Impacts of Petroleum Fuels on Fertilization and Development of the Antarctic Sea Urchin Sterechinus neumayeri.

Antarctic marine environments are at risk from petroleum fuel spills as shipping activities in the Southern Ocean increase. Knowledge of the sensitivity of Antarctic species to fuels under environmentally realistic exposure conditions is lacking. We determined the toxicity of 3 fuels, Special Antarctic Blend diesel (SAB), marine gas oil (MGO), and intermediate fuel oil (IFO 180) to a common Antarctic sea urchin, Sterechinus neumayeri. Sensitivity was estimated for early developmental stages from fertilization to the early 4-arm pluteus in toxicity tests of up to 24 d duration. The effects of the water accommodated fractions (WAFs) of fuels were investigated under different exposure scenarios to determine the relative sensitivity of stages and of different exposure regimes. Sensitivity to fuel WAFs increased through development. Both MGO and IFO 180 were more toxic than SAB, with median effect concentration values for the most sensitive pluteus stage of 3.5, 6.5, and 252 µg/L total hydrocarbon content, respectively. Exposure to a single pulse during fertilization and early embryonic development showed toxicity patterns similar to those observed from continuous exposure. The results show that exposure to fuel WAFs during critical early life stages affects the subsequent viability of larvae, with consequent implications for reproductive success. The sensitivity estimates for S. neumayeri that we generated can be utilized in risk assessments for the management of Antarctic marine ecosystems. Environ Toxicol Chem 2020;39:2527-2539. © 2020 SETAC.

The Effect of Photobiomodulation on the Sea Urchin Paracentrotus lividus (Echinodermata) Using Higher-Fluence on Fertilization, Embryogenesis, and Larval Development: An In Vitro Study.

OBJECTIVE: The aim of this study was to investigate the photobiomodulation (PBM) effect of the 808 nm diode laser irradiation on spermatozoa, eggs, fertilized eggs, embryos, and larvae of Paracentrotus lividus, using two different power settings. BACKGROUND DATA: Studies have shown the possible use of PBM in artificial insemination. These have shown the potential effect of low-power laser irradiation on spermatozoa, while there are few studies on the effect of laser photonic energy on oocytes and almost no reports on the influence of lasers in embryogenesis. METHODS: P. lividus gametes, zygotes, embryos, and larvae were irradiated using the 808 nm diode laser (fluence 64 J/cm2 using 1 W or 192 J/cm2 with 3 W) with a flat-top hand-piece delivery, compared to a control without laser irradiation (0 J/cm2-0 W). The fertilization rate and the early developmental stages were investigated. RESULTS: The fertilization ability was not affected by the sperm/egg irradiation. At the gastrula stage, no significant differences were observed compared with the control samples. In the late pluteus stage, there were no differences in the developmental percentage observed between the control and the treated samples (1 W), with the exception of larvae from gastrulae and larvae, which were irradiated at 3 W. CONCLUSIONS: This study has demonstrated that both the 64 J/cm2-1 W and the 192 J/cm2-3 W do not induce morphological damage on the irradiated P. lividus gametes whose zygotes generate normal embryos and larvae. Our data therefore support the assumption to use higher fluence in preliminary studies on in vitro fertilization.

Biogeographic and phylogenetic effects on feeding resistance of generalist herbivores toward plant chemical defenses.

Many terrestrial and most marine herbivores have generalist diets, yet the role that evolutionary history plays in their foraging behaviors is poorly documented. On tropical hard-bottom reefs, generalist fishes and sea urchins readily consume seaweeds that produce lipophilic secondary metabolites. In contrast, herbivores on temperate reefs less commonly encounter seaweeds with analogous metabolites. This biogeographic pattern suggests that tropical herbivores should evolve greater feeding resistance to lipophilic defenses relative to temperate herbivores, but tests of this biogeographic pattern are rare. We offered lipophilic extracts from nine subtropical seaweeds at two concentrations to sea urchins (four subtropical and three cold-temperate populations) and quantified urchin feeding resistance. Patterns of feeding resistance toward lipophilic defenses were more similar within genera than across genera of urchins, indicating a substantial role for phylogenetic history in the feeding ecology of these generalist herbivores. The biogeographic origin of urchins also influenced feeding resistance, as subtropical species displayed greater feeding resistance than did temperate species. Similarly, a subtropical population of Arbacia punctulata had greater feeding resistance for Dictyota and Stypopodium extracts relative to temperate A. punctulata. We conclude that evolutionary history plays a more central role in the foraging ecology of generalist herbivores than is currently appreciated.

Terpenoid metabolites from Spongia spp. and their effects on nucleic acid biosynthesis in sea urchin eggs.

19-Norspongia-13(16),14-diene-3-one (1) was isolated for the first time from a natural source, along with a series of known spongiane diterpenoids (2-11) and sesquiterpene (12) from two unidentified species belonging to the genus Spongia. The effects of 1, 4, 5, 8-12 on biosynthesis of nucleic acids and embryonic development of the sea urchin Strongylocentrotus intermedius have been studied. All the compounds inhibit sea urchin embryo development at concentration of 20 microg/mL and above and DNA biosynthesis at the dose of 10 microg/mL. The inhibitory effect of diterpenoids at least partly may be explained by the inhibition of thymidine kinase activity.

Metal content and toxicity of produced formation water (PFW): study of the possible effects of the discharge on marine environment.

A preliminary chemical and ecotoxicological assessment was performed on the produced formation water (PFW) and superficial sediment around a gas platform (Fratello Cluster), located in the Adriatic Sea (Italy), in order to evaluate the effects of PFW discharged from the installation. The ecotoxicological bioassays, with the marine bacterium Vibrio fischeri and the sea urchin Paracentrotus lividus, were associated with chemical data to estimate the possible effects on living organisms. PFW collected on the platform was toxic, but no significant effect was recorded on marine sediment. Only the sediment station nearest to the discharge point showed higher values of some contaminants (zinc and arsenic) in comparison to other sites and only some stations showed low toxicity.

Synaptotagmin I is involved in the regulation of cortical granule exocytosis in the sea urchin.

Cortical granules are stimulus-dependent secretory vesicles found in the egg cortex of most vertebrates and many invertebrates. Upon fertilization, an increase in intracellular calcium levels triggers cortical granules to exocytose enzymes and structural proteins that permanently modify the extracellular surface of the egg to prevent polyspermy. Synaptotagmin is postulated to be a calcium sensor important for stimulus-dependent secretion and to test this hypothesis for cortical granule exocytosis, we identified the ortholog in two sea urchin species that is present selectively on cortical granules. Characterization by RT-PCR, in-situ RNA hybridization, Western blot and immunolocalization shows that synaptotagmin I is expressed in a manner consistent with it having a role during cortical granule secretion. We specifically tested synaptotagmin function during cortical granule exocytosis using a microinjected antibody raised against the entire cytoplasmic domain of sea urchin synaptotagmin I. The results show that synaptotagmin I is essential for normal cortical granule dynamics at fertilization in the sea urchin egg. Identification of this same protein in other developmental stages also shown here will be important for interpreting stimulus-dependent secretory events for signaling throughout embryogenesis.

Tolerance to biodegraded crude oil in marine invertebrate embryos and larvae is associated with expression of a multixenobiotic resistance transporter.

The toxicity of water-soluble fractions of biodegraded crude oil (BWSF) to embryos and larvae of two marine invertebrates, the white sea urchin (Lytechinus anamesus) and the fat innkeeper (Urechis caupo), was studied. Santa Barbara Channel crude oil was artificially weathered and subjected to biodegradation using a mixed microbe culture obtained from natural oil seep sites. The degradation culture inoculated with seep sediment microbes accumulated 43.7 microg/l water-soluble hydrocarbons. In contrast water-soluble fractions from the non-degraded cultures (NWSF) only accumulated 3.05 microg/l. BWSF proved deleterious to Lytechinus embryo development at low concentrations (EC50 = 0.33 mg/l) but was essentially non-toxic to Urechis embryos/larvae up to 3.0 mg/l. An established mechanism for handling of a wide array of xenobiotics in Urechis embryos is the multixenobiotoic resistance transporter multixenobiotic response (MXR, also known as multidrug resistance, MDR). This mechanism is primarily mediated by ATP-dependent, efflux pumps that extrude a wide array of xenobiotic compounds. In this study, we show that Lytechinus larvae do not appear to express MXR efflux protein nor MXR mediated dye efflux capacity. In contrast, BWSF acts as a competitive inhibitor of MXR transport-mediated dye efflux in Urechis larvae. These results suggest that MXR may be an important mechanism for extrusion of the by-products of crude oil degradation by microbes, and that the level of its expression may determine the susceptibility of organisms to degraded oil hydrocarbons.

