Methods for toxicology studies in echinoderm embryos and larvae.

Sea urchin embryos have been used in toxicological studies for many decades as they are an accepted model system for investigations of chemicals that impact development. Here we describe methods for using pulse-chase experiments to study the impacts of environmental chemicals on early development as well as development of larvae. This includes the application of fluorescence plate assays with living embryos and fluorescent probes to assess cell functions (mitochondrial membrane potential, lysosome abundance, reactive oxygen species, and esterase activity) based on total cell numbers. We also describe how to use some of these fluorescent probes in embryos/larvae with confocal microscopy for the localization of cellular damage in response to toxics exposure. Finally, we assess skeleton formation in sea urchin larvae and present methods for using polarized light microscopy to examine spicule morphology.

Sediment quality assessment in tidal salt marshes in northern California, USA: an evaluation of multiple lines of evidence approach.

The objective of this study was to evaluate the efficacy of integrating a traditional sediment quality triad approach with selected sublethal chronic indicators in resident species in assessing sediment quality in four salt marshes in northern California, USA. These included the highly contaminated (Stege Marsh) and relatively clean (China Camp) marshes in San Francisco Bay and two reference marshes in Tomales Bay. Toxicity potential of contaminants and benthic macroinvertebrate survey showed significant differences between contaminated and reference marshes. Sublethal responses (e.g., apoptotic DNA fragmentation, lipid accumulation, and glycogen depletion) in livers of longjaw mudsucker (Gillichthys mirabilis) and embryo abnormality in lined shore crab (Pachygrapsus crassipes) also clearly distinguished contaminated and reference marshes, while other responses (e.g., cytochrome P450, metallothionein) did not. This study demonstrates that additional chronic sublethal responses in resident species under field exposure conditions can be readily combined with sediment quality triads for an expanded multiple lines of evidence approach. This confirmatory step may be warranted in environments like salt marshes in which natural variables may affect interpretation of toxicity test data. Qualitative and quantitative integration of the portfolio of responses in resident species and traditional approach can support a more comprehensive and informative sediment quality assessment in salt marshes and possibly other habitat types as well.

Potent phototoxicity of marine bunker oil to translucent herring embryos after prolonged weathering.

Pacific herring embryos (Clupea pallasi) spawned three months following the Cosco Busan bunker oil spill in San Francisco Bay showed high rates of late embryonic mortality in the intertidal zone at oiled sites. Dead embryos developed to the hatching stage (e.g. fully pigmented eyes) before suffering extensive tissue deterioration. In contrast, embryos incubated subtidally at oiled sites showed evidence of sublethal oil exposure (petroleum-induced cardiac toxicity) with very low rates of mortality. These field findings suggested an enhancement of oil toxicity through an interaction between oil and another environmental stressor in the intertidal zone, such as higher levels of sunlight-derived ultraviolet (UV) radiation. We tested this hypothesis by exposing herring embryos to both trace levels of weathered Cosco Busan bunker oil and sunlight, with and without protection from UV radiation. Cosco Busan oil and UV co-exposure were both necessary and sufficient to induce an acutely lethal necrotic syndrome in hatching stage embryos that closely mimicked the condition of dead embryos sampled from oiled sites. Tissue levels of known phototoxic polycyclic aromatic compounds were too low to explain the observed degree of phototoxicity, indicating the presence of other unidentified or unmeasured phototoxic compounds derived from bunker oil. These findings provide a parsimonious explanation for the unexpectedly high losses of intertidal herring spawn following the Cosco Busan spill. The chemical composition and associated toxicity of bunker oils should be more thoroughly evaluated to better understand and anticipate the ecological impacts of vessel-derived spills associated with an expanding global transportation network.

Unexpectedly high mortality in Pacific herring embryos exposed to the 2007 Cosco Busan oil spill in San Francisco Bay.

In November 2007, the container ship Cosco Busan released 54,000 gallons of bunker fuel oil into San Francisco Bay. The accident oiled shoreline near spawning habitats for the largest population of Pacific herring on the west coast of the continental United States. We assessed the health and viability of herring embryos from oiled and unoiled locations that were either deposited by natural spawning or incubated in subtidal cages. Three months after the spill, caged embryos at oiled sites showed sublethal cardiac toxicity, as expected from exposure to oil-derived polycyclic aromatic compounds (PACs). By contrast, embryos from the adjacent and shallower intertidal zone showed unexpectedly high rates of tissue necrosis and lethality unrelated to cardiotoxicity. No toxicity was observed in embryos from unoiled sites. Patterns of PACs at oiled sites were consistent with oil exposure against a background of urban sources, although tissue concentrations were lower than expected to cause lethality. Embryos sampled 2 y later from oiled sites showed modest sublethal cardiotoxicity but no elevated necrosis or mortality. Bunker oil contains the chemically uncharacterized remains of crude oil refinement, and one or more of these unidentified chemicals likely interacted with natural sunlight in the intertidal zone to kill herring embryos. This reveals an important discrepancy between the resolving power of current forensic analytical chemistry and biological responses of keystone ecological species in oiled habitats. Nevertheless, we successfully delineated the biological impacts of an oil spill in an urbanized coastal estuary with an overlapping backdrop of atmospheric, vessel, and land-based sources of PAC pollution.

