Fertilization and development of Arbacia lixula eggs are affected by osmolality conditions.

The sea urchin Arbacia lixula coexist with Paracentrotus lividus in the Mediterranean, but the two sea urchin species are quite different from each other. Concerning the female gamete, A. lixula eggs are much darker than those of P. lividus due to the characteristic pigmentation. Upon insemination, the fertilization envelope formed by A. lixula eggs is remarkably thinner than that of P. livius eggs, which implies that the cortical organization of the eggs in the two species may be quite different. In this communication, we examined the phenotypic plasticity of A. lixula eggs in the changing osmolality. The plasma membrane, cortical actin cytoskeleton and vesicles are extensively altered in the eggs exposed to 40% seawater for 15 min. When fertilized, the Ca2+ response in these eggs was significantly compromised and the sperm often failed to enter the eggs. Remarkably, the pattern of the Ca2+ response was restored when these eggs were transferring back to the natural seawater before fertilization, while the actin cytoskeleton partially reverted to the original state. Nonetheless, these eggs restored in seawater failed to regain the innate sperm receptivity that allows only one sperm to enter in natural seawater. Thus, the ability to guide monospermic fertilization is lost by water entry into the eggs, and the eggs incorporated either multiple or no sperm. On the other hand, eggs briefly exposed to hypertonic seawater exhibited no evident morphological anomaly. Nonetheless, the monospermic eggs that experienced a brief exposure (15 min) to hypertonic seawater prior to fertilization in natural seawater displayed a subtly altered sperm-induced Ca2+ response and morpho-functional anomaly around the pluteus stage. Our results suggest that A. lixula eggs attain only a limited extent of cytological plasticity, and that the osmolality shock affects the physical nature of the egg surface which in turn affects the developmental programming.

Short-Term Exposure to Storm-Like Scenario Microplastic and Salinity Conditions Does not Impact Adult Sea Urchin (Arbacia punctulata) Physiology.

The effects of microplastic pollution on sea urchins has received little attention despite their ecological and economical importance. This is the first study to focus on adult sea urchins (Arbacia punctulata). These organisms were exposed to storm-like sediment resuspension of microplastic concentrations (9-µm polystyrene 25,000 spheres L-1) combined with salinity reductions (salinity 25 vs. 33) associated with high precipitation. Urchins were exposed to these parameters for 24 h before assessing righting times and for 48 h before assessing oxygen consumption rates. No significant impacts on urchin physiology were observed showing resilience to short-term exposures of storm-like induced microplastics and salinity. No microplastic particles blocked the madreporite pores indicating the active removal of particles by cilia and pedicellariae. Gut tissue samples indicated consumption of microplastics. Studies on more species are urgently required to determine their responses to plastic pollution to inform management decision-making processes.

Copper oxide nanoparticles induce the transcriptional modulation of oxidative stress-related genes in Arbacia lixula embryos.

Copper oxide nanoparticles (CuO NPs) are widely used in various industrial applications, i.e. semiconductor devices, batteries, solar energy converter, gas sensor, microelectronics, heat transfer fluids, and have been recently recognized as emerging pollutants of increasing concern for human and marine environmental health. Therefore, the toxicity of CuO NPs needs to be thoroughly understood. In this study, we evaluated the potential role of oxidative stress in CuO NP toxicity by exploring the molecular response of Arbacia lixula embryos to three CuO NP concentrations (0.7, 10, 20 ppb) by investigating the transcriptional patterns of oxidative stress-related genes (catalase and superoxide dismutase) and metallothionein, here cloned and characterized for the first time. Time- and concentration-dependent changes in gene expression were detected in A. lixula embryos exposed to CuO NPs, up to pluteus stage (72 h post-fertilization, hpf), indicating that oxidative stress is one of the toxicity mechanisms for CuO NPs. These findings provide new insights into the comprehension of the molecular mechanisms underlying copper nanoparticle toxicity in A. lixula sea urchin and give new tools for monitoring of aquatic areas, thus corroborating the suitability of this embryotoxicity assay for future evaluation of impacted sites.

