Nervous system characterization during the development of a basal echinoderm, the feather star Antedon mediterranea

Neural development of echinoderms has always been difficult to interpret, as larval neurons degenerate at metamorphosis and a tripartite nervous system differentiates in the adult. Despite their key phylogenetic position as basal echinoderms, crinoids have been scarcely studied in developmental research. However, since they are the only extant echinoderms retaining the ancestral body plan of the group, crinoids are extremely valuable models to clarify neural evolution in deuterostomes. Antedon mediterranea is a feather star, endemic to the Mediterranean Sea. Its development includes a swimming lecithotrophic larva, the doliolaria, with basiepithelial nerve plexus, and a sessile filter-feeding juvenile, the pentacrinoid, whose nervous system has never been described in detail. Thus, we characterized the nervous system of both these developmental stages by means of immunohistochemistry and, for the first time, in situ hybridization techniques. The results confirmed previous descriptions of doliolaria morphology and revealed that the larval apical organ contains two bilateral clusters of serotonergic cells while GABAergic neurons are localized under the adhesive pit. This suggested that different larval activities (e.g., attachment and metamorphosis) are under the control of different neural populations. In pentacrinoids, the analysis showed the presence of a cholinergic entoneural system while the ectoneural plexus appeared more composite, displaying different neural populations. The expression of three neural-related microRNAs was described for the first time, suggesting that these are evolutionarily conserved also in basal echinoderms. Overall, our results set the stage for future investigations that will reveal new information on echinoderm evo-devo neurobiology

Effects of exposure to microplastics on the development and metamorphosis of Ciona robusta

Microplastics are plastic particles produced to be of small size, as sandblasting media, virgin pellet, cosmetics (Fendall and Sewell, 2009) or that originate from degradation of bigger plastic debris, as polyester fibers, polyethylene plastic bags and polystyrene particles from buoys and floats (Browne et al., 2011; Davidson, 2012; O\u2019Brine and Thompson, 2010). An incredible amount of plastic reaches the marine environment and its ecological impacts are of particular interest. In fact, after ingestion, they may cause damage and block of feeding appendages and digestive system, can migrate from alimentary canal to other body tissues, limit the food intake and transfer pollutants in living organisms (Cole et al., 2013). We evaluated microplastics effects on development and metamorphosis of the solitary ascidian Ciona robusta. We used microbeads with 10 \u3bcm diameter made of polystyrene, because it is highly stable with a negligible styrene migration (Cohen et al., 2002). We tested four different concentrations (0.125, 1.25, 12.5 and 25 \u3bcg/ml) and analyzed the effects on larval development and survival. Then we exposed attached larvae for five days to test the effects on metamorphosis. Juvenile survival was calculated exposing metamorphosed individuals to microplastics for eight days. We also estimated the ingestion rate as the number of particles ingested in one minute and the expulsion capability measuring the localization of particles in stomach or fecal pellets after 24 hours. Metamorphosis was the process most affected by the microplastics. In fact, the percentage of individuals that completed metamorphosis was statistically lower in all the exposed groups, compared to control. Feeding individuals were also fed with microalgae. The co-ingestion of algae and beads probably reduced the amount of algae assimilated by the juveniles. Moreover, they might spend energy to expel microplastics thus reducing the energy available for the metamorphosis. After 8 days, juvenile survival was not affected, even if the particles persisted longer in the stomach of individuals exposed to the highest concentration, suggesting that further detrimental effects could be observed after a longer exposure

Effects of bisphenol A on early development of the ascidian Phallusia mammillata (Chordata, Tunicata)

