Cellular aspects of allorecognition in the compound ascidian Botryllus schlosseri Francesca Cima, Armando Sabbadin, Loriano Ballarin * Dipartimento di Biologia, Universita ` di Padova, Via U. Bassi 58/B, 35100 Padova, Italy Received 9 December 2003; revised 11 January 2004; accepted 6 February 2004 Abstract We studied changes in the morphology of morula cells, a common haemocyte type in botryllid ascidians, during both the rejection reaction (occurring between contacting, genetically incompatible colonies) and fusion (occurring between compatible colonies), and in short-term cultures of haemocytes incubated with heterologous or autologous blood plasma. In both the rejection reaction and haemocyte cultures in the presence of heterologous blood plasma, we observed alterations in morula cells, consistent with a degranulation event, and their expression of molecules recognised by anti-IL-1-a- and anti- TNF-a-antibodies. Anti-cytokine-antibodies markedly reduced the extent of the in vitro cytotoxicity, when haemocytes were exposed to heterologous blood plasma. In addition, the increase in the production of nitrite ions and the decrease of the in vitro cytotoxicity by the nitric oxide synthase inhibitor N v -nitro-L-arginine methyl ester, suggest the role of nitric oxide in cell death. These results provide new clues to understand the process of rejection reaction in botryllid ascidians. q 2004 Elsevier Ltd. All rights reserved. Keywords: Ascidian; Botryllus; Allorecognition; Haemocytes; Morula cells 1. Introduction Colonies of the cosmopolitan ascidian species Botryllus schlosseri are formed of several zooids grouped in star-shaped systems, which share a vascular network, branching out in the common tunic, from which many blind, sausage-like ampullae depart. When the leading edges of different colonies contact each other, either fusion of genetically compatible colonies, with the consequent sharing of tunic and circulation, or a rejection reaction between genetically incompatible colonies occurs. The latter is characterised by the appearance of a series of dark brown, necrotic spots along the touching borders [1–4]. According to Sabbadin et al. [3], the rejection reaction is preceded by partial fusion of the contacting tunics, in front of the opposite, facing marginal ampullae, after local disappearance of the respective cuticles. This allows the diffusion of soluble factors from the circulation of one colony to the blood vessels 0145-305X/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2004.02.001 Developmental and Comparative Immunology 28 (2004) 881–889 www.elsevier.com/locate/devcompimm * Corresponding author. Tel.: þ39-049-827-6197; fax: þ 39-049- 827-6199. E-mail address: loriano.ballarin@unipd.it (L. Ballarin). Abbreviations: BA, Botryllus agglutinin; BP, blood plasma; L-NAME, N v -nitro-L-arginine methyl ester; FSW, filtered sea water; TEM, transmission electron microscope.