Comparative toxicity of seven rare earth elements in sea urchin early life stages.

The widespread use of rare earth elements (REEs) in a number of technological applications raises unanswered questions related to REE-associated adverse effects. We have previously reported on the multiple impact of some REEs on the early life stages of the sea urchin Paracentrotus lividus. The present investigation was to evaluate REE toxicity to early life stages in two unrelated sea urchin species, Sphaerechinus granularis and Arbacia lixula. The comparative toxicities were tested of seven REEs, namely yttrium, lanthanum, cerium, neodymium, samarium, europium and gadolinium as chloride salts at concentrations ranging from 10-7 to 10-4 M. The evaluated endpoints included developmental defects and cytogenetic anomalies in REE-exposed embryos/larvae, and decreased fertilization success and offspring damage following sperm exposure. The results showed different toxicity patterns for individual REEs that varied according to test species and to treatment protocol, thus showing toxicity scaling for the different REEs. Further, the observed effects were compared with those reported for P. lividus either following embryo or sperm exposures. S. granularis showed a significantly higher sensitivity both compared to A. lixula and to P. lividus. This study provides clear-cut evidence for distinct toxicity patterns among a series of REEs. The differences in species sensitivity at micromolar REE levels may warrant investigations on species susceptibility to impacts along polluted coasts.

Alteration of neurotransmission and skeletogenesis in sea urchin Arbacia lixula embryos exposed to copper oxide nanoparticles.

The extensive use of copper oxide nanoparticles (CuO NPs) in many applications has raised concerns over their toxicity on environment and human health. Herein, the embryotoxicity of CuO NPs was assessed in the black sea urchin Arbacia lixula, an intertidal species commonly present in the Mediterranean. Fertilized eggs were exposed to 0.7, 10 and 20ppb of CuO NPs, until pluteus stage. Interferences with the normal neurotransmission pathways were observed in sea urchin embryos. In detail, evidence of cholinergic and serotoninergic systems affection was revealed by dose-dependent decreased levels of choline and N-acetyl serotonin, respectively, measured by nuclear magnetic resonance (NMR)-based metabolomics, applied for the first time to our knowledge on sea urchin embryos. The metabolic profile also highlighted a significant CuO NP dose-dependent increase of glycine, a component of matrix proteins involved in the biomineralization process, suggesting perturbed skeletogenesis accordingly to skeletal defects in spicule patterning observed previously in the same sea urchin embryos. However, the expression of skeletogenic genes, i.e. SM30 and msp130, did not differ among groups, and therefore altered primary mesenchyme cell (PMC) migration was hypothesized. Other unknown metabolites were detected from the NMR spectra, and their concentrations found to be reflective of the CuO NP exposure levels. Overall, these findings demonstrate the toxic potential of CuO NPs to interfere with neurotransmission and skeletogenesis of sea urchin embryos. The integrated use of embryotoxicity tests and metabolomics represents a highly sensitive and effective tool for assessing the impact of NPs on aquatic biota.

Heavy rare earth elements affect early life stages in Paracentrotus lividus and Arbacia lixula sea urchins.

