Experimental transmission of Leishmania (Mundinia) parasites by biting midges (Diptera: Ceratopogonidae)

Abstract

Leishmania parasites, causative agents of leishmaniasis, are currently divided into four subgenera: Leishmania, Viannia, Sauroleishmania and Mundinia. The recently established subgenus Mundinia has a wide geographical distribution and contains five species, three of which have the potential to infect and cause disease in humans. While the other Leishmania subgenera are transmitted exclusively by phlebotomine sand flies (Diptera: Psychodidae), natural vectors of Mundinia remain uncertain. This study investigates the potential of sand flies and biting midges of the genus Culicoides (Diptera: Ceratopogonidae) to transmit Leishmania parasites of the subgenus Mundinia. Sand flies (Phlebotomus argentipes, P. duboscqi and Lutzomyia migonei) and Culicoides biting midges (Culicoides sonorensis) were exposed to five Mundinia species through a chicken skin membrane and dissected at specific time intervals post bloodmeal. Potentially infected insects were also allowed to feed on ear pinnae of anaesthetized BALB/c mice and the presence of Leishmania DNA was subsequently confirmed in the mice using polymerase chain reaction analyses. In C. sonorensis, all Mundinia species tested were able to establish infection at a high rate, successfully colonize the stomodeal valve and produce a higher proportion of metacyclic forms than in sand flies. Subsequently, three parasite species, L. martiniquensis, L. orientalis and L. sp. from Ghana, were transmitted to the host mouse ear by C. sonorensis bite. In contrast, transmission experiments entirely failed with P. argentipes, although colonisation of the stomodeal valve was observed for L. orientalis and L. martiniquensis and metacyclic forms of L. orientalis were recorded. This laboratory-based transmission of Mundinia species highlights that Culicoides are potential vectors of members of this ancestral subgenus of Leishmania and we suggest further studies in endemic areas to confirm their role in the lifecycles of neglected pathogens.

Fig 1

(A) Phylogenetic relationships of the four genera of the subfamily Leishmaniinae and subgenera of the genus Leishmania, based on [3,6,37]. (B) Geographical distribution of L. (Mundinia) species, based on [–,–,,–17]. The blank map source - https://commons.wikimedia.org/wiki/Atlasof the_world/. (C) Female sand fly Phlebotomus duboscqi feeding on the mouse demonstrating prediuresis which allows rapid concentration of proteins and restoring water and weight balance (a) and Culicoides biting midge feeding on a human host while also performing prediuresis (b).

Fig 2. Mundinia development in the sand fly Lutzomyia migonei.

Intensity (A) and localization (B) of L. enriettii (ENR), L. macropodum (MAC) and L. infantum (INF) infections. SV, stomodeal valve; AMG, abdominal midgut; TMG, thoracic midgut; E. sp., endoperitrophic space; PBM, post blood meal. Intensity of infection (parasite load) was categorized as light, <100 parasites per gut; moderate, 100–1000 parasites per gut and heavy, >1000 parasites per gut. Numbers of dissected females are displayed above the columns. Statistical differences in intensities of infection among Leishmania species were not significant on day 1 PBM (P = 0.860, X² = 2.571, d.f. = 6) while significant on day 5 PBM (P < 0.0001, X² = 49.922, d.f. = 6) and day 8 PBM (P < 0.0001, X² = 44.950, d.f. = 6).

Fig 3. Mundinia development in the sand fly Phlebotomus duboscqi.

Intensity (A) and localization (B) of L. sp. strain GH5 from Ghana (GHA) and L. major (MAJ) infections. SV, stomodeal valve; AMG, abdominal midgut; TMG, thoracic midgut; E. sp., endoperitrophic space; PBM, post blood meal. Intensity of infection (parasite load) was categorized as light, <100 parasites per gut; moderate, 100–1000 parasites per gut and heavy, >1000 parasites per gut. Numbers of dissected females are displayed above the columns. Differences among Leishmania species were significant and increased from day 3 PBM (P = 0.039, X² = 8.352, d.f. = 3) to day 4 PBM (P < 0.0001, X² = 34.008, d.f. = 3), 7 PBM (P < 0.0001, X² = 54.884, d.f. = 3) and day 11 PBM (P < 0.0001, X² = 57.711, d.f. = 3).

Fig 4. Leishmania development in the sand fly P. argentipes.

Intensity (A) and localization (B) of L. martiniquensis (MAR; MAR1, Cu1, Cu2, Aig1), L. orientalis (ORI) and L. donovani (DON) infections assessed by light microscopy. SV, stomodeal valve; AMG, abdominal midgut; TMG, thoracic midgut; E. sp., endoperitrophic space; PBM, post blood meal. Intensity of infection (parasite load) was categorized as light, <100 parasites per gut; moderate, 100–1000 parasites per gut and heavy, >1000 parasites per gut. Numbers of dissected females are displayed above the columns. Differences among Leishmania species/strains were significant and increased from day 1 PBM (P < 0.0001, X² = 97.997, d.f. = 15) to day 4 PBM (P < 0.0001, X² = 207.642, d.f. = 15) and day 8 PBM (P < 0.0001, X² = 214.778, d.f. = 15).

Fig 5. Mundinia development in the biting midge C. sonorensis.

Intensity (A, C, E) and localization (B, D, F) of L. enriettii (ENR), L. macropodum (MAC), L. sp. strain GH5 from Ghana (GHA), L. orientalis (ORI) and L. martiniquensis (MAR/MAR1, MAR/Cu1, MAR/Cu2, MAR/Aig1) infections assessed by light microscopy. SV, stomodeal valve; AMG, abdominal midgut; TMG, thoracic midgut; E. sp., endoperitrophic space; PBM, post blood meal. Intensity of infection (parasite load) was categorized as light, <100 parasites per gut; moderate, 100–1000 parasites per gut and heavy, >1000 parasites per gut. Numbers of dissected females are written above the columns. Differences among Leishmania species/strains were significant on day 3 PBM (P < 0.0001, X² = 181.173, d.f. = 21), day 6 PBM (P < 0.0001, X² = 70.048, d.f. = 21) and day 10 PBM (P < 0.0001, X² = 117.932, d.f. = 21).

Fig 6. Localisation and morphology of Mundinia in biting midge C. sonorensis on day 10 PBM.

A-B, colonization of the stomodeal valve with the part of the thoracic midgut filled with parasites, the region is marked by a red dotted line: (A) mature infection with L. martiniquensis Cu1; (B) mature infection with L. orientalis. C-G, various morphological forms present in the midgut on day 10 PBM: (C) metacyclic form of L. martiniquensis Cu2; (D) metacyclic form of L. orientalis; (E) nectomonad form of L. martiniquensis MAR1; (F) leptomonad form of L. martiniquensis Cu2; (G) haptomonad form of L. martiniquensis Cu2. Scale bar = 20μm.