Sea Urchin Larvae Research Articles

Effects of fine grain environmental variability on morphological plasticity

AbstractVirtually all studies investigating morphological plasticity have focused on how organisms change in response to treatments that are constant throughout the experiment but which have different means. In this study, we investigated the possibility that organisms can morphologically respond to other environmental parameters like the amount of environmental variability or environmental maximum. Sea urchin larvae adjust the length of their feeding structure, a band of cilia, in response to different mean food concentrations. We investigated whether sea urchin larvae are also capable of responding to environmental variability or maxima by rearing larvae on four fluctuating diets, where all treatments had the same mean concentration of food. Larvae reared on a low variable diet produced longer larval arms (i.e. a longer ciliary band) than larvae reared on more variable diets. This response is similar to the morphological change that occurs when the mean food concentration is reduced (small mean = long arms), and indicates that organisms can morphologically respond to environmental parameters other than the environmental mean – such as the amount of environmental variability or the environmental maxima. We also quantified the shape of the relationship between larval arm length and fixed food concentrations to determine whether our results might be explained by nonlinearity in this relationship (i.e. Jensen's inequality). The shape of the relationship was inconsistent with a Jensen's inequality explanation. In addition, sea urchin larvae were unable to track fluctuations in food concentrations. This inability to track our imposed environmental fluctuations indicates that there was a time delay greater than 2 days in the response of larvae to changes in food concentrations. Since plutei likely experience fluctuations in food concentrations at least once a day, it is possible that larvae cannot track natural fluctuations in food concentration. We discuss the importance of our results in light of adaptive interpretations of plasticity and predictions of morphological response.

The localization of occluded matrix proteins in calcareous spicules of sea urchin larvae

The sea urchin embryo forms calcareous endoskeletal spicules composed of calcite and an occluded protein matrix. Though the latter is approximately 0.1% of of the mass, the composite has substantially altered material properties, e.g., conchoidal fracture planes and increased hardness. Experiments were conducted to examine the localization of matrix proteins occluded in the mineral by use of immunocytochemistry coupled with scanning electron microscopy (SEM). The isolated, unfixed spicules were etched under relatively gentle conditions and exposed to affinity purified antibodies made against two different matrix proteins, as well as an antibody to the entire constellation of matrix proteins. Immunogold tagged secondary antibody was used to observe antibody localization in the back scatter mode of SEM. All proteins examined were very widely distributed throughout the calcite, supporting a model of the structure in which a multiprotein assemblage is woven with fine texture around microcrystalline domains of calcite. Gentle etching revealed a laminar arrangement of calcite solubility, consistent with a stepwise deposition of matrix and mineral to increase girth of the spicule.

The 5-HT receptor cell is a new member of secondary mesenchyme cell descendants and forms a major blastocoelar network in sea urchin larvae

A gene encoding the serotonin (5-hydroxytryptamine, 5-HT) receptor ( 5-HT-hpr) was identified from the sea urchin, Hemicentrotus pulcherrimus. Partial amino acid sequence deduced from the cDNA showed strong similarity to Aplysia californica 5-HT2 receptor. Immunoblotting analysis of this 5-HT-hpr protein (5-HT-hpr) with an antibody raised against a deduced peptide showed two bands. Their relative molecular masses were 69 and 53 kDa, respectively. The larger band alone disappeared after N-glycopeptidase F digestion, indicating the protein was N-glycosylated. Immunolocalization analysis showed that cells expressing the 5-HT-hpr (SRC) first appeared near the tip of the archenteron in 33-h post-fertilization (33 hpf) prism larvae. Their cell number doubled in 2 h, and 5-HT-hpr protein expression increased further without cell proliferation. SRC spread ventrally on the basal surface of the oral ectoderm in 36 hpf prism larvae, and then clockwise on the ventral ectoderm to the posterior region to complete formation of the SRC network in 48 hpf early plutei. The SRC network was comprised of 7 main tracts: 4 spicule system-associated tracts and 3 spicule system-independent tracts. The network extended short fibers to the larval body surface through the ectoderm, implicating a signal transmission system that receives exogenous signal. Double-stain immunohistochemistry with antibodies to primary mesenchyme cells showed that SRC were not stained by the antibody. In embryos deprived of secondary mesenchyme cell (SMC) by microsurgery, the number of SRC decreased considerably. These two data indicate that SRC are SMC descendants, adding a new member to the SMC lineage.

