Leishmania parasites alternate in their life cycle between promastigote stages that develop in the gut of phlebotomine sand flies and amastigotes residing inside phagocytic cells of vertebrate hosts. For experimental infections of sand flies, promastigotes are frequently used as this way of infection is technically easier although ingestion of promastigotes by sand flies is unnatural. Here we aimed to answer a critical question, to what extent do promastigote-initiated experimental infections differ from those initiated with intracellular amastigotes. We performed side-by-side comparison of Leishmania development in Phlebotomus argentipes females infected alternatively with promastigotes from log-phase cultures or amastigotes grown ex vivo in macrophages. Early stage infections showed substantial differences in parasite load and representation of morphological forms. The differences disappeared along the maturation of infections; both groups developed heavy late-stage infections with colonization of the stomodeal valve, uniform representation of infective metacyclics and equal efficiency of transmission. The results showed that studies focusing on early phase of Leishmania development in sand flies should be initiated with intracellular amastigotes. However, the use of promastigote stages for sand fly infections does not alter significantly the final outcome of Leishmania donovani development in P. argentipes and their transmissibility to the vertebrate host.

Department of Parasitology Faculty of Science Charles University Vinicna 7 128 44 Prague 2 Czech Republic

Department of Pathogen Molecular Biology Faculty of Infectious and Tropical Diseases London School of Hygiene and Tropical Medicine Keppel Street WC1E 7HTLondon UK

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Anjili C., Langat B., Lugalia R., Mwanyumba P., Ngumbi P., Mbati P. A., Githure J. and Tonui W. K. (2006). Estimation of the minimum number of Leishmania major amastigotes required for infecting Phlebotomus duboscqi (Diptera: Psychodidae). East African Medical Journal 83, 68–71. PubMed

Bates P. A. (1993). Axenic culture of Leishmania amastigotes. Parasitology Today 9, 143–146. PubMed

Bates P. A. (2007). Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies. International Journal for Parasitology 37, 1097–1106. PubMed PMC

Borovsky D. and Schlein Y. (1987). Trypsin and chymotrypsin-like enzymes of the sandfly Phlebotomus papatasi infected with Leishmania and their possible role in vector competence. Medical and Veterinary Entomology 1, 235–242. PubMed

Chang K. P. (1980). Human cutaneous leishmania in a mouse macrophage line: propagation and isolation of intracellular parasites. Science 209, 1240–1242. PubMed

Charest H. and Matlashewski G. (1994). Developmental gene expression in Leishmania donovani: differential cloning and analysis of an amastigote-stage-specific gene. Molecular and Cellular Biology 14, 2975–2984. PubMed PMC

Dostalova A. and Volf P. (2012). Leishmania development in sand flies: parasite–vector interactions overview. Parasites & Vectors 5, 276. PubMed PMC

Freitas V. C., Parreiras K. P., Duarte A. P., Secundino N. F. and Pimenta P. F. (2012). Development of Leishmania (Leishmania) infantum chagasi in its natural sandfly vector Lutzomyia longipalpis. The American Journal of Tropical Medicine and Hygiene 86, 606–612. PubMed PMC

Gupta N., Goyal N. and Rastogi A. K. (2001). In vitro cultivation and characterization of axenic amastigotes of Leishmania. Trends in Parasitology 17, 150–153. PubMed

Holzer T. R., McMaster W. R. and Forney J. D. (2006). Expression profiling by whole-genome interspecies microarray hybridization reveals differential gene expression in procyclic promastigotes, lesion-derived amastigotes, and axenic amastigotes in Leishmania mexicana. Molecular and Biochemical Parasitology 146, 198–218. PubMed

Kimblin N., Peters N., Debrabant A., Secundino N., Egen J., Lawyer P., Fay M. P., Kamhawi S. and Sacks D. (2008). Quantification of the infectious dose of Leishmania major transmitted to the skin by single sand flies. Proceedings of the National Academy of Sciences of the United States of America 105, 10125–10130. PubMed PMC

Lehane M. J. (1997). Peritrophic matrix structure and function. Annual Review of Entomology 42, 525–550. PubMed

Maia C., Seblova V., Sadlova J., Votypka J. and Volf P. (2011). Experimental transmission of Leishmania infantum by two major vectors: a comparison between a viscerotropic and a dermotropic strain. PLoS Neglected Tropical Disieses 5, e1181. PubMed PMC

Mary C., Faraut F., Lascombe L. and Dumon H. (2004). Quantification of Leishmania infantum DNA by a real-time PCR assay with high sensitivity. Journal of Clinical Microbiology 42, 5249–5255. PubMed PMC

McCall L. I. and Matlashewski G. (2012). Involvement of the Leishmania donovani virulence factor A2 in protection against heat and oxidative stress. Experimental Parasitology 132, 109–115. PubMed

Myskova J., Votypka J. and Volf P. (2008). Leishmania in sand flies: comparison of quantitative polymerase chain reaction with other techniques to determine the intensity of infection. Journal of Medical Entomology 45, 133–138. PubMed

Pescher P., Blisnick T., Bastin P. and Späth G. F. (2011). Quantitative proteome profiling informs on phenotypic traits that adapt Leishmania donovani for axenic and intracellular proliferation. Cellular Microbiology 13, 978–991. PubMed

Pimenta P. F., Modi G. B., Pereira S. T., Shahabuddin M. and Sacks D. L. (1997). A novel role for the peritrophic matrix in protecting Leishmania from the hydrolytic activities of the sand fly midgut. Parasitology 115, 359–369. PubMed

Pruzinova K., Sadlova J., Seblova V., Homola M., Votypka J. and Volf P. (2015). Comparison of bloodmeal digestion and the peritrophic matrix in four sand fly species differing in susceptibility to Leishmania donovani. PLoS ONE 10, e0128203. PubMed PMC