Sperm motility-initiating substance in newt egg-jelly induces differential initiation of sperm motility based on sperm intracellular calcium levels.

Sperm motility-initiating substance (SMIS), a novel motility inducer from newt egg-jelly, is activated by the release from associated jelly substances at the beginning of internal fertilization and affects female-stored sperm. We examined motility initiation kinetics of newt sperm in response to SMIS by monitoring the changes of sperm intracellular calcium ([Ca²(+)](i)). In quiescent non-motile sperm loaded with the Ca²(+) indicator Fluo-4, intracellular free Ca²(+) was observed around mitochondria using confocal scanning laser microscopy. A slight increase in [Ca²(+)](i) occurred simultaneously and transiently at motility initiation in sperm treated with either heated jelly extract (hJE) containing activated SMIS, or a low osmotic solution, which naturally initiates motility in externally-fertilizing amphibians and can initiate motility in urodele sperm. When the increase of [Ca²(+)](i) at motility-initiation was monitored using spectrofluorometry, large increases in [Ca²(+)](i) occurred immediately in the low osmotic solution and within 1.5 min in the hJE. In the intact jelly extract (no heating), small increases of [Ca²(+)](i) irregularly occurred from around 1 min and for about 4 min, during which motility was differentially initiated among sperm. These results indicate that the SMIS induces differential initiation of sperm motility depending on the activational states of the SMIS and its overall activity. The motility initiation in the jelly extract was delayed in sperm whose intracellular Ca²(+) had been chelated with BAPTA-AM. The relative levels of [Ca²(+)](i) were variable with a mean of 414 ± 256 nmol/L among resting sperm, suggesting that the level of [Ca²(+)](i) in the resting sperm modulates the responsiveness to the SMIS.

Impacts of suspended sediments on fertilization, embryonic development, and early larval life stages of the pacific herring, Clupea pallasi.

Pacific herring reproduce in the San Francisco Bay estuary during times of the year when suspended sediment loads are highest due to freshwater input, yet little is known about the effects of sediment on herring early life stages. During the first 2 h after eggs contacted water, embryos were adhesive and susceptible to having sediment particles attach permanently to the chorion. Treatment with suspended San Francisco Bay dredged sediments at ecologically relevant concentrations of 250 or 500 mg/l during this time period increased self-aggregation of the eggs and led to sublethal and lethal effects. After the first 2 h in water, sediments that contacted embryos did not attach to chorions and did not have an observable impact. Sediment treatment during the first 2 h was not linked statistically to declines in fertilization or total larval hatch rate, but it did produce significant sublethal effects that included increases in precocious larval hatch and higher percentages of abnormal larvae, as well as an increase in larval mortality.

Two egg-derived molecules in sperm motility initiation and fertilization in the Pacific herring (Clupea pallasi).

Sperm of the Pacific herring are immotile at spawning. Two egg-derived molecules are capable of initiating sperm motility. One is herring sperm activating protein(s) (HSAPs) and the other is sperm motility initiation factor (SMIF). These two motility initiators differ in their location and association with the chorion, and in their isoelectric points and molecular weights. In this study we have investigated the roles of these two inducers with respect to motility and fertilization. Using computer analysis of sperm motility, we found that HSAPs, as well as the C-terminal HSAPs peptide, elicit a linear motility pattern, while SMIF induced a highly circular and asymmetric pattern. HSAPs induced a two-fold increase in intracellular calcium, whereas SMIF induced a four-fold increase of motility initiation. SMIF-exposed sperm, preloaded with BAPTA-AM, showed a more linear motility and this motility trajectory decreased with their fertilizing capability. The difference in intracellular calcium levels between HSAPs and SMIF is consistent with the observed linear and circular motility. In the absence of SMIF, HSAPs do not support fertilization. Fertilization is rescued in these experiments if SMIF is reintroduced. We propose that diffusible HSAPs are not essential for fertilization, but enhance sperm-egg collisions via linear motility. SMIF, which is bound to the micropylar region of the chorion, is required for fertilization and induces circular motility that is a prerequisite for sperm to enter the micropylar canal and fertilize the egg.

Integrating contaminant responses in indicator saltmarsh species.