Same Difference: Sea Urchins or Starfish

DEVELOPMENT Evolutionary forces are known to work on the gene at many levels, from sequence to organization. In the sea urchin, there have been extensive efforts to map the network responsible for endoderm formation. Despite their differences—including the ability of sea urchin larvae to form a skeleton during embryogenesis—endoderm formation shows similarities between sea urchins and starfish. Hinman et al. studied the conservation and changes in embryonic gene regulatory networks during the course of metazoan evolution. They compared the expression patterns of genes involved in endoderm formation in embryo development and the effects of perturbing expression profiles. A crucial feedback loop involving three transcription factors was found to be almost identical in both of these evolutionarily distant species. — BJ Proc. Natl. Acad. Sci. U.S.A. 100, 13356 (2003).

Identification and developmental expression of new biomineralization proteins in the sea urchin Strongylocentrotus purpuratus.

The endoskeleton of the sea urchin larva is a network of calcareous rods secreted by primary mesenchyme cells (PMCs). In this study, we identified seven new biomineralization-related proteins through an analysis of a large database of gene products expressed by PMCs. The proteins include three new spicule matrix proteins (SpSM29, SpSM32, and SpC-lectin), two proteins related to the PMC-specific cell surface glycoprotein MSP130 (MSP130-related-1 and -2), and two novel proteins (SpP16 and SpP19). The genes encoding these proteins are expressed specifically by cells of the large micromere-PMC lineage and are activated zygotically beginning at the blastula stage, prior to PMC ingression. Several of the mRNAs show regulated patterns of expression within the PMC syncytium that correlate with the pattern of skeletal rod growth. This work identifies new proteins that may regulate the process of biomineralization in this tractable model system.

Physiological and induced apoptosis in sea urchin larvae undergoing metamorphosis.

Paracentrotus lividus embryos at the early pluteus stage undergo spontaneous apoptosis. Using a TUNEL (TdT-mediated dUTP Nick-End Labelling) assay on whole mount embryos, we showed that there was a different distribution of the apoptotic cells in different optical sections. Not more than 20% of cells in plutei were spontaneously apoptotic, as confirmed by the counts of dissociated ectoderm and intestine cells. Observation of larva stages closer to metamorphosis confirmed that apoptosis is a physiological event for the development of the adult. In particular, larvae at different developmental stages showed apoptotic cells in the oral and aboral arms, intestine, ciliary band and both apical and oral ganglia. Moreover, we found that the number of apoptotic cells decreased in later larva stages, possibly because in the organism approaching metamorphosis, a smaller number of cells needs to be eliminated. Furthermore, combined phorbol ester (TPA) and heat shock treatment enhanced apoptosis by increasing the number of cells involved in the phenomenon.

The effect of diet type and quantity on the development of common sea urchin larvae Echinus esculentus

