Search

This chapter was inadvertently published with an incorrect spelling of the author's name as V. Arriza whereas it should be V. Arizza. In addition to this the affiliation of one of the chapter authors Elisse Sutton was published incorrectly and it has now been corrected to read as Department of Biological Sciences, Macquarie University, Sydney, NSW, Australia.

In invertebrates, the recognition of non-self molecules frequently leads to the release of phenoloxidase (PO) by circulating granulocytes, in the form of its precursor, pro-PO, which is quickly activated by soluble serine proteases. This response represents a powerful weapon against potentially pathogenic microorganisms and, in several taxa, independently of their phylogenetic position, the pro-PO activation system is responsible of a massive production of melanin. The latter is supported by the formation of amyloid fibrils as well as other concurrent events, such as ACTH production, NEP overexpression and α-MSH formation. In the colonial ascidian Botryllus schlosseri, allorecognition between contacting, genetically incompatible colonies, leads to the recruitment of haemocytes in the contact region and their degranulation with the consequent release of PO and the production of melanin which is deposited in the necrotic spots along the colony contacting border.

Nano-scale nipple array on the body surface has been described from various invertebrates including endoparasitic and mesoparasitic copepods, but the functions of the nipple array is not well understood. Using the hydrophilized nanopillar sheets made of polystyrene as a mimetic material of the nipple arrays on the parasites’ body surface, we assayed the cell spreading and phagocytosis of the hemocytes of the colonial ascidian Botryllus schlosseri. On the pillared surface, the number of spreading amebocytes and the number of phagocytizing hemocytes per unit area were always smaller than those on the flat surface (Mann-Whitney test, p < 0.05 - 0.001), probably because the effective area for the cell attachment on the pillared surface is much smaller than the area on the flat sheet. The present results supports the idea that the nipple array on the parasites' body surface reduces the innate immune reaction from the host hemocytes.

Morula cells (MCs) represent the most abundant circulating hemocyte of the compound ascidian Botryllus schlosseri. They are cytotoxic cells involved in the rejection reaction between contacting, genetically incompatible colonies. Upon the recognition of foreign substances, they degranulate and release their content, which contribute to the cell death along the contact borders. A major role in MC-related cytotoxicity is exerted by the enzyme phenoloxidase (PO) that converts polyphenol substrata to quinones which, then, polymerize to form melanins. During this reaction, reactive oxygen species are formed which are the cause of MC-related cytotoxicity. Here, we carried out new analyses to investigate further the nature of MC content and its role in cytotoxicity. Results confirm that PO is located inside MC vacuoles together with arylsulfatase, iron and polyphenols/quinones, the latter probably representing ready-to-use cytotoxic molecules, deriving from the oxidation of DOPA-containing proteins. In addition, small DOPA-containing peptides, called tunichromes, are also present inside MCs. MC degranulation and PO-mediated cytotoxicity are prevented by secretion inhibitors and by H89 and calphostin C. The observation that PO activity is always detectable in MCs in the absence of protease treatment, and its inhibition by sulfites and sulfates, suggest a non-classical pathway of PO modulation in botryllid ascidians.

When colonies of the compound ascidian Botrylloides simodensis contact each other at their cut surfaces, either fusion or rejection occurs. Contact between genetically compatible colonies leads to the complete fusion of their tunics and vasculature within 24 h. Conversely, the rejection reaction between incompatible colonies is characterized by the appearance of a melanic, necrotic band along the contact border. In the case of fusion, limited crowding of cytotoxic morula cells (MCs) was observed in the ampullae near the contact border. In rejection, limited tunic fusion occurred in the contact region and MCs were selectively recruited inside the ampullae near the cut surface: most of them leaked into the tunic where they changed their morphology and contributed to the formation of the necrotic region. Granular amebocytes, like MCs, have granules well stained by eosin and were also seen inside the ampullae involved in the rejection reaction and along the contact border between incompatible colonies. Immunohistochemical analysis using antibodies raised against Botryllus schlosseri phenoloxidase (PO) and mammalian IL-1-α and TNF-α indicate that MCs were the only cells recognized by the anti-PO antibody; they resulted immunopositive also to the anti-cytokine antibodies in both fusion and rejection, whereas granular amoebocytes were recognized by the latter antibodies only during the rejection reaction.

