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In order to study the defense strategies activated by Paracentrotus lividus embryos in response to sub-lethal doses of CdCl2, we compared the induced transcripts to that of control embryos by suppression subtractive hybridization technique. We isolated five metallothionein (MT) cDNAs and other genes related to detoxification, to signaling pathway components, to oxidative, reductive and conjugative biotransformation, to RNA maturation and protein synthesis. RT-qPCR analysis revealed that two of the five P. lividus MT (PlMT7 and PlMT8) genes appeared to be constitutively expressed and upregulated following cadmium treatment, whereas the other three genes (PlMT4, PlMT5, PlMT6) are specifically switched-on in response to cadmium treatment. Moreover, we found that this transcriptional induction is concentration dependent and that the cadmium concentration threshold for the gene activation is distinct for every gene. RT-qPCR experiments showed in fact that, among induced genes, PlMT5 gene is activated at a very low cadmium concentration (0.1 μM) whereas PlMT4 and PlMT6 are activated at intermediate doses (1-10 μM). Differently, PlMT7 and PlMT8 genes increase significantly their expression only in embryos treated with the highest dose (100 μM CdCl2). We found also that, in response to a lethal dose of cadmium (1 μM), only PlMT5 and PlMT6 mRNA levels increased further. These data suggest a hierarchical and orchestrated response of the P. lividus embryo to overcome differential environmental stressors that could interfere with a normal development.

Ascidian swimming larvae are entirely surrounded by a hyaline extracellular coat, called as tunic or test, on which numerous test cells adhere. The functional role played by test cells in larvae of various ascidian species consists in depositing submicroscopic structures known as ornaments and/or substances of proteoglycan nature in the larval test surface. The deposition of ornaments would render the larval test hydrophilic and thus allow the larvae to swim being immersed in sea water. Ultrastructural investigations reported in literature on larvae of Cionidae and Ascididae families have not evidenced the presence of ornaments in the swimming larval test. For these Ascididae families it has been hypothesized that test cells secrete an amorphous substance that would let them to adhere to larval tunic. In order to clarify the functional role played by test cells of swimming larvae of the Ascididae family, ultrastructural and cytochemical investigations have been carried out on test cells of Ascidia malaca swimming larvae. The ultrastructural observations have evidenced that these cells are metabolically active and show an amoeboidic behaviour as they mainly adhere to the surface of the test. Their cytoplasm is characterized by the presence of a Golgi and large granules that gradually empty their contents and release the same on the test surface. The cytochemical investigastions carried out at ultrastructural level have evidenced that the substances secreted by test cells and deposited on the larval test consisting of glycosaminoglycans. According to the data reported in literature the results of the present investigations confirm that the deposition of glycosaminoglycans enables the adhesion of test cells on the surface of larval tunic and would render the Ascidia malaca larva hydrophilic and able to swim being immersed in sea water.

Ascidians represent an intriguing candidate experimental system for studying the effects of environmental stress. We studied TBT effects and probable related pathways were investigated on ascidian embryos by using Western immunoblotting. Among the various signal transduction pathways involved in response to environmental stress, both tyrosine kinase signalling and MAPKs have been played a significant role. To better understand molecular mechanisms after exposure to TBT we studied the two signal transduction pathways above mentioned. Attempting to unravel the molecular effects of TBT-induced on ascidians embryogenesis, TBT treatments carried out in Phallusia mammillata embryos at gastrula stage. We found different levels of tyrosine protein phosphorylation in response to the incubation with TBT in µM range and a remarkable ERK phosphorylation inhibition dose-dependent with 10, 50 and 100 µM of TBT solution. Based on these data, the use of tyrosine phosphorylation leves and MAPK signal transduction could be considered as biomarkers in the response of marine organisms to pollutant.

