Cytogenetic and developmental toxicity of bisphenol A and bisphenol S in Arbacia lixula sea urchin embryos.

Bisphenol S (BP-S) is one of the most important substitutes of bisphenol A (BP-A), and its environmental occurrence is predicted to intensify in the future. Both BP-A and BP-S were tested for adverse effects on early life stages of Arbacia lixula sea urchins at 0.1 up to 100 µM test concentrations, by evaluating cytogenetic and developmental toxicity endpoints. Embryonic malformations and/or mortality were scored to determine embryotoxicity (72 h post-fertilization). It has been reported in academic dataset that bisphenols concentration reached µg/L in aquatic environment of heavily polluted areas. We have chosen concentrations ranging from 0.1-100 µM in order to highlight, in particular, BP-S effects. Attention should be paid to this range of concentrations in the context of the evaluation of the toxicity and the ecological risk of BP-S as emerging pollutant. Cytogenetic toxicity was measured, using mitotic activity and chromosome aberrations score in embryos (6 h post-fertilization). Both BP-A and BP-S exposures induced embryotoxic effects from 2.5 to 100 µM test concentrations as compared to controls. Malformed embryo percentages following BP-A exposure were significantly higher than in BP-S-exposed embryos from 0.25 to 100 µM (with a ~5-fold difference). BP-A, not BP-S exhibited cytogenetic toxicity at 25 and 100 µM. Our results indicate an embryotoxic potential of bisphenols during critical periods of development with a potent rank order to BP-A vs. BP-S. Thus, we show that BP-A alternative induce similar toxic effects to BP-A with lower severity.

Spatio-temporal patterns of genetic variation in Arbacia lixula, a thermophilous sea urchin in expansion in the Mediterranean.

The genetic structure of 13 populations of the amphiatlantic sea urchin Arbacia lixula, as well as temporal genetic changes in three of these localities, were assessed using ten hypervariable microsatellite loci. This thermophilous sea urchin is an important engineer species triggering the formation of barren grounds through its grazing activity. Its abundance seems to be increasing in most parts of the Mediterranean, probably favoured by warming conditions. Significant genetic differentiation was found both spatially and temporally. The main break corresponded to the separation of western Atlantic populations from those in eastern Atlantic and the Mediterranean Sea. A less marked, but significant differentiation was also found between Macaronesia (eastern Atlantic) and the Mediterranean. In the latter area, a signal of differentiation between the transitional area (Alboran Sea) and the rest of the Mediterranean was detected. However, no genetic structure is found within the Mediterranean (excluding Alboran) across the Siculo-Tunisian Strait, resulting from either enough gene flow to homogenize distance areas or/and a recent evolutionary history marked by demographic expansion in this basin. Genetic temporal variation at the Alboran Sea is as important as spatial variation, suggesting that temporal changes in hydrological features can affect the genetic composition of the populations. A picture of genetic homogeneity in the Mediterranean emerges, implying that the potential expansion of this keystone species will not be limited by intraspecific genetic features and/or potential impact of postulated barriers to gene flow in the region.

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.

Expression and functional activity of neurotransmitter system components in sea urchins' early development.

Reverse-transcription polymerase chain reaction (RT-PCR) investigation of the expression of the components supposedly taking part in serotonin regulation of the early development of Paracentrotus lividus has shown the presence of transcripts of five receptors, one of which has conservative amino acid residues characteristic of monoaminergic receptors. At the early stages of embryogenesis the expressions of serotonin transporter (SERT) and noradrenaline transporter (NET) were also recognized. The activities of the enzymes of serotonin synthesis and serotonin transporter were shown using immunohistochemistry and incubation with para-chlorophenylalanine (PСРА) and 5-hydroxytryptophan (HTP). Pharmacological experiments have shown a preferential cytostatic activity of ligands characterized as mammalian 5-hydroxytryptamine (5-HT)1-antagonists. On the basis of the sum of the data from molecular biology and embryo physiological experiments, it is suggested that metabotropic serotonin receptors and membrane transporters take part in the regulatory processes of early sea urchin embryogenesis.

Identification of ABC transporter genes in gonad tissue of two Mediterranean sea urchin species: black, Arbacia lixula L., and rocky, Paracentrotus lividus L.

Multixenobiotic resistance (MXR) represents an important cellular detoxification mechanism in aquatic organisms as it provides them robustness toward natural and man-made contaminants. Several ABC transporters have major roles in the MXR phenotype - P-gp/ABCB1, MRP1-3/ABCC1-3 and BCRP/ABCG2. In this study, we identified the presence of ABC transporters involved in the MXR mechanism of Arbacia lixula and Paracentrotus lividus. AlABCB1/P-gp, AlABCC3/MRP3, AlABCC9/SUR-like and AlABCG-like transcripts were identified in A. lixula; and PlABCC1/P-gp, PlABCC3/MRP3, PlABCC5/MRP5, and PlABCC9/SUR-like transcripts in P. lividus. For each of the new partial sequences, we performed detailed phylogenetic and identity analysis as a first step toward full characterization and understanding of the ecotoxicological role of these ABC transporters.

Optimizing preservation protocols to extract high-quality RNA from different tissues of echinoderms for next-generation sequencing.

