Date Published: August 19, 2010
Publisher: Public Library of Science
Author(s): Virginie Rougeron, Thierry De Meeûs, Sandrine Kako Ouraga, Mallorie Hide, Anne-Laure Bañuls, Glenn F. Rall.
Leishmaniases remain a major public health problem today (350 million people at risk, 12 million infected, and 2 million new infections per year). Despite the considerable progress in cellular and molecular biology and in evolutionary genetics since 1990, the debate on the population structure and reproductive mode of Leishmania is far from being settled and therefore deserves further investigation. Two major hypotheses coexist: clonality versus sexuality. However, because of the lack of clear evidence (experimental or biological confirmation) of sexuality in Leishmania parasites, until today it has been suggested and even accepted that Leishmania species were mainly clonal with infrequent genetic recombination (see  for review). Two recent publications, one on Leishmania major (an in vitro experimental study) and one on Leishmania braziliensis (a population genetics analysis), once again have challenged the hypothesis of clonal reproduction. Indeed, the first study experimentally evidenced genetic recombination and proposed that Leishmania parasites are capable of having a sexual cycle consistent with meiotic processes inside the insect vector. The second investigation, based on population genetics studies, showed strong homozygosities, an observation that is incompatible with a predominantly clonal mode of reproduction at an ecological time scale (∼20–500 generations). These studies highlight the need to advance the knowledge of Leishmania biology. In this paper, we first review the reasons stimulating the continued debate and then detail the next essential steps to be taken to clarify the Leishmania reproduction model. Finally, we widen the discussion to other Trypanosomatidae and show that the progress in Leishmania biology can improve our knowledge of the evolutionary genetics of American and African trypanosomes.
A major challenge with parasitic pathogens is determining disease epidemiologies and transmission cycles. In addition to the different biotic and abiotic factors involved in the infectious pattern, the pathogen’s reproductive system is one of the basic biological processes that condition the microorganism’s ecology and disease spread. It has been largely demonstrated that improved knowledge on the population structure and reproductive strategy of pathogens provides key information to gaining a better understanding of transmission patterns and indispensable information for diagnostic and epidemiological inquiries as well as drug and vaccine development –. Nevertheless, it is often difficult to obtain such information on these microorganisms because the experimental investigations are often difficult to conduct and sometimes even almost impossible to develop. For a majority of pathogens, including the Trypanosomatidae family, the reproductive strategy was mainly deduced from population genetics analysis. The main goal of this review is to detail the progress made in the knowledge of the Leishmania reproduction model over the past 20 years.
In the Leishmania genus, asexual reproduction was proposed long ago as the main mechanism of reproduction. Since 1990, Tibayrenc et al. have proposed the clonal theory for all or most Leishmania species , –. This theory was principally based on the concept that offspring are genetically identical to their parents. Consequently, several criteria have been proposed to test this theory (e.g., fixed heterozygosity, absence of recombinant genotypes, deviation from Hardy-Weinberg expectations, widespread identical genotypes, linkage disequilibrium, and a correlation between two independent sets of genetic markers (see glossary in Box 1)) . Nevertheless, based on several observations of the interspecific hybrid profiles –, the occurrence of infrequent recombination events was not excluded for Leishmania. At the same time, this clonal hypothesis has been challenged by other authors. Indeed, based on molecular karyotype analysis using the pulse field gel electrophoresis (PFGE) technique, Bastien, Blaineau, and Pagès argued for a predominance of automixis (corresponding in this case to the definition of autogamy; see Box 1) , . In this study, for each chromosome analyzed, they observed a variation in the size of subtelomeric regions (which corresponds to different chromosome forms). Moreover, the results showed that, in each individual strain, only one chromosome form was observed and that, at the population level, various recombinations were observed across these different chromosome forms. To explain these results, authors proposed the existence of a predominantly autogamic sexual reproductive mode. Similarly, the existence of genetic recombination was brought back to the debate in 2007 by Kuhls et al. , who proposed the possible existence of selfing (producing an excess of homozygosity; see Box 1) on the basis of multilocus microsatellite typing performed on allopatric (see Box 1) Leishmania samples (91 strains collected in 18 different countries). However, because of the lack of clear evidence (experimental or biological confirmation) of sexuality in Leishmania, it was largely accepted that Leishmania species were mainly clonal with infrequent genetic events (see  for review).
