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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.

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.