and Psammechinus miliaris, snapping sound of the snapping shrimp Athanas nitescens, and feeding and other sounds of the spider crab Maja brachydactyla. Estimated detection distances ranged from a few metres for sea urchin feeding sounds up to about 40 m for A. nitescens snaps and spider crab feeding sounds. These invertebrates, particularly A. nitescens, probably make a substantial contribution to the maerl ambient noise. This invertebrate sound library sets a basis for in situ acoustic studies.

Unexpected conversion of echinochrome to brominated spinochrome D. Synthesis of 2-amino-3,6,7-trihydroxynaphthazarin produced by the sea urchins Strongylocentrotus nudus and Psammechinus miliaris

Two approaches to the total synthesis of 2-amino-3,6,7-trihydroxynaphthazarin, a metabolite of the sea urchins Strongylocentrotus nudus and Psammechinus miliaris are described. The first was based on the conversion of 2,3-dichloro-6,7-dimethoxynaphthazarin into the corresponding 3-hydroxy-2-nitro derivative and subsequent reduction to give 2-amino-3-hydroxy-6,7-dimethoxynaphthazarin. The basis for the second method was the conversion of echinochrome into brominated spinochrome D and the introduction of an amino group via direct substitution of the bromine atom by an azido group.

Extraction and identification of antioxidant polyhydroxynaphthoquinone pigments from the sea urchin, Psammechinus miliaris

Pigments were extracted from tests of individual sea urchins, Psammechinus miliaris, collected from a sea loch in Scotland. The extracts were red brown-to-orange in colour and yielded UV spectra with maxima at 475, 320 and 270 nm characteristic of polyhydroxylated naphthoquinone pigments (PHNQs) identified in other sea urchins. The yield of total phenols from the tests was relatively low (1–2 mg/g dry weight) but in line with levels found in tests from other sea urchin species. The extracts displayed reasonable antioxidant capacity assayed using the FRAP assay.After selection by solid phase extraction, the extracts were analysed by liquid chromatography-mass spectrometry (LC–MS). The LC–MS data suggested that the pigmentation was due to a range of spinochrome and echinochrome PHNQ components. Most of these components have been noted previously but there was evidence for sulphated forms which have not been identified before. The role and potential utility of these pigments in food applications is discussed.

Experimental influence of pH on the early life-stages of sea urchins II: increasing parental exposure times gives rise to different responses

Many studies into the responses of early life-stages to ocean acidification utilise offspring obtained from parents reared under present-day conditions. Their offspring are directly introduced to altered-pH conditions. This study determined whether this approach is suitable by pre-exposing parent sea urchins (Psammechinus miliaris) to altered seawater pH (~1000 μatm) for several durations, spawning them and rearing their offspring to settlement. Parents acclimated when exposed to low seawater pH for extended periods (>42 d). Longer adult pre-exposures reduced larval survival and less competent offspring were removed from populations earlier than in controls. Control offspring were larger during earlier development stages (2–7 d), but smaller during later development stages (14 + d) than offspring reared under low pH conditions. Juvenile settlement levels were similar across all treatments. After 17 d, offspring sourced from parents pre-exposed to low pH for 42 and 70 d were larger than those pre-exposed for 28 d and ambient sourced offspring directly transferred to low pH. These different responses show that the use of ambient derived offspring utilised in many studies is likely not an ideal approach when assessing larval development responses via morphometric measurements and survivorship prior to settlement. This study also suggests that calcifying organisms have capacities to acclimate and possibly adapt towards conditions beyond natural rates of ocean acidification.

Experimental influence of pH on the early life-stages of sea urchins I: different rates of introduction give rise to different responses

Many early life-stage response studies to ocean acidification utilize gametes/offspring obtained from ambient-sourced parents, which are then directly introduced to experimentally altered seawater pH. This approach may produce a stress response potentially impacting development and survival. Hence, this study determined whether this approach is suitable by subjecting embryos/larvae to different rates of introduction to lowered seawater pH to assess larval success under acute and staggered experimental pH scenarios. Embryos and 4-armed larvae of the sea urchin Psammechinus miliaris were introduced to pH conditions, widely used in ocean acidification studies, from ambient conditions utilizing 380, 470, 560, 700 and 840 ppm CO2 changed at incremental steps at two rates: fast (every 3rd hour) or slow (every 48th hour). Direct transfers from ambient to low seawater pH gave rise to dramatic negative impacts (smaller size and low survival), but slower rates of introductions gave rise to lesser negative responses (low survival). There was no treatment effect on settled juveniles. Fast introductions utilized in many studies are likely not ideal approaches when assessing pre-settlement larval developmental responses. Therefore, careful consideration of the pattern of response is needed when studies report the responses of offspring, derived from ambient conditions, introduced directly to forecasted ocean acidification conditions.