Sea Urchin Psammechinus Miliaris Research Articles

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.

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.

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.

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.

Early life developmental effects of marine persistent organic pollutants on the sea urchin Psammechinus miliaris

A new 16-day echinoid early life stage (ELS) bioassay was developed to allow for prolonged observation of possible adverse effects during embryogenesis and larval development of the sea urchin Psammechinus miliaris. Subsequently, the newly developed bioassay was applied to study the effects of key marine persistent organic pollutants (POPs). Mortality, morphological abnormalities and larval development stages were quantified at specific time points during the 16-day experimental period. In contrast to amphibians and fish, P. miliaris early life development was not sensitive to dioxin-like toxicity in the prolonged early life stage test. Triclosan (TCS) levels higher than 500 nM were acutely toxic during embryo development. Morphological abnormalities were induced at concentrations higher than 50 nM hexabromocyclododecane (HBCD) and 1000 nM tetrabromobisphenol A (TBBPA). Larval development was delayed above 25 nM HBCD and 500 nM TBBPA. Heptadecafluorooctane sulfonic acid (PFOS) exposure slightly accelerated larval development at 9 days post-fertilization (dpf). However, the accelerated development was no longer observed at the end of the test period (16 dpf). The newly developed 16-day echinoid ELS bioassay proved to be sensitive to toxic effects of POPs that can be monitored for individual echinoid larvae. The most sensitive and dose related endpoint was the number of developmental penalty points. By manipulation of the housing conditions, the reproductive season could be extended from 3 to 9 months per year and the ELS experiments could be performed in artificial sea water as well.

The effect of artificial diets on gonad colour and biomass in the edible sea urchin Psammechinus miliaris

During two 12 week trials, groups of edible sea urchins, Psammechinus miliaris, were fed artificial diets containing carotenoid pigments. The aim was to improve both the biomass and the colour of the sea urchin gonad in terms of its acceptability as a human food-stuff in the European market place. The pigmented artificial diets, based on the formula used by Robinson et al. (2002), increased gonad index (GI), pigment deposition and improved gonad colour from that of the initial samples. In Trial I gonad β-carotene levels increased > 2 fold, echinenone and total carotenoid > 7 fold. Trial II showed greater increases. Diets containing high levels of β-carotene (500 mg per kg dry weight of diet) gave rise to the highest percentages of marketable gonad colours (61–73%), and GI of 17.87–19.81%, (Trial I and II respectively). There was some variation in the results for this particular diet treatment across the two trials presumably reflecting individual urchins varying capacity to ingest, deposit or express the carotenoids in their diet. Providing additional lipids in the diets gave no improvement to gonad colour (56% acceptable) or GI (15.95%) suggesting the lipid content of the basic formulation is adequate. Utilizing an esterified form of lutein and zeaxanthin as a pigment source gave no significant improvement in gonad colour (30–63% acceptable) suggesting that this form of xanthophyll cannot be assimilated by P. miliaris. Female urchins had acceptable gonad colouration more often than males. The dominant carotenoid in the successful diets was β-carotene and this was successfully metabolized into echinenone, the dominant carotenoid in all gonad samples. Total levels of echinenone positively correlated with acceptable gonad colour scores. This study demonstrates that 12 weeks is sufficient to effect the desired change in gonad biomass and colour in cultivated P. miliaris.

Colonization of an artificial reef in south-west England—ex-HMS ‘Scylla’

An ex-Royal Navy frigate, HMS ‘Scylla’, was placed on the seabed in Whitsand Bay, south Cornwall on 27 March 2004. After five years, the reef supported a mature steel wreck community. The colonization of the reef showed wide fluctuations in species abundance in the first two years but, by 2006, most species that dominated or characterized the reef after five years had settled. Significant colonization events included settlement of barnacles, tubeworms and hydroids within a month and remarkably high settlements of the sea urchin Psammechinus miliaris and the queen scallop Aequipecten opercularis in the first year together with starfish Asterias rubens, solitary sea squirts and ephemeral algae. The plumose anemone Metridium senile, a characteristic species of wrecks, arrived in late summer 2004 but the widely distributed dead man's fingers Alcyonium digitatum was not observed until spring 2005. Wrasse were slow to colonize the reef but were established in small numbers by the end of 2007. Sea fans, Eunicella verrucosa, were first observed in August 2007. The species count for the reef stood at 263 taxa by the end of March 2009. The inside of the reef remained poorly colonized even after five years. Areas coated with tributyltin (TBT) antifouling paint only had colonization where the paint had flaked-off or on non-toxic paint markings, but with some indication that colonization may be occurring by a very few species especially near to non-TBT areas. Many species characteristic of natural reefs had not settled and neither do they occur on older wrecks including branching axinellid sponges, some cushion sponges and the yellow cluster anemone Parazoanthus axinellae. The artificial reef developed a community that was distinctly different to nearby natural rock reefs and such artificial structures should not be considered as a replacement for damaged or destroyed natural habitats.

