Occurrence of d-alanine in the eggs and the developing embryo of the sea urchin Paracentrotus lividus

• 1.1. d-Alanine has been found in appreciable amounts in the eggs and embryos of the sea urchin Paracentrotus lividus.
• 2.2. The content of d-alanine, expressed as pmol/egg or embryo, is 1.32 in the egg, 0.81 in the blastula, 0.54 in the gastrula and 0.60 in the pluteus.
• 3.3. The percentage of d-alanine with respect to the total alanine (d + l) decreases during embryonic development.
• 4.4. d-Amino acid oxidase, d-alanine transaminase and d-alanine racemase activities were found neither in eggs nor in embryos.
• 5.5. Therefore, it does not appear likely that d-alanine is subject to oxidative metabolism.
• 6.6. The decrease in this d-amino acid during development may be due to its utilization in the synthesis of a more complex molecule.

     

Separation and partial characterization of DNA polymerases in sea urchin Paracentrotus lividus eggs.

$\text{Paracentrotus lividus}$ eggs contain three separable DNA polymerases (deoxynucleoside triphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7.). The two main peaks of activity, designated form I and form II, differ in the following features: 1) form I is able to use poly(dA) as primer-template more efficiently than form II; 2) the initial rate of incorporation of dTTP or dCTP in the absence of other deoxynucleosidetriphosphates (dNTPs) is higher with form I than with form II when the template is DNA or poly(dA,dT); 3) form II is preferentially inhibited by KCl; 4) the two forms show a different optimal Mn²⁺ concentration for their maximal activity.     

Mifepristone affects fertility and development in the sea urchin Paracentrotus lividus

Drugs such as oral contraceptives and hormone replacement therapies are known to find their way into rivers, lakes and seas, and have the potential to affect reproduction and development of the wildlife. The knowledge of the reproductive mechanisms and their regulation in aquatic species is of fundamental importance for predicting and preventing the damage by the increasing release of such chemicals in the environment. Mifepristone, a synthetic steroid used as a drug for chemical abortion, works by blocking the effects of progesterone. Its presence in fresh and salt water has been reported, representing a danger for aquatic species. In this frame, we evaluated in both acute and chronic exposures, the effects of mifepristone on the reproductive performance of the sea urchin P. lividus. In both acute and chronic exposures, mifepristone did not affect the histological structure of the gonads. However, mifepristone administered to females caused the decrease of the percentage of normal developed plutei larvae compared with the control, whereas it did not alter sperm motility parameters and fertilization success in males. The immunohistological localization of progesterone receptor‐like immunoreactivity on the plasma membrane of oocytes and ova and the molecular weight of a progesterone receptor‐like immunoband identified by western blotting, are in agreement with a membrane progesterone receptor deducted from the genome sequence of the sea urchin Strongylocentrotus purpuratus and suggest that in P. lividus mifepristone actions may be mediated by a progesterone receptor.     

Defensome against toxic diatom aldehydes in the sea urchin Paracentrotus lividus

Many diatom species produce polyunsaturated aldehydes, such as decadienal, which compromise embryonic and larval development in benthic organisms. Here newly fertilized Paracentrotus lividus sea urchins were exposed to low concentration of decadienal and the expression levels of sixteen genes, implicated in a broad range of functional responses, were followed by Real Time qPCR in order to identify potential decadienal targets. We show that at low decadienal concentrations the sea urchin Paracentrotus lividus places in motion different classes of genes to defend itself against this toxic aldehyde, activating hsp60 and two proteases, hat and BP10, at the blastula stage and hsp56 and several other genes (14-3-3ε, p38 MAPK, MTase, and GS) at the prism stage. At this latter stage all genes involved in skeletogenesis (Nec, uni, SM50 and SM30) were also down-expressed, following developmental abnormalities that mainly affected skeleton morphogenesis. Moreover, sea urchin embryos treated with increasing concentrations of decadienal revealed a dose-dependent response of activated target genes. Finally, we suggest that this orchestrated defense system against decadienal represents part of the chemical defensome of P. lividus affording protection from environmental toxicants.     

Gender differences in the immune system activities of sea urchin Paracentrotus lividus

In the immune system of vertebrates, gender-specific differences in individual immune competence are well known. In general, females possess more powerful immune response than males. In invertebrates, the situation is much less clear. For this purpose we have chosen to study the immune response of the two sexes of the echinoderm Paracentrotus lividus in pre- and post-spawning phases. The coelomic fluid from the echinoderms contains several coelomocyte types and molecules involved in innate immune defenses. In this article we report that the degree of immune responses in the P. lividus differs according to sex in both pre- and post-spawning phases. We found in all tests that females were more active than males. The results indicate that females possess a significant higher number of immunocytes consisting of phagocytes and uncolored spherulocytes. Since the immunological activity is mainly based on immunocytes, it was not surprising that females possessed the highest values of cytotoxicity and hemolysis activity and showed a greater ability to uptake neutral red and phagocyte yeasts cells, while the average number of ingested particles per active phagocyte was not significantly different. Furthermore, agglutinating activity was more evident in the coelomocyte lysate and coelomic fluid of females than in those of males. Finally we found that the acidic extract of female gonads possessed greater antimicrobial activity than that of male gonads. These results make it very likely that gender differences in the immune response are not restricted to vertebrates; rather, they are a general evolutionary phenomenon.     

