Flow cytometry analysis of the circulating haemocytes from Biomphalaria glabrata and Biomphalaria tenagophila following Schistosoma mansoni infection R. L. MARTINS-SOUZA 1 , C. A. J. PEREIRA 1 , P. M. Z. COELHO 2 , O. A. MARTINS-FILHO 2 and D. NEGRA ˜ O-CORRE ˆ A 1 * 1 Departamento de Parasitologia, Instituto de Cie ˆncias Biolo ´gicas (ICB), Universidade Federal de Minas Gerais (UFMG), Belo Horizonte – MG. Brasil 2 Centro de Pesquisas Rene ´ Rachou (CPRR-Fiocruz), Belo Horizonte, Minas Gerais, Brasil (Received 9 July 2008; revised 5 September and 23 September 2008; accepted 24 September 2008) SUMMARY Aiming to further characterize the haemocyte subsets in Biomphalaria snails, we have performed a detailed flow cytometric analysis of whole haemolymph cellular components using a multiparametric dual colour labelling procedure. Ethidium bromide/acridine orange fluorescence features were used to first select viable haemocytes followed by flow cytometric morphometric analysis based on the laser scatter properties (forward scatter-FSC and side scatter-SSC). Our findings demonstrated that B. glabrata (BG-BH, highly susceptible to S. mansoni) and 2 strains of B. tenagophila (BT-CF, mod- erately susceptible and BT-Taim, resistant to S. mansoni) have 3 major circulating haemocyte subsets, referred to as small, medium and large haemocytes. The frequency of small haemocytes was higher in BG-BH, while medium haemocytes were the most abundant cell-type in both B. tenagophila strains. Schistosoma mansoni infection resulted in early reduction of large and medium circulating haemocytes followed by an increase of small haemocytes. Although parasite infection induced haemocyte alterations in all Biomphalaria strains, the response was particularly intense in BT-Taim, the parasite-resistant snail. Interestingly, the trematode infection induces changes in haemocytes with less granular rather than in those with more granular profile. The results indicated that, in B. tenagophila of Taim strain, circulating haemocytes, especially the medium and high subset with less granular profile, are very reactive cells upon S. mansoni infection, suggesting that this cell subset would participate in the early parasite destruction observed in this snail strain. Key words: Biomphalaria, Schistosoma mansoni, haemocyte, flow cytometry, defence system, molluscs. INTRODUCTION Biomphalaria glabrata and B. tenagophila are Brasilian fresh water Planorbids of great medical relevance as intermediate hosts of Schistosoma man- soni, a trematode parasite that causes human schisto- somiasis, a disease that affects about 8 million people in Brazil (Paraense, 2001). B. glabrata has an internal defence system (IDS) consisting of soluble components of haemolymph and circulating cells, termed haemocytes, which work in association during the snail responses against infectious agents (van der Knaap and Loker, 1990). In snails, circulating haemocytes, especially the phagocytic cell population, are the principal line of cellular defence involved in destruction of S. mansoni larvae inside the intermediate host (Bayne et al. 1980 ; Noda and Loker, 1989 a ; Zelck and Becker, 1992; Ottaviani, 1992 ; Adema et al. 1994 ; Sapp and Loker 2000 ; Negra ˜o-Corre ˆa et al. 2007). Therefore, the characterization of circulating haemocytes is fun- damental for understanding differences in the resist- ance of Biomphalaria species to S. mansoni. Most of the studies (Harris, 1975; Lo Verde et al. 1982; Lie et al. 1987 ; Barraco et al. 1993) have reported that B. glabrata circulating haemocytes are composed of at least 2 cell populations, based mainly on morphologi- cal and functional aspects : the hyalinocytes and the granulocytes. However, ultrastructural (Matricon- Gondran and Letorcart, 1999) and biochemical (Granath and Yoshino, 1983) analyses indicated that circulating granulocytes are very heterogeneous cells and could be involved in different processes during snail infection. In agreement with the studies on B. glabrata, previous studies reported that circulating haemo- cytes from B. tenagophila are also composed of hyalinocytes and granulocytes (Martins-Souza et al. 2003). Further analysis showed that injection of silica into B. tenagophila resulted in temporary reduction of the granulocyte subset and increase in the snail * Corresponding author : Departamento de Parasitologia, Instituto de Cie ˆncias Biolo ´ gicas (ICB), Universidade Federal de Minas Gerais (UFMG), Av. Presidente Anto ˆ nio Carlos 6627, Campus Pampulha, 31270-901, Belo Horizonte, MG, Brasil. Tel: +55 31 3409 2855. Fax: +55 31 3409 2970. E-mail : denegrao@icb.ufmg.br 67 Parasitology (2009), 136, 67–76. f 2009 Cambridge University Press doi:10.1017/S0031182008005155 Printed in the United Kingdom