Measuring Ca 2+-signalling at fertilization in the sea urchin Psammechinus miliaris: Alterations of this Ca 2+-signal by copper and 2,4,6-tribromophenol

During fertilization, eggs undergo a temporary rise in the intracellular concentration of free Ca 2+ ions. Using the membrane permeable acetoxymethylester of the fluorescent calcium indicator dye Fura-2, Fura-2 AM, the Ca 2+-signal at fertilization was not detectable in eggs of the sea urchin Psammechinus miliaris. However, after treatment of the eggs with Fura-2 AM in combination with MK571, an inhibitor for multidrug resistance associated proteins, clear Ca 2+-signals at fertilization could be measured without microinjection of the dye. We used this methodology to detect possible alterations of Ca 2+-signalling at fertilization by exposure of eggs to environmental pollutants. For this purpose, the heavy metal copper, the bromophenol 2,4,6-tribromophenol, the organic compound bisphenol A and the polycyclic aromatic hydrocarbon phenanthrene were tested for their potential to inhibit fertilization success of P. miliaris. Copper and 2,4,6-tribromophenol showed a dose-dependent effect on fertilization rates of P. miliaris and significantly inhibited fertilization at 6.3 µM Cu 2+ and 1 µM 2,4,6-tribromphenol. Bisphenol A significantly inhibited fertilization success at 438 µM while phenanthrene had no effect up to 56 µM. 6.3 µM copper and 100 µM 2,4,6-tribromophenol significantly increased the Ca 2+-signal at fertilization. This alteration may contribute to the reduced fertilization rates of P. miliaris after exposure to copper and 2,4,6-tribromophenol.

Gonadal lesions of female sea urchin ( Psammechinus miliaris) after exposure to the polycyclic aromatic hydrocarbon phenanthrene

Histopathological alterations in the reproductive tissue of the female sea urchin Psammechinus miliaris after exposure to the polycyclic aromatic hydrocarbon (PAH) phenanthrene were investigated using light microscopy. Sexually mature sea urchins were exposed to 500 μg L −1 phenanthrene for 20 days. A severe disorganisation of the acinal structure of the gonads was observed in phenanthrene-treated animals, mainly as a result of aggregates of degenerating previtellogenic oocytes. Growth and maturation of previtellogenic oocytes were inhibited, whereas there were no obvious effects on vitellogenic oocytes and mature ova. The acinal wall showed signs of fibrosis as well as dilation of the two epithelial layers. Phenanthrene severely affected the reproductive function of the sea urchins since a proper oogenesis of the ovarian tissue is very unlikely. Generally, the observed alterations are similar to histopathological changes in reproductive tissue in other invertebrates after exposure to PAHs and oil.

Carotenoids in the gonad and gut of the edible sea urchin Psammechinus miliaris

The carotenoid content and composition of the gonad and gut of the edible sea urchin Psammechinus miliaris were determined. The dominant carotenoid of the gut wall was fucoxanthinol, indicating the degradation of the main carotenoid found in the Phaeophycean algae. Also present in the gut wall was the carotenoid echinenone, a pigment absent from the diet of wild urchins. Its presence in the gut wall indicates that this is a site for metabolism of dietary, algal, β-carotene. Fucoxanthinol and related products are not found in the gonad and instead this organ was found to selectively accumulate echinenone and β-carotene together with a small number of related compounds. Both echinenone and β-carotene were found in two geometric forms, namely all- trans and 9′- cis or 9- cis respectively. Echinenone is predominantly deposited in the gonad as the 9′- cis form, whilst β-carotene is mainly found in the same form as that seen in the algae that, in part, make up its diet in the wild, i.e., all- trans. This indicates that isomerisation of dietary β-carotene occurs as part of the process of accumulating echinenone in the gonad. The colour and overall appearance of the gonads of this wild and food limited population were generally poor. No correlation between CIE L ⁎ a ⁎ b chromameter readings or a visual assessment of gonad colour with carotenoid content as determined by HPLC was observed.