Transcriptomic information provides fundamental insights into biological processes. Extraction of quality RNA is a challenging step, and preservation and extraction protocols need to be adjusted in many cases. Our objectives were to optimize preservation protocols for isolation of high-quality RNA from diverse echinoderm tissues and to compare the utility of parameters as absorbance ratios and RIN values to assess RNA quality. Three different tissues (gonad, oesophagus and coelomocytes) were selected from the sea urchin Arbacia lixula. Solid tissues were flash-frozen and stored at -80 °C until processed. Four preservation treatments were applied to coelomocytes: flash freezing and storage at -80 °C, RNAlater and storage at -20 °C, preservation in TRIzol reagent and storage at -80 °C and direct extraction with TRIzol from fresh cells. Extractions of total RNA were performed with a modified TRIzol protocol for all tissues. Our results showed high values of RNA quantity and quality for all tissues, showing nonsignificant differences among them. However, while flash freezing was effective for solid tissues, it was inadequate for coelomocytes because of the low quality of the RNA extractions. Coelomocytes preserved in RNAlater displayed large variability in RNA integrity and insufficient RNA amount for further isolation of mRNA. TRIzol was the most efficient system for stabilizing RNA which resulted on high RNA quality and quantity. We did not detect correlation between absorbance ratios and RNA integrity. The best strategies for assessing RNA integrity was the visualization of 18S rRNA and 28S rRNA bands in agarose gels and estimation of RIN values with Agilent Bioanalyzer chips.

Natural or naturalized? Phylogeography suggests that the abundant sea urchin Arbacia lixula is a recent colonizer of the Mediterranean.

We present the global phylogeography of the black sea urchin Arbacia lixula, an amphi-Atlantic echinoid with potential to strongly impact shallow rocky ecosystems. Sequences of the mitochondrial cytochrome c oxidase gene of 604 specimens from 24 localities were obtained, covering most of the distribution area of the species, including the Mediterranean and both shores of the Atlantic. Genetic diversity measures, phylogeographic patterns, demographic parameters and population differentiation were analysed. We found high haplotype diversity but relatively low nucleotide diversity, with 176 haplotypes grouped within three haplogroups: one is shared between Eastern Atlantic (including Mediterranean) and Brazilian populations, the second is found in Eastern Atlantic and the Mediterranean and the third is exclusively from Brazil. Significant genetic differentiation was found between Brazilian, Eastern Atlantic and Mediterranean regions, but no differentiation was found among Mediterranean sub-basins or among Eastern Atlantic sub-regions. The star-shaped topology of the haplotype network and the unimodal mismatch distributions of Mediterranean and Eastern Atlantic samples suggest that these populations have suffered very recent demographic expansions. These expansions could be dated 94-205 kya in the Mediterranean, and 31-67 kya in the Eastern Atlantic. In contrast, Brazilian populations did not show any signature of population expansion. Our results indicate that all populations of A. lixula constitute a single species. The Brazilian populations probably diverged from an Eastern Atlantic stock. The present-day genetic structure of the species in Eastern Atlantic and the Mediterranean is shaped by very recent demographic processes. Our results support the view (backed by the lack of fossil record) that A. lixula is a recent thermophilous colonizer which spread throughout the Mediterranean during a warm period of the Pleistocene, probably during the last interglacial. Implications for the possible future impact of A. lixula on shallow Mediterranean ecosystems in the context of global warming trends must be considered.

New sea urchin phylogeography reveals latitudinal shifts associated with speciation.

Where do new species arise? When do they form and how do they diverge from a common ancestor? A new comprehensive study of Arbacia sea urchins provides surprising answers to these questions. By combining mtDNA phylogeographic markers with a nuclear locus(encoding the sperm acrosomal protein bindin) known to be susceptible to high rates of adaptive codon evolution, Lessios et al. (2012) show that new species and lineages arose relatively recently, most often in association with latitudinal shifts between the temperate zones and the tropics, and in one case, in association with a significant geological barrier to gene flow (the rise of the Isthmus of Panama). In addition to the 'where' and 'when' of Arbacia speciation, these new data resolve an important question about 'who' Arbacia species are by revealing extensive allele sharing at both loci between a pair of broadly sympatric nominal species (that should perhaps be considered a single taxon). 'How' Arbacia diverge from each other is less easily resolved: there is no evidence for reinforcement (via selection on bindin) as an important source of divergence between nominal species, and there are few other data to decide among the alternative hypotheses to explain Arbacia speciation.

Phylogeography and bindin evolution in Arbacia, a sea urchin genus with an unusual distribution.

Among shallow water sea urchin genera, Arbacia is the only genus that contains species found in both high and low latitudes. In order to determine the geographical origin of the genus and its history of speciation events, we constructed phylogenies based on cytochrome oxidase I and sperm bindin from all its species. Both the mitochondrial and the nuclear gene genealogies show that Arbacia originated in the temperate zone of the Southern Hemisphere and gave rise to three species in the eastern Pacific, which were then isolated from the Atlantic by the Isthmus of Panama. The mid-Atlantic barrier separated two additional species. The bindin data suggest that selection against hybridization is not important in the evolution of this molecule in this genus. Metz et al. in a previous publication found no evidence of selection on bindin of Arbacia and suggested that this might be due to allopatry between species, which obviated the need for species recognition. This suggestion formed the basis of the conclusion, widely spread in the literature, that the source of selection on sea urchin bindin (where it does occur) was reinforcement. However, the range of Arbacia spatuligera overlaps with that of two other species of Arbacia, and our data show that it is hybridizing with one of them. We found that even in the species that overlap geographically, there are no deviations from selective neutrality in the evolution of bindin.