Anopheles darlingi polytene chromosomes: revised maps including newly described inversions and evidence for population structure in Manaus

Salivary gland polytene chromosomes of 4th instar Anopheles darlingi Root were examined from multiple locations in the Brazilian Amazon. Minor modifications were made to existing polytene photomaps. These included changes to the breakpoint positions of several previously described paracentric inversions and descriptions of four new paracentric inversions, two on the right arm of chromosome 3 and two on the left arm of chromosome 3 that were found in multiple locations. A total of 18 inversions on the X (n = 1) chromosome, chromosome 2 (n = 7) and 3 (n = 11) were scored for 83 individuals from Manaus, Macapá and Porto Velho municipalities. The frequency of 2Ra inversion karyotypes in Manaus shows significant deficiency of heterozygotes (p < 0.0009). No significant linkage disequilibrium was found between inversions on chromosome 2 and 3. We hypothesize that at least two sympatric subpopulations exist within the An. darlingi population at Manaus based on inversion frequencies

Skin parasite landscape determines host infectiousness in visceral leishmaniasis

Increasing evidence suggests that the infectiousness of patients for the sand fly vector of visceral leishmaniasis is linked to parasites found in the skin. Using a murine model that supports extensive skin infection with Leishmania donovani, spatial analyses at macro-(quantitative PCR) and micro-(confocal microscopy) scales indicate that parasite distribution is markedly skewed. Mathematical models accounting for this heterogeneity demonstrate that while a patchy distribution reduces the expected number of sand flies acquiring parasites, it increases the infection load for sand flies feeding on a patch, increasing their potential for onward transmission. Models representing patchiness at both macro- and micro-scales provide the best fit with experimental sand fly feeding data, pointing to the importance of the skin parasite landscape as a predictor of host infectiousness. Our analysis highlights the skin as a critical site to consider when assessing treatment efficacy, transmission competence and the impact of visceral leishmaniasis elimination campaigns.Parasitemia has been considered the main determinant of visceral leishmaniasis transmission. By combining imaging, qPCR and experimental xenodiagnoses with mathematical models, Doehl et al. argue that the patchy landscape of parasites in the skin is necessary to explain infectiousness

Peritrophic matrix of Phlebotomus duboscqi and its kinetics during Leishmania major development

Light microscopy of native preparations, histology, and electron microscopy have revealed that Phlebotomus duboscqi belongs to a class of sand fly species with prompt development of the peritrophic matrix (PM). Secretion of electron-lucent fibrils, presumably chitin, starts immediately after the ingestion of a blood meal and, about 6 h later, is followed by secretion of amorphous electron-dense components, presumably proteins and glycoproteins. The PM matures in less than 12 h and consists of a thin laminar outer layer and a thick amorphous inner layer. No differences have been found in the timing of the disintegration of the PM in females infected with Leishmania major. In both groups of females (infected and uninfected), the disintegration of the PM is initiated at the posterior end. Although parasites are present at high densities in the anterior part of the blood meal bolus, they escape from the PM at the posterior end only. These results suggest that L. major chitinase does not have an important role in parasite escape from the PM. Promastigotes remain in the intraperitrophic space until the PM is broken down by sand-fly-derived chitinases and only then migrate anteriorly. Disintegration of the PM occurs simultaneously with the morphological transformation of parasites from procyclic forms to long nectomonads. A novel role is ascribed to the anterior plug, a component of the PM secreted by the thoracic midgut; this plug functions as a temporary barrier to stop the forward migration of nectomonads to the thoracic midgut

Cooperation between Apoptotic and Viable Metacyclics Enhances the Pathogenesis of Leishmaniasis

