Relative clonal proportions over time in mixed-genotype infections of the lizard malaria parasite Plasmodium mexicanum Alice Flynn Ford 1 , Jos J. Schall ⇑ Department of Biology, University of Vermont, Burlington, VT 05405, USA article info Article history: Received 31 October 2010 Received in revised form 20 January 2011 Accepted 25 January 2011 Available online 9 March 2011 Keywords: Plasmodium Lizard malaria Malaria life history Mixed-clone infections Clonal proportions Microsatellites abstract Vertebrate hosts of malaria parasites (Plasmodium) often harbour two or more genetically distinct clones of a single species, and interaction among these co-existing clones can play an important role in Plasmodium biology. However, how relative clonal proportions vary over time in a host is still poorly known. Experimental mixed-clone infections of the lizard malaria parasite, Plasmodium mexicanum, were followed in its natural host, the western fence lizard using microsatellite markers to determine the rel- ative proportions of two to five co-existing clones over time (2–3 months). Results for two markers, and two PCR primer pairs for one of those, matched very closely, supporting the efficacy of the method. Of the 54 infections, 67% displayed stable relative clonal proportions, with the others showing a shift in propor- tions, usually with one clone outpacing the others. Infections with rapidly increasing or slowly increasing parasitemia were stable, showing that all clones within these infections reproduced at the same rapid or slow rate. Replicate infections containing the same clones did not always reveal the same growth rate, final parasitemia or dominant clone; thus there was no clone effect for these life history measures. The rate of increase in parasitemia was not associated with stable versus unstable relative proportions, but infections with four to five clones were more likely to be unstable than those with two to three clones. This rare look into events in genetically complex Plasmodium infections suggests that parasite clones may be interacting in complex and unexpected ways. Ó 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. 1. Introduction An individual vertebrate organism (mammal, bird or squamate reptile) may be infected with parasites of two or more species of Plasmodium, or multiple genetically distinct clones of a single par- asite species (Anderson et al., 2000; Mayxay et al., 2004; Vardo and Schall, 2007; Havryliuk and Ferreira, 2009). Researchers for many years have suspected that interaction both among species and among conspecific clones could play a significant role in the biol- ogy of Plasmodium (Wenyon, 1926; Richie, 1988). Recent theoreti- cal studies and empirical evidence highlight the importance of clonal interaction within individual hosts for the ecology and evo- lution of malaria parasites. These results include cross-species cycling in dominance within an infection (Bruce et al., 2000; Bruce and Day, 2003), as well as intraspecific clonal competition (de Roode et al., 2003, 2005a; Wargo et al., 2007), infection virulence (Read and Taylor, 2001; Mackinnon and Read, 2004; de Roode et al., 2005b; Bell et al., 2006; Vardo-Zalik and Schall, 2009), life history traits including the parasite’s rate of replication and game- tocyte sex ratio (Schall, 2009; Vardo-Zalik and Schall, 2009) and transmission success into the vector (Taylor et al., 1997; Vardo- Zalik, 2009). Lacking in these studies on single-species infections, however, is detailed information on the relative proportions of clones in genetically complex infections and the changes in these relative proportions over time. For single-species Plasmodium infections, information on the rel- ative abundance of clones and the changes in relative abundance over time will certainly offer critical insights into the infection dynamics of malaria parasites. Most interesting for studies of relative abundance of clones in complex infections would be the possible nature of interaction among genotypes, ranging from com- petition to co-operation, and how clonal interaction influences life history traits. For example, sex ratio theory predicts that both the number of co-existing clones and their relative proportions will drive the ratio of male to female gametocyte cells that is optimal for the fitness of the parasite (Reece et al., 2008). If clonal propor- tions commonly shift over time in infection, this would present a for- midable challenge to the parasite (and to researchers attempting to test theory!). Studies on the relative proportions of co-existing parasite species can use microscopic examination of blood films (Bruce et al., 2000), but determining the relative proportions of conspecific genetic clones has been possible only with recent devel- opment of powerful new tools of molecular biology (Wargo et al., 0020-7519/$36.00 Ó 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2011.01.010 ⇑ Corresponding author. Tel.: +1 802 656 0448; fax: +1 802 656 2914. E-mail address: jschall@zoo.uvm.edu (J.J. Schall). 1 Present address: School of Medicine, University of Pennsylvania, Stemmler Hall, Box 47, PA 19104-6087, USA. International Journal for Parasitology 41 (2011) 731–738 Contents lists available at ScienceDirect International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara