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

Significance

Protozoan parasites of the genus Leishmania are transmitted by sand flies and produce diseases in humans ranging from localized cutaneous lesions to fatal visceral infection. Although these clinical outcomes have clear parasite species associations, the genes controlling these differences are not known. We provide, to our knowledge, the first experimental demonstration of genetic exchange in the sand fly vector between different Leishmania species: a cutaneous strain of Leishmania major and a visceral strain of Leishmania infantum. Eleven full genomic hybrids were generated that displayed differences in their ability to grow in the skin or viscera of mice, indicating that the genes controlling these traits may be polymorphic within the parental species and are potentially amenable to identification by classical linkage analysis.

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.

Kinetoplastid protozoan parasites of the genus Leishmania are transmitted by a sand fly bite into the skin of the mammalian host. More than 20 different species of Leishmania are known to produce disease in humans, ranging from localized, self-limiting cutaneous lesions that develop at the site of inoculation to visceralizing infections that are fatal in the absence of treatment (1). These diverse clinical outcomes have generally clear parasite species and strain associations, although the ability of visceral strains to occasionally produce cutaneous disease, and vice versa, is also well described. The specific contribution of parasite genotype to disease outcome remains largely unknown. Studies by Zhang et al. demonstrated that the introduction of Leishmania donovani-specific genes into Leishmania major increased their ability to grow in the viscera of BALB/c mice, but in no case reached the level of infection produced by the visceral strains (2, 3), suggesting that these tissue tropisms are under multigenic control.

The Leishmania genome is organized in 36 chromosomes for the Old World species, such as L. major, L. donovani, and Leishmania infantum, and in 35 and 34 for the New World species Leishmania braziliensis and Leishmania mexicana, respectively (4, 5). Although approximately a diploid organism, aneuploidy, including mosaic aneuploidy, is now known to be widespread (6–10); we refer to the nearly diploid state as “2n” here. A series of population genetic studies have suggested that clonal lineages have experienced at least occasional bouts of genetic exchange both within and between species (11–16), and the frequency of these recombination events underlies the continuing clonality vs. sexuality debate (17).

By using drug-resistance markers, the first direct demonstration of genetic exchange between different L. major strains was reported (18) and possibly between L. donovani lines using fluorescent markers (19). 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. By using L. major and L. infantum parental lines bearing independent drug-resistance markers and that reproduce in mice their respective human cutaneous and visceral tropisms, hybrid progeny were recovered from coinfected Lutzomyia longipalpis sand flies. Detailed genotyping and phenotyping of 11 progeny clones are described.

Results

Generation of Hybrids.

To generate hybrids between L. major and L. infantum, we used Lu. Longipalpis, a sand fly species that is experimentally permissive to both strains (22). We tested L. major strain Friedlin clone V1 (LmjF) expressing a drug-resistance gene for nourseothricin (SAT) (18) and L. infantum LLM-320 (LinL) expressing a drug-resistance gene for hygromycin B (HYG), each inserted into the ribosomal RNA locus located on chromosome (Chr) 27 (Materials and Methods). As shown in Fig. S1, clonal lines of each were able to establish and maintain midgut infections during and after blood-meal digestion and excretion, although the intensity of the infection was 1.6–6.7 times greater for L. major compared with L. infantum.

The Lu. longipalpis sand flies were coinfected with 4 × 10⁶ and 8 × 10⁶ parasites per mL of blood of L. major–SAT (Lm) and L. infantum–HYG (Li), respectively. A total of 446 sand flies from five independent coinfections were dissected at different times postinfection (p.i.; 5–11 d p.i.) to recover double–drug-resistant parasites (Table S1). Of the cultures established from the midgut homogenates of these flies, 345 were free of bacterial or fungal contamination, and of these a total of 11 yielded doubly drug-resistant parasites (3.2%) from which 11 progeny clones were generated, each representing an independent crossing event. The percent of flies yielding hybrids was less than that seen in our previous studies involving intraspecies mating of L. major [7–26% (18, 20)], which may be related to the greater genetic distance between the Lm–Li parents vs. different geographic L. major isolates. Because the low frequency of mating requires selection for both parental markers, only 25% of the successful mating events involving the heterozygous parents can be recovered in 2n progeny

SNP Analysis of the Lm/Li Hybrids.

Using specific primers, we confirmed for each progeny clone the presence of both parental antibiotic-resistance genes (Fig. 1A). The inheritance patterns of nine additional unlinked marker loci located on nine different chromosomes (Table S2) were determined by SNP–cleaved amplification polymorphic site (SNP-CAPS) analysis (representative data shown in Fig. 1 B–E). The SNPs were identified by comparing gene alignments determined from the genome data bases of L. major and L. infantum. Table 1 summarizes the nuclear markers genotyping. Ten of the 11 hybrids inherited both parental alleles at all of the loci analyzed, whereas LimH1 inherited both parental alleles at all marker loci tested except for the marker on Chr 29, for which only the Lm parental allele was observed (Fig. 1B and Table 1). These genotypes were maintained following passage of each of the hybrid progeny through mice and their subsequent serial subculture as promastigotes, except for LimH2, which became homozygous for the Lm parental allelic marker on Chr 29 (Fig. 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).

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).

Table 1.

Summary of nuclear markers genotyping by SNP-CAPS

Parasite	Chromosome no.
	5	7	9	21	22	25	29	31	35
Lm	M	M	M	M	M	M	M	M	M
Li	I	I	I	I	I	I	I	I	I
LimH1	H	H	H	H	H	H	M	H	H
LimH2	H	H	H	H	H	H	H/M*	H	H
LimH3	H	H	H	H	H	H	H	H	H
LimH4	H	H	H	H	H	H	H	H	H
LimH5	H	H	H	H	H	H	H	H	H
LimH6	H	H	H	H	H	H	H	H	H
LimH7	H	H	H	H	H	H	H	H	H
LimH9	H	H	H	H	H	H	H	H	H
LimH10	H	H	H	H	H	H	H	H	H
LimH11	H	H	H	H	H	H	H	H	H
LimH12	H	H	H	H	H	H	H	H	H

H, heterozygous; I, Li; M, Lm.

^*Genotype pre-/postpassage in mice.

We included in our nuclear DNA genotyping an analysis of the A2 loci because the expression of these amastigote-specific genes is associated with the capacity of L. donovani and L. infantum to visceralize (23). In these species, multiple copies of the A2 genes are arranged in tandem on Chr 22, whereas in L. major, the corresponding sequences have been found functionally expressed as a single copy gene (24). Amplification of these sequences in Li and Lm confirmed a full-length and truncated copy of the A2 gene(s), respectively, and revealed that each of the hybrids had inherited at least one A2 locus from each parent (Fig. S2). The hybrids were also comparable in the stress-induced expression of the A2 genes following exposure of the promastigotes to elevated temperature, as described (25).

Hybrids Show 2n, 3n, and 4n DNA Contents.

The DNA contents of all of the progeny clones were measured relative to those of the parental lines at the cell population level by flow cytometry (Fig. S3 and Table S3). Both the parental Lm and Li lines showed similar profiles; here we refer to the major G1 peak as 2n, because despite the occurrence of aneuploidy in the two parents, most of the parental chromosomes are disomic. Staining with propidium iodide does not distinguish kinetoplast (mitochondrial DNA) and nuclear DNA, which are both included as “total” DNA. By this standard, five hybrids showed profiles for 2n DNA content, whereas five bore 3n and one bore 4n DNA content. The DNA content of each of the 3n and 4n hybrids appeared stable after their isolation as tissue amastigotes from infected mice and their subsequent serial subculture as promastigotes.

To identify the origin of the extra chromosomes, we determined the relative intensity of SNP-CAPS digestion products for two loci in the 3n progeny. Analysis of a SNP in gene LmjF.25.2420 results in the presence of a BstEII restriction site in the Lm allele, but not in the Li allele. The ratio between the intensity of the middle band from Lm and the uncut upper band from Li are shown for selected hybrids on the bar graph in Fig. 1D and in comparison with controls generated by mixing the respective parental DNA Lm:Li at 1:1; 2:1, and 1:2 ratios. A similar analysis was carried out on a SNP in LmjF.35.0050 that results in a PstI restriction site in both the Li and Lm alleles (Fig. 1E). From both analyses, we conclude that LimH1 and LimH5 inherited the extra trisomic Chrs 25 and 35 from Lm, whereas for LimH9 and LimH12, the extra trisomic chromosome genome is from Li. The differences in the Lm/Li ratios particularly likely reflect mosaic aneuploidy in the progeny, where trisomy would be present in a variable proportion of each cell population. Note that discordant results for the two markers were obtained for LimH3, suggesting that both parents were able to transmit an extra chromosome to this hybrid.

