Hydrobiologia 383: 207–214, 1998. E. Schockaert, N. Watson & J.-L. Justine (eds), Biology of the Turbellaria. ©1998 Kluwer Academic Publishers. Printed in the Netherlands. 207 Acoel spermatozoa: ultrastructure and immunocytochemistry of tubulin Olga I. Raikova 1,2 , Ludmila P. Flyatchinskaya 1 & Jean-Lou Justine 2,∗ 1 Zoological Institute, 199034 St.-Petersburg, Russia 2 Laboratoire de Biologie Parasitaire, Protistologie, Helminthologie, Mus´ eum National d’Histoire Naturelle, 61 rue Buffon, F-75231 Paris cedex 05, France; E-mail:justine@mnhn.fr ( ∗ author for correspondence) Key words: Platyhelminthes, Acoela, spermatozoa, immunocytochemistry, tubulin, microtubules Abstract Acoel spermatozoa are filiform and contain two parallel axonemes, which do not show the trepaxonematan 9 + ‘1’ pattern, but instead, another kind of 9 + ‘1’ pattern, or a 9 + 0 or 9 + 2 pattern. Spermatozoa have either cortical singlet microtubules or central microtubules. Identification of these groups of microtubules and recognition of homologies between species is difficult with electron microscopy. In addition to conventional electron microscopy, indirect immunofluorescence of tubulin was performed on three species (Symsagittifera schultzei, Symsagittifera psammophila, and Actinoposthia beklemischevi). This technique facilitated understanding of the general mor- phology of the filiform spermatozoon and of the arrangement of the microtubular organelles along its length. We have found that different monoclonal antibodies (anti-alpha-, anti-alpha-acetylated- and anti-beta-tubulin) can distinguish distinct subcellular populations of microtubules. The axonemes were labelled by the three antibodies in all species. The cortical microtubules (in Actinoposthia beklemischevi) were labelled by the three antibodies. The central microtubules (in Symsagittifera schultzei and S. psammophila) were labelled with the anti-beta-tubulin antibody and not labelled by the anti-alpha- and anti-alpha-acetylated-tubulin. Similar experiments were performed on other Platyhelminthes and indicated that immunocytochemistry of spermatozoa may provide new characters for phylogenetic studies. Abbreviations: A – Axonemes; GR – Granules; MCH – Mitochondria; MT – Microtubules; N – Nucleus Introduction The Acoela are primitive free-living Platyhelminthes which display a great variety in most morphological and ultrastructural features (Ehlers, 1985; Mamkaev, 1986). Among other features, spermatozoa seem to be of special importance for phylogenetic considerations (Raikova, 1991; Raikova & Justine, 1994, 1997, 1998; Watson & Rohde, 1995). Most Platyhelminthes have highly specific elon- gated sperm cells with two flagella that are either free or incorporated into the spermatid shaft (Jus- tine, 1995; Watson & Rohde, 1995). All Acoela examined have two incorporated axonemes running along the cytoplasmic region and a part of the nu- clear region of the filiform spermatozoon (Raikova & Justine, 1994). These axonemes have a reverse ori- entation: modified centrioles, also termed centriolar derivatives, are observed at the extremity opposite to the nucleus (Hendelberg, 1969, 1977). The axonemes do not show the trepaxonematan 9 + ‘1’ pattern, but instead a classic 9 + 2 pattern or sometimes a 9 + 0 pattern or 9 + ‘1’ structure, different from that of the Trepaxonemata. In addition to the axonemes, the cytoskeleton of acoel spermatozoa includes other microtubular organelles: either cortical singlet micro- tubules or a central structure of singlet microtubules (for references see Raikova & Justine, 1994). Identifi- cation of these groups of microtubules and recognition of homologies between species is difficult with elec- tron microscopy alone. In this study, we combined conventional transmission electron microscopy with indirect immunocytochemistry of tubulin. Tubulin is the protein constituting the micro- tubules, and therefore is one of the major proteins in spermatozoa which have axonemes and/or micro- tubules. It is a dimer with two constituents, namely alpha- and beta-tubulin (Cleveland, 1993; Mandelkow