Influence of dietary protein on essential fatty acids in the gonadal tissue of the sea urchins Psammechinus miliaris and Paracentrotus lividus (Echinodermata)

The elevated accumulation of certain essential long-chain polyunsaturated fatty acids (PUFA) during reproductive maturation can place a significant demand on lipid metabolism. As lipid metabolism is closely linked with protein metabolism, protein intake during maturation may significantly influence transport proteins and enzyme activity which is responsible for supplying the essential fatty acids for gamete development. The present study investigated the influence of increased protein intake on the accumulation of certain long-chain PUFA in two species of commercially valuable sea urchin, Paracentrotus lividus and Psammechinus miliaris during maturation. Sea urchins were fed three macroalgal diets over a 90-day period. These diets included the red alga Palmaria palmata, which had been grown in seawater enriched with two levels of ammonia-N, which produced a high protein content (41%) or low protein content (32%). The third diet (or reference diet) used the brown macroalga Laminaria saccharina with a 23% protein content. The fatty acid composition of the gonads was determined by GC. Feeding the high protein diet had no effect on the maturation and total lipid in the gonad compared with the low protein diet, however, it did significantly increase the levels of docosahexaenoic acid DHA 22:6n-3 in the mature gonad tissue in P. miliaris and P. lividus. In contrast, only P. miliaris exhibited higher levels of arachidonic acid ARA 20:4n-6 when fed the high protein diet. The present study suggests that elevated protein levels can promote the accumulation of DHA and ARA in gonadal tissue during maturation, although the extent of the influence is species-specific and highlights the importance of understanding the complexities and inter-connectivity between protein and lipid metabolism when producing commercially valuable species.

Effect of variation in the protein value of the red macroalga Palmaria palmata on the feeding, growth and gonad composition of the sea urchins Psammechinus miliaris and Paracentrotus lividus (Echinodermata)

The sea urchins Psammechinus miliaris and Paracentrotus lividus were fed three macroalgal diets with varying protein content over a 90-day period. These diets were the red alga Palmaria palmata, which had been grown in seawater enriched with two levels of ammonia-N and contained a protein content of either 41% (High-N) or 32% (Low-N) and the brown alga Laminaria saccharina (23% protein). A significantly different response to the macroalgal diets by the two sea urchin species was found in terms of food consumption rate, food conversion efficiency, gonadal growth and gonad nutritional composition. P. miliaris was able to efficiently utilise the High-N P. palmata, whereas P. lividus exhibited a higher food conversion efficiency (FCE) and specific growth rate (SGR) when fed the Low-N compared with the High-N P. palmata. P. miliaris had a significantly higher food consumption rate and higher gonad index (GI) compared with P. lividus, irrespective of diet type. The lowest FCE, SGR and GI were shown by both species fed L. saccharina. No interspecific difference in gonadal nitrogen content was observed, although a positive relationship was shown between dietary and gonadal nitrogen content for both species. All three macroalgal diets produced bright orange or yellow orange gonad colour in P. lividus. High-N and Low-N P. palmata improved gonad colouration in P. miliaris compared with L. saccharina. The results suggest that P. miliaris can efficiently assimilate high protein plant-based diet, whereas P. lividus is less capable of benefiting from high dietary protein levels and this should be taken into consideration when culturing this species either in land-based or sea-based polyculture systems.

How prey size, type and abundance affects foraging decisions by the regular echinoid Psammechinus miliaris

The influence of size and density of the blue mussel, Mytilus edulis and the common barnacle, Semibalanus balanoides on the foraging behaviour of the sea urchin Psammechinus miliaris was examined in laboratory studies. Urchins of two size-classes offered six size-categories of barnacles exhibited a constant preference for the smallest category ([les ]7.5 mm plate length). Ingestion rates were independent of the size–frequency distribution of mussels presented to large urchins of 38–46 mm test diameter, which showed a preference for small–medium sizes (>13.4–17.8 mm shell length). Large urchins ingested a maximum of six mussels a day and increased their consumption in a density dependent manner until reaching a plateau once more than ten mussels were offered. In most cases, the rate of successful attacks decreased with mussel size.Pairwise comparison experiments to determine the ingestion rate of fronds of the algae Laminaria saccharina showed that P. miliaris had no preference for old fronds with or without epifauna, but young fronds of the algae were frequently rejected. These results highlight the omnivorous nature and semi-selective behaviour of this urchin and demonstrate the potential for it to have an important role in structuring algal and epifaunal communities.

