Comparative biochemical analysis of the major yolk protein in the sea urchin egg and coelomic fluid

The major yolk protein (MYP) is localized to the egg and coelomic fluid of the adult sea urchin. While the egg‐localized MYP has been extensively studied, much less is known about the coelomic fluid‐localized protein. Therefore, we have conducted a comparative biochemical analysis of these proteins. Sucrose density gradient ultracentrifugation revealed unique elution profiles for the MYP species present in the egg, 170‐ and 240 kDa, and the coelomic fluid, 180‐ and 250 kDa. Fractionation in polyacrylamide gels revealed that under reducing conditions both species were present in each location. However, in the absence of reducing agent only one species was present in each fraction: 240 kDa in the egg and 250 kDa in the coelomic fluid. In addition, V8 peptide mapping indicated that all four species have very similar primary structures. Circular dichroic spectral analysis and endogenous tryptophan measurements of the purified 170‐ and 180 kDa species revealed distinctive secondary and tertiary structural features with notable differences in their responses to calcium: apparent Kds of 245‐ and 475 μmol/L were measured for the 170‐ and 180 kDa species, respectively. Further analysis revealed that both species have differing calcium requirements for binding to membranes as well as protein‐dependent, membrane‐membrane interaction. We discuss the functional implications arising from the structural differences which exist between the egg and coelomic fluid resident MYPs.     

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.     

Ege A,a novel C2 domain containing protein,is essential for GPCR-mediated gene expression in dictyostelium

The major yolk protein (MYP) in sea urchins has historically been classified as a vitellogenin based on its abundance in the yolk platelets. Curiously, it is found in both sexes of sea urchins where it is presumed to play a physiological role in gametogenesis, embryogenesis, or both. Here we present the primary structure of MYP as predicted from cDNAs of two sea urchins species, Strongylocentrotus purpuratus and Lytechinus variegatus. The sequence from these two species share identity to one another, but bear no resemblance to other known vitellogenins. Instead the sequence shares identity to members of the transferrin superfamily of proteins. In vitro iron binding assays, including both (59)Fe overlay assays of MYP enriched coelomic fluid and immunoprecipitation of native iron-bound MYP from coelomic fluid, support this classification. We suggest that one of MYP's transferrin-like properties is to shuttle iron to developing germ cells.     

Detection of two immunochemically identical forms of mannan-binding lectin in the sea urchin <Emphasis Type="Italic">Strongylocentrotus nudus</Emphasis>

This study revealed a new lectin (MBL-SN) in the coelomic fluid of the sea urchin Strongylocentrotus nudus. Based on the peculiarities of molecular structure and carbohydrate specificity, MBL-SN can be assigned to the mannan-binding lectin family. Using polyclonal monospecific rabbit antibodies against MBL-SN, the presence of MBL-SN in the sea urchin was detected in two forms: a soluble form dissolved in the coelomic fluid and an extracellular matrix-bound form. The biosynthesis site of this lectin may be one of the subpopulations of morula cells-coelomic fluid cells that perform heterosynthesis. Our results demonstrate the similarity of the sea urchin lectin MBL-SN to the previously investigated MBLs of the holothurians Cucumaria japonica and Apostichopus japonicus, and suggest a similarity to MBLs of vertebrates, which also have soluble and bound forms.     

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).     

The major yolk protein is synthesized in the digestive tract and secreted into the body cavities in sea urchin larvae

Major yolk protein (MYP), a transferrin superfamily protein contained in yolk granules of sea urchin eggs, also occurs in the coelomic fluid of male and female adult sea urchins regardless of their reproductive cycle. MYP in the coelomic fluid (CFMYP; 180 kDa) has a zinc-binding capacity and has a higher molecular mass than MYP in eggs (EGMYP; 170 kDa). CFMYP is thought to be synthesized in the digestive tract and secreted into the coelomic fluid where it is involved in the transport of zinc derived from food. To clarify when and where MYP synthesis starts, we investigated the expression of MYP during larval development and growth in Pseudocentrotus depressus. MYP mRNA was detected using RT-PCR in the early 8-arm pluteus stage and its expression persisted until after metamorphosis. Real-time RT-PCR revealed that MYP mRNA increased exponentially from the early 8-arm stage to metamorphosis. Western blotting showed that maternal EGMYP disappeared by the 4-arm stage and that newly synthesized CFMYP was present at and after the mid 8-arm stage. In the late 8-arm larvae, MYP mRNA was detected in the digestive tract using in situ hybridization, and the protein was found in the somatocoel and the blastocoel-derived space between the somatocoel and epidermis using immunohistochemistry. These results suggest that CFMYP is synthesized in the digestive tract and secreted into the body cavities at and after the early 8-arm stage. We assume that in larvae, CFMYP transports zinc derived from food via the body cavities to various tissues, as suggested for adults.     

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.     

The pH of body fluids from marine intertidal invertebrates

The pH of the body fluids of 25 species of marine invertebrates from nine phyla was determined at temperatures to which these animals were acclimated. The gastrovascular fluid of several coelenterates and one species of flatworm was found to have pH values approximating to that of sea water. The coelomic fluid of nemertine, sipinculid, echiuroid, and annelid worms, as well as the heart fluid of several molluscs and the sternal sinus fluid of decapod crustaceans, were characterized by more alkaline pH values. Echinoderm polian vesicle fluid was characterized by slightly acidic pH values. The importance of the pH of the body to the respiratory physiology of marine invertebrates is discussed.     

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.     

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...     

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.     

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.     