Bisphenol A (BPA) is an organic compound used in the manufacture of polycarbonate plastic and epoxy resins that is released into the environment from sewage treatment effluent, landfill leachate and degradation of plastic products. BPA can act both as a teratogenic substance and as an endocrine disruptor. The phylogenetic position of tunicates as sister group of vertebrates and their cosmopolitan distribution in marine ecosystems coupled with their ecology and easy manipulability make them reliable model organisms for ecotoxicology bioassays. Here we evaluated the effects of different concentration of BPA (0.1, 0.5, 1, 5, 10, and 20 \ub5M) on ontogenetic processes sensitive to environmental pollutants in the ascidian Phallusia mammillata. To test the effects of this substance on sperm viability we fertilized eggs with sperm pre-exposed for 30 minutes to BPA. One hour post fertilization (hpf) we calculated the percentage of eggs that reached the 2-cell stage compared to control. Then we analyzed the consequences of a fertilization performed directly in BPA solutions, counting the percentage of 2 cell-stage embryos 1 hpf. To test the effects on embryogenesis, we analyzed hatched larvae developed from 2-cell embryos exposed to BPA. We evaluated the number and type of malformations under a dissection microscope and we performed immunostaining of central nervous system (CNS). Exposure of sperm to BPA did not influence fertilization rate. Coexposure of eggs and sperm at concentrations higher than 5 \ub5M caused incomplete division of eggs, producing heart shaped embryos that did not develop further. Regarding embryogenesis, 10 \ub5M BPA caused specific malformations at central nervous system, with an increased distance between otolith and ocellus, or an extrusion of ocellus from the sensory vesicle. Embryonic development was ignificantly altered at 20 \ub5M concentration. 97% of the larvae presented a severely affected phenotype with short and kinked tail. Immunostainig with anti \u3b2-tubulin antibody showed an altered pattern of fibers in CNS. These results showed that the most sensitive process to BPA is the first cell division. When the 2-cell stage embryos are treated, higher concentration are required to alter the correct development of CNS. BPA confirmed its teratogenic effects on ascidians and its interference with CNS development even if the mechanism of action in this group is still to be clarified

Effects of polystyrene microplastics on early stages of two marine invertebrates with different feeding strategies

Nowadays, microplastics represent one of the main threats to marine ecosystems, being able to affect organisms at different stages of their life cycle and at different levels of the food web. Although the presence of plastic debris has been reported in different habitats and the ability to ingest it has been confirmed for different taxa, few studies have been performed to elucidate the effects on survival and development of marine animals. Thus, we explored the effects of different environmental concentrations of polystyrene microbeads on the early stages of two invertebrate species widespread in the Mediterranean shallow waters: the pelagic planktotrophic pluteus larvae of the sea urchin Paracentrotus lividus and the filter-feeding sessile juveniles of the ascidian Ciona robusta. We evaluated the effects on larvae and juvenile development and determined the efficiency of bead ingestion. The feeding stages of both species proved to be extremely efficient in ingesting microplastics. In the presence of microbeads, the metamorphosis of ascidian juveniles was slowed down and development of plutei altered. These results prompted the necessity to monitor the populations of coastal invertebrates since microplastics affect sensitive stages of life cycle and may have consequences on generation recruitment

Effects of Bisphenol A on early development of two ascidian species

Bisphenol A(BPA) is an organic compound present in plastic products that is released into the environment after degradation. BPA is both a teratogenic substance and an endocrine disruptor. The phylogenetic position of tunicates as sister group of vertebrates and their cosmopolitan distribution in marine ecosystems make them reliable model organisms for ecotoxicology bioassays. We tested the effects of different concentration of BPA (0.1, 0.5, 1, 5, 10, and 20 \u3bcM) on sperm viability, fertilization and embryogenesis of two ascidian species, Phallusia mammillata and Ciona intestinalis. We evaluated the type and the incidence of induced malformations. Then we focused on the effects on the nervous system performing animmunostaining of central nervous system (CNS) and whole mount in situ hybridization (WISH) with neural specific markers. Exposure of sperm to BPA did not influence fertilization rate. Co-exposure of eggs and sperm to concentrations higher than 5\u3bcM caused incomplete division of zygotethat did not develop further. In P. mammillata, embryonic development was altered by 20\u3bcM BPA causing a severe phenotype with malformed sensory organs in almost all treated larvae. In C. intestinalis 20 \u3bcM BPA was lethal, while 10 \u3bcM concentration caused alteration to the sensory organs, indicating that C. intestinalis is less tolerant to BPA. Nervous system is a target of BPA action that caused an altered pattern of neural fibers. WISH with Ci-GAD and Ci-TH showed an alterationof dopaminergic and GABAergic cells after exposureto 10 \u3bcM BPA. These results showed that the most sensitive process to BPA is the first cell division. After 2-cell stage, higher concentrations are required to alter the development. BPA confirmed its teratogenic effects on ascidians and its interference with CNS development

Genetic structure of the long-snouted seahorse, Hippocampus guttulatus, in the Central-Western Mediterranean Sea

