External amebocytes guard the pharynx entry in a tunicate (Ascidiacea) Francesca Cima * , Loriano Ballarin, Fabio Gasparini, Paolo Burighel Dipartimento di Biologia, Universita ` di Padova, Via U. Bassi 58/B, 35100 Padova, Italy Received 27 June 2005; revised 4 July 2005; accepted 22 July 2005 Available online 24 August 2005 Abstract In the present report, we describe the identification of unusual free amebocytes, completely exposed to seawater, which inhabit the inner surface of the oral and atrial siphons of the compound ascidian Botryllus schlosseri (Urochordata). The origin and biological role of these cells were studied by cytochemical and ultrastructural analysis. These amebocytes are mononucleate cells, with numerous round granules, varying in content, and long filopodia, which contact the cuticle protrusions of the tunic in the siphon. Histochemical, histoenzymatic and immunohistochemical assays were carried out under light microscopy on sections and on living and fixed cultured hemocytes. Results showed that the phagocytic blood cells and the free amebocytes of the siphons shared: (i) affinity for the a-mannose specific agglutinin of Narcissus pseudonarcissus (NPA), (ii) occurrence of hydrolytic activities of acid phosphatase and non-specific esterases inside lysosomal vesicles and large vacuoles, (iii) membrane labeling with the lipophilic dye PKH26 specific for phagocytic cells, (iv) anti-CD39 immunocytochemical labeling specific for lysosomes of mammalian macrophages. All histochemical data support the hypothesis that these cells are ‘sentinel cells’ belonging to the hyaline amebocyte population of the phagocytic differentiation line of the immunocytes, since they can also recognize and phagocytize carmine experimentally administered as target particles. q 2005 Elsevier Ltd. All rights reserved. Keywords: Ascidian; Botryllus; Wandering cells; External immunocytes; Phagocytes; Pharynx 1. Introduction Ascidians are marine filter-feeding organisms in which a flow of seawater enters the pharynx through the oral siphon and exits through the cloacal siphon. The siphons are not only simple openings of the filtering and digestive apparatus, but also complex organs endowed with musculature, innervation and blood lacunae, in the form of two cylindrical dorso-lateral projections of the animal’s body. In the colonial species Botryllus schlosseri, each zooid has its own oral siphon which opens independently at the periphery, whereas the atrial siphon converges together with the siphons of nearby zooids, grouped in star-shaped systems, in a central cloacal chamber which opens externally into a common cloacal siphon. A current of seawater created by the movement of the cilia covering the voluminous branchial pharynx enters across the oral siphon Developmental and Comparative Immunology 30 (2006) 463–472 www.elsevier.com/locate/devcompimm 0145-305X/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2005.07.004 * Corresponding author. Tel.: C39 049 827 6196; fax: C39 049 827 6199. E-mail address: francesca.cima@unipd.it (F. Cima).