We studied the effects of various TBT concentrations by assaying the activity of ERK 1/2 (p44/42) and phospho-ERK1/2 (phospho-p44/42), proteins with a key role in ascidian development, and tyrosine kinase-dependent pathway. The effects of this xenobiotic and the role of some signalling mechanisms on ascidian embryos were examined by using Western immunoblotting. The tyrosine phosphorylation pattern in the ascidians Ciona intestinalis and Phallusia mammillata development was examined and different levels of protein phosphorylation were found as a response to TBT at μM range. To determine whether another key signalling pathway was activated, the effects of TBT on the phosphorylation state of a component of tyrosine kinase-mediated signal transduction MAPK, ERK 1/2 (p44/42) were evaluated. Embryos of Ciona intestinalis exposed to 0.1, 0.25 and 0.5 μM TBT showed a slight decrement in the level of phosphorylated ERK, while a remarkable decrement in level of phopshorylated ERK were observed at higher TBT concentrations (0.5 μM to 10 μM). These data indicated that exposures to TBT induced changes in the total pattern of phosphotyrosine and in the phosphorylation levels of ERK 1/2 but there were no changes on the overall level of total ERK in ascidian embryos.

This paper presents a preliminary report on the papillae of the swimming larvae of three ascidian species: Ascidia malaca, Phallusia mammillata and Ciona intestinalis. The investigations, carried out at ultrastructural level and at confocal laser microscope, have evidenced, in the adhesive papillae of the three studied species, three different cell-types: axial columnar cells, collocytes, sensory cells respectively. The adhesive papillae of A. malaca and P. mammillata show central axial columnar cells with long microvilli emerging from the apical edge and extending throughout the hyaline cap. Collocytes are elongated secreting cells, lying in middle-lateral side. Sensory cells have a cilium at the apical side and an axon proceeding from the basal side. The adhesive papillae of C. intestinalis present some differences in the ultrastructure of the axial columnar cells, which bear a big digitiform protrusion, extending throughout the hyaline cap and a lot of microtubules along the cell axis. The investigations, carried out at confocal microscopy, have evidentiated a clear fluorescence in the papillae of the three studied species and a network of nervous fibers projecting from the papillar base up to cerebral vesicle of the cephalenteron. The characteristic of simple and coniforme type and the adhesive and sensorial functions of adhesive papillae of three ascidian species examined are confirmed.

Ascidian swimming larvae bear three peculiar organs of ectodermic origin, named “palps” or adhesive papillae, located in the anterior region of cephalenteron. Term “adhesive” is correlated to one of the function of these structure based on secretion of an adhesive substance which enables swimming larvae to adhere to a substratum. Recently a sensory function has also been described in some Phlebobranchia papillae with a simple morpho-functional organization. There are few ultrastructural investigations in literature, sometimes disputed, able to make clear papillae cells functions. To clarify this problem, a comparative investigation has been carried out, just in this work, about ultrastructural and morpho-functional organizations of adhesive papillae of three ascidian swimming larvae, Ascidia malaca, Phallusia mammillata and Ciona intestinalis. The investigations has been carried out by transmission electronic microscopy and confocal microscopy. Papillae of above-mentionated ascidian swimming larvae, are three in number and they are located at the vertices of a triangular field. According to a recent classification scheme by Burighel and Cloney, they are coniform and non-eversible simple type papillae. In the apex of each papilla there is an hyaline cap with an electron-dense substance made of proteoglycan component. It’s quite certain that this substance is used by ascidian larvae to adhere to the substratum. Ultrastructural investigations carried out on the adhesive papillae of Ascidia malaca and Phallusia mammillata have not emphasize significant ultrastructural differences in the papillae cells body. Ascidia malaca and Phallusia mammillata papillae are made of three types of cells that characterize their functions: a) collocytes, b) axial columnar cells, c) sensory cells. Collocytes, whose ultrastructure is typical of cells with secretory activity, are certainly deputed to form adhesive secretion. They lie in a median or semilateral side of the papillar body. Axial columnar cells that show an elongated shape, lie in the mid-central papillae region and they are characterized by the presence, in their apical part of long microvilli that run along the whole of hyaline cap length. Above a structural supporting role, it is supposed a possible sensory function concerning these cells. Sensory cells presents an ovoidal and elongated shape holding on tight at their base like a funnel lying a lateral and marginal position in papillar body. These type of cells have been described in Ascidia malaca which can be classified as primary sensory neurons. These are characterized by the presence of a single cilium in the apical end and by an axonicprocess at their base extending over papilla. Ultrastructural investigations on the adhesive papillae of Ciona intestinalis confirmed, even if showing some differences, ultrastructural likeness the two above-mentioned species of adhesive papillae. Collocytes with secretory activity, sensory cells and columnar axial cells come into sight also in Ciona intestinalis papillae. The columnar axial cells ultrastructure is different from the Ascidia malaca and Phallusia mammillata papillae. In the apical part of these cells there are not microvilli but digitiform protusions with numerous microtubules inside running paralleling along longitudinal cell axis. The ultrastructure of these cells suggest their lengthening ability to allow digitiform protrusions to put into contact with the cuticular layer of hyaline cap apex, during step before the adhesion. Ultrastructural characteristics suggest that they can also perform a sensorial duty but their mechanism is still unclear. The observation was carried out by confocal microscopy on the ascidian larvae just before or at the beginning of adhesion to the substratum (6-7 hours from hatching). The use of anti-β-tubulin antibody, have shown an extensive nervous network in all three Ascidiae species starting from the papillar base and converging into only one nerve extending up to the cerebral vescicle. These data confirm previous observations carried out with confocal microscopy on Ascidia malaca and Phallusia mammillata. larvae. In Ciona intestinalis adhesive papillae marked with anti-β- tubulin antibody, the fluorescence is more bright than Phallusia mammillata and Ascidia malaca. Probably this result is correlated with apical protrusion rich in microtubule. Ultrastructure observations of some neurons being in the anterior region of cephalenteron, extending from the base of papilla to the cerebral vescicle, have been emphasized in this work. These neurons are in the same position of the ones described like RTEN (rostral trunk epidermal neuron) by Imai and Meinertzhagen and they are formed by cellular ovoidal body extending into a long axon. Ultrastructural of these neurons have pointed out likeness with vertebrate neurons. Concluding, ultrastructural investigations have emphasize only some difference between papillar cells of Ascidia malaca, Phallusia mammillata and Ciona intestinalis larvae, confirming adhesive and sensory functions.

Among the class of organotin compounds, the most well known is tributyltin (TBT). Organotin have many applications, which include use in PVC, as catalyst in chemical reactions, agricultural pesticides and antifungal treatments for textile polymers. In particular TBT is used in marine antifoulant paints to prevent the growth of organisms such as barnacles on the hull of ships. Extensive use in antifouling paints led to the widespread distribution of TBT and its breakdown products in the global marine, sediment and biota. High levels of TBT in the waters were found to have impaired reproduction, by inhibiting embryogenesis and larval development in a variety of marine organisms. Symptoms of the exposure to high levels of TBT in some invertebrates includes the development of male sexual characteristics as a penis and vas deferens by females (imposex). Ascidians are a good model for the study of embryonal development. They are also sensitive bioindicators of habitat degradation. The effects of tributyltin (IV) chloride (TBT chloride) solutions on ascidian embryos of Ciona intestinalis at different stages of development have been described. Previously, we carried out observations with both the light and the electron microscope on Ciona intestinalis embryos and larvae incubated in TBT solutions. This studies showed morphological and ultrastructural modifications of the embryos and larvae after incubation in TBT chloride at different concentrations. To understand molecular effects of TBT-induced on ascidians embryogenesis we have set out to study the effects of TBT at different concentrations, testing the activity of some protein with a basic role in embryonic development. In ascidian embryos, a fibroblast growth factor (FGF)-like signal has been proposed to be involved in induction of notochord and mesoderm formation. A main pathway is a protein kinase transduction pathway, which includes Ras, Raf, mitogenactivated protein (MAP) kinase and extracellular signal-regulated kinase/MAP kinase (ERK). The aim of this work in progress is to understand whether the TBT exposure on ascidian embryos at different stage of development cause alterations in tyrosine phosphorylation pattern and in MAPK activity. Tyrosine phosphorylation promotes cell growth, differentiation and apoptosis, due to activity of receptor tyrosine kinases and furthermore different stressors are known to stimulate tyrosine kinase activity. At first we focused our attention on tyrosine phosphorylation pattern after ascidian embryos to different stage of development TBT treatment. Phosphorylated proteins pattern is evaluated by SDS-PAGE electrophoresis and Western blotting on protein extract of ascidianembryos incubated with TBT, using anti-phosphotyrosine-antibody directed against mammalian phosphotyrosine. Preliminary results showed a different pattern on protein phosphorylation in response to the incubation with TBT in μM range. Since MAPKs play a key role in animal responses to a wide variety of environmental stresses, we have thought to test the role of MAPK pathway proteins such as MAPK p38 (Thr 180 and Tyr 182), p44/42 (Thr 202/Tyr 204) and c-Jun Nterminal kinases (JNK) after TBT treatment.