A challenge in environmental management is to provide both methodology and a framework for assessing effects of pollutants in resident species and then applying the findings to management. The Pacific Estuarine Ecosystem Indicator Research (PEEIR) consortium advocates the development of an integrated portfolio of techniques using indicator species selected for various habitat types. We developed such a portfolio for California salt marsh ecosystems and evaluated the feasibility of our approach in management applications. PEEIR is employing a suite of biomarker responses in two indigenous species, the lined shore crab (Pachygrapsus crassipes) and the longjaw mudsucker (Gillichthys mirabilis). Detrimental effects such as apoptosis, endocrine disruption, and ovarian tumors have been observed in G. mirabilis at a site where toxicity test responses were relatively low. With P. crassipes, developmental abnormalities and several markers of decreased reproductive performance were quantified at the same site. Multivariate statistical techniques are used to examine the relationships between the responses and multiple contaminant and natural stressors. For the fish, findings are related to population-level parameters using dynamic energy budget (DEB) models.

Polycyclic aromatic hydrocarbons disrupt axial development in sea urchin embryos through a beta-catenin dependent pathway.

Sea urchin (Lytechinus anemesis) embryos were used as an experimental system to investigate the mechanisms of the developmental toxicity of creosote, one of the most widely used wood preserving chemicals, as well as some of its polycyclic aromatic hydrocarbon (PAH) constituents (phenanthrene, fluoranthene, fluorene, pyrene and quinoline). Data suggest that creosote and PAHs affect axial development and patterning in sea urchin embryos by disrupting the regulation of beta-catenin, a crucial transcriptional co-activator of specific target genes in the Wnt/wg signaling pathway. When ciliated blastula stage embryos were exposed to these compounds, they developed into exogastrulae with completely evaginated archentera, demonstrating that these chemicals disrupt axial development and patterning. This response occurred in a dose-dependent fashion, with the EC(50) of creosote for complete exogastrulation being 1.57 ppm, while the EC(50)s of the PAHs ranged from 0.41 ppm (2.0 microM) to 4.33 ppm (33.5 microM). Morphologically, the exogastrulae that developed from embryos exposed to creosote and PAHs appeared to be identical to those that resulted from exposure to lithium chloride, a classical agent known to induce vegetalization and exogastrulation in sea urchin embryos. Immunological studies using antibodies against beta-catenin, a multi-functional protein known to be involved in cell-cell adhesion and cell fate specification during embryonic development, revealed high levels of nuclear accumulation of beta-catenin by cells of creosote- and PAH-exposed embryos, irrespective of their positions in the developing embryo. Dissociated embryonic cells cultured in the presence of these agents rapidly responded in a similar fashion. Since beta-catenin accumulation occurs in nuclei of several types of cancer cells, it is possible this may be a general mechanism by which PAHs affect a variety of different cell types.

Motility initiation in herring sperm is regulated by reverse sodium-calcium exchange.

Sperm of the Pacific herring, Clupea pallasi, are unique in that they are immotile upon spawning in the environment. Herring sperm have evolved to remain motionless for up to several days after spawning, yet are still capable of fertilizing eggs. An egg chorion ligand termed "sperm motility initiation factor" (SMIF) induces motility in herring sperm and is required for fertilization. In this study, we show that SMIF induces calcium influx, sodium efflux, and a membrane depolarization in herring sperm. Sperm motility initiation by SMIF depended on decreased extracellular sodium (<350 mM) and could be induced in the absence of SMIF in very low sodium seawater. Motility initiation depended on > or =1 mM extracellular calcium. Calcium influx caused by SMIF involved both the opening of voltage-gated calcium channels and reverse sodium-calcium (Na(+)/Ca(2+)) exchange. Membrane depolarization was slightly inhibited by a calcium channel blocker and markedly inhibited by a Na(+)/Ca(2+) exchange inhibitor. Sodium efflux caused by SMIF-initiated motility was observed when using both extracellular and intracellular sodium probes. A Na(+)/Ca(2+) exchange antigen was shown to be present on the surface of the sperm, primarily over the midpiece, by using an antibody to the canine Na(+)/Ca(2+) exchanger. This antibody recognized a 120-kDa protein that comigrated with the canine myocyte Na(+)/Ca(2+) exchanger. Sperm of Pacific herring are now shown to use reverse Na(+)/Ca(2+) exchange in motility initiation. This mechanism of regulation of motility initiation may have evolved for both maintenance of immotility after spawning as well as ligand-induced motility initiation.

Sperm motility initiation factor is a minor component of the Pacific herring egg chorion.

Polyclonal antibodies were generated to the 105 kDa herring sperm motility initiation factor (SMIF) and used to explore the role of SMIF in sperm-egg interaction. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting with SMIF antibodies, it was demonstrated that SMIF is present as a minor (4-7% of total chorion protein) component of the chorion. The major polypeptides in the chorion migrated at 117 kDa and in a grouping between 48-54 kDa, with other minor bands above and below. The only detectable glycosylated component was the 105 kDa band, which was resolved at two isoelectric points (8.22 and 8.31) after isoelectric focusing gel electrophoresis. Using antibodies to SMIF, fertilization was blocked, sperm motility was inhibited in vitro in the presence of solubilized SMIF and SMIF binding sites on sperm were localized. Lastly, SMIF was localized to the region of the herring egg that encircles the micropyle.