Maxicircle Inheritance.

We then assessed the inheritance of the kinetoplast maxicircle DNA by sequence and SNP-CAPS analysis of SNPs identified within three kinetoplast DNA (kDNA) genes: 12s rRNA, CYTB, and ND5 (Table S3 and Fig. S4). All of the hybrid clones were homozygous for each kDNA gene analyzed, with three having inherited their kDNA only from the Lm parent and eight only from the Li parent, suggesting uniparental retention of maxicircle kDNA. A summary of the ploidy and maxicircle inheritance patterns of the 11 progeny clones, and their associations with the phenotypes described below, is presented in Table 2.

Table 2.

Summary of hybrid genotypes and phenotypes in mice

Parasite	Genotype				Phenotype
	Ploidy	CAPS	kDNA		s.c.*	i.d.†	i.d. spl‡	i.d.liv‡	i.v. spl‡	i.v. liv‡
Lm	2n	1:1	M		M	M	M	M	M	M
Li	2n	1:1	I		I	I	I	I	I	I
LimH2	2n	1:1	I		I	M	M	Int	Int	Int
LimH4	2n	1:1	I		I	I	I	I	I	I
LimH6	2n	1:1	M		I	I	M	I	Int	I
LimH7	2n	1:1	I		I	M	M	M	Int	Int
LimH11	2n	1:1	I		I	I	I	I	I	I
LimH1	3n	M > I	I		I	M	M	M	M	M
LimH5	3n	M > I	M		I	M	M	M	Int	Int
LimH3	3n	n.d.	I		I	I	M	M	M	Int
LimH9	3n	M < I	M		I	I	I	I	I	I
LimH12	3n	M < I	I		I	I	I	I	I	I

n.d., not determined.

^*M (Lm), progressive footpad lesion in BALB/c mice; I (Li), no lesion.

^†I, transient ear lesion in C57BL/6 mice; M, persistent lesion >5 wk.

^‡I, parasite load significantly different from Lm; M, parasite load significantly different from Li; Int, intermediate parasite load significantly different from Li and Lm.

Phenotype Analysis of the Lm/Li Hybrids.

Infection in Phlebotomus duboscqi.

P. duboscqi is a natural vector of L. major transmission in west Africa, and both in nature and experimentally, its vector competence is restricted to L. major (26). Survival of Leishmania in the sand fly midgut is tightly controlled by the polymorphic structure of the surface lipophosphoglycan (LPG) that mediates promastigote adhesion to midgut epithelial cells and prevents their elimination during excretion of the digested blood meal. Thus, Li promastigotes, synthesizing LPG lacking in β1,3-galactosyl side chains recognized by the monoclonal antibody (mAb) WIC79.3 (Fig. 2A), grow adequately in the blood-fed midgut of P. duboscqi during the first 2 d p.i., but are eliminated from the midgut along with the blood meal remnants by day 5 (Fig. 2B and Fig. S5). By contrast, Lm promastigotes expressing a poly-galactosylated LPG and strongly agglutinated by WIC79.3 are retained in the midgut after blood meal excretion. The Li/Lm hybrids expressed intermediate levels of galactosylated LPG and accordingly displayed a variable, although generally intermediate, phenotype with respect to the percentage of flies retaining infections after blood meal excretion. The 3n hybrids for which the extra chromosomes were transmitted by the Li parent were the least fit hybrids for survival in P. duboscqi, suggesting a gene-dosage effect.

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).

Cutaneous leishmaniasis in BALB/c and C57BL/6 mice.

Infection of BALB/c mice with L. major in the footpad represents a classical experimental model of nonhealing cutaneous leishmaniasis. As shown in Fig. 3A, footpad inoculation of 10⁶ metacyclics of the Lm parental line produced rapidly progressing lesions, whereas the Li parent produced no footpad swelling, although low numbers of viable organisms could be recovered from the inoculation site at even late time points. All of the hybrids behaved identically to the Li parent and failed to produce any lesions for up to 9 wk p.i. Thus, the inability to grow or to produce cutaneous pathology in BALB/c mice is inherited from the Li parent as a fully dominant trait.

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).

