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Background  The  Leishmania  protozoan parasites cause devastating human diseases.  Leishmania  have been considered to replicate clonally, without genetic exchange. However, an accumulation of evidence indicates that there are inter-specific and intra-specific hybrids among natural populations. The first and so far only experimental proof of genetic exchange was obtained in 2009 when double drug resistant  Leishmania major  hybrids were produced by co-infecting sand flies with two strains carrying different drug resistance markers. However, the location and timing of hybridisation events in sand flies has not been described.    Methodology/Principal Findings  Here we have co-infected  Phlebotomus perniciosus  and  Lutzomyia longipalpis  with transgenic promastigotes of  Leishmania donovani  strains carrying hygromycin or neomycin resistance genes and red or green fluorescent markers. Fed females were dissected at different times post bloodmeal (PBM) and examined by fluorescent microscopy or fluorescent activated cell sorting (FACS) followed by confocal microscopy. In mixed infections strains LEM3804 and Gebre-1 reached the cardia and stomodeal valves more rapidly than strains LEM4265 and LV9. Hybrids unequivocally expressing both red and green fluorescence were seen in single flies of both vectors tested, co-infected with LEM4265 and Gebre-1. The hybrids were present as short (procyclic) promastigotes 2 days PBM in the semi-digested blood in the endoperitrophic space. Recovery of a clearly co-expressing hybrid was also achieved by FACS. However, hybrids could not sustain growth in vitro.    Conclusions/Significance  For the first time, we observed  L. donovani  hybrids in the sand fly vector, 2 days PBM and described the morphological stages involved. Fluorescence microscopy in combination with FACS allows visualisation and recovery of the progeny of experimental crosses but on this occasion the hybrids were not viable in vitro. Nevertheless, genetic exchange in  L. donovani  has profound epidemiological significance, because it facilitates the emergence and spread of new phenotypic traits.

Visualisation of Leishmania donovani Fluorescent Hybrids during Early Stage Development in the Sand Fly Vector