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Additional file 1: Table S1. Number of developed oocytes of Sergentomyia minuta females feeding on different hosts. TAR, Tarentola mauritanica; HEM, Hemidactylus turcicus; POD, Podarcis siculus; HUM, human volunteer. Table S2. Comparison of Sergentomyia minuta fecundity after feeding on different hosts. TAR, Tarentola mauritanica; HEM, Hemidactylus turcicus; POD, Podarcis siculus; HUM, human volunteer.

Additional file 3: Table S5. Representation of individual morphological forms of Leishmania (Sauroleishmania) adleri developing in Phlebotomus orientalis on days 5 to 9 post blood meal. Table S6. Detailed measurements of individual forms of Leishmania (Sauroleishmania) adleri developing in Phlebotomus orientalis on days 5, 7 and 9 post blood meal. Table S7. Representation of individual morphological forms of Leishmania (Sauroleishmania) hoogstraali developing in Phlebotomus orientalis on days 5 to 9 post blood meal. Table S8. Detailed measurements of individual forms of Leishmania (Sauroleishmania) hoogstraali developing in Phlebotomus orientalis on days 5, 7 and 9 post blood meal

Additional file 1: Table S1. Localization of Leishmania (Sauroleishmania) adleri promastigotes in three sand fly species differing in vector competence to Leishmania. Table S2. Localization of Leishmania (Sauroleishmania) hoogstraali promastigotes in three sand fly species differing in vector competence to Leishmania

Additional file 4: Table S9. Xenodiagnoses of Hemidactylus turcicus geckos experimentally infected with Leishmania (Sauroleishmania) adleri and Leishmania (Sauroleishmania) hoogstraali

Visceral leishmaniasis caused by Leishmania donovani is regarded as mostly anthroponotic, but a role for animal reservoir hosts in transmission has been suggested in East Africa. Field studies in this region have shown the presence of this parasite in several mammalian species, including rodents of the genera Arvicanthis and Mastomys. Further, the natural reservoirs of Leishmania (Mundinia) sp. causing human cutaneous disease in Ghana, West Africa, are unknown. This study assessed the potential role of the Sub-Saharan rodents Arvicanthis neumanni, A. niloticus and Mastomys natalensis as hosts of L. donovani and L. sp. from Ghana, based on experimental infections of animals and xenodiagnoses. The distribution and load of parasites were determined post mortem using qPCR from the blood, skin and viscera samples. The attractiveness of Arvicanthis and Mastomys to Phlebotomus orientalis was tested by pair-wise comparisons. None of the animals inoculated with L. donovani were infectious to P. orientalis females, although, in some animals, parasites were detected by PCR even 30 weeks post infection. Skin infections were characterized by low numbers of parasites while high parasite burdens were present in spleen, liver and lymph nodes only. Therefore, wild Arvicanthis and Mastomys found infected with L. donovani, should be considered parasite sinks rather than parasite reservoirs. This is indirectly supported also by results of host choice experiments with P. orientalis in which females preferred humans over both Arvicanthis and Mastomys, and their feeding rates on rodents ranged from 1.4 to 5.8% only. Therefore, the involvement of these rodents in transmission of L. donovani by P. orientalis is very unlikely. Similarly, poor survival of Leishmania parasites in the studied rodents and negative results of xenodiagnostic experiments do not support the involvement of Arvicanthis and Mastomys spp. in the transmission cycle of L. sp. from Ghana. Keywords: Visceral leishmaniasis, Reservoir hosts, Xenodiagnosis, Grass rats, Multimammate mice, Mundinia

