Mannan-Binding Lectin of the Sea Urchin Strongylocentrotus nudus

A novel lectin specific to low-branched mannans (MBL-SN) was isolated from coelomic plasma of the sea urchin Strongylocentrotus nudus by combining anion-exchange liquid chromatography on DEAE Toyopearl 650 M, affinity chromatography on mannan-Sepharose and gel filtration on the Sephacryl S-200. The molecular mass of MBL-SN was estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis under non-reducing conditions to be about 34 kDa. MBL-SN was shown to be a dimer with two identical subunits of about 17 kDa. The native MBL-SN exists as a tetramer. The physico-chemical properties of MBL-SN indicate that it belongs to C-type mannan-binding lectins. The cDNA encoding MBL-SN was cloned from the total cDNA of S. nudus coelomocytes and encodes a 17-kDa protein of 144 amino acid residues that contains a single carbohydrate-recognition domain of C-type lectins. Prediction of the MBL-SN tertiary structure using comparative modelling revealed that MBL-SN is an α/β-protein with eight β-strands and two α-helices. Comparison of the MBL-SN model with available three-dimensional structures of C-type lectins revealed that they share a common fold pattern.     

Essential oil of Lippia alba as a sedative and anesthetic for the sea urchin Echinometra lucunter (Linnaeus, 1758)

The sea urchin, Echinometra lucunter, is consumed in restaurants in Europe and Asia for high prices. This study evaluated the use of the essential oil of Lippia alba (EOL) as a sedative and anesthetic in this sea urchin. Different EOL concentrations were tested (50, 100 and 150 μL L^?1), in addition to a group that received ethanol and a control group. After the anesthesia and recovery from the different treatments, the coelomic fluid, gonads and intestine were collected for the analysis of oxidative stress parameters. The highest concentration tested (150 μL L^?1) was determined to be the optimal concentration for anesthesia. Results suggest that EOL improved the response to oxidative stress, since a lower level of thiobarbituric acid-reactive substances and greater superoxide dismutase and catalase activities were observed in the coelomic fluid and E. lucunter gonads, making the EOL a promising anesthetic for sea urchins.     

Measurement of oxygen consumption and sampling of body fluids of echinoderms in situ

A number of echinoderm species common to the coast of Malta have been subjected to respirometry measurement and coelomic fluid sampling. Experiments were carried out in situ by SCUBA divers and the results compare favourably with parallel laboratory investigations. One species of sea urchin had low coelomic fluid pO₂ when consuming more oxygen at night. The pH of the coelomic fluid of all the species was acid to the sea water. The value of making in situ measurements is discussed.     

Echinoid phagocytes in vitro

A method is described for obtaining pure monolayers of phagocytes from the sea urchin Strongylocentrotus droebachiensis in vitro. The coelomic fluid contains four types of cells. About 67% of the cells are phagocytes, the rest is comprised of the red and white morula cells and the vibratile cells. The different cell types could be separated by centrifugation on a discontinuous gradient of sodium metrizoate. Release of granula from the vibratile cells was found to be responsible for rapid and extensive clotting of the coelomic fluid immediately after its removal from the animal. Clotting was prevented by adding a mixture of 50 mM mercaptoethanol, 3 mM caffeine and 2 mM TAME (p-tosyl- -arginine methyl ester) to the coelomic fluid. The phagocytes were isolated from other cell types by their attachment to glass, and were grown at 10 °C in a simple peptone-sea water medium. The phagocytes are very motile cells and spread rapidly on glass, accompanied by a complete change of their morphology to flattened cells with peripheral ruffling. After few hours in vitro the cells fuse to form monolayer-syncytia, and later still cell clusters and free floating balls of cells are formed. During a culture period of 10 days there was no change in the DNA content per culture, while a small increase in protein was found.     

Zinc-binding property of the major yolk protein in the sea urchin - implications of its role as a zinc transporter for gametogenesis

Major yolk protein (MYP), a transferrin superfamily protein that forms yolk granules in sea urchin eggs, is also contained in the coelomic fluid and nutritive phagocytes of the gonad in both sexes. MYP in the coelomic fluid (CFMYP; 180 kDa) has a higher molecular mass than MYP in eggs (EGMYP; 170 kDa). Here we show that MYP has a zinc-binding capacity that is diminished concomitantly with its incorporation from the coelomic fluid into the gonad in the sea urchin Pseudocentrotus depressus. Most of the zinc in the coelomic fluid was bound to CFMYP, whereas zinc in eggs was scarcely bound to EGMYP. Both CFMYP and EGMYP were present in nutritive phagocytes, where CFMYP bound more zinc than EGMYP. Saturation binding assays revealed that CFMYP has more zinc-binding sites than EGMYP. Labeled CFMYP injected into the coelom was incorporated into ovarian and testicular nutritive phagocytes and vitellogenic oocytes, and the molecular mass of part of the incorporated CFMYP shifted to 170 kDa. Considering the fact that the digestive tract is a major production site of MYP, we propose that CFMYP transports zinc, essential for gametogenesis, from the digestive tract to the ovary and testis through the coelomic fluid, after which part of the CFMYP is processed to EGMYP with loss of zinc-binding site(s).     