Mimicking mammalian apoptotic cells by exposing phosphatidylserine (PS) is a strategy used by virus and parasitic protozoa to escape host protective inflammatory responses. With Leishmania amazonensis (La), apoptotic mimicry is a prerogative of the intramacrophagic amastigote form of the parasite and is modulated by the host. Now we show that differently from what happens with amastigotes, promastigotes exposing PS are non-viable, non-infective cells, undergoing apoptotic death. As part of the normal metacyclogenic process occurring in axenic cultures and in the gut of sand fly vectors, a sub-population of metacyclic promastigotes exposes PS. Apoptotic death of the purified PS-positive (PSPOS) sub-population was confirmed by TUNEL staining and DNA laddering. Transmission electron microscopy revealed morphological alterations in PSPOS metacyclics such as DNA condensation, cytoplasm degradation and mitochondrion and kinetoplast destruction, both in in vitro cultures and in sand fly guts. TUNELPOS promastigotes were detected only in the anterior midgut to foregut boundary of infected sand flies. Interestingly, caspase inhibitors modulated parasite death and PS exposure, when added to parasite cultures in a specific time window. Efficient in vitro macrophage infections and in vivo lesions only occur when PSPOS and PS-negative (PSNEG) parasites were simultaneously added to the cell culture or inoculated in the mammalian host. The viable PSNEG promastigote was the infective form, as shown by following the fate of fluorescently labeled parasites, while the PSPOS apoptotic sub-population inhibited host macrophage inflammatory response. PS exposure and macrophage inhibition by a subpopulation of promastigotes is a different mechanism than the one previously described with amastigotes, where the entire population exposes PS. Both mechanisms co-exist and play a role in the transmission and development of the disease in case of infection by La. Since both processes confer selective advantages to the infective microorganism they justify the occurrence of apoptotic features in a unicellular pathogen

The midgut transcriptome of Phlebotomus (Larroussius) perniciosus, a vector of Leishmania infantum: comparison of sugar fed and blood fed sand flies

<p>Abstract</p> <p>Background</p> <p>Parasite-vector interactions are fundamental in the transmission of vector-borne diseases such as leishmaniasis. <it>Leishmania </it>development in the vector sand fly is confined to the digestive tract, where sand fly midgut molecules interact with the parasites. In this work we sequenced and analyzed two midgut-specific cDNA libraries from sugar fed and blood fed female <it>Phlebotomus perniciosus </it>and compared the transcript expression profiles.</p> <p>Results</p> <p>A total of 4111 high quality sequences were obtained from the two libraries and assembled into 370 contigs and 1085 singletons. Molecules with putative roles in blood meal digestion, peritrophic matrix formation, immunity and response to oxidative stress were identified, including proteins that were not previously reported in sand flies. These molecules were evaluated relative to other published sand fly transcripts. Comparative analysis of the two libraries revealed transcripts differentially expressed in response to blood feeding. Molecules up regulated by blood feeding include a putative peritrophin (<it>PperPer1</it>), two chymotrypsin-like proteins (<it>PperChym1 </it>and <it>PperChym2</it>), a putative trypsin (<it>PperTryp3</it>) and four putative microvillar proteins (<it>PperMVP1</it>, <it>2</it>, <it>4 </it>and <it>5</it>). Additionally, several transcripts were more abundant in the sugar fed midgut, such as two putative trypsins (<it>PperTryp1 </it>and <it>PperTryp2</it>), a chymotrypsin (<it>PperChym3</it>) and a microvillar protein (<it>PperMVP3</it>). We performed a detailed temporal expression profile analysis of the putative trypsin transcripts using qPCR and confirmed the expression of blood-induced and blood-repressed trypsins. Trypsin expression was measured in <it>Leishmania infantum</it>-infected and uninfected sand flies, which identified the <it>L. infantum</it>-induced down regulation of <it>PperTryp3 </it>at 24 hours post-blood meal.</p> <p>Conclusion</p> <p>This midgut tissue-specific transcriptome provides insight into the molecules expressed in the midgut of <it>P. perniciosus</it>, an important vector of visceral leishmaniasis in the Old World. Through the comparative analysis of the libraries we identified molecules differentially expressed during blood meal digestion. Additionally, this study provides a detailed comparison to transcripts of other sand flies. Moreover, our analysis of putative trypsins demonstrated that <it>L. infantum </it>infection can reduce the transcript abundance of trypsin <it>PperTryp3 </it>in the midgut of <it>P. perniciosus</it>.</p