Neoparamoeba branchiphila infections in moribund sea urchins Diadema aff. antillarum in Tenerife, Canary Islands, Spain

A total of 109 sea urchins from 3 species collected in 2 localities off the coast of Tenerife Island, Spain, were examined for the presence of free-living amoebae in their coelomic fluid. Amoeba trophozoites were isolated exclusively from moribund individuals of long-spined sea urchins Diadema aff. antillarum (Philippi) (Echinoidea, Echinodermata) that manifested lesions related to sea urchin bald disease on their tests (16 out of 56 examined). No amoebae were detected in Arbacia lixula (L.) and Paracentrotus lividus (Lamarck). From the former sea urchin species, 8 strains, established from 10 primary isolates, were identified as Neoparamoeba branchiphila Dyková et al., 2005 using morphological and molecular methods. Results of this study (limited to the screening for free-living amoebae) together with data on agents of sea urchin mortalities reported to date justify the hypothesis that free-living amoebae play an opportunistic role in D. aff. antillarum mortality. The enlargement of the dataset of SSU rDNA sequences brought new insight into the phylogeny of Neoparamoeba species.     

Functional anatomy of the respiratory system of Branchipolynoe species (Polychaeta, Polynoidae), commensal with Bathymodiolus species (Bivalvia, Mytilidae) from deep-sea hydrothermal vents

 The gills of three species of Branchipolynoe have been studied in order to better understand the morphological and anatomical adaptations of their respiratory system. These Polynoidae live commensally inside the pallial cavity of different species of Bathymodiolus (Mytilidae), found clustered near deep-sea hydrothermal vents and cold seeps, and which harbor chemolithoautotrophic bacteria in their gills. As the mussels exploit hydrothermal fluid, the pallial cavity is perfused with a sulfide-rich hydrothermal water. The gills of Branchipolynoe species are well-developed branched outgrows of the body wall, located on the parapodia, and filled with coelomic fluid. They do not contain blood vessels. Living animals are red, due to the presence of extracellular hemoglobins in the coelom. The gill epidermis is made of supporting cells and a few ciliated cells arranged in longitudinal rows along the branches. Myoepithelial and ciliated cells line the interior of the coelomic cavity which contains the respiratory pigments. Coelomic fluid circulation inside the gills and body cavity is probably facilitated by both the cilia and myoepithelial contractions. The cuticle, the epidermis, and the coelomic epithelium are completely devoid of bacteria. The gill surface areas per unit body weight and the minimum diffusion distances, between external milieu and coelomic hemoglobins, have been calculated and compared with data already obtained on vascular gills of littoral or hydrothermal species of Polychaeta. In Branchipolynoe species, the respiratory surface area is very large, similar to that of a free-living hydrothermal species Alvinella pompejana, and the minimum diffusion distance is short, similar to that of the littoral species Arenicola marina. Although the organization of these coelomic gills in Branchipolynoe species is totally different from that of usual vascular gills, their characteristics represent a unique and effective respiratory system in Polynoidae which has adapted to the hypoxic and sulfide-rich micro-habitat which probably holds in the mantle cavity of vent mussels. In the gill epidermis, numerous secondary and large compound lysosomes are present which might be involved in sulfide detoxification. Accepted: 5 August 1998     

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.     

Expression of SpC3, the sea urchin complement component,in response to lipopolysaccharide

The homologue of the vertebrate complement component C3 that is expressed in the coelomocytes of the purple sea urchin, Strongylocentrotus purpuratus, designated SpC3, was investigated for changes in response to immune challenge or injury. Immunoquiescent animals were used in this study because they have reduced or no detectable SpC3 in their coelomocytes or coelomic fluid (CF). Animals were injected with lipopolysaccharide (LPS) or sterile sea water (SSW, injury control). Changes in the amounts of SpC3 in coelomic fluid and in coelomocytes were then followed over time by Western blots and ELISA. Changes in mRNA from the SpC3 gene (Sp064) were also followed by RT-PCR. Although all animals responded to injury with increased levels of SpC3 in the coelomic fluid, those challenged with LPS had greater amounts of SpC3 in both CF and coelomocytes than those receiving SSW. In most of the animals receiving LPS, initial increases in SpC3 were observed within 1 h post-injection, while the earliest response in the animals receiving SSW was 6 h. The appearance of SpC3 in the coelomocytes was delayed compared to its appearance in CF, and was first detected several days after challenge. Changes in mRNA from the Sp064 gene paralleled the appearance of SpC3 in the coelomic fluid. Increases in the number of coelomocytes per milliliter of CF and in the percentage of coelomocytes that were SpC3+ also occurred after challenge with LPS or in response to injury, with a slightly greater increase in response to LPS. Although the changes in SpC3 were not as great as those identified previously for human C3 expressed in macrophages, the kinetics of the response are similar to that of acute-phase reactants in mammals.     

Hyperosmotic properties of the fluids of the perivisceral coelom and water vascular system of starfish kept under stable conditions

• 1.1. Osmotic measurements were made on the perivisceral coelomic and water vascular fluids of 4 species of northwest Pacific starfish and their stable sea-water media.
• 2.2. Mean levels of both fluids were hyperosmotic in every species, often at statistically significant levels.
• 3.3. For all species combined, mean hyperosmolality (mosmol/kg ± SE) of perivisceral coelomic fluid was 1.49 ± 0.17, and water vascular fluid 6.07 ± 0.74.
• 4.4. The hyperosmotic nature of these fluids contributes to water balance, working in conjunction with madreporitic inflow and other factors.

     

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.