Mechanical properties of sea-urchin lantern muscles: a comparative investigation of intact muscle groups in Paracentrotus lividus (Lam.) and Stylocidaris affinis (Phil.) (Echinodermata, Echinoidea)

The jaw apparatus, or lantern, of sea-urchins contains five pairs of retractor and protractor muscles which are responsible for lantern displacement. Using intact retractor or protractor groups, the force-length relations of these muscles were compared in two taxonomically distant species, Paracentrotus lividus and Stylocidaris affinis. The total contractile forces generated by the muscles can be resolved into vertical and horizontal components. It was found that the vertical component of the retractors is maximal at a lantern position which is significantly lower (i.e. more protruded) in Paracentrotus than in Stylocidaris. Total forces generated by the retractors were in both species maximal at or above the lantern `resting positions'. In Paracentrotus alone, the total force-displacement curves tended to be bimodal. It is hypothesized that the retractors of Paracentrotus contain two populations of muscle fibres, one adapted for jaw opening and one for lantern retraction. No significant differences in the properties of the protractors of the two species could be identified. The lantern of Paracentrotus is more mobile than that of Stylocidaris and is able to exploit a wider range of food sources. This investigation has shown that the force-length relations of the lantern muscles match their differing working conditions. Accepted: 3 November 1997     

Cloning and characterization of the ribosomal genes of the sea-urchin Paracentrotus lividus. Heterogeneity of the multigene family

A Paracentrotus lividus genomic library was constructed using sperm DNA prepared from a single animal. The DNA was fragmented by partial digestion with DNase II, sized on a preparative agarose gel and inserted in the Pst I site of pBR 322 by the dG X dC tailing method. Recombinant plasmids containing ribosomal DNA were isolated, a restriction map of the gene was determined and the 18S and 26S transcribed sequences were located by S1 protection mapping. The organization of the ribosomal genes in genomic DNA of individual animals and of a pool of animals was studied by blot-hybridization of the restriction fragments, using as probes nick-translated 32P-labelled cloned ribosomal DNA fragments or 18S and 26S sea-urchin ribosomal RNA. The repeat length of the ribosomal unit was about 10.5 X 10(3) bases. A comparison of the restriction patterns of DNA from different animals showed a marked sequence heterogeneity in the spacer region of these genes. Variations of about 200 base pairs were detectable in the length of the spacer of some individuals.     

Embryotoxicity of fenbendazole in Paracentrotus lividus

The aim of the present work was to evaluate the embryotoxicity of Fenbendazole, a benzimidazole carbamate-derived anthelmintic drug widely employed in Veterinary Medicine, by using the embryonal development of Paracentrotus lividus (sea urchin) as a experimental model. Embryos were obtained by in vitro eggs fertilization and cultured in seawater. Five embryo suspensions were added by Fenbendazole reaching a final concentration of 5 micrograms/l, 7.5 micrograms/l, 10 micrograms/l, 12.5 micrograms/l and 25 micrograms/l; a suspension was kept drug-free as a control. Embryo development was evaluated by microscopical examination of suspensions at 3 and 40 hours. Our results show that a concentration of 5 micrograms/l of the drug determines a considerable delay of the embryonal development in the 95 percent of the elements observed, and a concentration of 25 micrograms/l produces a block of the embryogenesis at the phase of morula and blastula in all embryos. Results confirm that the effects observed are probably due to an extended inhibition of several enzyme complexes of the embryo cells.     

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.     

Rate of protein synthesis in oocytes of Paracentrotus lividus

The rate of protein synthesis of Paracentrotus lividus oocytes in comparison with the rate in unfertilized eggs and embryos has been analyzed, both in vivo and after oocyte and egg isolation. It is suggested that oocytes synthesize proteins at the same rate as unfertilized eggs.     

Cortical modifications in Paracentrotus lividus eggs after ammonia activation

We have shown by electron microscopy that ammonia activation of Paracentrotus lividus eggs alters the inner ultrastructure of cortical granules. If activated eggs are inseminated, they fail to undergo a typical cortical reaction.     

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.     

Cell cooperation in coelomocyte cytotoxic activity of Paracentrotus lividus coelomocytes

The coelomic fluid from the sea urchin Paracentrotus lividus contains several coelomocyte types including amoebocytes and uncoloured spherulocytes involved in immune defences. In the present paper, we show a Ca(2+)-dependent cytotoxic activity for the unfractionated coelomocytes assayed in vitro, with rabbit erythrocytes and the K562 tumour cell line. In a plaque-forming assay, whole coelomocyte preparations as well as density gradient separated coelomocyte populations revealed that cell populations enriched in uncoloured spherulocytes, exerted high cytotoxic activity by releasing lysins in the presence of amoebocytes. This cooperative effect could be dependent on soluble factors released by amoebocytes. With regard to this, we show that an enhanced cytotoxic activity was found by adding the supernatant from sonicated amoebocytes or hemocyte culture medium into spherulocyte preparations.     

The Aristotle's lantern of the sea-urchin Stylocidaris affinis (Echinoida,Cidaridae): functional morphology of the musculo-skeletal system

Summary The Aristotle's lantern, or masticatory apparatus, of regular sea-urchins is a complex musculo-skeletal system which is thought to have contributed significantly to the evolutionary success of these animals. This paper gives an account of the antomical relationships and functional morphology of both skeletal and soft tissue components in the lantern and related structures of the sea-urchin Stylocidaris affinis (Cidaridae), and compares these features with their equivalent in the previously described lantern of the sea-urchin Paracentrotus lividus (Echinidae, Camarodonta). There are major differences in the skeletons of these lanterns which involve mostly the arrangement and morphology of elements participating in movement, i.e. joints and articular surfaces, and which highlight the generally heavier and less mobile nature of the lantern in the Cidaridae. There are remarkably few differences, however, in the microstructure of the skeletal stereom. Significant dissimilarities were found in the anatomical arrangement of muscles and ligamentous structures and in their macro- and microstructure. The implications of these morphological features for the functioning of the lantern of the Cidaridae are discussed in the context of an integrated model of lantern biomechanics.     

Localization of putative nicotinic cholinoreceptors in the early development of Paracentrotus lividus

Experiments are described, showing the presence of putative nicotinic cholinoreceptors in the egg after fertilization. The experiments were carried out on gametes and early embryos of the sea urchin Paracentrotus lividus, by using nicotinic agonists and antagonists. 1 mM Acetylcholine (ACh), 100 microM nicotine, 100 nM alpha-bungarotoxin (alpha-BuTx) and 100 microM curare inhibit sperm motility and fertilization, while they have no effect on unfertilized eggs. The drugs added within 1 min. after the raising of the fertilization layer had stronger effects on cleavage and development; when added more than 15 min. after the raising of the fertilization layer, they had lesser effects on further development up to pluteus stage. In all the experiments, nicotine was the most effective drug. The binding of fluorescein-labelled alpha-BuTx did not point out any affinity sites on unfertilized eggs, while they were localized on the sperms and on the eggs fertilized by sperms, but not on the eggs activated artificially. The binding was prevented by pretreatment of sperms and activated eggs with 10 nM native alpha-BuTx and 10 microM curare. We conclude that, in the fertilized egg, putative nicotinic cholinoreceptors are present, which are able to bind alpha-BuTx and curare. Fertilization by sperms is needed to trigger the formation of alpha-BuTx receptors.