The seahorse Hippocampus guttulatus reaches its highest abundance in confined environments, where it has unique biological and ecological traits that suggest significant genetic differentiation among populations. In the present study, we aimed to reveal the genetic structure of this species by analysing eight microsatellite loci and a mitochondrial DNA region (cytochrome b) of eight populations from the Central-Western Mediterranean Sea, including lagoon sites. Levels of genetic diversity, as measured by the total number of alleles, number of private alleles, allelic richness and heterozygosity, ranged from low to moderate. The overall value of inbreeding was high, indicating a deficiency in heterozygotes. The haplotype network had a star-like construction, with the most common haplotype present in all populations. Data from the two molecular markers congruently displayed a similar pattern and revealed low genetic differentiation, notwithstanding predictions based on species traits. The observed genetic structure is probably the result of both historical population demographic events and current gene flow. The investigated lagoons, however, revealed a unique genetic profile, which is especially highlighted by the Taranto population. At this site, the results also showed altered values of observed/expected heterozygosity and allelic richness, a characteristic of marginal populations. Our study suggests that lagoon populations should be managed as distinct genetic units

Half versus full vacuum suction drainage after modified radical mastectomy for breast cancer- a prospective randomized clinical trial[ISRCTN24484328]

BACKGROUND: Suction drains are routinely used after modified radical mastectomy and are an important factor contributing to increased hospital stay as the patients are often discharged only after their removal. Amongst various factors that influence the amount of postoperative drainage, the negative suction pressure applied to the drain has been reported to be of great significance. While a high negative suction pressure is expected to drain the collection and reduce the dead space promptly, it may also prevent the leaking lymphatics from closing and lead to increased drainage from the wound. Against this background a prospective randomized clinical study was conducted to compare the amount and duration of drainage between a half negative suction and full vacuum suction drainage in patients following modified radical mastectomy. The associated postoperative morbidity was also compared between the two groups. METHODS: 85 FNAC (fine needle aspiration cytology) proven cases of locally advanced breast cancer were randomized. (Using randomly ordered sealed envelops, which were opened immediately before the closure of the wound) in to 50 patients with full vacuum suction (pressure = 700 g/m2) and 35 cases in to half vacuum suction drainage (pressure = 350 g/m2) groups. The two groups were comparable in respect of age, weight, and technique of operation and extent of axillary dissection. Surgery was performed by the same surgical team comprising of five surgeons (two senior and three resident surgeons) using a standardized technique with electrocautery. External compression dressing was provided over the axilla for first 48 hrs and following that patients were encouraged to do active and passive shoulder exercises. The outcomes measured were postoperative morbidity and the length of hospital stay. Statistical methods used: Descriptive studies were performed with SPSS version 10 and group characteristics were compared using student t-test. RESULTS: Half vacuum suction drains were removed earlier than the full suction vacuum suction drains. There was no significant difference in the incidence of seroma formation in the two groups and there was a significant reduction in the total hospital stay in patients with half vacuum suction drainage systems as compared to the full suction drainage group (p < 0.001) without any added morbidity. CONCLUSIONS: Half negative suction drains provide an effective compromise between no suction and full or high suction drainage after modified radical mastectomy by reducing the hospital stay and the post operative morbidity including post operative seromas

EFFECTS OF POLLUTANTS ORIGINATING FROM PLASTICS ON ASCIDIANS:DEVELOPMENT AND SURVIVAL

Marine pollution was defined as \u201cthe introduction by man of substances or energy into the marine environment, which results or is likely to result in deleterious effects such as harm to marine life\u201d (UNCLOS, 1982). To develop management protocols for pollutants present in marine environment it is important to fill the gap of knowledge about their effects on environmental and human health. This work centers on the effects of different kinds of pollutants that originate from plastics on marine invertebrates. The first part of this study focused on toxic and teratogenic effects of a chemical produced by plastic degradation, Bisphenol A (BPA), particularly considering the effects on nervous system and sensory organs of ascidians. The second part is centered on the impact of microplastics (MPs), on ascidians and sea urchin larvae. BPA is an organic compound used in the manufacturing of polycarbonate plastics and epoxy resins. After the degradation of food and beverage containers made of BPA, it can be released in food and water and be ingested, or reach the environment. Many different negative effects have been reported on human health and on wildlife. In this study, the effects of different concentrations of BPA have been analyzed on ontogenetic processes, embryogenesis, nervous system development and sensory organ differentiation in different ascidian species, namely Ciona robusta, Ciona intestinalis and Phallusia mammillata. The most sensitive developmental step was the first cell division both in C. robusta and P. mammillata. The teratogenic effect of BPA was confirmed in ascidians and the main alterations reported involved GABAergic and dopaminergic neural populations and pigment cells differentiation. Specific alterations of pigmented organs were reported, namely abnormal deposition of pigments, absence of one or both pigmented organs or presence of supernumerary ones. Since similar effects on nervous system and sensory organs were already reported in different vertebrate species, ascidian species confirmed their value as invertebrate animal model for preliminary screenings of effects of pollutants. In C. intestinalis, exposure to BPA altered genes expression of transcription factors involved in central nervous system and pigment cells specification, as Ci-Six3/6 and Ci-Pax6. Similar alteration were previously described as a consequence of perturbation of FGF and Wnt signaling pathways leading to hypothesize that BPA act at some point of the cascade activated by these pathways. At last, the co-exposure to BPA and an inverse agonist of estrogen related receptor (ERR), 4-hydroxytamoxifen (4-OHT), during embryogenesis produced a rescue of normal pigmented organs phenotype in P. mammillata. In zebrafish, it was previously demonstrated that BPA 3 exposure determines otolith malformations through its binding to ERR\u3b3. Our results support the idea that also in ascidians the effects of BPA were mediated by ERR. Plastic debris that reach the marine environment can become a threat to wildlife not only because they release chemicals compound in the seawater, but also because the fragmentation and degradation of these products produce small particles named microplastics (MPs). In this study, the effects of polystyrene micro particles of 1 and 10 \u3bcm of diameter were analyzed on embryonic development of ascidians and sea urchins and on metamorphosis and survival rate of ascidian juveniles. Ascidian lecitotrophic larvae were not affected by the presence of microsphere in culture medium. Filter feeders ascidian juveniles and suspension feeders plutei were both able to ingest MPs. Exposure during the metamorphosis produced a delay of growth in ascidians, since the presence of MPs in the digestive system reduced the food intake. Moreover, the effects on metamorphosis were more strong in presence of 10 \u3bcm beads than of 1 \u3bcm ones, probably because smallest ones can be more easily expelled with feces. Body growth was altered also in sea urchin. When MPs are present at low concentrations (0.125 and 1.25 \u3bcg/ml) juveniles were able to expel them in 24 hours and plutei in 6 hours. However at higher concentrations (12.5 and 25 \u3bcg/ml), MPs persisted longer in the digestive system. Moreover, comparing the intake efficiency of the ascidian and plutei, the sessile filtering organisms appeared more vulnerable to MPs effects than pelagic suspension feeders. At last, 1 \u3bcm MPs were able to translocate from digestive system of ascidian juveniles. Histological analysis revealed that some particles crossed the stomach wall, and some were phagocytized by blood cells. These data highlight that MPs not only negatively impact the feeding ability of marine species, but probably further affect the physiology of organisms after translocation. The wide range of alterations and the possible subsequent negative effects of BPA and MPs make clear that a monitoring of concentrations and distributions of these pollutants in natural environment is required. Moreover, a reduction of the use of plastic products and new policies on plastic wastes are desirable to avoid the increase of distribution of these new pollutants that could negatively impact marine wildlife

Effects of bisphenol A on the development of pigmented organs in the ascidian Phallusia mammillata

Bisphenol A (BPA) is an organic compound that is used in the manufacture of polycarbonate plastic and epoxy resins and is increasingly being released into the environment. BPA can act as a teratogenic substance and an endocrine disruptor, raising concerns about its impact on humans and wildlife. Thus, in the present work, we evaluated the effects of different concentrations of BPA on ontogenetic processes in the ascidian Phallusia mammillata. The phylogenetic position of ascidians, their cosmopolitan distribution in marine ecosystems, and multiple technical advantages associated with their biology make ascidians reliable model organisms for ecotoxicology bioassays. Our investigations showed that BPA did not affect the capability of ascidian sperm to fertilize eggs, but it impaired embryonic development and caused a phenotype that was characterized by a short and kinked tail. Larvae developed from BPA-exposed embryos also presented malformations of pigmented organs such as altered pigment deposition, absence of one or both pigmented organs, and supernumerary organs. The co-exposure with 4-hydroxytamoxifen, an estrogen-related receptor (ERR) inverse agonist, rescued the normal phenotype of pigmented organs, thus supporting the hypothesis that, in ascidians, BPA exerts its teratogenic effects mainly by binding to ERR, as in vertebrate models