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This study shows that high methylmercury concentrations are cytotoxic for Styela plicata hemocytes, whereas sublethal concentrations affect immunocyte responses. Moreover, hemocytes exposed to the xenobiotic present a significantly enhanced phenoloxidase activity as revealed in the hemocyte lysate supernatant compared with the control. Although the cytotoxic activity of S. plicata hemocytes toward rabbit erythrocytes is a PO-dependent cell-target reaction due to quinone products, it was significantly decreased by suitable methylmercury concentrations in the medium. The same xenobiotic concentrations decreased the hemocyte phagocytic activity toward yeast. In both the responses cell-target contacts could be affected by methylmercury, whereas the releasing capacity appeared to be unchanged, as indicated by hemocyte PO-release in the medium. Finally, changes in hemocyte shape and spreading capacity were shown. On the basis of the present results, Styela plicata hemocyte responses could be an additional immunotoxicology test using a microplate method that reveals cell morphological changes and spreading capacity. Copyright © 2007 John Wiley & Sons, Ltd.

Un’attenzione particolare è stata rivolta, negli anni più recenti, allo studio degli effetti di un organo metallo contenente stagno, il TBT (IV) chloride, sugli stadi iniziali dello sviluppo embrionale di animali marini. Gli effetti di questo potente inquinante sullo sviluppo e sulla sopravvivenza degli embrioni sono stati proposti come parametri utili per il controllo e il monitoraggio delle acque. Nel presente lavoro sono riportati gli effetti tossici del TBT (IV) chloride sulla neurulazione sull’ascidia Ciona intestinalis, stadio molto importante durante il quale avviene la determinazione del sistema nervoso e la formazione del tubo neurale. L’esposizione degli embrioni allo stadio di neurula iniziale per 1-4 h. con TBT (IV) chloride ad una concentrazione compresa tra 10-5 e 10-7 mol. dm-3 determina l’arresto dello sviluppo. Le osservazioni morfologiche ed ultrastrutturali riportate nel presente lavoro suggeriscono che le cause del blocco della neurulazione possono essere dovute in gran parte nei danni che subiscono i mitocondri e le proteine del citoscheletro dei blastomeri dell’ectoderma neurale. L’inibizione della polimerizzazione e/o la degradazione delle proteine che costituiscono i microtubuli e i microfilamenti del citoscheletro impediscono di fatto i cambiamenti di forma e la mobilità delle cellule, processi che stanno alla base della neurulazione. Anche le gravi anomalie ultrastrutturali riscontrate nei mitocondri debbono essere considerate una causa importante dell’arresto dello sviluppo embrionale in quanto, con la compromissione dell’attività funzionale di questi organuli, vengono inibiti i processi di fosforilazione ossidativa e di sintesi di ATP.

This paper reports the cytotoxic effects of tri-n-butyltin (IV) chloride, TBTCl, on the neurulation process of the ascidian Ciona intestinalis. Exposure of the embryos at early neurula stage in 10−5 and 10−7M TBT (IV) chloride solutions for 1–2 h provoked the irreversible arrest of their development. Morphological and ultrastructural observations suggested that most probably there are two principal causes determining the neurulation process block. The first is due to the TBT effects of inhibiting the polymerization and/or degradation of microfilaments and microtubules, proteins that constitute the cytoskeleton. The lack of orientation and extension of both microtubules and microfilaments of actin prevent the shape changes and mobility of neural plate blastomeres indispensable to the neurulation process. The second cause is certainly determined by the ultrastructural modification which mitochondria undergo. The ultrastructural anomalies showed by these organules are so serious as to impede their proper functionality with consequent inhibition of oxidative phosphorylation and ATP synthesis, remarkable metabolic processes that occur during ascidian neurulation. Copyright © 2005 John Wiley & Sons, Ltd.

