Sand fly transmitted Leishmania species are responsible for severe, wide ranging, visceral and cutaneous leishmaniases. Genetic exchange can occur among natural Leishmania populations and hybrids can now be produced experimentally, with limitations. Feeding Phlebotomus orientalis or Phlebotomus argentipes on two strains of Leishmania donovani yielded hybrid progeny, selected using double drug resistance and fluorescence markers. Fluorescence activated cell sorting of cultured clones derived from these hybrids indicated diploid progeny. Multilocus sequence typing of the clones showed hybridisation and nuclear heterozygosity, although with inheritance of single haplotypes in a kinetoplastid target. Comparative genomics showed diversity of clonal progeny between single chromosomes, and extraordinary heterozygosity across all 36 chromosomes. Diversity between progeny was seen for the HASPB antigen, which has been noted previously as having implications for design of a therapeutic vaccine. Genomic diversity seen among Leishmania strains and hybrid progeny is of great importance in understanding the epidemiology and control of leishmaniasis. As an outcome of this study we strongly recommend that wider biological archives of different Leishmania species from endemic regions should be established and made available for comparative genomics. However, in parallel, performance of genetic crosses and genomic comparisons should give fundamental insight into the specificity, diversity and limitations of candidate diagnostics, vaccines and drugs, for targeted control of leishmaniasis.

Department of Cell and Molecular Biology Karolinska Institute Stockholm Sweden

Department of Parasitology Faculty of Science Charles University Prague Czech Republic

Faculty of Infectious and Tropical Diseases London School of Hygiene and Tropical Medicine London United Kingdom

Genome Institute of Singapore Biomedical Sciences Institutes Agency for Science Technology and Research Singapore

Laboratory of Confocal and Fluorescence Microscopy Faculty of Science Charles University Prague Czech Republic

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World Health Organization, Leishmaniasis, Geneva, Switzerland, 2023, January.

World Health Organization. Control of the leishmaniases Technical Report Series 949; WHO: Geneva, Switzerland, 2010; pp1–173.3. PubMed

Espinosa OA, Serrano MG, Camargo EP, Teixeira MMG, Shaw JJ. An appraisal of the taxonomy and nomenclature of trypanosomatids presently classified as Leishmania and Endotrypanum. Parasitology. 2018;145: 430–442. doi: 10.1017/S0031182016002092 PubMed DOI

Dostálová A, Volf P. Leishmania development in sand flies: parasite-vector interactions overview. Parasit Vectors. 2012;5: 276. doi: 10.1186/1756-3305-5-276 PubMed DOI PMC

Lukes J, Mauricio IL, Schönian G, Dujardin JC, Soteriadou K, Dedet JP, et al.. Evolutionary and geographical history of the Leishmania donovani complex with a revision of current taxonomy. Proc. Natl. Acad. Sci. USA 2007;104: 9375–9380. doi: 10.1073/pnas.0703678104 PubMed DOI PMC

Van den Broeck F, Savill NJ, Imamura H, Sanders M, Maes I, Cooper S, et al.. Ecological divergence and hybridization of Neotropical Leishmania parasites. Proc. Natl. Acad. Sci. USA. 2020; 117: 25159–25168. doi: 10.1073/pnas.1920136117 PubMed DOI PMC

Cotton JA, Durrant C, Franssen SU, Gelanew T, Hailu A, Mateus D, et al.. Genomic analysis of natural intra-specific hybrids among Ethiopian isolates of Leishmania donovani. PLoS Negl. Trop. Dis. 2020; 14: e0007143. doi: 10.1371/journal.pntd.0007143 PubMed DOI PMC

Glans H, Lind Karlberg M, Advani R, Bradley M, Alm E, Andersson B, Downing T. High genome plasticity and frequent genetic exchange in Leishmania tropica isolates from Afghanistan, Iran and Syria. PLoS Negl. Trop. Dis. 2021; 15: e0010110. doi: 10.1371/journal.pntd.0010110 PubMed DOI PMC

Dumetz F, Imamura H, Sanders M, Seblova V, Myskova J, Pescher P, et al.. Modulation of Aneuploidy in Leishmania donovani during Adaptation to Different In Vitro and In Vivo Environments and Its Impact on Gene Expression. mBio 2017; 8: e00599–17. doi: 10.1128/mBio.00599-17 PubMed DOI PMC

Akopyants NS, Kimblin N, Secundino N, Patrick R, Peters N, Lawyer P, et al.. Demonstration of genetic exchange during cyclical development of Leishmania in the sand fly vector. Science 2009; 324: 265–268. PubMed PMC