Several species of sea urchins are now being cultivated for commercial purposes and with the continued increased demand for sea urchin gonads as a food product, new species are being assessed for their aquaculture and market potential. This study focussed on establishing protocols for the production of common sea urchin Echinus esculentus larvae and juveniles to assess its potential as an echinoculture species. Two trials were carried out, the first trial evaluated the influence of three microalgal diets (D= Dunaliella tertiolecta only, mixed D/P= D. tertiolecta plus Phaeodactylum tricornutum and P= P. tricornutum only) on larval morphology. Larval length, width, post-oral arm length and rudiment length were significantly effected by diet. Diets D and D/P prompted more rapid metamorphosis. In the second trial, the effects of different rations of D. tertiolecta were tested. The food ration, standard ration (SR; 1000, 3000, and 5000 cells ml −1 ) and high ration (HR; 3000, 9000, and 15,000 cells ml −1 ) were increased as the larvae acquired the 3rd and 4th pair of larval arms. Larvae fed the SR were significantly larger (longer and wider) and had significantly longer rudiments than those in the HR treatment. The number of larvae metamorphosing and settling onto substrates was significantly higher in treatment SR compared to HR. Optimising the larval diet shortened the larval stage from 21–23 days in the first trial to 16 days in the second trial. The maximum percentage of metamorphosing individuals which survived to post-larvae or juveniles (10 days after they were first judged competent to settle) was 46.6%, suggesting E. esculentus is a viable aquaculture candidate.

Effect of humic acids on speciation and toxicity of copper to Paracentrotuslividus larvae in seawater

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 (EC 50 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.

Induction of larval settlement and metamorphosis of the sea urchin Strongylocentrotus intermedius by glycoglycerolipids from the green alga Ulvella lens

In aquaculture centers of the northern region of Japan, "Nami-ita" (waved polycarbonate plates), on which the green alga Ulvella lens Crouan frat. (Chaetophoraceae: Chaetophorales) was cultured, are used to promote larval settlement and metamorphosis of the sea urchin species Strongylocentrotus intermedius (A. Agassiz) and S. nudus (A. Agassiz). We investigated chemical inducer(s) for larval settlement and metamorphosis of these sea urchins with extracts of U. lens. Bioassay-guided separation of the methanol extract using a combination of column and thin-layer chromatography led to the isolation of several active compounds, the chemical structures of which were determined by spectral and chemical methods. These active compounds were identified as glycoglycerolipids, all comprising several molecular species: sulfoquinovosyl monoacylglycerols (SQMGs), sulfoquinovosyl diacylglycerols (SQDGs), monogalactosyl monoacylglycerols (MGMGs), monogalactosyl diacylglycerols (MGDGs), digalactosyl monoacylglycerols (DGMGs) and digalactosyl diacylglycerols (DGDGs). Among these glycolipids, SQMGs, MGMGs, MGDGs and DGMGs induced larval metamorphosis of the sea urchin S. intermedius. SQMGs and MGDGs induced larval metamorphosis at a concentration of 5 µg ml–1, whereas SQDGs and DGDGs only induced larval settlement. These glycoglycerolipids are new congeners of chemical inducers to settlement and metamorphosis of planktonic larvae of sea urchins. The findings would provide a better understanding of larval settlement and metamorphosis in sea urchins.

Chemical defense and antifouling activity of three Mediterranean sponges of the genus Ircinia.

The defense roles and the antifouling activity of the organic extracts and the major metabolites of the sponges Ircinia oros, I. variabilis and I. spinosula were investigated. The antifeedant activity was tested in experimental aquaria on the generalist predator fish Thalassoma pavo as well as in coastal ecosystems rich in fishes. Some of the major metabolites exhibited high levels of antifeedant activity. The antifouling activity was tested in laboratory assays, against representatives of the major groups of fouling organisms (marine bacteria, marine fungi, diatoms, macroalgae and mussels). All extracts showed promising levels of activity. As was expected, no single extract was active in all tests and some fractions that were effective against one organism showed little or no activity against the others. The high but variable level of antifouling activity in combination with the absence of toxicity (tested on the development of oyster and sea urchin larvae) shows the potential of these metabolites to become ingredients in environmentally friendly antifouling preparations.

Role of syndecan in the elongation of postoral arms in sea urchin larvae.

