Clonal propagation and the fast generation of karyotype diversity : an in vitro Leishmania model J.-C. DUJARDIN 1 *, S. DE DONCKER 1 , D. JACQUET 1 , A.-L. BAN ˜ ULS 2 , M. BALAVOINE 2 , D. VAN BOCKSTAELE 3 , M. TIBAYRENC 2 , J. AREVALO 4 and D. LE RAY 1 1 Unit of Molecular Parasitology, Instituut voor Tropische Geneeskunde, 155 Nationalestraat, B-2000 Antwerpen, Belgium 2 Ge ´ne ´tique et Evolution des Maladies Infectieuses, UMR CNRS/IRD 2724, Montpellier, France 3 Department of Medicine, University Hospital of Antwerp, Wilrijkstraat 10, B-2650 Edegem, Belgium 4 Departamento de Bioquimica, Biologia Molecular y Farmacologia, Facultad de Ciencias y Filosofia and Instituto de Medicina Tropical ‘ Alexander von Humboldt ’, Universidad Peruana Cayetano Heredia, A.P. 5045, Lima 100, Peru (Received 2 June 2006; revised 26 June 2006; accepted 26 June 2006; first published online 18 September 2006) SUMMARY In the present work we studied the karyotype stability during long-term in vitro maintenance in 3 cloned strains of Leishmania (Viannia) peruviana, Leishmania (Viannia) braziliensis and a hybrid between both species. Only the L. (V.) peruviana strain showed an unstable karyotype, even after subcloning. Four chromosomes were studied in detail, each of them characterized by homologous chromosomes of different size (heteromorphy). Variations in chromosome patterns during in vitro maintenance were rapid and discrete, involving loss of heteromorphy or appearance of additional chromosome size variants. The resulting pattern was not the same according to experimental conditions (subinoculation rate or incubation temperature), and interestingly, this was associated with differences in growth behaviour of the respective parasites. No change in total ploidy of the cells was observed by flow cytometry. We discuss several mechanisms that might account for this variation of chromosome patterns, but we favour the occurrence of aneuploidy, caused by aberrant chromosome segregation during mitosis. Our results provide insight into the generation of karyotype diversity in natural conditions and highlight the relativity of the clone concept in parasitology. Key words: Leishmania (Viannia) peruviana, karyotype, hybrid, selection, ploidy, pseudo-sexuality. INTRODUCTION Leishmania (Kinetoplastida, Trypanosomatids) are parasitic protozoa causing a spectrum of clinical forms in humans and animals. These organisms may be considered as very successful parasites, when considering the number of different ecological niches they have colonized. Leishmania life-cycle occurs in 88 countries, in biotopes ranging from primary forest to xerophytic biotopes, from sylvatic to domestic environment, from lowlands to highlands. More than 50 species of sandflies are reported vectors and many mammals may act as reservoir, including edentates, monkeys, rodents, canids and humans (Shaw and Lainson, 1987). The genetic mechanisms underlying such a successful adaptation and spreading deserve attention. Sexual recombination, a major actor of innovation and adaptive evolution, can occur in Leishmania as demonstrated by the observation of hybrids in wild populations (Dujardin et al. 1995 a ; Ban ˜ uls et al. 1997). However, it is likely too rare to have implications on the parasite population structure, which is essentially clonal (Tibayrenc and Ayala, 1999). Besides point mutations, Leishmania have developed a series of asexual mechanisms that might contribute efficiently to parasite fitness. Many genes involved in host–parasite relationships are tandemly repeated and prone to mitotic recombination, which may occur between and within tandem repeats, amplifying genes and generating diversity of geno- types (Victoir and Dujardin, 2002). Under stress conditions, the parasites can also amplify sets of genes in the form of circular amplicons or small chromosomes (Segovia and Ortiz, 1997). Last but not least, it was shown during gene knock-out ex- periments that Leishmania – normally diploid para- sites (Beverley, 2003) – can undergo polyploidy and aneuploidy (Cruz et al. 1993). It was hypothesized that this might constitute another mechanism lead- ing to genetic polymorphism, during reversion to the normal ploidy. For instance, a heterozygote AB changed to AABB could generate AA, AB or BB offspring during reversion, having an effect similar to recombination (Cruz et al. 1993). However, to our knowledge, this hypothesis was never explored experimentally in Leishmania. * Corresponding author : Unit of Molecular Parasitology, Instituut voor Tropische Geneeskunde, 155 Nationales- traat, B-2000 Antwerpen, Belgium. Tel : +32 3 2476358. Fax: +32 3 2476359. E-mail : jcdujard@itg.be 33 Parasitology (2007), 134, 33–39. f 2006 Cambridge University Press doi:10.1017/S0031182006001156 Printed in the United Kingdom