Louradour I, Ferreira TR, Duge E, Karunaweera N, Paun A, Sacks D. Stress conditions promote Leishmania hybridization in vitro marked by expression of the ancestral gamete fusogen HAP2 as revealed by single-cell RNA-seq. Elife. 2022; 11: e73488. doi: 10.7554/eLife.73488 PubMed DOI PMC

Monte-Neto RL, Fernandez-Prada C, Moretti NS. Sex under pressure: stress facilitates Leishmania in vitro hybridization. Trends Parasitol. 2022;38: 274–276. doi: 10.1016/j.pt.2022.02.001 PubMed DOI

https://protect-eu.mimecast.com/s/6EHxC0LKzCMZyRDhWQZml?domain=pubmed.ncbi.nlm.nih.gov Ferreira TR, Sacks DL. Experimental Hybridization in Leishmania: Tools for the Study of Genetic Exchange. Pathogens 2022; 11: 580. doi: 10.3390/pathogens11050580 PubMed DOI PMC

Gutiérrez-Corbo C, Domínguez-Asenjo B, Pérez-Pertejo Y, García-Estrada C, Bello FJ, Balaña-Fouce R, et al.. Axenic interspecies and intraclonal hybrid formation in Leishmania: Successful crossings between visceral and cutaneous strains. PLoS Negl. Trop. Dis. 2022; 16: e0010170. doi: 10.1371/journal.pntd.0010170 PubMed DOI PMC

Peacock L, Bailey M, Carrington M, Gibson W. Meiosis and haploid gametes in the pathogen Trypanosoma brucei. Curr Biol. 2014; 24: 181–6. doi: 10.1016/j.cub.2013.11.044 PubMed DOI PMC

Volf P, Volfova V. Establishment and maintenance of sand fly colonies. J. Vector Ecol. 2011; 36: Suppl 1, S1–S9. doi: 10.1111/j.1948-7134.2011.00106.x PubMed DOI

Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012; 9: 671–5. doi: 10.1038/nmeth.2089 PubMed DOI PMC

Sadlova J, Myskova J, Lestinova T, Votypka J Yeo M, Volf P. Leishmania donovani development in Phlebotomus argentipes: comparison of promastigote- and amastigote-initiated infections. Parasitology 2017; 144: 403–410. doi: 10.1017/S0031182016002067 PubMed DOI PMC

Yeo M, Lewis MD, Carrasco HJ, Acosta N, Llewellyn M, da Silva Valente SA, et al.. Resolution of multiclonal infections of Trypanosoma cruzi from naturally infected triatomine bugs and from experimentally infected mice by direct plating on a sensitive solid medium. Int J Parasitol. 2007; 37:111–20. doi: 10.1016/j.ijpara.2006.08.002 PubMed DOI

Steinbiss S, Silva-Franco F, Brunk B, Foth B, Hertz-Fowler C, Berriman M, et al.. Companion: a web server for annotation and analysis of parasite genomes. Nucleic Acids Res. 2016: 44: W29–W34, doi: 10.1093/nar/gkw292 PubMed DOI PMC

Li H, Durbin R. Fast an accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 2009;25:1754–60. doi: 10.1093/bioinformatics/btp324 PubMed DOI PMC

Danecek P, Bonfield JK, Liddle J, Marshall J, Valeriu O, Pollard MO, et al.. Twelve years of SAMtools and BCFtools. Gigascience. 2021;10: giab008. doi: 10.1093/gigascience/giab008 PubMed DOI PMC

<Poplin R, Ruano-Rubio V, De Pristo MA, Tim J, Fennell MO, Carneiro, Van der Auwera GA et al. Scaling accurate genetic variant discovery to tens of thousands of samples. doi: 10.1101/201178 DOI

Osman M, Mistry A, Keding A, Gabe R, Cook E, Forrester S et al.. A third generation vaccine for human visceral leishmaniasis and post kala azar dermal leishmaniasis: First-in-human trial of ChAd63-KH. PLoS Negl Trop Dis. 2017; 11: e0005527. doi: 10.1371/journal.pntd.0005527 PubMed DOI PMC

Mauricio IL, Yeo M, Baghaei M, Doto D, Pratlong F, Zemanova E et al.. Towards multilocus sequence typing of the Leishmania donovani complex: resolving genotypes and haplotypes for five polymorphic metabolic enzymes (ASAT, GPI, NH1, NH2, PGD). Int J Parasitol. 2006; 36: 757–769. doi: 10.1016/j.ijpara.2006.03.006 PubMed DOI

