Paola Cirino

Cryptic speciation in a model invertebrate chordate

We applied independent species concepts to clarify the phylogeographic structure of the ascidian Ciona intestinalis, a powerful model system in chordate biology and for comparative genomic studies. Intensive research with this marine invertebrate is based on the assumption that natural populations globally belong to a single species. Therefore, understanding the true taxonomic classification may have implications for experimental design and data management. Phylogenies inferred from mitochondrial and nuclear DNA markers accredit the existence of two cryptic species: C. intestinalis sp. A, genetically homogeneous, distributed in the Mediterranean, northeast Atlantic, and Pacific, and C. intestinalis sp. B, geographically structured and encountered in the North Atlantic. Species-level divergence is further entailed by cross-breeding estimates. C. intestinalis A and B from allopatric populations cross-fertilize, but hybrids remain infertile because of defective gametogenesis. Although anatomy illustrates an overall interspecific similarity lacking in diagnostic features, we provide consistent tools for in-field and in-laboratory species discrimination. Finding of two cryptic taxa in C. intestinalis raises interest in a new tunicate genome as a gateway to studies in speciation and ecological adaptation of chordates.

Accumulation and embryotoxicity of polystyrene nanoparticles at early stage of development of sea urchin embryos Paracentrotus lividus.

Nanoplastic debris, resulted from runoff and weathering breakdown of macro- and microplastics, represents an emerging concern for marine ecosystems. The aim of the present study was to investigate disposition and toxicity of polystyrene nanoparticles (NPs) in early development of sea urchin embryos (Paracentrotus lividus). NPs with two different surface charges where chosen, carboxylated (PS-COOH) and amine (PS-NH2) polystyrene, the latter being a less common variant, known to induce cell death in several in vitro cell systems. NPs stability in natural seawater (NSW) was measured while disposition and embryotoxicity were monitored within 48 h of postfertilization (hpf). Modulation of genes involved in cellular stress response (cas8, 14-3-3ε, p-38 MAPK, Abcb1, Abcc5) was investigated. PS-COOH forms microaggregates (PDI > 0.4) in NSW, whereas PS-NH2 results are better dispersed (89 ± 2 nm) initially, though they also aggregated partially with time. Their respectively anionic and cationic nature was confirmed by ζ-potential measurements. No embryotoxicity was observed for PS-COOH up to 50 μg mL(-1) whereas PS-NH2 caused severe developmental defects (EC50 3.85 μg mL(-1) 24 hpf and EC50 2.61 μg mL(-1) 48 hpf). PS-COOH accumulated inside embryos digestive tract while PS-NH2 were more dispersed. Abcb1 gene resulted up-regulated at 48 hpf by PS-COOH whereas PS-NH2 induced cas8 gene at 24 hpf, suggesting an apoptotic pathway. In line with the results obtained with the same PS NPs in several human cell lines, also in sea urchin embryos, differences in surface charges and aggregation in seawater strongly affect their embryotoxicity.

A mutational approach to the study of development of the protochordate Ciona intestinalis (Tunicata, Chordata)

Abstract We have developed a protocol to perform a genetic screen for zygotic mutations affecting embryogenesis on the protochordate Ciona intestinalis. The choice of this taxon, whose phylogenetic position places it at the basis of the chordates as one the most primitive vertebrate relatives, could allow to address several evolutionary questions. The protochordates share many morphological features with the vertebrates, in primis the presence of a notochord. Ciona intestinalis shows several ideal features for a mutational analysis, such as external development and larvae made of a limited number of cells and cell types. Detailed cell lineage studies are available. The haploid genome size is comparable to the size of the Drosophila haploid genome. We have optimised conditions for chemical mutagenesis studying the efficiency at which different concentration of N-ethyl-N-nitrosourea (ENU) can induce mutations. Because the adult Ciona are hermaphrodites, we are performing a one-generation screen. The induced mutations are identified by visual inspection of developmental stages. We report the preliminary results from our screen including examples of the different classes of mutant phenotypes found so far.

Natural variation of model mutant phenotypes in Ciona intestinalis.

Background The study of ascidians (Chordata, Tunicata) has made a considerable contribution to our understanding of the origin and evolution of basal chordates. To provide further information to support forward genetics in Ciona intestinalis, we used a combination of natural variation and neutral population genetics as an approach for the systematic identification of new mutations. In addition to the significance of developmental variation for phenotype-driven studies, this approach can encompass important implications in evolutionary and population biology. Methodology/Principal Findings Here, we report a preliminary survey for naturally occurring mutations in three geographically interconnected populations of C. intestinalis. The influence of historical, geographical and environmental factors on the distribution of abnormal phenotypes was assessed by means of 12 microsatellites. We identified 37 possible mutant loci with stereotyped defects in embryonic development that segregate in a way typical of recessive alleles. Local populations were found to differ in genetic organization and frequency distribution of phenotypic classes. Conclusions/Significance Natural genetic polymorphism of C. intestinalis constitutes a valuable source of phenotypes for studying embryonic development in ascidians. Correlating genetic structure and the occurrence of abnormal phenotypes is a crucial focus for understanding the selective forces that shape natural finite populations, and may provide insights of great importance into the evolutionary mechanisms that generate animal diversity.