In contrast to the nonhealing infections in BALB/c mice, L. major infection in C57BL/6 mice results in self-limiting lesions, and the ear dermis has become a favored inoculation site to study cutaneous leishmaniasis in this model. Fig. 3B shows the development of large, chronic lesions in the ear dermis p.i. with the Lm parent and the minimal and transient cutaneous pathology produced by Li. The progeny clones in this case did not behave uniformly like the Li parent, with both 3n hybrids with the extra chromosomes from Lm and two of the 2n hybrids (LimH2 and LimH7) producing larger and persisting lesions. By contrast, the 3n hybrids with the extra chromosomes from Li and the 2n LimH11 and LimH4 hybrids produced minimal pathology, similar to the Li parent. The 3n LimH3 hybrid also produced minimal pathology in the skin.

Visceral leishmaniasis in C57BL/6 mice.

Inoculation of mice in the ear dermis with a high dose of L. infantum metacyclic promastigotes has been introduced as an experimental model of visceral leishmaniasis that better reflects the conditions of natural infection compared with i.v. inoculation (27). Fig. 3C shows the parasite loads in the liver and spleen 6 wk p.i. with 2 × 10⁶ metacyclics in the ear dermis. The Li parental line established low but reproducible infections in the spleen (10²) and liver (10³) following intradermal (i.d.) inoculation, whereas the Lm parent was minimally detectable in these organs (<10). The 3n hybrids bearing the extra chromosomes from Lm behaved like the Lm parent, with virtually no parasites detectable in the liver or spleen, whereas the two 2n hybrids (LimH2 and LimH7) that produced the greatest cutaneous pathology were minimally detected in the spleen and were significantly different from both parental phenotypes in the liver. The two 3n hybrids with the extra chromosomes from Li, and the 2n LimH11 and LmH4 hybrids that produced minimal pathology in the skin, behaved comparably to the Li parent in both the liver and spleen. LimH3 failed to disseminate to and/or grow in the viscera, but because it also grew poorly in the sand fly midgut and produced minimal pathology in the skin, this hybrid appears to have a generalized growth defect that is unrelated to developmental stage or tissue preference.

The visceral dissemination and growth phenotypes of the hybrids were largely replicated by using the more conventional visceral leishmaniasis infection model involving high-dose i.v. inoculation (Fig. 3D). The 3n hybrids with the extra Lm chromosomes again showed the least growth in both organs, whereas 2n LimH2 and LimH7 hybrids also displayed reduced growth in both organs compared with the Li parent, although significantly greater than the Lm parent. The 2n LimH6 hybrid showed significantly reduced growth only in the spleen. By contrast, the 3n hybrids with the extra chromosomes from Li and the 2n LimH11 and LimH4 hybrids again behaved most like the Li parent, with comparable growth in both the liver and spleen. Together, these results indicate that the origin of the extra chromosomes in the 3n progeny correlates to the phenotype, whereas for the 2n progeny, a number of mechanisms, including differential allelic inheritance, may control their tissue tropisms.

Discussion

This study, to our knowledge, represents the first experimental confirmation of cross-species mating in Leishmania. Eleven hybrid progeny were generated between L. major and L. infantum in Lu. longipalpis. The genetic analysis of multiple allelic markers distributed across the genome indicates that every progeny is likely a full genomic hybrid, with occasional loss of heterozygosity at some loci. The conclusion that the apparent uniparental inheritance of a marker locus in two of the progeny clones was in fact the result of a postmating process leading to the loss of heterozygosity is supported by the sequential genotyping of one of the hybrids (LimH2) showing the loss of one the parental alleles after mouse passage. 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. 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).

Our attempts to cross-hybridize Leishmania species was deliberately explored by using L. infantum and L. major because the biology of these species is so clearly distinguishable in both their invertebrate and vertebrate hosts. In the sand fly, the natural as well as experimental vector competence of P. duboscqi is restricted to L. major, whereas infections with other Leishmania species, including L. infantum, are lost during excretion of the blood-meal remnants (26). The hybrids displayed variable, but more or less intermediate, phenotypes compared with either parent, both in the expression of poly-galactosylated LPG that mediates midgut attachment and their persistence in the midgut after blood-meal excretion. The increased fitness of some of the hybrids compared with the Li parent for potential transmission by a normally refractory vector replicates the findings reported for natural genetic hybrids between L. infantum and L. major (29). The experimental hybrids validate that these genotypes can arise via genetic exchange.