Ac-SYN is the core protein of a cell surface proteoglycan of the sea urchin Anthocidaris crassispina. To examine the functions of Ac-SYN, embryos were cultured in the presence of affinity-purified antibody against Ac-SYN. At the late pluteus stage, severe inhibition of elongation of the postoral arms was seen in treated embryos compared with control embryos. Blastocoeleic microinjection of the antibody did not affect morphogenesis. The relationship between the number of cells in the postoral arms and the length of the postoral rods was investigated in normal embryos. This showed that postoral arm elongation has two phases: the first phase accompanies the increase in cell numbers while the second does not. The syndecan antibody inhibited the increase in cell numbers in the postoral arms. Furthermore, in the treated embryos, cell numbers continued to increase normally until 31 h post fertilization (hpf), while cell division stopped after 31 hpf. These results suggest that Ac-SYN participates in postoral arm formation via cell division in sea urchin embryos.

Experiments with small animals in BIOLAB and EMCS on the international space station

Two ESA facilities will be available for animal research and other biological experiments on the International Space Station: the European Modular Cultivation System (EMCS) in the US Lab “Destiny” and BIOLAB in the European “Columbus” Laboratory. Both facilities use standard Experiment Containers, mounted on two centrifuge rotors allowing either research in microgravity or acceleration studies with variable g-levels from 0.001 to 2.0x g. Standard interface plates provide each container with power and data lines, gas supply (controlled CO 2, O 2 concentration and relative humidity), and -for EMCS only- connectors to fresh and waste water reservoirs. The experiment hardware inside the containers will be developed by the user, but ESA conducted a feasibility study for several kinds of Experiment Support Equipment with potential use for research on small animals: design concepts for experiments with insects, with aquatic organisms like rotifers and nematodes, and with small aquatic animals (sea urchin larvae, tadpoles, fish youngsters) are described in detail in this presentation. Also ESA's initial steps to support experiments with rodents on the Space Station are presented.

Screening of Marine Algal Extracts for Anti-settlement Activities against Microalgae and Macroalgae

The ban on the use of TBT-based antifouling paints for boats under 25 m in length has lead to a search for new non-toxic antifoulants. One of the most promising alternative technologies to heavy metal based antifouling paint is the development of antifouling coatings whose active ingredients are naturally occurring compounds from marine organisms. This is based on the principle that marine organisms also face the problem of the presence of epibionts on their own surfaces. In this study, the antifouling activity of a series of aqueous, ethanolic and dichloromethane extracts from thirty algae from the North East Atlantic coast was investigated. The extracts were tested in laboratory assays against species representative of two major groups of fouling organisms, viz . macroalgae and microalgae. The activity of several extracts was comparable to that of heavy metals and biocides (such as TBTO and CuSO 4 ) currently used in antifouling paints and their lack of toxicity with respect to the larvae of oysters and sea urchins suggests a potential for novel active ingredients.

Left-right positioning of the adult rudiment in sea urchin larvae is directed by the right side

Indirect-developing sea urchins eventually form an adult rudiment on the left side through differential left-right development in the late larval stages. Components of the adult rudiment, such as the hydropore canal, the hydrocoel and the primary vestibule, all develop on the left side alone, and are the initial morphological traits that exhibit left-right differences. Although it has previously been shown that partial embryos dissected in cleavage stages correctly determine the normal left-right placement of the adult rudiment, the timing and the mechanism that determine left-right polarity during normal development remain unknown. In order to determine these, we have carried out a series of regional operations in two indirect-developing sea urchin species. We excised all or a part of tissue on the left or right side of the embryos during the early gastrula stage and the two-armed pluteus stage, and examined the left-right position of the adult rudiment, and of its components. Excisions of tissues on the left side of the embryos, regardless of stage, resulted in formation of a left adult rudiment, as in normal development. By contrast, excisions on the right side of the embryos resulted in three different types of impairment in the left-right placement of the adult rudiment in a stage-dependent manner. Generally, when the adult rudiment was definitively formed only on the right side of the larvae, no trace of basic development of the components of the adult rudiment was found on the left side, indicating that a right adult rudiment results from reversal of the initial left-right polarity but not from a later inhibitory effect on the development of an adult rudiment. Thus, we suggest that determination of the left-right placement of the adult rudiment depends on a process, which is directed by the right side, of polarity establishment during the gastrula and the prism stages; however, but commitment of the cell fate to initiate formation of the adult rudiment occurs later than the two-armed pluteus stage.

