Inhibitory effects of neurotoxin ß-N-methylamino-L-alanine on fertilization and early development of the sea urchin Lytechinus pictus.

Neurotoxin ß-N-methylamino-L-alanine (BMAA) has been widely detected in diverse aquatic organisms and hypothesized as an environmental risk to neurodegenerative diseases in humans. However, the knowledge of its toxicity to marine organisms requires attention. In the present study, embryos and sperm of the sea urchin, Lytechinus pictus, were used to assess the toxicity of BMAA. Effects of BMAA on fertilization and development of sea urchin embryos were measured, and its impacts on efflux transport of sea urchin blastula were also assayed. Results demonstrated that the fertilization and development of embryos were significantly inhibited by high concentrations of BMAA above 300 µg L-1. The EC50 values indicated by active swimming larvae and total larvae numbers at 96 HPF (hours post fertilization) were 165 µg L-1 (1.4 µmol L-1) and 329 µg L-1 (2.8 µmol L-1), respectively. Additionally, sperm exposed to BMAA for 10 min significantly reduced the fertilization ratio of sea urchin eggs. However, the ABC transport activity on the cytomembrane of sea urchin blastula was not inhibited by the presence of BMAA at 50 µg L-1, even up to 500 µg L-1. Abnormal division and developmental malformations occurred at different developmental stages for sea urchin embryos exposed to BMAA at 500 µg L-1. The inhibitory effects of BMAA on sea urchin embryos were reported at the first time in this study, for which the toxicological mechanisms will be explored in future studies.

Colorimetric method for determining viability of sea urchin sperm applied in toxicity tests.

The aim of this study was to improve the methodological procedure for the evaluation of sea urchin (Lytechinus variegatus) sperm sensitivity in MTT (3-(4,5-dimethylthiazol-2yl)-2,5 diphenyltetrazolium bromide) enzyme reduction assays with the formation of formazan (purple color) in the interior of viable cells. Assays were carried out with the reference toxicants sodium dodecyl sulfate (SDS), copper, zinc, cadmium and ammonium, using a sperm solution previously activated in sea water and a sperm solution prepared in sea water containing 400 µg L-1 verapamil, which enabled activation of the sperm to occur only when exposed to the toxicants. The assays performed with sperm in verapamil presented similar sensitivity to that shown in the fertilization tests with copper, zinc and SDS, while the assays carried out with the pre-activated sperm solution presented greater resistance to the action of the toxicants. It appears that the action of verapamil involves an intracellular effect on the distribution of Ca2+ ions and that the toxicants used prevent the metabolic reactivation of the sperm.

Effects of the toxic benthic dinoflagellate Ostreopsis cf. ovata on fertilization and early development of the sea urchin Lytechinus variegatus.

Blooms of the benthic dinoflagellate Ostreopsis cf. ovata have been recorded with increasing frequency, intensity and geographic distribution. This dinoflagellate produces potent toxins that may cause mortality of marine invertebrates. Adults of sea urchins are commonly affected by O. cf. ovata exposure with evidence of spines loss and high mortality during periods of high dinoflagellate abundances. Here, we report on the effects of the toxic dinoflagellate O. cf. ovata on fertilization and early development of the sea urchin Lytechinus variegatus, a key ecological herbivore. Lytechinus variegatus eggs and sperm were experimentally exposed to different concentrations of Ostreopsis cf. ovata (4, 40, 400, and 4000 cells ml-1) to test the hypothesis that fertilization success, embryonic and larval development of the sea urchin are negatively affected by the toxic dinoflagellate even at low abundances. Reduced fertilization, developmental failures, embryo and larval mortality, and occurrence of abnormal offspring were evident after exposure to O. cf. ovata. Fertilization decreased when gametes were exposed to high O. cf. ovata abundances (400 and 4000 cells ml-1), but just the exposure to the highest abundance significantly reduced fertilization success. Sea urchin early development was affected by O. cf. ovata in a dose-dependent way, high dinoflagellate abundances fully inhibited the early development of L. variegatus. Ostreopsis cf. ovata significantly increased the mortality of sea urchin eggs and embryos in the first hours of exposure (∼1-3 h), regardless of dinoflagellate abundance. Abundances of 400 and 4000 O. cf. ovata cells ml-1 induced significantly higher mortality on sea urchin initial stages in the first hours, and no egg or embryo was found in these treatments after 18 h of incubation. The early echinopluteus larva was only reached in the control and in treatments with low Ostreopsis cf. ovata abundances (4 and 40 cells ml-1). The exposure to O. cf. ovata led to significantly higher occurrence of skeletal anomalies in the early larva of L. variegatus. Interactions of sea urchin gametes and Ostreopsis cells may naturally occur in coastal areas due to the match between O. cf. ovata blooms and L. variegatus reproductive period. Reduced larval density and increased larval abnormalities were observed even at low abundances (4 and 40 cells ml-1) frequently found in tropical environments all year round. The chronic exposure to O. cf. ovata could significantly impact larval fitness, thus compromising recruitment success, and highlight the negative effects of benthic HABs on sea urchin populations and its possible broader ecological implications.