Purification of the most abundant protein in the coelomic fluid of a sea urchin which immunologically cross reacts with 23S glycoprotein in the sea urchin eggs

The most abundant glycoprotein in the coelomic fluid of sea urchin Anthocidaris crassispina was purified and its subunit structure, molecular form in the native state, amino acid composition, and electron micrographic image were studied. The results showed that the protein in its native state was basically a tetramer with a total molecular weight of about 700,000, which was in equilibrium with a high molecular weight form corresponding to an octamer. The electron micrograph of the tetramer showed two ellipsoidal units aligned in parallel with a wide gap in between. The subunits all had the same molecular weight of 180,000 +/- 10,000 and were disulfide bonded in pairs. The carbohydrate content was about 16% with mannose and fucose as the two most abundant sugars. Although this protein accounted for 70% of the total protein in the coelomic fluid, it did not take part in the known activities of the fluid, namely hemagglutination and coagulation. Despite its structural similarity to the mammalian alpha-2-macroglobulin or reptilian and avian ovomacroglobulins it did not interact with bovine trypsin or chymotrypsin. This protein showed immunological cross reactivity with 23S glycoprotein purified from sea urchin eggs which, we believe, corresponds to the previously described 22-27S protein particles in eggs.     

The importance of inter‐individual variation in predicting species' responses to global change drivers

Inter‐individual variation in phenotypic traits has long been considered as “noise” rather than meaningful phenotypic variation, with biological studies almost exclusively generating and reporting average responses for populations and species’ average responses. Here, we compare the use of an individual approach in the investigation of extracellular acid–base regulation by the purple sea urchin Paracentrotus lividus challenged with elevated pCO₂ and temperature conditions, with a more traditional approach which generates and formally compares mean values. We detected a high level of inter‐individual variation in acid–base regulation parameters both within and between treatments. Comparing individual and mean values for the first (apparent) dissociation constant of the coelomic fluid for individual sea urchins resulted in substantially different (calculated) acid–base parameters, and models with stronger statistical support. While the approach using means showed that coelomic pCO₂ was influenced by seawater pCO₂ and temperature combined, the individual approach indicated that it was in fact seawater temperature in isolation that had a significant effect on coelomic pCO₂. On the other hand, coelomic [HCO₃^?] appeared to be primarily affected by seawater pCO₂, and less by seawater temperature, irrespective of the approach adopted. As a consequence, we suggest that individual variation in physiological traits needs to be considered, and where appropriate taken into account, in global change biology studies. It could be argued that an approach reliant on mean values is a “procedural error.” It produces an artefact, that is, a population's mean phenotype. While this may allow us to conduct relatively simple statistical analyses, it will not in all cases reflect, or take into account, the degree of (physiological) diversity present in natural populations.     

Biochemical and pathogenic properties of the natural isolate of Shewanella algae from Peter the Great Bay, Sea of Japan

Pathogenic properties of the natural isolate of Shewanella algae from the coelomic fluid of the sea cucumber Apostichopus japonicus (Peter the Great Bay, Sea of Japan) were investigated. The isolate had oxydative metabolism, was positive for ornithine decarboxylase, cytochrome oxidase, catalase, DNase and gelatinase, hemolytically active, did not produce acid from carbohydrates, and did not hydrolyze urea and esculin. The strain was resistant to penicillin, amoxicillin, and ampicillin and susceptible to tetracycline and carbenicillin. Among cellular fatty acids, 13:0-i, 15:0-i, 16:0, 16:1(n-7), 17:0-i, and 17:0-ai dominated. These biochemical properties made it possible to attribute the isolated bacteria to the genus Shewanella and identified as S. algae. The cells of this bacterium were introduced into the coelomic cavity of another echinoderm, the sea urchin Strongylocentrotus nudus. As a result, in about 24 h the animals became slow and 3-8 days after the inoculation died. Dividing bacteria were being found during the experiment in the coelomic fluid as well as in the phagosomes of amoebocytes, i.e. cells acting as phagocytes in the coelomic fluid. The studies of the invasive properties of strain 156 showed that bacterial cells entered the subcuticular space of S. nudus and A. japonicus through the cuticle and stayed there for a long time without penetrating epithelium and exerting toxic effect upon the organisms of the laboratory animals. Pathogenic effect of S. algae can be manifested only if the cutaneous epithelium is destroyed permitting it to penetrate the lower tissue layers. The toxicity of S. algae is confirmed by in vitro experiments. The inoculation of the embryonic cells of S. nudus with samples of this bacterium caused the death of 10% of cells within an hour and 100% of cells within 12 h after inoculation. The results of the investigations demonstrate that S. algae could produce opportunistic infection in the sea cucumber A. japonicus and the sea urchin S. nudus, which may be natural reservoirs of this human pathogen.     