In the ascidian Botryllus schlosseri, we studied the effects of hemocyte incubation with foreign cells, such as bacteria, bacterial spores and yeast. In the presence of yeast and bacterial spores, morula cells, a common cell type in botryllid ascidians, changed their morphology, release phenoloxidase in the medium, thus causing an increase in cytotoxicity, and express molecules recognised by anti-IL-1-a- and anti-TNF-a-antibodies. These effects were not observed when hemocytes were incubated with both gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria. Considering that morula cells are the main source of molecules recognised by anti-cytokine-antibodies we suggest an immunosurveillance role of these cells, which may influence immune responses such as phagocytosis.

In the present work, we investigated, in the colonial ascidian Botryllus schlosseri, the role of complement C3 (BsC3) in phagocytosis. We studied the modulation of BsC3 transcription in the course of the colonial blastogenetic cycle, with particular reference to the takeover, when apoptotic cells in the tissues of old zooids are cleared by circulating phagocytes. In situ hybridisation with BsC3 riboprobes labelled only morula cells, the most abundant haemocytes. Anti-hC3 antibody recognised morula cells and also phagocytes when haemocytes were previously incubated with zymosan. The inhibition of C3 activation prevented the labelling of phagocytes. In phagocytosis assays with haemocytes from colonies injected with anti-hC3 antibody or bsc3 iRNA, the capability to ingest target cells was significantly (p < 0.001) reduced. Therefore, our results strongly support a key role of BsC3 in phagocytosis and open to new investigations on the nature of the receptors of the products of BsC3 activation.

Environmental stress conditions are ultimately related to the induction of oxidative stress in organisms, as a consequence of an increased production of reactive oxygen species (ROS). This could be exploited to study sub-lethal effects induced by the environment in the organisms. In the present work, we evaluate the possibility to use the colonial ascidian Botryllus schlosseri as a bioindicator, to assess the environmental quality in the Lagoon of Venice. Three colony batches were immersed, for 22 days, at two sites (1 and 2) with different grades of hydrodynamics and anthropogenic impact and physico-chemical features of seawater; a control batch was kept in a large tank with continuous seawater flow at the Marine Station of the Department of Biology, University of Padova, in Chioggia (site 3). Seawater at site 2 had higher pH and temperature than site 1. Colonies were then retrieved, their mRNA was extracted and the level of transcription of genes involved in oxidative stress response (glutathione synthase, γ-glutamyl-cysteine ligase, modulatory subunit, two isoforms of glutathione peroxidases and Cu/Zn superoxide dismutase) was evaluated. In colonies from sites 1 and 2, most genes showed significantly increased transcriptional levels with respect to control values. Spectrophotometric analyses of colony homogenates revealed that the enzymatic activity of superoxide dismutase and catalase was higher in colonies from site 2 as compared to site 1, allowing us to speculate that colonies in site 2 were under higher stress level than those in site 1. Overall, we can conclude that B. schlosseri seems a good indicator of the ecological status of the Lagoon environment, within a range of pH and temperature in which colonies are used to live.

Antioxidant enzymes are known to protect living organisms against the oxidative stress risk, also induced by metals. In the present study, we describe the purification and molecular characterization of two Cu,Zn superoxide dismutases (SODs), referred to as Ci-SODa and Ci-SODb, from Ciona intestinalis, a basal chordate widely distributed in temperate shallow seawater. The putative amino acid sequences were compared with Cu,Zn SODs from other metazoans and phylogenetic analyses indicate that the two putative Ci-SODs are more related to invertebrate SODs than vertebrate ones. Both phylogenetic and preliminary homology modeling analyses suggest that Ci-SODa and Ci-SODb are extracellular and intracellular isoform, respectively. The mRNA of the two Cu,Zn SODs was localized in hemocytes and in ovarian follicular cells, as revealed by in situ hybridization. The time course of SOD mRNA levels in the presence of three different metals showed upregulation of ci-soda and inhibition of ci-sodb. Spectrophotometric analysis confirms the presence of SOD activity in Ciona tissues. Our in silico analyses of the ci-soda promoter region revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE), suggesting that the transcription of these genes directly depends on metals. These data emphasize the importance of complex metal regulation of ci-soda and ci-sodb transcription, as components of an efficient detoxification pathway allowing the survival of C. intestinalis in continued, elevated presence of metals in the environment.