Induction of innate immune gene expression following methyl methanesulfonate-induced DNA damage in sea urchins.

Sea urchins are noted for the absence of neoplastic disease and represent a novel model to investigate cellular and systemic cancer protection mechanisms. Following intracoelomic injection of the DNA alkylating agent methyl methanesulfonate, DNA damage was detected in sea urchin cells and tissues (coelomocytes, muscle, oesophagus, ampullae and gonad) by the alkaline unwinding, fast micromethod. Gene expression analyses of the coelomocytes indicated upregulation of innate immune markers, including genes involved in NF-κB signalling. Results suggest that activation of the innate immune system following DNA damage may contribute to the naturally occurring resistance to neoplastic disease observed in sea urchins.

Developmental effects of two different copper oxide nanomaterials in sea urchin (Lytechinus pictus) embryos.

Copper oxide nanomaterials (nano-CuOs) are widely used and can be inadvertently introduced into estuarine and marine environments. We analyzed the effects of different nano-CuOs (a synthesized and a less-pure commercial form), as well as ionic copper (CuSO4) on embryo development in the white sea urchin, a well-known marine model. After 96 h of development with both nano-CuO exposures, we did not detect significant oxidative damage to proteins but did detect decreases in total antioxidant capacity. We show that the physicochemical characteristics of the two nano-CuOs play an essential role in their toxicities. Both nano-CuOs were internalized by embryos and their differential dissolution was the most important toxicological parameter. The synthesized nano-CuO showed greater toxicity (EC50 = 450 ppb of copper) and had increased dissolution (2.5% by weight over 96 h) as compared with the less-pure commercial nano-CuO (EC50 = 5395 ppb of copper, 0.73% dissolution by weight over 96 h). Copper caused specific developmental abnormalities in sea urchin embryos including disruption of the aboral-oral axis as a result in changes to the redox environment caused by dissolution of internalized nano-CuO. Abnormal skeleton formation also occurred.

4α-Acetoxyamijidictyol - A New Antifeeding Dolastane Diterpene from the Brazilian Brown Alga Canistrocarpus cervicornis.

Chemical investigation of the CH2 Cl2 crude extract from the brown alga Canistrocarpus cervicornis (Dictyotaceae) led to isolation of one new (1) and four previously reported dolastane diterpenes (2-5). Their structures were characterized by 1D- and 2D-NMR spectroscopic techniques, including a full single crystal X-ray diffraction analysis for 1, 2, and 4. In addition, the new structure 1 was assayed as chemical defense inhibiting the feeding by the sea urchin Lytechinus variegatus. This study constitutes an additional report broadening the known spectrum of action and defensive roles of secondary metabolites of the C. cervicornis and Dictyotales species.

EXTENDING THE VIABILITY OF SPERMATOZOA AND EGGS OF THE SEA URCHIN LYTECHINUS VARIEGATUS.

BACKGROUND: The storage of spermatozoa and eggs of the sea urchin Lytecninus variegatus can meet the demand of different human activities. OBJECTIVE: To develop a protocol easy to reproduce for spermatozoa cryopreservation and cooling of the eggs of the sea urchin. MATERIALS AND METHODS: Different formulations of artificial sea water were tested for their effectiveness in the freezing of sea urchin spermatozoa and storage of the eggs. RESULTS: Protocol for freezing of spermatozoa in liquid nitrogen presented the positive results when the cryoprotectant solution was diluted in artificial seawater free of calcium and magnesium. For the conservation of the eggs by cooling, the calcium-free artificial sea water, the calcium- and magnesium-free sea water, and the low-sodium water proved more efficient in preserving the integrity of the eggs. CONCLUSION: The results showed success in the freezing protocol of spermatozoa and cooling of the eggs mainly in artificial calcium- and magnesium-free sea water.