Ramalho-Ortigão J. M. and Traub-Csekö Y. M. (2003). Molecular characterization of Llchit1, a midgut chitinase cDNA from the leishmaniasis vector Lutzomyia longipalpis. Insect Biochemistry and Molecular Biology 33, 279–287. PubMed

Ramalho-Ortigão J. M., Kamhawi S., Joshi M. B., Reynoso D., Lawyer P. G., Dwyer D. M., Sacks D. L. and Valenzuela J. G. (2005). Characterization of a blood activated chitinolytic system in the midgut of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi. Insect Molecular Biology 14, 703–712. PubMed

Rochette A., Raymond F., Corbeil J., Ouellette M. and Papadopoulou B. (2009). Whole-genome comparative RNA expression profiling of axenic and intracellular amastigote forms of Leishmania infantum. Molecular and Biochemical Parasitology 165, 32–47. PubMed

Rogers M. E., Chance M. L. and Bates P. A. (2002). The role of promastigote secretory gel in the origin and transmission of the infective stage of Leishmania mexicana by the sandfly Lutzomyia longipalpis. Parasitology 124, 495–507. PubMed

Rogers M. E., Hajmova M., Joshi M. B., Sadlova J., Dwyer D. M., Volf P. and Bates P. A. (2008). Leishmania chitinase facilitates colonization of sand fly vectors and enhances transmission to mice. Cellular Microbiology 10, 1363–1372. PubMed PMC

Sacks D. L. and Perkins P. V. (1985). Development of infective stage Leishmania promastigotes within phlebotomine sand flies. The American Journal of Tropical Medicine and Hygiene 34, 456–459. PubMed

Schlein Y. and Jacobson R. L. (1998). Resistance of Phlebotomus papatasi to infection with Leishmania donovani is modulated by components of the infective bloodmeal. Parasitology 117, 467–473. PubMed

Seblova V., Volfova V., Dvorak V., Pruzinova K., Votypka J., Kassahun A., Gebre-Michael T., Hailu A., Warburg A. and Volf P. (2013). Phlebotomus orientalis sand flies from two geographically distant Ethiopian localities: biology, genetic analyses and susceptibility to Leishmania donovani. PLoS Neglected Tropical Diseases 7, e2187. PubMed PMC

Stamper L. W., Patrick R. L., Fay M. P., Lawyer P. G., Elnaiem D. E., Secundino N., Debrabant A., Sacks D. L. and Peters N. C. (2011). Infection parameters in the sand fly vector that predict transmission of Leishmania major. PLoS Neglected Tropical Disieses 5, e1288. PubMed PMC

Stierhof Y. D., Bates P. A., Jacobson R. L., Rogers M. E., Schlein Y., Handman E. and Ilg T. (1999). Filamentous proteophosphoglycan secreted by Leishmania promastigotes forms gel-like three-dimensional networks that obstruct the digestive tract of infected sandfly vectors. European Journal of Cell Biology 78, 675–689. PubMed

Sadlova J. and Volf P. (2009). Peritrophic matrix of Phlebotomus duboscqi and its kinetics during Leishmania major development. Cell and Tissue Research 337, 313–325. PubMed PMC

Sadlova J., Price H. P., Smith B. A., Votypka J., Volf P. and Smith D. F. (2010). The stage-regulated HASPB and SHERP proteins are essential for differentiation of the protozoan parasite Leishmania major in its sand fly vector, Phlebotomus papatasi. Cellular Microbiology 12, 1765–1779. PubMed PMC

Sadlova J., Yeo M., Seblova V., Lewis M. D., Mauricio I., Volf P. and Miles M. A. (2011). Visualisation of Leishmania donovani fluorescent hybrids during early stage development in the sand fly vector. PLoS ONE 6, e19851. PubMed PMC

Volf P. and Volfova V. (2011). Establishment and maintenance of sand fly colonies. Journal of Vector Ecology 36, S1–S9. PubMed

Walters L. L. (1993). Leishmania differentiation in natural and unnatural sand fly hosts. Journal of Eukaryotic Microbiology 40, 196–206. PubMed

Warburg A. and Schlein Y. (1986). The effect of post-bloodmeal nutrition of Phlebotomus papatasi on the transmission of Leishmania major. The American Journal of Tropical Medicine and Hygiene 35, 926–930. PubMed

The development of L. major, L. donovani and L. martiniquensis, Leishmania currently emerging in Europe, in the sand fly species Phlebotomus perniciosus and P. tobbi

Effect of Phlebotomus papatasi on the fitness, infectivity and antimony-resistance phenotype of antimony-resistant Leishmania major Mon-25

Comparative genomics of Leishmania donovani progeny from genetic crosses in two sand fly species and impact on the diversity of diagnostic and vaccine candidates

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Three types of Leishmania mexicana amastigotes: Proteome comparison by quantitative proteomic analysis

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Host competence of the African rodents Arvicanthis neumanni, A. niloticus and Mastomys natalensis for Leishmania donovani from Ethiopia and L. (Mundinia) sp. from Ghana

Comparative Study of Promastigote- and Amastigote-Initiated Infection of Leishmania infantum (Kinetoplastida: Trypanosomatidae) in Phlebotomus perniciosus (Diptera: Psychodidae) Conducted in Different Biosafety Level Laboratories

Lutzomyia longipalpis TGF-β Has a Role in Leishmania infantum chagasi Survival in the Vector

Refractoriness of Sergentomyia schwetzi to Leishmania spp. is mediated by the peritrophic matrix

Leishmania mortality in sand fly blood meal is not species-specific and does not result from direct effect of proteinases