Leishmania (Sauroleishmania) tarentolae is transmitted by reptile-biting sand flies of the genus Sergentomyia, but the role of Phlebotomus sand flies in circulation of this parasite is unknown. Here, we compared the development of L. (S.) tarentolae strains in three Phlebotomus species: P. papatasi, P.&nbsp, sergenti, and P. perniciosus. Laboratory-bred sand flies were membrane-fed on blood with parasite suspension and dissected on days 1 and 7 post blood meal. Parasites were measured on Giemsa-stained gut smears and five morphological forms were distinguished. In all parasite-vector combinations, promastigotes were found in Malpighian tubules, often in high numbers, which suggests that this tissue is a typical location for L. (S.) tarentolae development in sand flies. All three studied strains colonized the hindgut, but also migrated anteriorly to both parts of the midgut and colonized the stomodeal valve. Significant differences were demonstrated between sand fly species: highest infection rates, high parasite loads, and the most frequent anterior migration with colonization of the stomodeal valve were found in P. perniciosus, while all these parameters were lowest in P. sergenti. In conclusion, the peripylarian type of development was demonstrated for three L. (S.) tarentolae strains in three Phlebotomus sand flies. We suggest paying more attention to Phlebotomus species, particularly P. perniciosus and P. papatasi, as potential secondary vectors of Sauroleishmania.

The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown. Here we address this important open question applying experimental evolution and integrative systems approaches on parasites adapting to in vitro culture. Phenotypic analyses of parasites from early and late stages of culture adaptation revealed an important fitness tradeoff, with selection for accelerated growth (fitness gain) impairing infectivity (fitness costs). Comparative genomics, transcriptomics and proteomics analyses revealed a complex regulatory network driving parasite fitness, with genome instability causing highly reproducible, gene dosage-dependent changes in protein linked to post-transcriptional regulation. These in turn were associated with a gene dosage-independent reduction in flagellar transcripts and a coordinated increase in abundance of coding and non-coding RNAs known to regulate ribosomal biogenesis and protein translation. We correlated differential expression of small nucleolar RNAs (snoRNAs) with changes in rRNA modification, providing first evidence that Leishmania fitness gain may be controlled by post-transcriptional and epitranscriptomic regulation. Our findings propose a novel model for Leishmania fitness gain, where differential regulation of mRNA stability and the generation of fitness-adapted ribosomes may potentially filter deleterious from beneficial gene dosage effects and provide proteomic robustness to genetically heterogenous, adapting parasite populations. This model challenges the current, genome-centric approach to Leishmania epidemiology and identifies the Leishmania non-coding small RNome as a potential novel source for biomarker discovery.

Gating strategy. a Gating on single GFP+ cells on GFP-A/GFP-H dotplot. b Gating on Leishmania cells on FSC-A/SSC-A dotplot. c Analysis of DAPI fluorescence on histogram – enumeration of % DAPI+ (i.e. dead) Leishmania cells. Figure S2. Different Leishmania donovani stages were incubated with midguts of P. argentipes dissected at 24 h post-blood meal. Analysis of dead Leishmania was performed using flow cytometry. Percentage of dead cells was assessed on histogram of DAPI fluorescence on gated single GFP-positive Leishmania. Figure S3. Different Leishmania donovani stages were incubated with midguts of P. orientalis dissected at 24 h post-blood meal. Analysis of dead Leishmania was performed using flow cytometry. Percentage of dead cells was assessed on histogram of DAPI fluorescence on gated single GFP-positive Leishmania. Figure S4. Different Leishmania donovani stages were incubated with midguts of P. papatasi dissected at 24 h post-blood meal. Analysis of dead Leishmania was performed using flow cytometry. Percentage of dead cells was assessed on histogram of DAPI fluorescence on gated single GFP-positive Leishmania. Figure S5. Different Leishmania donovani stages were incubated with midguts of S. schwetzi dissected at 24 h post-blood meal. Analysis of dead Leishmania was performed using flow cytometry. Percentage of dead cells was assessed on histogram of DAPI fluorescence on gated single GFP-positive Leishmania. Figure S6. Negative control; different Leishmania donovani stages were incubated with saline. Analysis of dead Leishmania was performed using flow cytometry. Percentage of dead cells was assessed on histogram of DAPI fluorescence on gated single GFP-positive Leishmania. Figure S7. Positive control; parasites killed by 1% formaldehyde and permeabilised by 0.5% Triton X-100. Percentage of dead cells was assessed on histogram of DAPI fluorescence on gated single GFP-positive Leishmania. (PPTX 346 kb)

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.