BACKGROUND: Heavy rare earth elements (HREEs) have been scarcely studied for their toxicity, in spite of their applications in several technologies. Thus HREEs require timely investigations for their adverse health effects. METHODS: Paracentrotus lividus and Arbacia lixula embryos and sperm were exposed to trichloride salts of five HREEs (Dy, Ho, Er, Yb and Lu) and to Ce(III) as a light REE (LREE) reference to evaluate: 1) developmental defects (% DD) in HREE-exposed larvae or in the offspring of HREE-exposed sperm; 2) mitotic anomalies; 3) fertilization success; and 4) reactive oxygen species (ROS) formation, and nitric oxide (NO) and malondialdehyde (MDA) levels. Nominal HREE concentrations were confirmed by inductively coupled plasma mass spectrometry (ICP-MS). RESULTS: HREEs induced concentration-related DD increases in P. lividus and A. lixula larvae, ranging from no significant DD increase at 10-7M HREEs up to ≅100% DD at 10-5M HREE. Larvae exposed to 10-5M Ce(III) resulted in less severe DD rates compared to HREEs. Decreased mitotic activity and increased aberration rates were found in HREE-exposed P. lividus embryos. Significant increases in ROS formation and NO levels were found both in HREE-exposed and in Ce(III) embryos, whereas only Ce(III), but not HREEs resulted in significant increase in MDA levels. Sperm exposure to HREEs (10-5-10-4M) resulted in a concentration-related decrease in fertilization success along with increase in offspring damage. These effects were significantly enhanced for Dy(III), Ho(III), Er(III) and Yb(III), compared to Lu(III) and to Ce(III). CONCLUSION: HREE-associated toxicity affected embryogenesis, fertilization, cytogenetic and redox endpoints showing different toxicities of tested HREEs.

TRICAINE METHANESULFONATE (MS-222) SEDATION AND ANESTHESIA IN THE PURPLE-SPINED SEA URCHIN (ARBACIA PUNCTULATA).

The purple-spined sea urchin ( Arbacia punctulata ) is commonly found in shallow waters of the western Atlantic Ocean from the New England area of the United States to the Caribbean. Sea urchins play a major role in ocean ecology, echinoculture, and biomedical research. Additionally, sea urchins are commonly displayed in public aquaria. Baseline parameters were developed in unanesthetized urchins for righting reflex (time to regain oral recumbency) and spine response time to tactile stimulus. Tricaine methanesulfonate (MS-222) was used to sedate and anesthetize purple-spined sea urchins and assess sedation and anesthetic parameters, including adhesion to and release from a vertical surface, times to loss of response to tactile stimulus and recovery of righting reflex, and qualitative observations of induction of spawning and position of spines and pseudopodia. Sedation and anesthetic parameters were evaluated in 11 individuals in three circumstances: unaltered aquarium water for baseline behaviors, 0.4 g/L MS-222, and 0.8 g/L MS-222. Induction was defined as the release from a vertical surface with the loss of righting reflex, sedation as loss of righting reflex with retained tactile spine response, anesthesia as loss of righting reflex and loss of tactile spine response, and recovery as voluntary return to oral recumbency. MS-222 proved to be an effective sedative and anesthetic for the purple-spined sea urchin at 0.4 and 0.8 g/L, respectively. Sodium bicarbonate used to buffer MS-222 had no measurable sedative effects when used alone. Anesthesia was quickly reversed with transfer of each individual to anesthesia-free seawater, and no anesthetic-related mortality occurred. The parameters assessed in this study provide a baseline for sea urchin anesthesia and may provide helpful comparisons to similar species and populations that are in need of anesthesia for surgical procedures or research.

Effect of silver nanoparticles on Mediterranean sea urchin embryonal development is species specific and depends on moment of first exposure.

With the ever growing use of nanoparticles in a broad range of industrial and consumer applications there is increasing likelihood that such nanoparticles will enter the aquatic environment and be transported through freshwater systems, eventually reaching estuarine or marine waters. Due to silver's known antimicrobial properties and widespread use of silver nanoparticles (AgNP), their environmental fate and impact is therefore of particular concern. In this context we have investigated the species-specific effects of low concentrations of 60 nm AgNP on embryonal development in Mediterranean sea urchins Arbacia lixula, Paracentrotus lividus and Sphaerechinus granularis. The sensitivity of urchin embryos was tested by exposing embryos to nanoparticle concentrations in the 1-100 µg L(-1) range, with times of exposure varying from 30 min to 24 h (1 h-48 h for S. granularis) post-fertilisation which corresponded with fertilized egg, 4 cell, blastula and gastrula development phases. The most sensitive species to AgNP was A. lixula with significant modulation of embryonal development at the lowest AgNP concentrations of 1-10 µg L(-1) with high numbers of malformed embryos or arrested development. The greatest impact on development was noted for those embryos first exposed to nanoparticles at 6 and 24 h post fertilisation. For P. lividus, similar effects were noted at higher concentrations of 50 µg L(-1) and 100 µg L(-1) for all times of first exposure. The S. granularis embryos indicated a moderate AgNP impact, and significant developmental abnormalities were recorded in the concentration range of 10-50 µg L(-1). As later post-fertilisation exposure times to AgNP caused greater developmental changes in spite of a shorter total exposure time led us to postulate on additional mechanisms of AgNP toxicity. The results herein indicate that toxic effects of AgNP are species-specific. The moment at which embryos first encounter AgNP is also shown to be an important factor in the development of abnormalities, and future applications of the sea urchin embryo development test for nanoparticle toxicity testing should carefully address the specific phase of development of embryos when nanoparticles are first introduced.