Galanew T, Hailu AJ, Schönian G, Lewis MD, Miles MA, Yeo M. Multilocus sequence and microsatellite identification of intra-specific hybrids and ancestor-like donors among natural Ethiopian isolates of Leishmania donovani. Int J Parasitol. 2014; 44: 751–757. PubMed PMC

Nolder D, Roncal N, Davies CR, Llanos-Cuentas A, Miles MA. Multiple hybrid genotypes of Leishmania (Viannia) in a focus of mucocutaneous Leishmaniasis. Am J Trop Med Hyg. 2007; 76: 573–578. PubMed

Rogers MB, Downing T, Smith BA, Imamura H, Sanders M, Svobodova M et al.. Genomic confirmation of hybridisation and recent inbreeding in a vector-isolated Leishmania population. PLoS Genet. 2014; 10: e1004092. doi: 10.1371/journal.pgen.1004092 PubMed DOI PMC

Imamura H, Downing T, Van den Broeck F, Sanders MJ, Rijal S, Sundar S, et al.. Evolutionary genomics of epidemic visceral leishmaniasis in the Indian subcontinent. Elife. 2016; 5:e12613. doi: 10.7554/eLife.12613 PubMed DOI PMC

Franssen SU, Durrant C, Stark O, Moser B, Downing T, Imamura H, et al.. Global genome diversity of the Leishmania donovani complex. Elife. 2020;25: 9:e51243. doi: 10.7554/eLife.51243 PubMed DOI PMC

Inbar E, Shaik J, Lantorno SA, Romano A, Nzelu CO, Owens K, et al.. Study on the Occurrence of Genetic Exchange Among Parasites of the Leishmania mexicana Complex. Front Cell Infect Microbiol. 2020;10: 607253. doi: 10.3389/fcimb.2020.607253 PubMed DOI PMC

Schwabl P, Boité MC, Bussotti G, Jacobs A, Andersson B, Moreira O et al.. Colonization and genetic diversification processes of Leishmania infantum in the Americas. Commun Biol. 2021;4: 139. doi: 10.1038/s42003-021-01658-5 PubMed DOI PMC

Miles MA, Yeo M, Mauricio IL. Genetics. Leishmania exploit sex. Science. 2009; 324(5924):187–189. doi: 10.1126/science.1172789 PubMed DOI

Volf P, Sadlova J. Sex in Leishmania. Science 2009;324(5935):1644. doi: 10.1126/science.324_1644b PubMed DOI

Volf P, Benkova I, Myskova J, Sadlova J, Campino L, Ravel C. Increased transmission potential of Leishmania major/Leishmania infantum hybrids. Int J Parasitol. 2007; 37: 589–593. PubMed PMC

Sádlová J, Price HP, Smith BA, Votýpka J, Volf P, Smith DF. The stage-regulated HASPB and SHERP proteins are essential for differentiation of the protozoan parasite Leishmania major in its sand fly vector, Phlebotomus papatasi. Cell Microbiol. 2010;12: 1765–79. doi: 10.1111/j.1462-5822.2010.01507.x PubMed DOI PMC

Sadlova J, Yeo M, Seblova V, Lewis MD, Mauricio I, Volf P and Miles MA. Visualisation of Leishmania donovani fluorescent hybrids during early stage development in the sand fly vector. PLoS One. 2011; 6: e19851. PubMed PMC

Inbar E, Akopyants NS, Charmoy M, Romano A, Lawyer P, Elnaiem DE et al.. The mating competence of geographically diverse Leishmania major strains in their natural and unnatural sand fly vectors. PLoS Genet. 2013;9: e1003672. doi: 10.1371/journal.pgen.1003672 PubMed DOI PMC

Bhattacharyya T., Boelaert M., Miles MA. Comparison of Visceral Leishmaniasis Diagnostic Antigens in African and Asian Leishmania donovani Reveals Extensive Diversity and Region-specific Polymorphisms. PLoS Neglected Diseases. 2013; 7: e2057. doi: 10.1371/journal.pntd.0002057 PubMed DOI PMC

Inbar E, Shaik J, Lantorno SA, Romano A, Nzelu CO, Owens K, et al.. Whole genome sequencing of experimental hybrids supports meiosis-like sexual recombination in Leishmania. PLoS Genet. 2019;15: e1008042. doi: 10.1371/journal.pgen.1008042 PubMed DOI PMC