Maternal Exposure to Cadmium and Manganese Impairs Reproduction and Progeny Fitness in the Sea Urchin Paracentrotus lividus

Metal contamination represents one of the major sources of pollution in marine environments. In this study we investigated the short-term effects of ecologically relevant cadmium and manganese concentrations (10-6 and 3.6 x 10-5 M, respectively) on females of the sea urchin Paracentrotus lividus and their progeny, reared in the absence or presence of the metal. Cadmium is a well-known heavy metal, whereas manganese represents a potential emerging contaminant, resulting from an increased production of manganese-containing compounds. The effects of these agents were examined on both P. lividus adults and their offspring following reproductive state, morphology of embryos, nitric oxide (NO) production and differential gene expression. Here, we demonstrated that both metals differentially impaired the fertilization processes of the treated female sea urchins, causing modifications in the reproductive state and also affecting NO production in the ovaries. A detailed analysis of the progeny showed a high percentage of abnormal embryos, associated to an increase in the endogenous NO levels and variations in the transcriptional expression of several genes involved in stress response, skeletogenesis, detoxification, multi drug efflux processes and NO production. Moreover, we found significant differences in the progeny from females exposed to metals and reared in metal-containing sea water compared to embryos reared in non-contaminated sea water. Overall, these results greatly expanded previous studies on the toxic effects of metals on P. lividus and provided new insights into the molecular events induced in the progeny of sea urchins exposed to metals.

Subtle reproductive impairment through nitric oxide-mediated mechanisms in sea urchins from an area affected by harmful algal blooms

The health of the sea urchin Paracentrotus lividus, a key species in the Mediterranean Sea, is menaced by several pressures in coastal environments. Here, we aimed at assessing the reproductive ability of apparently healthy P. lividus population in a marine protected area affected by toxic blooms of Ostreospsis cf. ovata. Wide-ranging analyses were performed in animals collected prior to and during the bloom, as well as at several times thereafter, during the reproductive season. Adults showed a low fertilization rate, along with high nitric oxide (NO) levels in the gonads and the nitration of the major yolk protein toposome, which is an important player in sea urchin development. Serious developmental anomalies were observed in the progeny, which persist several months after the bloom. NO levels were high in the different developmental stages, which also showed variations in the transcription of several genes that were found to be directly or indirectly modulated by NO. These results highlight subtle but important reproductive flaws transmitted from the female gonads to the offspring with the NO involvement. Despite a recovery along time after the bloom, insidious damages can be envisaged in the local sea urchin population, with possible reverberation on the whole benthic system.

Immune and reproductive system impairment in adult sea urchin exposed to nanosized ZnO via food

In marine environment the release and the consequent sedimentation of ZnO NPs, mainly used in sunscreens, could provoke toxic effects in particular in grazer organisms, like sea urchins. In this work, a first evaluation of DNA and cellular effects on adult sea urchins Paracentrotus lividus exposed through the diet to different sizes (100 and 14nm) ZnO NPs, was performed. Moreover, the consequent impact upon offspring quality was evaluated. Preliminarily results showed that the assumption of food containing ZnO NPs 100nm provoked in adult echinoids damages to immune cells (33% of damaged nucleus) and transmissible effects to offspring (75.5% of malformed larvae). Instead food with ZnO NPs 14nm provoked 64% of damaged nucleus in immune cells and 84.7% of malformed larvae.

Investigating sperm cryopreservation in a model tunicate, Ciona intestinalis sp. A.

In cryopreservation procedures, the capacity to protect the cells from freezing and thawing processes is sensitive to the choice of the cryoprotective agent (CPA) and to its optimal concentration. The advancement of research on Tunicate model species has raised interest in liquid nitrogen cryopreservation for the storage and distribution of genetic resources. Ciona intestinalis (Linnè, 1767) consists of a complex of cryptic taxa that are central to several areas of investigation, from comparative genomics to invasive biology. Here we investigated how five CPAs, three chilling rates and two freezing rates influence semen cryopreservation in C. intestinalis sp. A. By using larval morphology and motility as endpoints, we estimated that long term semen storage requires 10% dimethyl sulfoxide as a protective agent, -1°C/min chilling rate (18°C to 5°C) and -13°C/min freezing rate (5°C to -80°C), followed by immersion in liquid nitrogen.

Control of feeding behaviour in Aplysia depilans by haemolymphatic glucose

Glucose solutions (160 mg/kg and 320 mg/kg body weight) were injected into the haemolymphatic sinuses of Aplysia depilans through an implanted cannula, and the effects on feeding behaviour were noted. The feeding parameter examined was the ingestion velocity of a standard weed ribbon (Ulva): it was evaluated from the time taken to ingest each of ten successive centimetres of the ribbon. The ingestion velocity was found to decrease in a dose‐dependent manner following glucose injection. No irregularities or alterations in the shape of the feeding curves were induced. The effect became progressively larger during the 60 minutes following injection, and appeared to level off afterwards.

Further investigations into sex reversal of Coris julis L. (Pisces, Labridae)

Abstract The morpho-histological analysis of 102 Coris julis kept under standard environmental conditions for more than three months and used for behavioural investigations has shown correlations between colour phases and gonadic state. Stages in transformation from ovary to testis are described.