The functional role played by test cells in larvae of various ascidian species consists in depositing submicroscopic structures known as ornaments and/or proteoglycan substances on the larval test surface. According to the data reported in the literature, the deposition of ornaments together with proteoglycan substances on the larval test would render the latter hydrophilic and thus allow the larva to swim being immersed in water. Ornament deposition on the larval test does not occur in all the ascidian species. Ultrastructural investigations made on larvae belonging to the Cionidae and Ascididae families, for instance, have failed to evidence the presence of ornaments on the test. For these ascidian families it has been hypothetized that in swimming larvae test cells secrete an amorphous substance that would allow them to adhere to the larval test. In order to ascertain the functional role played by test cells in swimming larvae of the Ascididae family, the presently reported ultrastructural and cytochemical investigations have been made on larvae of Ascidia malaca. Besides suggesting that test cells, tightly adherent to the test surface, present an amoeboidic behaviour, the ultrastructural investigations have evidenced that these cells are still metabolically active. Their cytoplasm, characterized by the presence of a Golgi apparatus actively involved in synthesis, is almost entirely filled with very large granules; some of them gradually empty their contents turning into vacuoles containing scarce residues of electrondense particles. The present ultrastructural observations support the hypothesis that the adhesion of test cells on the larval test could be very likely eased by the secretion of substances synthetized by the Golgi and released through pseudopodes which test cells then wedge into the test. The cytochemical investigations were based on a reaction (fixation in glutaraldehyde-tannic acid) which evidences the presence, at the ultrastructural level, of proteoglycan substances such as glycosaminoglycans (Singley and Solursh, 1980). The reaction has given positive results in test cell granules undergoing emptying, on the outer membrane of the same cells, and on the outer cuticular layer C1 of the larval test. The present investigations, besides confirming the absence of ornament deposition on the test surface by test cells of Ascidia malaca swimming larvae, have evidenced that the secretion products deposited on the larval test surface by test cells consist of glycosaminoglycans, i.e. proteoglycan substances. In agreement with the data reported in the literature, it is hypothetized that the deposition of glycosaminoglycans on the surface of Ascidia malaca larval test makes the larval tunic hydrophilic and thus the larva is able to swim being immersed in water.

The swimming larvae of ascidians are entirely covered by a hyalin coat called tunic, or test. This covering consists of two cuticular layers, C 1 and C 2 , which surround an inner compartment composed of an amorphous hyalin matrix with numerous fibrils dispersed inside. Data from the literature agree on the key role played by the cells of the larval ectodermic layer in the synthesis and secretion of larval test components. In the present article are reported ultrastructural and cytochemical investigations made during test formation in the swimming larva of Ascidia malaca . Besides confirming the role played by ectodermic cells during the early stages of test formation, the investigations highlight the way in which the fibrillar component of the test is synthetized and secreted. At the ultrastructural level it has been evidenced that the C 1 and C 2 cuticular layers originate from the tight packing of fibrils. Based on the data reported in the present study, it is hypothesized that while a relevant part of the fibrils, once secreted, remains dispersed inside the matrix of the inner compartment of the test, quite likely in order to increase its consistency, packing of the remaining fibrils leads to the formation of the C 1 and C 2 cuticular layers. Packing of the fibrils in C 1 and C 2 could be favoured by their chemically adhesive nature. This hypothesis is strongly supported by the herewith reported results of the cytochemical investigations carried out on the test of the swimming larva of A. malaca . The cytochemical PA–TCH–SP reaction has in fact evidenced that both fibril types, i.e. those dispersed inside the inner compartment and those packed in the C 1 and C 2 cuticular layers, are constituted by glycoproteins and/or proteoglycans substances whose adhesive properties are well documented in the literature.