A putative precursor to the major yolk protein of the sea urchin

We detected by electrophoresis, several glycoproteins in the eggs of three species of sea urchin. The major glycoprotein band disappears as development of the embryo proceeds. This protein is enriched in the yolk fraction obtained by zone sedimentation in 2.5–30% sucrose gradients. A fractionally larger glycoprotein has been found to be the major protein in the coelomic fluid of male and female gravid sea urchins. Partial proteolysis peptide mapping shows that the major coelomic fluid protein and the major yolk protein are related, presumably in a precursor-product relationship.     

Early development of coelomic structures in an echinoderm larva and a similarity with coelomic structures in a chordate embryo

Early coelomic development in the abbreviated development of the sea urchin Holopneustes purpurescens is described and then used in a comparison with coelomic development in chordate embryos to support homology between a single arm of the five-armed radial body plan of an echinoderm and the single bilateral axis of a chordate. The homology depends on a positional similarity between the origin of the hydrocoele in echinoderm development and the origin of the notochord in chordate development, and a positional similarity between the respective origins of the coelomic mesoderm and chordate mesoderm in echinoderm and chordate development. The hydrocoele is homologous with the notochord and the secondary podia are homologous with the somites. The homology between a single echinoderm arm and the chordate axis becomes clear when the aboral to oral growth from the archenteron in the echinoderm larva is turned anteriorly, more in line with the anterior–posterior axis of the early zygote. A dorsoventral axis inversion in chordates is not required in the proposed homology.     

Cellular Responses of Congenital Immunity in the Starfish <Emphasis Type="Italic">Asterias rubens</Emphasis>

The work deals with analysis of changes of cellular defense factors in the starfish Asterias rubens in response to injection of human erythrocytes (HE). The number of circulating coelomocytes, dynamics of their production of active oxygen forms, activity of peroxidase, and dynamics of elimination of human hemoglobin from coelomic fluid were estimated before immunization with HE as well as at 6–144 h. The number of coelomocytes was counted in Goryaev chamber, production of active oxygen forms was determined in the test of spontaneous and zymosan-induced reduction of Tetrazolium Nitro Blue, peroxidase activity—in a color enzymatic reaction. Time of human hemoglobin elimination from the coelomic fluid was determined by spectrophotometric method by hemoglobin binding with acetone cyanohydrin with formation of a colored product. It is revealed that injection of human erythrocytes into the starfish Asterias rubens leads to a decrease of the number of coelomocytes in 24–96 h and to an increase of their specific production of active oxygen forms in 96–120 h after the HE injection. In coelomic fluid of Asterias rubens the presence of peroxidase activity is established. The circulation time of human hemoglobin released from erythrocytes in coelomic fluid of these animals does not exceed 24 h. It is suggested that the cellular defense reactions are the major factor of the starfish congenital immunity.__________Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 2, 2005, pp. 107–113.Original Russian Text Copyright © 2005 by Kudryavtsev, D’yachkov, Kazakov, Kanaikin, Kharazova, Polevshchikov.     

Inhibition of spicule elongation in sea urchin embryos by the acetylcholinesterase inhibitor eserine

The activity of acetylcholinesterase (AchE) increases rapidly after the gastrula stage of sea urchin development. In this report, changes in activity and in the molecular differentiation of AchE were investigated. AchE activity increased slightly during gastrulation and rose sharply thereafter, and was dependent on new RNA synthesis. No activity of butyrylcholinesterase was found. Morphogenesis in sea urchin embryos was inhibited by the AchE inhibitor eserine, which specifically inhibited arm rod formation but not body rod formation. Spicule formation and enzyme activity in cultured micromeres were inhibited by eserine in a dose-dependent manner. During gastrulation, two molecular forms of AchE were detected with polyacrylamide gel electrophoresis. The appearance of an additional band on the gel was consistent with the occurrence of a remarkable increase in the enzyme activity. This additional band appeared as a larger molecular form in Anthocidaris crassispina, Hemicentrotus pulcherrimus, Stomopneustes variolaris, and Strongylocentrotus nudus, and as a smaller form in Clypeaster japonicus and Temnopleurus hardwicki. These results suggest that the change in the molecular form of AchE induced a change in enzymatic activity that in turn may play a role in spicule elongation in sea urchin embryos.     