The phenotype analysis of the Lm/Li hybrids for their tissue tropisms in the mammalian host was of obvious interest. Using the classical experimental model to study nonhealing forms of cutaneous leishmaniasis, we unexpectedly found that all of the hybrids, including the 3n hybrids bearing the extra genome from Lm, phenocopied the Li parent in producing no footpad lesions at all. A similar phenotype was previously observed by using cosmid-transfected L. major capable of expressing multiple copies of the L. donovani A2 gene (30). Because the development of nonhealing L. major lesions in BALB/c mice is largely dependent on a parasite-driven Th2 response, the effect of the Li genes, including A2, is likely due to an inhibition of this response.

Finally, we used i.d. inoculation of the hybrids in C57BL/6 mice to explore the heritability of the genes controlling the development of pathology associated with healing forms of cutaneous leishmaniasis and, more critically, the ability to disseminate from the skin and establish growth in the deep organs. The diploid hybrids appeared to differentially segregate the skin and viscera tropisms of the parents, with two hybrids producing only transient pathology in the skin but disseminating to and growing in the liver and/or spleen equivalent to the Li parent, and two behaving more like the Lm parent in producing stronger pathology in the skin but growing poorly in the viscera. Thus, even these few 2n progeny appear to have differentially inherited the genes controlling the respective tissue tropisms of their parents, suggesting that one or both of the parents are heterozygous for these genes(s). Alternatively, or in concert with specific inheritance patterns, epigenetic mechanisms involved in regulating transcription initiation or termination of the relevant genes (31, 32), or changes in gene dosage due to aneuploidy, might contribute to the phenotypic differences observed. A possible effect of increasing gene dosage due to polyploidy was clearly evident, because the triploid hybrids displayed distinct skin or viscera tropisms depending on the parental origin of the extra chromosomes, although differences in allelic inheritance might still be influencing the behavior of these clones.

Although there is strong evidence that A2 genes influence the ability of Leishmania to disseminate to and/or grow in the viscera (23), there were no apparent differences between the hybrids in A2 gene inheritance or inducible expression in vitro (Fig. S1). At least three additional L. donovani-specific orthologs of L. infantum genes were found to promote L. major survival in the viscera (2, 3). Quantitative trait loci mapping of the genes controlling these traits in a larger series of Lm/Li hybrids should advance our understanding of these basic characters beyond what has so far been possible using reverse genetic approaches.

Our formal demonstration that experimental hybridization between different Leishmania species can occur could be viewed as a challenge to the conventional species definition of these eukaryotic microbes, which in Leishmania and other microbes has largely devolved toward molecular distance criteria (33). However, it is important to note that the fertility of the offspring themselves has not yet been addressed, an important criterion because interspecies hybrids in metazoans are typically infertile, the classic example being the mule. Additionally, it is well known that taxa can differ in their ability to undergo hybridization over evolutionary distances (34). Thus, the ability of these Lm/Li hybrids to cross among themselves or with either parent—and the consequences of this information to understanding of Leishmania species boundaries and biology—is an important area for future study. Based on their high degree of synteny and low number of species-specific genes (35), it may not be surprising that these species can experimentally cross-hybridize, presented the opportunity. As a tool for experimental analysis, cross-species mating offers a powerful genetic approach to advance our understanding of the molecules controlling the remarkable biological diversity of the genus.

Materials and Methods

Parasite Strains.

L. major FV1 (SAT), containing a heterozygous nourseothricin-resistance marker integrated into one allele of the 18S rRNA locus located on Chr 27 (5), was derived from NIH Friedlin clone V1 (MHOM/IL/80/FN) as described (18). L. infantum (HYG) was derived from L. infantum (MHOM/ES/92/LLM-320; isoenzyme typed MON-1) (27), provided by Diane MacMahon-Pratt (Yale School of Public Health, New Haven, CT). Wild-type L. infantum promastigotes were transfected with the RFP/Hyg expression cassette and selected on hygromycin B-containing agar plates as described (36). The integration of the expression cassette into the 18S rRNA locus in selected drug-resistant clones was verified by PCR. LinL–HYG clone 7 also showed good survival in Lu. longipalpis and was chosen for the studies shown in this work. All parental and progeny lines were cultured in vitro at 26 °C in complete medium 199 (CM199) supplemented as described in SI Materials and Methods. Infective-stage metacyclic promastigotes were isolated from stationary cultures (4–6 d old) by centrifugation through a Ficoll-step gradient as described (37). LPG-mediated agglutination assay of parents and hybrids is described in SI Materials and Methods.

