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.

Author Summary

Leishmania are pathogenic protozoa characterized by substantial diversity in the sand fly species that can transmit them, in the mammalian species that can serve as their reservoir hosts, and in the disease forms and severity of the clinical outcomes they can produce in humans. The possibility that this diversity has arisen, at least in part, by a process involving genetic exchange was recently given experimental support by the recovery of hybrid parasites from sand flies co-infected with two strains of Leishmania major. Here, we demonstrate the sexual competency of L. major strains originating across the full geographic range of this parasite species, and in both natural and unnatural sand fly vectors. Our genotype analyses of a large number of hybrid clones confirmed that they inherited both parental alleles at the majority of chromosomal marker loci analyzed, consistent with a meiotic process, while kinetoplast DNA was inherited from only one parent. Surprisingly, a few nuclear loci were sometimes inherited from only one parent, suggesting loss of heterozygosity. The early timing of hybrid recovery suggests that nectomonad promastigotes are the most likely mating competent stage of the parasite. These studies provide the strongest evidence to date that sex is a component of the natural reproductive strategy of L. major.