Abstract
BackgroundLeishmania development in sand flies is confined to the alimentary tract and is closely connected with blood meal digestion. Previously, it has been published that activities of sand fly midgut proteases are harmful to Leishmania, especially to amastigote-promastigote transition forms. However, our experiments with various Leishmania-sand fly pairs gave quite opposite results.MethodsWe evaluated the effect of semi-digested midgut content on different life stages of Leishmania donovani and Leishmania majorin vitro. Various morphological forms of parasites, including macrophage-derived amastigotes and transition forms, were incubated 2 h with midguts dissected at various intervals (6–72 h) post-blood meal or with commercially available proteinase, and their viability was determined using flow cytometry. In parallel, using amastigote-initiated experimental infections, we compared development of L. donovani in sand flies that are either susceptible (Phlebotomus argentipes and P. orientalis) or refractory (P. papatasi and Sergentomyia schwetzi) to this parasite.ResultsIn vitro, sand fly midgut homogenates affected L. major and L. donovani in a similar way; in all sand fly species, the most significant mortality effect was observed by the end of the blood meal digestion process. Surprisingly, the most susceptible Leishmania stages were promastigotes, while mortality of transforming parasites and amastigotes was significantly lower. Parasites were also susceptible to killing by rabbit blood in combination with proteinase, but resistant to proteinase itself. In vivo, L. donovani developed late-stage infections in both natural vectors; in P. argentipes the development was much faster than in P. orientalis. On the other hand, in refractory species P. papatasi and S. schwetzi, promastigotes survived activity of digestive enzymes but were lost during defecation.ConclusionsWe demonstrated that Leishmania transition forms are more resistant to the killing effect of semi-digested blood meal than 24 h-old promastigotes. Data suggest that Leishmania mortality is not caused directly by sand fly proteases, we assume that this mortality results from toxic products of blood meal digestion. Survival of L. donovani promastigotes in refractory sand flies until blood meal defecation, together with similar mortality of Leishmania parasites incubated in vitro with midgut homogenates of susceptible as well as refractory species, contradict the previously raised hypotheses about the role of midgut proteases in sand fly vector competence to Leishmania.
Highlights
Leishmania development in sand flies is confined to the alimentary tract and is closely connected with blood meal digestion
All parasite forms tested of both Leishmania species were relatively resistant to killing effect of midgut homogenates of P. orientalis, P. papatasi and S. schwetzi; the mortality ranged between 1 and 8% in L. donovani (Fig. 1), 5–30% in L. major (Fig. 2) and was comparable to negative controls
Significant differences found in killing effect of various homogenates and time intervals (ANOVA, F(6,27) = 834, P < 0.0001) correlated with differences in the time course of blood meal digestion: Phlebotomus argentipes digest fast and, significant promastigote mortality (Tukey HSD, P < 0.0001) was observed in groups incubated with homogenates obtained at 24 h pbm, or 32 and 48 h pbm
Summary
Leishmania development in sand flies is confined to the alimentary tract and is closely connected with blood meal digestion. It has been published that activities of sand fly midgut proteases are harmful to Leishmania, especially to amastigote-promastigote transition forms. Protozoan parasites of the genus Leishmania (Kinetoplastida: Trypanosomatidae) have a digenetic life-cycle consisting of extracellular promastigotes developing in sand fly vectors (Diptera: Phlebotominae) and intracellular amastigotes in mammalian hosts [1, 2]. Leishmania major, which is mainly transmitted by P. papatasi and P. duboscqi, is the causative agent of cutaneous leishmaniasis (CL), and is widely distributed in arid and savannah areas of subSaharan and North Africa, Middle East and Indian subcontinent [4, 5]. As the majority of nutrients in the ingested blood is constituted by protein molecules, the proteolytic enzymes play the main role during blood meal digestion of sand flies, most of them being serine proteases, namely trypsin- and chymotrypsin-like molecules [7,8,9]. The midgut epithelial cells start to produce trypsin- and chymotrypsin-like proteases after the engorgement of the blood meal, activity levels are significantly increased from 6 h post-blood meal (pbm) and peak at 12–48 h pbm, depending on sand fly species [10,11,12,13]
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