Infection of Sand Flies and Hybrid Recovery.

Lu. longipalpis and P. duboscqi were obtained from field specimens collected in Brazil and Mali, respectively. Two- to 4-d-old female sand flies were infected by artificial feeding through a chick-skin membrane containing heparinized, heat-inactivated mouse blood and parasites. For the generation of hybrids in Lu. longipalpis, the blood was seeded with a mixture of 4 × 10⁶ L. major (SAT) and 8 × 10⁶ L. infantum (HYG) logarithmic phase promastigotes per milliliter of blood; relatively high concentrations were used to compensate for the suboptimal growth of these lines in the colonized flies. Infected flies were dissected at different times p.i., and midgut homogenates were prepared for selection of doubly drug-resistant hybrids as described in SI Materials and Methods. For the phenotyping of hybrid progeny in P. duboscqi, the flies were infected with 4 × 10⁶ per milliliter logarithmic phase promastigotes, and at different times p.i. midgut homogenates were prepared and deposited on a hemocytometer to count the numbers of parasites per midgut.

Hybrids Genotyping.

Total DNA contents were determined by flow cytometry as described (18) (SI Materials and Methods). For genotype analysis, total DNA was extracted by using the Wizard genomic DNA purification Kit. PCR amplifications were performed in 20-µL final volume, using 20 ng of DNA and 2× GeneAmp PCR Master Mix (Applied Biosystems) and 25 pmol of each primer specific for the marker genes listed in Table S1. DNA products were verified by electrophoresis on a 1.5% (wt/vol) agarose gel and visualized by ethidium bromide staining. PCR primers for SNP-CAPS analysis were described (8) and are summarized in Table S1. PCR products were cleaned with Wizard SV Gel and PCR Clean-Up System (Promega), and 6 µL of product was digested with 10 U of restriction enzyme (Fermentas) for 16 h, electrophoresed on 1.5% agarose gel, and visualized by ethidium staining. The relative intensity of the band corresponding to the digestion products was analyzed with ImageJ software (Version 1.46). PCR primers for gene sequencing are summarized in Table S1. PCR products were cleaned with ExoSAP-IT kit (USB), and sequences were confirmed with forward and/or reverse reads by Rocky Mountain Laboratory Genomics Unit DNA Sequencing Center, Division of Intramural Research (Hamilton, MT). The sequences were analyzed by using Lasergene software.

In Vivo Infections.

The 7- to 9-wk-old female BALB/c and C57BL/6 mice were purchased from Taconic Laboratories. All mice were maintained in the National Institute of Allergy and Infectious Diseases (NIAID) animal care facility under specific pathogen-free conditions and used under a study protocol approved by the NIAID animal care and use committee (protocol no. LPD 68E). All aspects of the use of animals in this research were monitored for compliance with the Animal Welfare Act; the Public Health Service Policy; the US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training; and the NIH Guide for the Care and Use of Laboratory Animals (38).

To compare the virulence of the parental and progeny clones in established models of cutaneous leishmaniasis, 2 × 10⁶ metacyclic promastigotes were injected s.c. in one hind footpad of BALB/c mice or i.d. in one ear of C57BL/6 mice. Lesion development was monitored weekly by measuring footpad width or the diameter of the induration in the ear by using a direct-reading vernier caliper. To study the dissemination to and growth in the deep organs, C57BL/6 mice infected in the ear dermis with 2 × 10⁶ metacyclic promastigotes were killed at 6 wk p.i., and the infected ear, spleen, and liver were harvested. Alternatively, C57BL/6 mice were infected i.v. with 3 × 10⁶ metacyclic promastigotes and 5 wk p.i. were euthanized, and spleen and liver were harvested. For quantification of parasite loads, the spleen and liver were cut with tweezers and homogenized with a syringe plunger, and the cell suspension was filtered through a 70-µm strainer. Red blood cells were lysed with ammonium-chloride-potassium lysing buffer for 5 min at room temperature, and cells from each tissue were resuspended in CM199. Ear tissue was prepared and parasite loads were determined as described (39).

Statistical Methods.

Parasite loads were compared by using an exact stratified Wilcoxon rank sum test, stratified by experiment to allow pooling of experiments. Comparisons in which the data represented replicate samples were carried out by using t tests. All P values are two-sided.