Developmental abnormalities and neurotoxicological effects of CuO NPs on the black sea urchin Arbacia lixula by embryotoxicity assay.

The embryotoxicity of CuO NPs was evaluated in the black sea urchin Arbacia lixula embryos, by using 24-well plates. Fertilized eggs were exposed to five doses of CuO NPs ranging from 0.07 to 20 ppb, until pluteus stage. CuO NPs suspensions in artificial seawater formed agglomerates of 80-200 nm size, and copper uptake was 2.5-fold up in larvae exposed to high NP concentrations in respect to control. Developmental delay and morphological alteration, including skeletal abnormalities, were observed, as well as impairment in cholinergic and serotonergic nervous systems. These findings suggest the potential of CuO NPs to interfere with the normal neurotransmission pathways, thus affecting larval morphogenesis. Overall, the embryotoxicity tests are effective for evaluation of nanoparticle effects on the health of aquatic biota. Furthermore, as the black sea urchin A. lixula demonstrated to be vulnerable to NP exposure, it may be a valid bioindicator in marine biomonitoring and ecotoxicological programmes.

Assessing the toxicity of chemical compounds associated with land-based marine fish farms: the sea urchin embryo bioassay with Paracentrotus lividus and Arbacia lixula.

In aquaculture, disinfection of facilities, prevention of fish diseases, and stimulation of fish growth are priority goals and the most important sources of toxic substances to the environment, together with excretory products from fish. In the present study, embryos of two species of sea urchin (Paracentrotus lividus and Arbacia lixula) were exposed to serial dilutions of six antibiotics (amoxicillin (AMOX), ampicillin, flumequine (FLU), oxytetracycline (OTC), streptomycin (ST), and sulfadiazine [SFD]) and two disinfectants (sodium hypochlorite (NaClO) and formaldehyde [CH(2)O]). Alterations in larval development were studied, and the effective concentrations (ECs) were calculated to evaluate the toxicity of the substances. Both species showed similar sensitivities to all substances tested. Disinfectants (EC(50) = 1.78 and 1.79 mg/l for CH(2)O; EC(50) = 10.15 and 11.1 mg/l for NaClO) were found to be more toxic than antibiotics. AMOX, OTC, and ST caused <20 % of alterations, even at the highest concentrations tested. FLU was the most toxic to P. lividus (EC(50) = 31.0 mg/l) and SFD to A. lixula (EC(50) = 12.7 mg/l). The sea urchin bioassay should be considered within toxicity assessment-monitoring plans because of the sensitivity of larvae to disinfectants.

Parental dietary effect on embryological development response to toxicants with the sea urchin Arbacia punctulata.

The role of echinoid parental nutrition in early-life stage toxicity is not well understood. Arbacia punctulata were fed either a fresh diet consisting of organic lettuce and carrots or a dry feed. Embryos from parents fed the dry feed exhibited lower sensitivity to copper, whereas the opposite occurred with 1,3,5-trinitrobenzene and sodium dodecyl sulfate (SDS). EC(50) values for the dry and fresh feed treatments, respectively, were 41.0 and 29.9 microg/L for copper, 0.5 and 1.8 mg/L for 1,3,5-trinitrobenzene, and 3.5 and 5.6 mg/L for SDS. The data suggests that nutritional standardization for sea urchins in ecotoxicological laboratories needs to be addressed and further investigated.

