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.
- MeSH
- Fluorescence MeSH
- Microscopy, Fluorescence MeSH
- Insect Vectors parasitology MeSH
- Hybridization, Genetic MeSH
- Crosses, Genetic MeSH
- Leishmania donovani cytology genetics growth & development pathogenicity MeSH
- Leishmaniasis, Visceral parasitology MeSH
- Humans MeSH
- Luminescent Proteins genetics metabolism MeSH
- Flow Cytometry MeSH
- Psychodidae parasitology MeSH
- Life Cycle Stages physiology MeSH
- Transfection MeSH
- Digestive System cytology parasitology MeSH
- Green Fluorescent Proteins genetics metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Acta chirurgica Scandinavica, ISSN 0301-1860 suppl. 525, 1985
155 s. : il., tab. ; 24 cm
