Journal of Cell Science 101, 109-123 (1992) 109 Printed in Great Britain © The Company of Biologists Limited 1992 How individual cells develop from a syncytium: merogony in Theileria pan/a (Apicomplexa) MICHAEL K. SHAW and LEWIS G. TILNEY* International Laboratory for Research on Animal Disease, P. O. Box 30709, Nairobi, Kenya *Present address: Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA Summary The central problem for Theileria parva during mero- development of an orderly array of tubules that gony is how to form numerous individual, uninucleate originate from the apical end and progressively form a merozoites from a syncytial schizont so that each longitudinal basket enclosing first the rhoptry complex, merozoite contains a single nucleus and a prescribed then the mitochondria and ribosomes, and finally the assortment of organelles. The way T. parva packages all nucleus. the requisite organelles into free merozoites is by binding The process of merogony is compared to sporogony these organelles to the nuclear envelope, which in turn within the tick salivary gland and with the differen- becomes associated, both directly and through the tiation of the intra-erythrocytic piroplasm stage. Be- rhoptry complex, with the schizont plasma membrane. cause all three processes occur by a morphologically Formation of the merozoites occurs in a synchronous similar mechanism, the possibility that the parasite uses manner by a budding process. The merozoites develop a single cassette of genes to perform each of these similar with the rhoptry complex at the apical end by the processes is discussed. progressive, outward evagination of the schizont plasma membrane. This evagination of the plasma membrane is Key words: Theileria parva, merogony, merozoite associated with, and presumably induced by, the formation, cellularization from a syncytium. Introduction daughter cell acquires at least one copy of each organelle, there are examples of unequal cell division In general eukaryotic cells cannot form cell organelles (e.g. during early embryonic differentiation) or cell de novo and require the presence of pre-existing copies divisions in which certain cellular components become within the cells either to act as templates or from which selectively segregated into only one daughter cell (e.g. new organelles can develop by growth and fission. In the preferential localization of P granules into only one the case of specialized organelles (e.g. secretory daughter cell during early embryogenesis in Caenorhab- granules or specialized invasive organelles such as ditis elegans, see Strome and Wood, 1983). It is thought rhoptries, microspheres etc.) that appear to be made de that this asymmetrical segregation of cell organelles novo, the cell must, however, contain the requisite involves the cytoskeleton. organelles from which to synthesize these specialized There is a third category, which is the antithesis of the cellular inclusions. Thus, the mechanism(s) whereby an unequal distribution of cellular components and is of organism distributes its cell organelles into daughter particular relevance to the present study. This is the cells during cell division is a fundamental problem in problem of how organisms are able to produce cells cell biology. In higher eukaryotic cells there appear to which contain either a fixed number of cell organelles or be enough copies of most cell organelles so that random a selected range of organelles (e.g. sperm of many distribution alone should ensure that at least one copy organisms). A particularly good example is the sperm of each organelle would be present in each daughter cell of Caenorhabditis elegans where ribosomes are after cytokinesis. In the case of organelles occurring in excluded from the mature sperm by a particular gene low copy numbers such as the Golgi complex and the product (see Ward, 1986). Of particular interest to us is endoplasmic reticulum, these organelles fragment, the problem of how syncytia become cellularized and prior to cell division, into numerous vesicular struc- produce large numbers of individual cells containing a tures, which then become randomly distributed be- precise and identical set of organelles. tween the daughter cells. Even though there may be no Theileria parva is a tick-borne protozoan parasite that special mechanism(s) required to ensure that each causes East Coast fever, an acute and often fatal disease 110 M. K. Shaw and L. G. Tilney must be the appearance of different molecules on the Sporogony in cell surface, related to the specificity of the subsequent Tick saBvary Merogony in host cell. gland Lymphoblasts In the present study we have described in detail the process of merogony in T. parva to determine how Sporozoites Lymphocyte uninucleate cells containing a fixed complement of organelles are formed from a syncytium. We will then relate these observations to what is known about the Schlzont similar and related processes of sporogony within the tick salivary gland (Fawcett et al. 1982, 1985) and the generally more limited development of the intra- Sporoblast erythrocytic piroplasm stage (Conrad et al. 1985, 1986; Fawcett et al. 1987). Until now there is only one brief Merozoltes and rather incomplete report describing the process of merogony in Theileria (see Schein et al. 1978). Materials and methods Erythroeyte Parasites and cells Piroplasm Theileria parva (Muguga stock) and the tick, Rhipicephalus differ errtiation appendiculatus maintained in the Tick Unit at ILRAD, were used. Ticks were infected as nymphs by feeding them on cattle Fig. 1. A highly simplified drawing of the life cycle of infected with ground up tick stabilate (3087), after which the Theileria parva. There are three stages (at sporogony, engorged nymphs were maintained at 23-25°C and 80% merogony and at the piroplasm stage) in which the parasite relative humidity and allowed to moult to the adult stage. undergoes karyokinesis without cytokinesis resulting in the Most of our observations were made on samples of formation of a multinucleate syncytium. To continue its life peripheral blood and lymph node cells collected from cycle the parasite has to form uninucleate cells from these stabilate-infected cattle. These cattle had very high piroplasm syncytia. For a detailed account of the life cycle of T. parasitemia (up to 70%) and low (<20%) packed cell parva, including the sexual stages within the tick gut, see volumes. Small samples (e.g. 5-10 f.A) of peripheral blood Norval et al. (1991). collected into heparin were fixed immediately for electron microsocpy as described below. Lymph node cells were aspirated from the prescapular lymph node into either RPMI in cattle, which is endemic in large areas of East and 1640 culture medium or directly into one of the fixatives Central Africa (see Norval et al. 1991). The life cycle of described below. T. parva is shown diagrammatically in Fig. 1. The Observations were also made on in vitro cultures of parasite is transmitted to cattle when the tick, Rhipi- schizont-infected lymphoblastoid cells. Bovine peripheral cephalus appendiculatus, feeds on a susceptible host. blood lymphocytes (PBL) were obtained from the defibri- Within the life cycle of T. parva there are three stages at nated blood of uninfected cattle (Lalor et al. 1986), isolated which the parasite undergoes a period of extensive on Ficoll-Hypaque (Pharmacia, Sweden) gradients, washed growth involving nuclear division without cytokinesis, three times in Alsever's solution and resuspended in RPMI prior to sporogony, merogony and to a lesser extent, at 1640 culture medium with 10 mM Hepes buffer supplemented the piroplasm stage (Fig. 1). This results in all three with 10% heat-inactivated foetal bovine serum (Gibco, stages in the formation of a multinucleate syncytium Paisley, Scotland, UK). Bovine PBLs were infected in vitro (Fig. 1). From these syncytia the parasite, in order to with T. parva by incubation with sporozoites derived from salivary glands of infected adult ticks fed on a rabbit for 4 days continue its life cycle, needs to form uninucleate cells (Brown, 1987). The infected cells were maintained in RPMI (the sporozoites and merozoites) each of which contain, 1640 culture medium with 10 mM Hepes buffer supplemented as a minimum, a nucleus and nuclear envelope, at least with 10% heat-inactivated foetal bovine serum, 5xlO~5 M one mitochondrion, ribosomes and the appropriate 2-mercaptoethanol, 2 mM L-glutamine and 50 ng ml"1 secretory organelles required by the parasite to invade gentamycin. the next host cell. Merogony was observed in in vitro infected PBLs as early as Until now individual investigators have studied day 10 post-infection and low levels (10-20%) of merogony separately either sporogony (Fawcett et al. 1982,1985), were observed throughout the period of observation to day merogony (Schein et al. 1978) or the piroplasm stage 48. (Conrad et al. 1985, 1986; Fawcett et al. 1987) in Theileria, and no one has taken an overview of all three Electron microscopy stages. As T. parva undergoes the same or a very similar Samples were fixed in suspension by the addition of an equal process three times during its life cycle it would seem volume of either (Method 1) a mixture of 2.5% glutaralde- hyde, 2% formaldehyde and 0.01% picric acid in 0.1 M reasonable to predict that the parasite might use the phosphate or cacodylate buffer (pH 7.2), or (Method 2) in a same mechanism to develop uninucleated cells contain- freshly prepared solution containing 1% glutaraldehyde (from ing a similar set of organelles from a syncytium. The an 8% stock solution supplied by Electron Microscope only major difference between these life cycle stages Sciences, Fort Washington, PA, USA), 1% OsO4 and 0.05% Cellularization in Theileria parva 111 M phosphate buffer (pH 6.2). In the case of the first fixative, bers of membrane-free ribosomes and some small samples were fixed at room temperature (22°C) for 2 h, clusters of polysomes.