Biomass dynamics of exotic Sargassum muticum and native Halidrys siliquosa in Limfjorden, Denmark—Implications of species replacements on turnover rates

The expansion of Sargassum muticum in the Danish estuary Limfjorden between 1984 and 1997 was followed by a decrease in abundance of native perennial macroalgae such as Halidrys siliquosa. Although commonly associated with the expansion of exotic species, it is unknown whether such structural changes affect ecosystem properties such as the production and turnover of organic matter and associated nutrients. We hypothesized that S. muticum possesses ‘ephemeral’ traits relative to the species it has replaced, potentially leading to faster and more variable turnover of organic matter. The biomass dynamics of S. muticum and H. siliquosa was therefore compared in order to assess the potential effects of the expansion of Sargassum. The biomass of Sargassum was highly variable among seasons while that of Halidrys remained almost constant over the year. Sargassum grew faster than Halidrys and other perennial algae and the annual productivity was therefore high (P/B = 12 year −1) and exceeded that of Halidrys (P/B = 5 year −1) and most probably also that of other perennial algae in the system. The major grazer on macroalgae in Limfjorden, the sea urchin Psammechinus miliaris, preferred Sargassum to Halidrys, but estimated losses due to grazing were negligible for both species and most of the production may therefore enter the detritus pool. Detritus from Sargassum decomposed faster and more completely than detritus from Halidrys and other slow-growing perennial macrophytes. High productivity and fast decomposition suggest that the increasing dominance of S. muticum have increased turnover of organic matter and associated nutrients in Limfjorden and we suggest that the ecological effects of the invasion to some extent resemble those imposed by increasing dominance of ephemeral algae following eutrophication.

Nursery-area function of maerl grounds for juvenile queen scallops Aequipecten opercularis and other invertebrates

The services provided by coastal ecosystems such as mangrove forests and sea-grass beds are becoming increasingly recognised, yet the functional role of maerl beds has not been ad- dressed. Maerl forms highly biodiverse habitats composed of loose-lying coralline red algae which build up over thousands of years. These carbonate-rich deposits occur in photic areas with strong water movement; they have a widespread global distribution yet remain one of the most overlooked shallow-water marine habitats, with little known about the ecosystem services maerl may provide. Our diving research in Scotland has shown that pristine live maerl (PLM) grounds fulfil nursery area prerequisites for commercial populations of queen scallops Aequipecten opercularis and other inver- tebrates, such as the soft clam Mya arenaria, the sea urchins Psammechinus miliaris and Echinus escu- lentus, and the starfish Asterias rubens, more effectively than impacted dead maerl and other common substrata. The complex architecture of maerl beds attracts high densities of these juvenile inverte- brates, which use PLM grounds as nursery areas in preference to adjacent substrata. Considering its global distribution, it is highly likely that ecosystem services provided by maerl are considerable. Maerl is easily damaged and killed by a variety of human activities, yet its protection would maintain vital nursery area function, benefiting commercial fishery yields and, pivotally, regional biodiversity.

Echinoderms as bioindicators, bioassays, and impact assessment tools of sediment-associated metals and PCBs in the North Sea.

The study assessed the occurrence, possible toxicity, and impact of sediment-associated metals and PCBs in the coastal zone of the southern North Sea using echinoderms as representatives of the macrobenthos. Metals and PCBs were analyzed in the sediments and in the body compartments of the starfish Asterias rubens from 11 stations. The general toxicity of sediment-associated contaminants was assessed by bioassays using embryonic and larval developments of both A. rubens and the sea urchin Psammechinus miliaris. The impact of contamination was assessed by measuring cellular immune responses of A. rubens collected in the same stations. Contamination of the starfish by metals and PCBs closely reflected that of the sediments. However, bioaccumulation was element-specific for metals and depended on the chlorination pattern for PCBs. The sediment-associated contaminants appeared to be toxic in both the A. rubens and P. miliaris developmental assays. Moreover, metals were shown to affect the immune responses of starfishes living in contaminated stations. The most significant effects on biological responses were recorded in the plumes of the Scheldt/Rhine/North Sea Canal and the Elbe/Weser Rivers.

