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

Abstract

Genetic exchange between Leishmania major strains during their development in the sand fly vector has been experimentally shown. To investigate the possibility of genetic exchange between different Leishmania species, a cutaneous strain of L. major and a visceral strain of Leishmania infantum, each bearing a different drug-resistant marker, were used to coinfect Lutzomyia longipalpis sand flies. Eleven double-drug-resistant progeny clones, each the product of an independent mating event, were generated and submitted to genotype and phenotype analyses. The analysis of multiple allelic markers across the genome suggested that each progeny clone inherited at least one full set of chromosomes from each parent, with loss of heterozygosity at some loci, and uniparental retention of maxicircle kinetoplast DNA. Hybrids with DNA contents of approximately 2n, 3n, and 4n were observed. In vivo studies revealed clear differences in the ability of the hybrids to produce pathology in the skin or to disseminate to and grow in the viscera, suggesting polymorphisms and differential inheritance of the gene(s) controlling these traits. The studies, to our knowledge, represent the first experimental confirmation of cross-species mating in Leishmania, opening the way toward genetic linkage analysis of important traits and providing strong evidence that genetic exchange is responsible for the generation of the mixed-species genotypes observed in natural populations.

Keywords: Leishmania; genetic exchange; sand fly.

Fig. 1.

Genotype analysis of the progeny clones. (A) PCR products for parental selectable markers SAT and HYG. (B and C) SNP-CAPS analysis for loci on Chrs 9 and 29 (B) and for the same marker on Chr 29 (C) following recovery of progeny clones from C57BL/6 mice. (D) Digestion with BsteII of the LmjF.25.2420 locus. The bar graph shows the ratio between the intensity of the middle band from Lm and the uncut upper band from Li. Values shown are the means ± SD of three independent experiments. *P < 0.002 (vs. 1:1 mix). (E) Digestion with PstI of the LmjF.35.0050 locus. The bar graph shows the ratio between the intensity of the upper band (519 bp) from Lm and the intermediate band (300 bp) from Li. Values shown are the means ± SD of two independent experiments. ^#P < 0.05 (vs. 1:1 mix).

Fig. 2.

LPG expression and P. duboscqi fitness phenotypes of parents and progeny clones. (A) WIC79.3-mediated agglutination profiles of parents (Left), diploid (Center), and triploid (Right) progeny clones. (B) P. duboscqi were infected with 4 × 10⁶ parasites per milliliter of mouse blood. The infection was monitored at 2 d (Left) and 5 d (Right) postblood meal (PBM), corresponding to the times before and just after passage of the digested blood. The percentage of flies (10–12 flies per group) harboring viable midgut promastigotes is shown. White bars represent the parental lines, black bars the 2n hybrids, and stippled bars the 3n hybrids. Values shown are the means ± SD of three independent experiments. ^#P < 0.05 (vs. Lm); *P < 0.05 (vs. Li).

Fig. 3.

Progeny virulence in mouse models of cutaneous and visceral leishmaniasis. (A) Two million metacyclic promastigotes were inoculated s.c. in the footpad of BALB/c mice. Footpad width (millimeters) was measured weekly. Results shown are means ± SD of three mice per group. The experiment was repeated once with identical results. (B) Four million metacyclic promastigotes were inoculated i.d. in the ear pinnae of C57BL/6 mice. Ear lesion diameter (millimeters) was measured weekly. Results shown are means ± SD of the pool of two independent experiments (n = 5 per group per experiment). (C) Parasite load in the spleen (Left) and liver (Right) of C57BL/6 mice 6 wk p.i. with 2 × 10⁶ metacyclic promastigotes in the ear. Results correspond to geometric mean + 95% confidence interval of the pool of two independent experiments (n = 4 per group per experiment). (D) C57BL/6 mice were infected i.v. with 3 × 10⁶ metacyclic promastigotes, and the parasite loads in the spleen (Left) and the liver (Right) were determined 5 wk p.i. Results correspond to geometric mean + 95% confidence interval of the pool of two independent experiments (n = 3 per group per experiment). ^#P < 0.05 (vs. Lm); *P < 0.05 (vs. Li); **P < 0.05 (vs. groups 4, 11, 9, and 12).