Assessment of microplastic toxicity to embryonic development of the sea urchin Lytechinus variegatus (Echinodermata: Echinoidea).

Apart from the physiological impacts on marine organisms caused by ingesting microplastics, the toxicity caused by substances leaching from these particles into the environment requires investigation. To understand this potential risk, we evaluated the toxicity of virgin (raw) and beach-stranded plastic pellets to the development of embryos of Lytechinus variegatus, simulating transfers of chemical compounds to interstitial water and water column by assays of pellet-water interface and elutriate, respectively. Both assays showed that virgin pellets had toxic effects, increasing anomalous embryonic development by 58.1% and 66.5%, respectively. The toxicity of stranded pellets was lower than virgin pellets, and was observed only for pellet-water interface assay. These results show that (i) plastic pellets act as a vector of pollutants, especially for plastic additives found on virgin particles; and that (ii) the toxicity of leached chemicals from pellets depends on the exposure pathway and on the environmental compartment in which pellets accumulate.

The effects of dietary silver on larval growth in the echinoderm Lytechinus variegatus.

Previous studies have demonstrated that the euryhaline copepod Acartia tonsa is extremely sensitive to dietborne silver (Ag) exposure, with a 20 % inhibition (EC(20)) of survival occurring when copepods are fed algae with 1.6 µg g(-1) dry weight (dw) Ag, corresponding to a waterborne Ag concentration of 0.46 µg l(-1) Ag. In contrast, 43 µg l(-1) Ag is required to elicit similar effects in copepods exposed to Ag by way of water. In the current study, we investigated whether another planktonic marine organism might also be sensitive to dietary Ag. Specifically, we tested larvae of the echinoderm, Lytechinus variegatus in an 18-day study in which larvae were continuously exposed to Ag-laden algae (Isochrysis galbana). After 7 days of exposure, no significant effects were observed on larval growth up to the highest concentration tested (10.68 µg g(-1) dw Ag in algae after exposure to 3.88 µg l(-1) waterborne Ag). After 18 days, significant effects were observed in all Ag treatments resulting in a lowest-observable effect concentration of 0.68 µg g(-1) dw Ag in algae and corresponding waterborne Ag concentration of 0.05-0.07 µg l(-1) Ag (depending on background Ag [see Results]). However, the dose-response relationship was quite flat with a similar level of growth inhibition (approximately 15 %) in all Ag treatments, resulting in an EC(20) of >10.68 µg g(-1) dw Ag in algae (>3.88 µg l(-1) Ag in water). This flat dose-response relationship is characteristic of dietary metal (silver, copper, cadmium, nickel, and zinc) toxicity to copepods as well, although the effect is slightly more robust (approximately 20-30 % inhibition of survival or reproduction). We conclude that echinoderm larvae may be similar to copepods in their sensitivity to dietary Ag, although a better understanding of the mechanisms underlying the apparent flat dose-response relationships is clearly needed.

Acetyl cholinesterase activity and muscle contraction in the sea urchin Lytechinus variegatus (Lamarck) following chronic phosphate exposure.

The common shallow-water sea urchin Lytechinus variegatus is capable of surviving inorganic phosphate exposures as high as 3.2 mg L(-1) and organic phosphate exposures of 1000 mg L(-1) . Nonetheless, chronic exposure to low, medium, and high-sublethal concentrations of organic phosphate inhibits the muscle enzyme acetyl cholinesterase (AChE), responsible for the break down of the neurotransmitter acetylcholine, as well as inhibiting contractions in the muscles associated with the Aristotle's lantern. AChE activity, measured in both a static enzyme assay and by vesicular staining, displayed concentration-dependent declines of activity in individuals maintained in organic phosphate for 4 weeks. The activity of AChE was not adversely affected by exposure to inorganic phosphate or seawater controls over the same time period. Maximum force of muscle contraction and rates of muscle contraction and relaxation also decreased with chronic exposure to increasing concentrations of organic phosphate. Chronic exposure to inorganic phosphates elicited no response except at the highest concentration, where the maximum force of muscular contraction increased compared to controls. These findings indicate that shallow-water populations of Lytechinus variegatus subjected to organic phosphate pollutants may display impaired muscular activity that is potentially related to the inhibition of the muscle relaxant enzyme AChE, and subsequently muscular overstimulation, and fatigue.