Morphological and ultrastructural characterization of sea urchin immune cells

The free circulating coelomocytes in the coelomic cavity of echinoderms are considered to be immune effectors by phagocytosis, encapsulation, cytotoxicity, and by the production of antimicrobial agents. Although echinoderms (especially sea urchin embryo) have been used as a model organisms in biology, no uniform criteria exist for classification of coelomocytes in echinoderms, and few studies have reported about the biological functions of their coelomocytes. Hence, we study the coelomocytes in the echinoid sea urchin, Paracentrotus lividus, and describe their morphological and ultrastructural features using light and transmission electron microscopes. We classify the coelomocytes of P. lividus into red spherule and colorless spherule cells, small cells, vibratile cells, and phagocytic cells; petaloid and filopodial cells. To our knowledge, this is the first report describing ultrastructural details of the coelomocytes of P. lividus. J. Morphol. 276:583–588, 2015. © 2015 Wiley Periodicals, Inc.     

The sea urchin major yolk protein is synthesized mainly in the gut inner epithelium and the gonadal nutritive phagocytes before and during gametogenesis

Major yolk protein (MYP), the predominant component of yolk granules in sea urchin eggs, is also contained in the coelomic fluid and nutritive phagocytes of the gonad in both sexes. MYP is stored in ovarian and testicular nutritive phagocytes prior to gametogenesis and is used during gametogenesis as material for synthesizing proteins and other components necessary for eggs and sperm. To reveal the expression profile and the main production site of MYP, we analyzed MYP mRNA expression in immature and maturing Pseudocentrotus depressus. Real‐time reverse‐transcribed polymerase chain reaction analysis showed that MYP mRNA was expressed predominantly in the digestive tract (stomach, intestine and rectum) and the gonad of both sexes. The total amounts of MYP mRNA in the whole digestive tract and in the whole gonad were at similar levels in both immature and maturing sea urchins. MYP mRNA was also detected in white morula cells and vibratile cells separated from the coelomic fluid by density gradient centrifugation, but the expression levels in these cells were very low compared with those in the digestive tract and the gonad. Using in situ hybridization analysis, MYP mRNA was detected in the inner epithelium of the digestive tract and in nutritive phagocytes of the ovary and testis, but was not detected in the germ cells. We conclude that the adult sea urchin has two predominant production sites for MYP regardless of sex and reproductive stage: the inner epithelium of the digestive tract and the nutritive phagocytes of the gonad. Mol. Reprod. Dev. 77: 59–68, 2010. © 2009 Wiley‐Liss, Inc.     

Impact of CO2-acidified seawater on the extracellular acid-base balance of the northern sea urchin Strongylocentrotus dröebachiensis

We investigated the effect of five day exposure to CO₂-acidified sea water treatments (pH_NBS = 7.89 [control], 7.44, 7.16 and 6.78, T = 9.5 °C) on the extracellular acid-base balance of the northern sea urchin Strongylocentrotus dröebachiensis. In each case there was an uncompensated respiratory acidosis which increased in intensity with decreasing environmental pH. This was very similar to results for another sea urchin species, Psammechinus miliaris (8 d exposure, T = 15 °C). However, there were some important differences in the response to low seawater pH between the two urchin species S. dröebachiensis and P. miliaris. At the lowest pH tested (6.78) there was a metabolic component to this acidosis recorded (correlated with a significant increase in l-lactate) in S. dröebachiensis but not P. miliaris. The acidosis was accompanied by a very small, but significant increase in coelomic fluid calcium. Also the water used in our study was (controlling for pH) markedly undersaturated with respect to carbonate compared with that used in the Psammechinus study, highlighting the need for the environmental context to be assessed in future comparative studies.     

Identité immunologique et métabolique entre le liquide coelomique,les coelomocytes et les ovocytes de Perinereis cultrifera (Annélide Polychète)

Immunological study of Perinereis cultrifera reveals the existence of an identical antigen in the coelomic fluid of females (oocyte diameter > 140 (μm), in oocytes, and in coelomocytes. This factor is not found in the body fluid of males or young females.

