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

Abstract

BackgroundThe 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 FindingsHere 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/SignificanceFor 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.

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

“…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.…”

The languages of parasite communication

“…Might this form of cell-cell communication occur in other flagellated parasites? Co-infection of sandflies with two tagged forms of Leishmania can give rise to double-positive parasites [59,60]. It is not always possible to isolate genetic hybrids, however [59], and one possibility is that some of them are products of protein exchange rather than mating.…”

“…Co-infection of sandflies with two tagged forms of Leishmania can give rise to double-positive parasites [59,60]. It is not always possible to isolate genetic hybrids, however [59], and one possibility is that some of them are products of protein exchange rather than mating. It is also worth noting that the Leishmania amastigote -so named because this life-cycle stage was supposed to lack a flagellum -has a flagellar pocket and a very short flagellum with a 9+0 axoneme structure that is more reminiscent of the sensory cilium of higher eukaryotes [61].…”

Epidemiological survey of Lutzomyia longipalpis infected by Leishmania infantum in an endemic area of Brazil

“…However, a limitation of this technique is the difficulty associated with the processing of a large number of specimens (PEREZ et al, 2007). The advantage of dissection should also be mentioned, i.e., the detection of developmental infectious stages with recording of metacyclic forms in the foregut (SADLOVA et al, 2011).…”