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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.