Benthic herbivores are not deterred by brevetoxins produced by the red tide dinoflagellate Karenia brevis.

Gulf of Mexico blooms of the dinoflagellate Karenia brevis produce neurotoxic cyclic polyethers called brevetoxins. During and after a red tide bloom in southwestern Florida, K. brevis cells lyse and release brevetoxins, which then sink to the benthos and coat the surfaces of seagrasses and their epiphytes. We tested the possibility that these brevetoxin-laden foods alter the feeding behavior and fitness of a common benthic herbivore within Floridean seagrass beds, the amphipod Ampithoe longimana. We demonstrated that coating foods with K. brevis extracts that contain brevetoxins at post-bloom concentrations (1 microg g(-1) drymass) does not alter the feeding rates of Florida nor North Carolina populations of A. longimana, although a slight deterrent effect was found at eight and ten-fold greater concentrations. During a series of feeding choice assays, A. longimana tended not to be deterred by foods coated with K. brevis extracts nor with the purified brevetoxins PbTx-2 and PbTx-3. Florida juveniles isolated with either extract-coated or control foods for 10 days did not differ in survivorship nor growth. A similar lack of feeding response to brevetoxin-laden foods also was exhibited by two other generalist herbivores of the southeastern United States, the amphipod A. valida and the urchin Arbacia punctulata. Given that benthic mesograzers constitute a significant portion of the diet for the juvenile stage of many nearshore fishes, we hypothesize that the ability of some mesograzers to feed on and retain brevetoxins in their bodies indicates that mesograzers may represent an important route of vertical transmission of brevetoxins through higher trophic levels within Gulf of Mexico estuaries.

Seasonal variation in the toxicity of sediment-associated contaminants in Corpus Christi Bay, TX.

Seasonal variation in pore water and sediment-water interface (SWI) toxicity at two sites of suspected contamination was investigated using sea urchin embryological development (Arbacia punctulata) and copepod hatching success (Schizopera knabeni). Site S1, located inside a marina, was fine-grained and S2, located near a neighboring stormwater outfall, was sandy. Both sites were cored in summer and winter, along with reference sites of equivalent grain sizes. Whereas the muddy contaminated site displayed a slight decrease in sea urchin toxicity from summer to winter, pore water from the sandy contaminated site exhibited an increase in sea urchin toxicity. The sandy sites displayed slight toxicity increase to copepod hatching success in winter, which occurred in the SWI of S2 and in the pore water from the reference site, R2, where it coincided with sedimentation. Cu was the most prevalent metal in the sediment samples. Although Cu concentration increased in the whole sediments and pore waters from summer to winter, its concentration in overlying SWI exposure decreased from summer to winter. Organic compounds exhibited opposite behavior, with smaller variety identified in the winter pore water samples, but more chemicals detected in the SWI samples in winter.

Embryotoxic effects of nonylphenol and octylphenol in sea urchin Arbacia lixula.

Nonylphenol (NP) and octylphenol (OP), both of which are biodegradation products of alkylphenols, are widely used in industrial applications and in some domestic products. These chemicals are found widely in surface water and aquatic sediments. We have carried out a comparative embryotoxicity analysis of the effects of increasing concentrations of NP (seven concentrations ranging from 0.937 to 18.74 microg/l) and OP (six concentrations ranging from 5 to 160 microg/l) on embryos of the sea urchin Arbacia lixula. The indicators evaluated were larval malformations, developmental arrest and embryonic/larval mortality. The results revealed that low concentrations of these chemicals (NP, OP) generally caused malformations in the skeletal system. High concentrations (18.74 microg NP/l, 160 microg OP/l) were found to inhibit the growth of embryos in the early life stages by preventing mitosis. We conclude that NP and OP present a major risk to the normal development of A. lixula at the low concentrations that have been recorded in the environment. These chemicals are therefore most likely to represent an ecological hazard at the population level given the cumulative effects of other environmental pollutants.