The Mating Competence of Geographically Diverse Leishmania major Strains in Their Natural and Unnatural Sand Fly Vectors

Abstract

Invertebrate stages of Leishmania are capable of genetic exchange during their extracellular growth and development in the sand fly vector. Here we explore two variables: the ability of diverse L. major strains from across its natural range to undergo mating in pairwise tests; and the timing of the appearance of hybrids and their developmental stage associations within both natural (Phlebotomus duboscqi) and unnatural (Lutzomyia longipalpis) sand fly vectors. Following co-infection of flies with parental lines bearing independent drug markers, doubly-drug resistant hybrid progeny were selected, from which 96 clonal lines were analyzed for DNA content and genotyped for parent alleles at 4–6 unlinked nuclear loci as well as the maxicircle DNA. As seen previously, the majority of hybrids showed ‘2n’ DNA contents, but with a significant number of ‘3n’ and one ‘4n’ offspring. In the natural vector, 97% of the nuclear loci showed both parental alleles; however, 3% (4/150) showed only one parental allele. In the unnatural vector, the frequency of uniparental inheritance rose to 10% (27/275). We attribute this to loss of heterozygosity after mating, most likely arising from aneuploidy which is both common and temporally variable in Leishmania. As seen previously, only uniparental inheritance of maxicircle kDNA was observed. Hybrids were recovered at similar efficiencies in all pairwise crosses tested, suggesting that L. major lacks detectable ‘mating types’ that limit free genetic exchange. In the natural vector, comparisons of the timing of hybrid formation with the presence of developmental stages suggest nectomonads as the most likely sexually competent stage, with hybrids emerging well before the first appearance of metacyclic promastigotes. These studies provide an important perspective on the prevalence of genetic exchange in natural populations of L. major and a guide for experimental studies to understand the biology of mating.

Cross-species genetic exchange between visceral and cutaneous strains of Leishmania in the sand fly vector

“…Furthermore, the 3n and 4n DNA contents were maintained in the hybrids throughout numerous in vitro passages as promastigotes and after recovery of tissue amastigotes from infected mice, suggesting that the polyploidy is not intrinsically unstable. As with all of the Leishmania intraspecies hybrids described previously (18,20), analysis of markers on the maxicircle DNA indicates that kDNA inheritance was uniparental and that either parent could contribute kDNA to the progeny. Although a similar conclusion was accorded the inheritance of maxicircle polymorphisms in hybrid progeny of Trypanosoma brucei, subsequent analyses at an earlier stage of hybrid generation indicated that kDNA inheritance was biparental, with segregation of maxicircles during subsequent mitotic divisions (28).…”

“…DNA content analysis of the hybrid clones revealed that the 11 independent crosses produced five 2n, five 3n, and a single 4n progeny. Triploid progeny have also been observed for many of the L. major intraspecies hybrids described (18,20). Based on the relative intensities of SNP-CAPS digestion products, we concluded that at least part of the extra set of chromosomes could be contributed by either parent.…”

“…1C). The simplest interpretation of these data is that the hybrids are full genomic hybrids, in which limited regions of the genome show one parental allele via mechanisms leading to loss of heterozygosity, as proposed previously (20).…”

“…In each case, the hybrids were generated between the extracellular promastigote stages in the sand fly vector. These and more recent studies (20,21) critically inform the population genetics data by formally demonstrating that Leishmania are capable of intraspecies and even intraclonal mating. The present studies were designed to provide, to our knowledge, the first experimental demonstration of cross-species mating and to explore the heritability of the genes controlling tissue-specific tropisms.…”

Is Predominant Clonal Evolution a Common Evolutionary Adaptation to Parasitism in Pathogenic Parasitic Protozoa, Fungi, Bacteria, and Viruses?

“…The results are especially convincing for Leishmania (Boité et al, 2012;Downing et al, 2011;Inbar et al, 2013;Lachaud et al, 2014;Mannaert et al, 2012;Rogers et al, 2011Rogers et al, , 2014Sterkers et al, 2011Sterkers et al, , 2012Sterkers et al, , 2014, but are also available for T. cruzi (Buscaglia et al, 2015;Minning et al, 2011;Reis-Cunha et al, 2015;Souza et al, 2011). Aneuploidy is also frequent in C. albicans and C. neoformans (Ene and Bennet, 2014;McManus and Coleman, 2014;Ni et al, 2013).…”