Nowadays, with the development of elaborate experimental techniques and sophisticated statistical tools, our understanding of the evolutionary processes that govern the propagation of these parasites is continuously improving. Recently, two papers once again challenged the clonal hypothesis on the basis of empirical and experimental data. On one hand, a publication reporting a population genetics study on 125 human strains of Leishmania (Viannia) braziliensis based on 12 microsatellite loci revealed very high levels of homozygosity within samples . The population structure studied at a finer scale by a Bayesian analysis demonstrated that a large part of this was explained by population substructuring (Wahlund effect). Nevertheless, there was still a substantial heterozygote deficit, which is theoretically incompatible with a predominant clonal mode of reproduction at ecological time scales, as explained above. Indeed, the high homozygosity obtained was more in agreement with the occurrence of mating between individuals from related strains (endogamy; see Box 1). We then proposed that L. (V.) braziliensis parasites could alternate between different modes of reproduction: clonality in both vertebrate host and insect vector and endogamy within the insect vector (as has been shown to occur for other kinetoplastid parasites, such as Trypanosoma brucei s.l. ), with occurrence of quite frequent recombination events between different (i.e., genetically divergent) individuals (see Figure 1) . On the other hand, in parallel, the occurrence of mating in Leishmania parasites has been validated by a recent experimental study . The authors provided evidence of genetic exchanges (i.e., recombination between individuals from two strains of the same species) within the vector host of Leishmania major. Using transgenic Leishmania strains resistant to different selective drugs, Akopyants et al. infected natural sandflies and then isolated parasites resistant to both drugs. They succeeded in producing hybrid progenies, characterized by full genomic complements from recombination between two related individuals, but with kinetoplast DNA maxicircles from only one parent. It should be noted that seven out of 18 hybrid progeny clones studied showed triploid DNA content, which could be explained by the capacity of Leishmania parasites to cope with aneuploidy (see Box 1). The authors proposed that recombination occurs only in the vector and that hybrid progeny are transmitted to the mammalian vertebrate host by sandfly bites. For the first time, this study directly and experimentally proved the existence of mating in L. major. These two studies combined challenge the assumption that the Leishmania parasites’ mode of reproduction is overwhelmingly clonal.
At this stage, it appears essential to extend population genetics approaches to different Leishmania species and various environments. Analysis of genetic variation at different hierarchical levels is often the only way to investigate natural population characteristics such as gene flow and reproductive strategies . Moreover, given the observation of high substructuring of the population observed in the empirical study described above , working at finer geographic scales to detect and limit the substructuring (Wahlund effect) of such pathogens would be a desirable goal for future studies . Thus, exploring the genetic patterns of these parasites (allelic frequencies, heterozygosity rates, or linkage disequilibrium) at microgeographic scales would be the next challenge. This will require samples taken in small geographic areas, within short time windows, and from different host species involved in the transmission cycle (i.e., symptomatic and asymptomatic hosts and different vector species). Very few studies fulfill these conditions, often because of problems arising in the field and/or limitations in molecular techniques (i.e., lack of sensitivity for characterizing parasites in asymptomatic hosts). Some as yet unpublished results from our team (S. Kako Ouraga, V. Rougeron, and A–L. Bañuls, unpublished data.) suggest that the demographic unit could reside in the individual host itself. We developed a pioneering experiment consisting of isolating each Leishmania cell by cell, providing a whole genome amplification on each cell, and then amplifying the microsatellite DNA of the parasite by a nested PCR (a technique developed by the team of F. Prugnolle for Plasmodium falciparum; personal communication, unpublished data). The preliminary tests were made on two different human strains of L. V. braziliensis, CH22B and CH25B, collected in Peru in 1994 from two different patients and known to be heterozygous at several loci . Single-cell genotypic characterization revealed the actual coexistence of multiple genetic entities (i.e., genetically distinct Leishmania individuals) within each of these strains (CH22B and CH25B). This unexpected result strongly suggests that for these parasites the most nested subpopulation unit (the smallest demographic unit) might be individual hosts, a too frequently neglected scale for infectious agents, as stressed in recent papers , . Thus, in future studies, it would be indispensable to attempt to analyze population genetic data from the most nested subpopulation. Ignoring these scales should invariably lead to more or less high-magnitude Wahlund effects, depending on the degree of isolation between these micro-subpopulations. We believe that this type of experiment should be generalized in order to improve our knowledge of Leishmania reproductive strategy.
In summary, we are still far from complete knowledge of the mechanisms that govern the multiplication, propagation, and diversity of Leishmania in particular and in kinetoplastids in general. It should be noted that we have not detailed the problem of aneuploidy here, which cannot be neglected since it has been largely demonstrated in previous investigations ,  and recently confirmed experimentally . Although no genetic data allow us to suspect a clear impact of aneuploidy for any of the microsatellite loci used so far in empirical approaches, this phenomenon should not be ignored in future studies. Additionally, although none of the published results on microsatellites suggests the existence of any gene conversion, the possibility of gene conversion has to be specifically studied in future population genetic studies. These parasites apparently have unsuspected resources to survive and propagate, and understanding why and how is of primary relevance for better knowledge of their epidemiology and evolutionary potential.