Flow-cytometric analyses of viability biomarkers in pesticide-exposed sperm of three aquatic invertebrates.

Toxicity studies on sperm often use fertilization success as the end point. This type of assay can be affected by sperm density, egg quality, and sperm-egg compatibility. Testing sperm viability biomarkers with flow cytometry is a fast, high-throughput technique for seminal analysis. In this study, we detected sperm viability biomarkers with several fluorescent reporter dyes using flow cytometry in three aquatic invertebrates (Crassostrea virginica, Dreissena polymorpha, and Lytechinus variegatus) after exposure to a pesticide and herbicide. The pesticide, Bayluscide, appeared to affect mitochondrial membrane potential in the sperm of all three species, as measured with MitoTracker Red CMXRos. A decrease in the percentage of sperm stained with SYBR-14 (indicating uncompromised plasma membrane) was observed in C. virginica and D. polymorpha sperm exposed to Bayluscide, but propidium iodide staining (indicating compromised plasma membranes) appeared to be inhibited by Bayluscide. Acrosome-reacted sperm, as measured by FITC-PNA, decreased after Bayluscide exposure in C. virginica and D. polymorpha sperm. The herbicide, Roundup Ready To-Use-Plus, did not affect the overall percentages of sperm stained with MitoTracker but did cause an increase in MitoTracker fluorescence intensity at 16 mg/L in D. polymorpha. Roundup also caused significant decreases in SYBR-14 staining, significant increases in propidium iodide staining, and significant increases in FITC-PNA staining in D. polymorpha sperm. By not having to rely on egg availability and optimal sperm density, sperm toxicity can be more accurately assessed with flow cytometry as being directly correlated to sperm viability rather than the possibility of altered toxicity results due to sperm-to-egg compatibility.

The effects of chronic inorganic and organic phosphate exposure on bactericidal activity of the coelomic fluid of the sea urchin Lytechinus variegatus (Lamarck) (Echinodermata: Echinoidea).

The sea urchin Lytechinus variegatus can survive chronic exposure to sodium phosphate (inorganic phosphate) concentrations as high as 3.2 mg L-1, and triethyl phosphate (organic phosphate) concentrations of 1000 mg L-1. However, chronic exposure to low (0.8 mg L-1 inorganic and 10 mg L-1 organic phosphate), medium (1.6 mg L-1 inorganic and 100 mg L-1 organic phosphate) or high (3.2 mg L-1 inorganic and 1000 mg L-1 organic phosphate) sublethal concentrations of these phosphates inhibit bactericidal clearance of the marine bacterium Vibrio sp. Bacteria were exposed to coelomic fluid collected from individuals maintained in either artificial seawater, or three concentrations of either inorganic phosphate or organic phosphate. Sterile marine broth, natural seawater and cell free coelomic fluid (cfCF) were employed as controls. Bacterial survival indices were measured at 0, 24 and 48 h periods once a week for four weeks. Bacteria were readily eliminated from the whole coelomic fluid (wCF) of individuals maintained in artificial seawater. Individuals maintained in inorganic phosphates were able to clear bacteria following a two week exposure period, while individuals maintained at even low concentrations of organic phosphates failed to clear all bacteria from their coelomic fluid. Exposure to phosphates represses antimicrobial defenses and may ultimately compromise survival of L. variegatus in the nearshore environment.

Exogenous hyalin and sea urchin gastrulation. Part III: biological activity of hyalin isolated from Lytechinus pictus embryos.

Hyalin is a large glycoprotein, consisting of the hyalin repeat domain and non-repeated regions, and is the major component of the hyaline layer in the early sea urchin embryo of Strongylocentrotus purpuratus. The hyalin repeat domain has been identified in proteins from organisms as diverse as bacteria, sea urchins, worms, flies, mice and humans. While the specific function of hyalin and the hyalin repeat domain is incompletely understood, many studies suggest that it has a functional role in adhesive interactions. In part I of this series, we showed that hyalin isolated from the sea urchin S. purpuratus blocked archenteron elongation and attachment to the blastocoel roof occurring during gastrulation in S. purpuratus embryos, (Razinia et al., 2007). The cellular interactions that occur in the sea urchin, recognized by the U.S. National Institutes of Health as a model system, may provide insights into adhesive interactions that occur in human health and disease. In part II of this series, we showed that S. purpuratus hyalin heterospecifically blocked archenteron-ectoderm interaction in Lytechinus pictus embryos (Alvarez et al., 2007). In the current study, we have isolated hyalin from the sea urchin L. pictus and demonstrated that L. pictus hyalin homospecifically blocks archenteron-ectoderm interaction, suggesting a general role for this glycoprotein in mediating a specific set of adhesive interactions. We also found one major difference in hyalin activity in the two sea urchin species involving hyalin influence on gastrulation invagination.

Acetylcholinesterase in the sea urchin Lytechinus variegatus: characterization and developmental expression in larvae.

Acetylcholinesterase (AChE) in the echinoid Lytechinus variegatus has been characterized. Kinetic parameters V(max), K(m), K(ss), and b were 2594+/-1048 nmol ATCh hydrolyzed/min/mg tissue wet weight, 185+/-11 microM, 308+/-100 mM, and 0.2, respectively for the substrate ATCh and 17.8+/-6.87 nmol BTCh hydrolyzed/min/mg tissue wet weight, 654+/-424 microM, 36+/-31 mM, and 0.6, respectively for BTCh. Pharmacologic analyses were performed with four inhibitors of cholinesterases, physostigmine, BW284c51, ethopropazine, and iso-OMPA, and yielded IC(50) values of 106+/-4 nM, 718+/-118 nM, 2.57+/-0.6 mM, and >0.0300 M, respectively. Both kinetic and pharmacologic results confirmed the existence of AChE in larval L. variegatus. Dimeric and tetrameric globular forms (determined by velocity sedimentation on sucrose gradients) were present in L. variegatus larvae. Activity of AChE increased significantly as larvae progressed in development from embryos to eight-arm larvae. Acetylcholinesterase activity of F1 larvae derived from sea urchins collected from wild populations and of F1 larvae derived from sea urchins cultured in the laboratory and fed two different diets suggest that the nutritional and/or environmental history of the adult sea urchin affect the developmental progression of AChE activity in the F1 offspring.

Inhibition of sea urchin fertilization by plant lectins.

Effects of plant lectins on sea urchin (Lytechinus variegatus) fertilization and a partial characterization of lectin-binding involved in the process were evaluated. IC50 doses for inhibition of fertilization varied from 4.1 to 135.5 microg/ml when the lectins were pre-incubated with sperms and from 0.7 to 33.4 microg/ml when pre-incubated with eggs. Such effects were reversed when the lectins were heat inactivated. FITC-labeled lectins bound egg surfaces while their denatured forms did not. Glucose/mannose specific lectins bound weaker to eggs when pre-incubated with the glycoprotein bovine lactotransferrin. None of the glycoproteins assayed diminished FITC patterns of the Gal/GalNAc binding lectins. Pre-incubation of Glucose/mannose binding lectins with eggs did not alter binding of Gal/GalNAc lectins. Lectins with distinct competencies for binding monosaccharide and glycoconjugates were able to inhibit sea urchin fertilization.

Cyclodextrin, a probe for studying adhesive interactions.

In this short communication, we introduce alpha-cyclodextrin as a new probe to study mechanisms of adhesive interactions. We show that this cyclic polysaccharide, that consisting of six glucosyl residues linked by alpha-1,4 bonds, was the only sugar of 22 tested that dramatically blocked a specific cellular interaction in the sea urchin embryo (p<0.001 compared with non-sugar controls). A total of 150-400 embryos were sampled for each concentration of each sugar tested. Mechanisms of cellular interactions have been studied for almost a century and they still remain poorly understood. Cyclodextrin is an exciting new tool that can be utilized for investigating these mechanisms.