The elution patterns obtained after Sephadex chromatography shows a similar glycoprotein fraction (fraction I) in the coelomic fluid, in coelomocytes, and in soluble oocyte extracts. This fraction includes the main part of the antigenic components of the coelomic fluid.

Gas chromatography reveals that identical monosaccharides are present, albeit in varying proportions, in samples of fraction I obtained from the different sources.

The metabolic interrelationships of coelomocytes, coelomic fluid and oocytes is discussed. Glycoprotein synthesized by coelomocytes may be discharged into the coelomic fluid and contribute to the development of the cortical alveoli of the oocytes. No evidence of an involvement of this material in yolk synthesis has been obtained.     

Humoral responses of congenital immunity in the starfish <Emphasis Type="Italic">Asterias rubens</Emphasis>

The work presents analysis of changes of humoral protective factors in the starfish Asterias rubens in response to injection of human erythrocytes (HE). The total protein concentration and the titers of hemagglutinins and hemolysins in starfish coelomic fluid, as well as the time of human hemoglobin elimination from circulation were estimated for 6–144 h of the experiment. The hemagglutinin titer was determined in hemagglutination reactions, the hemolysin titer—in hemolysis reaction. Time of human hemoglobin elimination from coelomic fluid was determined in a color enzymatic reaction. The starfish coelomic fluid was revealed to contain soluble factors that are able to interact with antigen— antibody complexes of mammals and have an opsonizing activity. It is established that injection of HE does not change the total protein concentration per 1 ml coelomic fluid, but affects dynamics of changes of the hemagglutinins titer. Time of hemoglobin elimination from circulation does not exceed 24 h. Humoral factors of coelomic fluid of the starfish Asterias rubens play an auxiliary role in congenital immunity reactions.Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 1, 2005, pp. 23–28.Original Russian Text Copyright © 2005 by Kudryavtsev, Dyachkov, Kazakov, Kanaikin, Kharazova, Polevshchikov.     

Changes in the Composition of Celomic Fluid Metabolites of the Black Sea Urchin Mesocentrotus nudus (Echinoidea) and the Starfish Asterina pectinifera (Asteroidea) under Conditions of Hypoxia Stress

Biology Bulletin - The composition of metabolites in the coelomic fluid of the starfish Asterina pectinifera and sea urchin Mesocentrotus nudus was studied under normal and hypoxic conditions using...     

The amino-acid sequence of multiple lectins of the acorn barnacle Megabalanus rosa and its homology with animal lectins

The amino-acid sequence of a lectin isolated from the coelomic fluid of the acorn barnacle Megabalanus rosa has been determined. The lectin (Mr 140,000) is a multimeric protein whose subunit consists of 173 amino acids and one carbohydrate chain attached to Asn-39. The amino-acid sequence was determined by the manual sequencing of peptides derived from the protein by digestion with Staphylococcus aureus V8 proteinase, lysine endopeptidase and chymotrypsin, as well as fragments produced by cleavage with cyanogen bromide. The amino-acid sequence of the lectin was compared with the sequence of one (Mr 64,000) of the multiple lectins of M. rosa. They are distinct molecules in spite of a significant homology in their amino-acid sequences. The amino-acid sequence includes some regions homologous to those in other invertebrate lectins, such as sea urchin and flesh fly lectins, and vertebrate lectins. This is the first report to show the amino-acid sequence of multiple lectins isolated from an invertebrate.     

MOLECULAR PROPERTIES AND DIFFERENTIATION OF ACETYLCHOLINESTERASE IN SEA URCHIN EMBRYOS*

The two molecular forms of acethylcholinesterase (EC 3.1.1.7) in sea urchin embryos were characterized by several physical methods. The sedimentation coefficients determined by sucrose gradient centrifugation are 7.6S and 10.6S. The Stokes radii determined by gel filtration are 65 Å and 91 Å. From these parameters, molecular weights were estimated as 190,000 and 380,000; the one is twice as large as the other. Both forms have similar electric property and buoyant density in a CsCl gradient. When the enzyme solution was concentrated, the 10.6S form became predominant. These results suggest that the two forms are monomer and dimer. The sea urchin enzymes resemble globular forms of acetylcholinesterase of the electric organ of fishes. The activity of the enzyme abruptly increases in post-gastrulation embryos. Inhibition of concomitant protein synthesis by a specific inhibitor, emetine, does not affect the increase in enzyme activity. The result suggests that post-translational processes may be involved in the differentiation of this enzyme in sea urchin development. The following sea urchins were used in the study: Strongylocentrotus purpuratus, Strongylocentrotus franciscanus, and Dendraster excentricus.