HPLC analysis of algal pigments to define diet of sea urchins

A method for a qualitative analysis of sea urchin diet is based on the characterization of the photosynthetic pigment indices of the major algal groups in the sea urchin gut. The pigments were separated by reverse-phase high performance liquid chromatography (RP-HPLC). This study demonstrated that HPLC is a better method to estimate chlorophyll-a in the gut contents than the conventional spectrophotometric methods which overestimate the amounts by including chlorophyll-a breakdown products. Three sea urchins species, Paracentrotus lividus, Psammechinus miliaris and Sphaerechinus granularis, settled on the loose-lying coralline algae (maerl) in the Bay of Brest (France), were used in this study. The algal pigments identified within the gut contents included chlorophylls-a, -b, -c, fucoxanthin, lutein, βε-carotene and ββ-carotene. The presence of chlorophylls and carotenoid biomarkers was used to characterize the three algal groups: Rhodophyceae, Chlorophyceae, Phaeophyceae in estimating sea urchin diet. The pigment analysis reported here demonstrated that the three species of sea urchins investigated mainly consumed Rhodophyceae which dominate the epibenthic flora in the study area.

A toxicity identification evaluation of silty marine harbor sediments to characterize persistent and non-persistent constituents

Sediment toxicity in silty marine harbor sediments is frequently dominated by ammonia or sulfide, leaving the adverse effects of persistent toxic substances unnoticed. To investigate the latter, we subjected interstitial water from three contaminated silty sediments to toxicity identification evaluation (TIE) phase I manipulations and tested for toxicity with four bioassays: the amphipod Corophium volutator (survival as an endpoint), the sea urchin Psammechinus miliaris (fertilization, embryo development) and the bacterium Vibrio fischeri (bioluminescence inhibition). The graduated pH manipulations identified the prominent toxicity of ammonia in the amphipod and sea urchin embryo tests, and also sulfide toxicity in the bacterium test. In two of the three samples tested with the amphipods, sea urchin embryos and bacteria, a small but significant reduction in interstitial water toxicity was achieved by removing persistent compounds through C 18 solid phase extraction. EDTA chelation resulted in a slight detoxification of the interstitial water for the amphipods and sea urchin embryos, but this was not related to any measured trace metals. Despite the presence of toxic levels of ammonia and sulfide in the harbor sediments, we established the adverse biological effects of persistent constituents by means of the TIE manipulations and in vivo interstitial water bioassays.

The dawn of gene isolation

Ever since it became clear through the work of Watson and Crick that the gene is a stretch of double stranded helical DNA and is understandable in chemical terms, biochemists have striven to get their hands on isolated genes. The isolation of the ribosomal genes of Xenopus laevis in 1966 provided a first instance where a purified DNA of known function could be investigated, long before the advent of gene cloning technologies. The second instance was the purification of the Lac operon from Escherichia coli. Later, but still before the gene cloning days the 5S RNA genes of X. laevis and the histone genes of the sea urchin Psammechinus miliaris were isolated by physico-chemical methods, but their isolation marked the end of an era. By 1975, gene cloning technology was well established and the isolation of genes quickly became an everyday occurrence.

Development of serotonin-like and SALMFamide-like immunoreactivity in the nervous system of the sea urchin Psammechinus miliaris.

The present immunocytochemical study utilizes serotonin and SALMFamide antisera, together with confocal laser scanning microscopy, to provide new information about the development of the nervous system in the sea urchin Psammechinus miliaris (Echinodermata: Echinoidea). Special attention is paid to the extent of the nervous system in later larval stages (6-armed pluteus to metamorphic competency), a characteristic that has not been well described in this and other species of sea urchin. An extensive apical ganglion appears by the 6-armed pluteus stage, forming a complex of 10-20 cells and fibers, including discrete populations of both serotonin-like and SALMF-amide-like immunoreactive cells. At metamorphosis this complex is large, comprising at least 40 cells in distinct arrays. Serotonin-like immunoreactivity is also particularly apparent in the lower lip ganglion of 6- to 8-armed plutei; this ganglion consists of 15-18 cells that are distributed around the mouth. The ciliary nerves that lie beneath the ciliary bands in the larval arms, the esophagus, and a hitherto undescribed network associated with the pylorus all show SALMFamide-like immunoreactivity. The network of cells and fibers in the pyloric area develops later in larval life. It first appears as one cell body and fiber, then increases in size and complexity through the 8-armed pluteus stage to form a complex of cells that encircles the pylorus. SALMFamide-like, but not serotonin-like, immunoreactivity is seen in the vestibule wall, tube feet, and developing radial nerve fibers of the sea urchin adult rudiment as the larva gains metamorphic competency.