Acta Zoologica (Stockholm) 82: 261–274 (October 2001) © 2001 The Royal Swedish Academy of Sciences  Abstract Cima, F., Perin, A., Burighel, P. and Ballarin, L. 2001. Morpho-functional characterization of haemocytes of the compound ascidian Botrylloides leachi (Tunicata, Acidiacea). — Acta Zoologica (Stockholm) 82: 261–274 A morpho-functional study of the colonial ascidian Botrylloides leachi haemocytes was carried out to propose their classification, relationships and specializations. This characterization was obtained by (i) investigations of both living and aldehyde- fixed cells by light and electron microscopy; (ii) cytochemical and cytoenzymatic assays; (iii) lectin-affinity assays; (iv) phagocytosis and haemagglutination assays; and (v) anti-CD34 immunocytochemical assay for vertebrate haematopoietic stem cells. Results indicate that the haemoblast is a circulating stem cell and there are at least five haemocyte differentiation pathways, the last two of which have never been described in botryllids: (i) phagocytic line (hyaline amoebocytes and macrophage-like cells) share ultrastructural features, the same hydrolytic enzymes and WGA lectin binding, and are involved in yeast phagocytosis and erythrocyte rosette formation; (ii) cytotoxic line (granular amoebocytes and morula cells) with vacuoles containing oxidative enzymes and polyphenolic substrates; (iii) vacuolated cell line (pigment cells and nephrocytes) involved in catabolite storage; (iv) compartment cell line (compartment amoebocytes and compartment cells) able to agglutinate erythrocytes and characterized by vacuoles with a moderately electron-dense content, positive to arylsulphatase activity and binding DBA, UEA-I, HPA lectins; and (v) granular cell line includes trophic cells, able to infiltrate the gut epithelium, showing a cytoplasm filled of PAS-positive vacuoles with arylsulphatase, chloroacetylesterase and β-glucuronidase activities. Francesca Cima, Department of Biology, University of Padova, Via Ugo Bassi 58/B, I-35121 Padova, Italy. E-mail: ascilab@civ.bio.unipd.it Blackwell Science, Ltd Morpho-functional characterization of haemocytes of the compound ascidian Botrylloides leachi (Tunicata, Ascidiacea) Francesca Cima, Andrea Perin, Paolo Burighel and Loriano Ballarin Department of Biology, University of Padova, Via Ugo Bassi 58/B, I-35121 Padova, Italy Keywords: Botrylloides leachi, haemocytes, ascidians, ultrastructure, enzymatic activity Accepted for publication: 30 November 2000 Introduction In the last few decades, interest in invertebrate immune responses has led to a great increase in the scientific production dealing with haemocytes and coelomic cells, their classification, mutual relationships and biological roles (see Ratcliffe and Rowley 1981; for reviews). As regards ascidians, although there is abundant literature describing the morphology of both living and fixed haemocytes (Pérès 1943; Endean 1955; Sabbadin 1955; Andrew 1961; Smith 1970a,b; Schlumpberger et al. 1984; Sawada et al. 1991, 1993; Dan-Sohkawa et al. 1995) and their ultrastructure (Overton 1966; Milanesi and Burighel 1978; Scippa et al. 1982; Burighel et al. 1983; Zhang et al. 1992; Azumi et al. 1993; Sawada et al. 1993; Sugino et al. 1993), many doubts and uncertainties still persist on both their classification and differentiation pathways, mainly due to the adoption of different classification criteria for each species studied. Unifying classification schemes have been proposed (Goodbody 1974; Wright 1981; Rowley et al. 1984; De Leo 1992; Burighel and Cloney; 1997). Many biological functions, other than defence reactions, have been ascribed to ascidian haemocytes, such as excretion, nutrition, budding, germ cell formation and tunic synthesis (Goodbody 1974; Wright 1981; Burighel and Cloney 1997). In botryllid ascidians, studies of the morphology and ultrastructure of haemocytes mainly focus on species of the