Three-dimensional mapping of cholinergic molecules by confocal laser scanning microscopy in sea urchin larvae

Confocal laser scanning microscopy (CLSM) was used to examine molecules related to the cholinergic neurotransmission system and detected at all the larval stages of Paracentrotus lividus, by histochemical and immunohistochemical methods. CLSM, providing spatial resolution of the cells located both at the larval surface and at depth, allows a complete mapping in a three-dimensional volumetric frame. At early larval stages acetylcholinesterase- as well as choline acetyltransferase-like molecules were found mainly in the gut wall cells, and along the ciliary bands of the arms, together with muscarinic acetylcholine receptors. At perimetamorphic stages, cholinergic molecules were present in the ciliate strands along the arms, in the larval body and in the rudiment. At metamorphosis, positivity to cholinergic molecules translocated to the juvenile, where a high frequency of mAChR- and ChAT-like positive cells was found.

Inhibition of marine bacteria by extracts of macroalgae: potential use for environmentally friendly antifouling paints

Although a total ban on the use of TBT coatings is not expected in the short term, there is a growing need for environmentally safe antifouling systems. A search for new non-toxic antifoulants has been carried out among marine macroalgae. Antifouling activity of aqueous, ethanolic and dichloromethane extracts from 30 marine algae from Brittany coast (France) was examined in vitro against 35 isolates of marine bacteria. About 20% of the extracts were found to be active. The high levels of inhibitory activities against bacteria recorded in some extracts and the absence of toxicity on the development of oyster and sea urchin larvae and to mouse fibroblast growth suggests a potential for novel active ingredients in antifouling preparations.

Spectacular Fluorescence Emission in Sea Urchin Larvae

Abstract Strong fluorescence emission occurred in sea urchin larvae when irradiated with blue light under a fluorescence microscope. The blue light irradiation first broke red granules in the pigment cells, releasing green fluorescent substance(s) into the cytoplasm of the pigment cells. The released and dispersed fluorescent substance(s) then made the entire pigment cell emit strong green fluorescence. With prolonged blue light irradiation, the pigment cell itself bursted, dispersing the fluorescent substance(s) into the body cavity or seawater. This resulted in “explosive emission” of the fluorescence. Cells that can emit such fluorescence first appeared at the late-blastula stage, proliferated with development, and changed into the red-pigmented cells. Similar fluorescence emission was observed in the larvae of Clypeaster japonicus, Hemicentrotus pulcherrimus, Anthocidaris crassispina and Pseudocentrotus depressus, of which C. japonicus larvae displayed the strongest fluorescence.

Theoretical and experimental dissection of gravity-dependent mechanical orientation in gravitactic microorganisms.

Mechanisms of gravitactic behaviors of aquatic microorganisms were investigated in terms of their mechanical basis of gravity-dependent orientation. Two mechanical mechanisms have been considered as possible sources of the orientation torque generated on the inert body. One results from the differential density within an organism (the gravity-buoyancy model) and the other from the geometrical asymmetry of an organism (the drag-gravity model). We first introduced a simple theory that distinguishes between these models by measuring sedimentation of immobilized organisms in a medium of higher density than that of the organisms. Ni2+-immobilized cells of Paramecium caudatum oriented downwards while floating upwards in the Percoll-containing hyper-density medium but oriented upwards while sinking in the hypo-density control medium. This means that the orientation of Paramecium is mechanically biased by the torque generated mainly due to the anterior location of the reaction center of hydrodynamic stress relative to those of buoyancy and gravity; thus the torque results from the geometrical fore-aft asymmetry and is described by the drag-gravity model. The same mechanical property was demonstrated in gastrula larvae of the sea urchin by observing the orientation during sedimentation of the KCN-immobilized larvae in media of different density: like the paramecia, the gastrulae oriented upwards in hypo-density medium and downwards in hyper-density medium. Immobilized pluteus larvae, however, oriented upwards regardless of the density of the medium. This indicates that the orientation of the pluteus is biased by the torque generated mainly due to the posterior location of the reaction center of gravity relative to those of buoyancy and hydrodynamic stress; thus the torque results from the fore-aft asymmetry of the density distribution and is described by the gravity-buoyancy model. These observations indicate that, during development, sea urchin larvae change the mechanical mechanism for the gravitactic orientation. Evidence presented in the present paper demonstrates a definite relationship between the morphology and the gravitactic behavior of microorganisms.