The cytotoxic and embryotoxic effects of kaurenoic acid, a diterpene isolated from Copaifera langsdorffii oleo-resin.

In this work, we studied the effects of kaurenoic acid, a diterpene isolated from the oleo-resin of Copaifera langsdorffii in developing sea urchin (Lytechinus variegatus) embryos, on tumor cell growth in microculture tetrazolium (MTT) test and on mouse and human erythrocytes in hemolysis assay. Continuous exposure of embryos to kaurenoic acid starting immediately after fertilization inhibited the first cleavage (IC(50): 84.2 microM) and progressively induced embryo destruction (IC(50): 44.7 microM and < 10 microM for blastulae and larvae stages, respectively). In MTT assay, kaurenoic acid at a concentration of 78 microM produced growth inhibition of CEM leukemic cells by 95%, MCF-7 breast and HCT-8 colon cancer cells by 45% each. Further, kaurenoic acid induced a dose-dependent hemolysis of mouse and human erythrocytes with an EC(50) of 74.0 and 56.4 microM, respectively. The destruction of sea urchin embryos, the inhibition of tumor cell growth and the hemolysis of mouse and human erythrocytes indicate the potential cytotoxicity of kaurenoic acid.

Ciliary protein turnover continues in the presence of inhibitors of golgi function: evidence for membrane protein pools and unconventional intracellular membrane dynamics.

The intimate association of the Golgi apparatus with cilia suggests a functional alliance. To explore the relationship between the synthesis and processing of membrane constituents and the turnover or regeneration of cilia, parallel cultures of gastrula-stage sea urchin embryos were pulse-chase labeled with (3)H-leucine in the presence of monensin, brefeldin A, or colchicine. Steady-state labeled cilia were isolated, and the embryos were allowed to regenerate cilia, which were then isolated after the equivalent of two normal regeneration times. Regeneration was absent in colchicine, minimal in monensin, and inhibited about 40% by brefeldin A. Both monensin and brefeldin A effectively inhibited the post-translational processing of prominent phosphatidylinositoylated and palmitoylated membrane proteins and the axoneme-associated transmembrane Spec3 protein, yet most other membrane plus matrix and 9+2 axonemal proteins were labeled to levels indistinguishable from untreated controls. However, total protein analysis of the membrane plus matrix fractions showed a substantial increase in glycoproteins and the calsequestrin-like protein ECaSt/PDI after treatment at steady-state with all three inhibitors and after regeneration in brefeldin A. Other constituents of this compartment, such as membrane-associated tubulin, calmodulin, and a 53-kDa calcium-binding protein, were unchanged. Therefore, inhibition of Golgi function via three different mechanisms left 9+2 protein turnover undiminished but resulted in an accumulation, in the cilium, of already-processed membrane pool constituents and a normally ER-resident protein. A disproportionate elevation of HSP70 suggests that a novel stress response may be involved in inhibiting ciliary regeneration or promoting glycoprotein augmentation.

EGTA treatment causes the synthesis of heat shock proteins in sea urchin embryos.

Paracentrotus lividus embryos, at post-blastular stage, when subjected to a rise in temperature from physiologic (20 degrees C) to 31 degrees C, synthesize a large group of heat shock proteins (hsps), and show a severe inhibition of bulk protein synthesis. We show, by mono- and two-dimensional electrophoresis, that also EGTA (ethylene glycol-bis[beta-aminoethyl ether] tetraacetic acid) treatment induces in sea urchin embryos both marked inhibition of bulk protein synthesis and the synthesis of the entire set of hsps. Furthermore, EGTA-treated sea urchin embryos are able to survive at a temperature otherwise lethal (35 degrees C) becoming thermotolerant. Because incubation with a different calcium-chelator, EDTA (ethylenediaminetetraacetic acid), or in calcium-free medium did not induce hsps synthesis we conclude that the stress response caused by EGTA is not related to its calcium chelator function.

Sea urchin ovoperoxidase: oocyte-specific member of a heme-dependent peroxidase superfamily that functions in the block to polyspermy.