Antimicrobial and antistaphylococcal biofilm activity from the sea urchin Paracentrotus lividus

Aims:  Staphylococcal biofilm-associated infections are resistant to conventional antibiotics. Consequently, new agents are needed to treat them. With this aim, we focused on the effector cells (coelomocytes) of the sea urchin Paracentrotus lividus immune system.
Methods and Results:  We tested the activity of the 5-kDa peptide fraction of the cytosol from coelomocytes (5-CC) against a group of Gram-positive, Gram-negative bacteria and fungi. We determined minimal inhibitory concentrations (MICs) ranging from 253·7 to 15·8 mg ml⁻¹. We observed an inhibitory activity and antibiofilm properties of 5-CC against staphylococcal biofilms of reference strains Staphylococcus epidermidis DSM 3269 and Staphylococcus aureus ATCC 29213. The antimicrobial efficacy of 5-CC against the biofilms of clinical strain Staph. epidermidis 1457 was also tested using live/dead staining in combination with confocal laser scanning microscopy. At a sub-MIC concentration (31·7 mg ml⁻¹) of 5-CC the formation of young (6-h old) and mature (24-h old) staphylococcal biofilms was inhibited.
Conclusions:  The biological activity of 5-CC could be attributed to three peptides belonging to the sequence segment 9–41 of a beta-thymosin of P. lividus .
Significance and Impact of the Study:  The effector cells of P. lividus represent an interesting source of marine invertebrates-derived antimicrobial agents in the development of new strategies to treat staphylococcal biofilms.     

Carotenoids in the sea urchin Paracentrotus lividus: occurrence of 9'-cis-echinenone as the dominant carotenoid in gonad colour determination

Regular sampling of wild Paracentrotus lividus was carried out over a 12-month period to examine seasonal effects on the pigment profile and content of the gonads, especially in comparison to gonad colour. The major pigments detected in the gut wall were breakdown products of fucoxanthin, namely fucoxanthinol and amarouciaxanthin A. Lower levels of other dietary carotenoids (lutein and β-carotene) together with some carotenoids not found in the diet, namely isozeaxanthin and echinenone ( 20% total carotenoid) were also detected in the gut wall. The presence of echinenone in the gut wall demonstrates that this organ acts as a major site of carotenoid metabolism. Echinenone is the dominant carotenoid in the gonads, accounting for approx. 50–60% of the total pigment. Both all-trans and 9′-cis forms of echinenone were detected in both the gut wall and in the gonad, with levels of the 9′-cis form typically 10-fold greater than the all-trans form in the gonad. The detection of large levels of 9′-cis-echinenone in wild sea urchins is unexpected due to the absence of 9- or 9′-cis forms of carotenoids in the natural, algal, diet. Whilst echinenone clearly contributes towards gonad pigmentation, levels of this carotenoid, cannot be directly linked to a qualitative assessment of gonad colour in terms of market acceptability. Indeed, unacceptable gonad colouration can be seen with both very low and high levels of echinenone and total carotenoid. The presence of 9′-cis-echinenone as the major carotenoid contributing to the pigmentation/colour of the gonad is an important observation in terms of developing artificial diets for urchin cultivation.     

Evidence for an acidic compartment in sea urchin eggs (Paracentrotus lividus): role at fertilization

The characteristics of [14C]methylamine accumulation by isolated cortices were measured in eggs from three species of sea urchins: Paracentrotus lividus, Arbacia lixula and Sphaerechinus granularis. In all cases, the results pointed to the existence of an acidic compartment in the cortical zone. In P. lividus eggs, cortical granules did not participate in proton storage which likely took place in pigment granules. [14C]Methylamine accumulation was dramatically reduced by monovalent cation ionophores (monensin and nigericin) and by NH4Cl, but not by valinomycin. ATP depletion only partially affected the isotope uptake. Simultaneous measurements of intracellular pH and of external titratable acidity during ammonia treatment of eggs, indicate that after fertilization, eggs increased their capacity to concentrate hydrogen ions in an intracellular store. Following insemination, cortices from P. lividus eggs exhibited a 3-fold increase in [14C]methylamine accumulation. It is concluded that the egg cortical area contains acidic organelles sequestering hydrogen ions by means of an electrogenic H+ pump, and that this mechanism, enhanced at fertilization, participates in a local alkalinization. The role of such a mechanism is discussed.     

Polymorphisms in the intergenic region of the sea urchin Paracentrotus lividus ribosomal DNA

Blot-hybridizations of the sea urchin Paracentrotus lividus genomic DNA with ribosomal DNA (rDNA) probes revealed individual variations in the length and in the sequence of the non-transcribed spacer (NTS) region. The number of rDNA repeat subclasses distinguishable within any individual sea urchin is usually limited (1 to 3) with respect to the widest polymorphism of the population as a whole. The heterogeneity in sequence is revealed by the presence or the absence of specific restriction sites in the spacer region. The data obtained by the intensity of the polymorphic bands indicate that different mechanisms bring about these two types of polymorphism. Preliminary data also indicate different methylation patterns in single individuals.     

Serotoninergic processes in cells of early embryos of the sea urchin Paracentrotus lividus

Agonists of serotonin receptors generate specific inward currents in the cells of early embryos of sea urchin Paracentrotus lividus. Dose-dependent current generated by 5-HT3-agonist 5-HTQ shows complex volt-amperic characteristics with reversal potential -25 and +15 mV. The 5-HTQ effect seems to be due to the activity of channels of mixed conductivity. The 5-HTQ effect is more obvious during cleavage furrow formation. The findings suggest presence of serotonin receptors in the surface membrane of blastomers and their activity play a certain role in regulation of cellular events during cleavage division in the early sea urchin embryo.