A large‐scale categorization of sites in San Francisco Bay, USA, based on the sediment quality triad, toxicity identification evaluations, and gradient studies

Sediment quality was assessed in San Francisco Bay, California, USA, using a two-tiered approach in which 111 sites were initially screened for sediment toxicity. Sites exhibiting toxicity were then resampled and analyzed for chemical contamination, recurrent toxicity, and, in some cases, benthic community impacts. Resulting data were compared with newly derived threshold values for each of the metrics in a triad-based weight-of-evidence evaluation. Sediment toxicity test results were compared with tolerance limits derived from reference site data, benthic community data were compared with threshold values for a relative benthic index based on the presence and abundance of pollution-tolerant and -sensitive taxa, and concentrations of chemicals and chemical mixtures were compared with sediment quality guideline-based thresholds. A total of 57 sites exceeded threshold values for at least one metric, and each site was categorized based on triad inferences. Nine sites were found to exhibit recurrent sediment toxicity associated with elevated contaminant concentrations, conditions that met program criteria for regulatory attention. Benthic community impacts were also observed at three of these sites, providing triad evidence of pollution-induced degradation. Multi- and univariate correlations indicated that chemical mixtures, heavy metals, chlordanes, and other organic compounds were associated with measured biological impacts in the Bay. Toxicity identification evaluations indicated that metals were responsible for pore-water toxicity to sea urchin larvae at two sites. Gradient studies indicated that the toxicity tests and benthic community metrics employed in the study predictably tracked concentrations of chemical mixtures in Bay sediments.

Mycosporine-like amino acids prevent UVB-induced abnormalities during early development of the green sea urchin Strongylocentrotus droebachiensis

Experiments were performed to determine how ultraviolet radiation (UVR) in the environmentally relevant range affects development of the sea urchin Strongylocentrotus droebachiensis (Muller) and whether mycosporine-like amino acids (MAAs), present in the early life stages, reduce UV-induced damage. Eggs, embryos, and larvae contained five MAAs having absorption maxima ranging from 320 to 334 nm. Eggs contained principally shinorine and porphyra-334, which absorb maximally at 334 nm and half-maximally at 312 and 348 nm, spanning much of the environmental range of biologically effective UVR. Concentrations of MAAs remained constant in unirradiated embryos through the gastrula stage, but decreased significantly in two-armed pluteus larvae. Daily exposure to combined photosynthetically active radiation (PAR, 400–700 nm) and UVR did not affect the concentration of MAAs in these embryos up to the two-armed pluteus stage. Prism larvae of sea urchins and the sand dollar Echinarachnius parma (Lamarck) did not accumulate shinorine from the surrounding seawater. Daily exposure of embryos to UVA (320–400 nm) and UVB (295–320 nm) radiation in the presence of PAR induced delays and abnormalities during development, and removing UVB eliminated this effect. Abnormalities in embryos included thickening of the blastoderm wall, filling of the blastocoel by abnormal cells, exogastrulation, and formation of abnormal spicules. The percentage of embryos that developed normally was lower in batches of embryos exposed to PAR + UVA + UVB, except in embryos from urchins maintained on MAA-rich diets. In all cases, the percentage of PAR + UVA + UVB-exposed embryos that developed normally was positively related to the concentration of MAAs in eggs from which the embryos developed. Thus, the MAAs found in S. droebachiensis embryos protect them against UVB-induced abnormalities during their development to at least the four-armed pluteus larval stage.