Ovoperoxidase is one of several oocyte-specific proteins that are stored within sea urchin cortical granules, released during the cortical reaction, and incorporated into the newly formed fertilization envelope. Ovoperoxidase plays a particularly important role in this process, crosslinking the envelope into a hardened matrix that is insensitive to biochemical and mechanical challenges and thus providing a permanent block to polyspermy. Here we present the primary structures of two ovoperoxidases as predicted from cDNAs cloned from the sea urchins Strongylocentrotus purpuratus (AF035380) and Lytechinus variegatus (AF035381). We also present a proposed scheme for the post-translational processing of ovoperoxidase based upon comparisons between the cDNA and protein structures and taking into account previously published reports. The sea urchin ovoperoxidase sequences conform to a profile shared by members of a heme-dependent animal peroxidase family, including the mammalian myelo-, lacto-, eosinophil, and thyroid peroxidases. Using in situ RNA hybridizations, we showed that the mRNA of S. purpuratus ovoperoxidase (4 kb) is present exclusively in oocytes, and is turned over rapidly following germinal vesicle breakdown. Taking into account our immunoblot and N-terminal sequencing data along with reports from similar peroxidases, we propose that ovoperoxidases are synthesized in a pre-pro form and proteolytically processed to result in the 70 and 50 kDa forms that are found in the fertilization envelope. The sequence and structural data presented here will facilitate our continuing studies of the biogenesis of cortical granules and the fertilization envelope. Additionally, since ovoperoxidase activities have been reported in a wide range of animals, these cDNAs will be useful in uncovering similar peroxidases used in the fertilization reactions of other metazoan eggs.

Identification of coelomocyte unconventional myosin and its association with in vivo particle/vesicle motility.

Sea urchin coelomocytes undergo an inducible structural transformation from petalloid to filopodial form during the 'clotting' response in sea urchins. Using a petalloid coelomocyte model, stimulated coelomocytes exhibited bidirectional particle/vesicle motility with a broad distribution of velocities, ranging from 0.02 to 0.12 microns s-1 in the outward bound direction. Coelomocytes treated with the microtubule-disrupting drug, nocodazole, continued to exhibit outward particle/vesicle movements along linear paths with an average velocity of 0.028 +/- 0.006 microns s-1. We partially purified a 110 kDa polypeptide possessing K+EDTA-, Ca2(+)-, Mg2(+)- and F-actin-activated Mg(2+)-ATPase activities characteristic of myosin-like motor proteins. The 110 kDa protein immuno-crossreacted with both affinity-purified, anti-brush border unconventional myosin-I polyclonal antibodies and anti-Acanthamoeba myosin head monoclonal antibodies. By indirect immunofluorescence, the 110 kDa unconventional myosin was localized to clusters of particles/vesicles within the perinuclear region of unstimulated coelomocytes, an area containing numerous mitochondria, acidic, lysosomal and Golgi organelles. Indirect immunofluorescence of partially transformed and filopodial coelomocytes detected a diminution of perinuclear staining with a concomitant appearance of stained linear arrays of particles/vesicles, enhanced staining of peripheral lamellae, and staining of the entire length of the filopodia. Subfractionation of unstimulated coelomocyte homogenates on linear sucrose gradients identified distinct peaks of ATPase activity associated with fractions containing conventional and 110 kDa unconventional myosin. Unconventional myosin-containing fractions were found to have numerous particles that stained with anti-brush border unconventional myosin-I antibodies and the lipophilic dye, DiOC6. Thus, coelomocytes demonstrate activatable movements of particles/vesicles in cells devoid of microtubules and possess an unconventional myosin, which may be the motor protein driving particle/vesicle translocation.

Measurement of intracellular pH in sea urchin eggs by 31P NMR.

The intracellular pH (pHi) of sea urchin eggs was measured using 31P nuclear magnetic resonance (NMR) spectroscopy. The mean value of pHi of unfertilized eggs was about 6.2 (H. pulcherrimus and A. crassispina) to 6.3 (P. depressus). In contrast to results of other studies, pHi of sea urchin eggs was not changed after fertilization. During exposure of the eggs to NH4Cl or procaine-containing natural sea water (NSW), however, pHi rose about 0.4-0.8 pH units; the pHi fell to its initial value upon washing the eggs with weak base-free NSW. These changes of pHi by weak base treatment agreed well with the data obtained by other workers. In order to understand the discrepancy of pHi changes in fertilized eggs between NMR data and other measuring procedures, we measured acid production and O2 uptake, so that CO2 accumulation and proton release did not result in alkalinization. the invariance of the fertilized eggs under anaerobic conditions; otherwise NMR showed a different answer from other measuring procedures, because of its particular characteristics such as non-destructivity and compartmentation of pH.