Sand Fly Gut Research Articles

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

Leishmania major is responsible for zoonotic cutaneous leishmaniasis. Therapy is mainly based on the use of antimony-based drugs; however, treatment failures and illness relapses were reported. Although studies were developed to understand mechanisms of drug resistance, the interactions of resistant parasites with their reservoir hosts and vectors remain poorly understood. Here we compared the development of two L. major MON-25 trivalent antimony-resistant lines, selected by a stepwise in vitro Sb(III)-drug pressure, to their wild-type parent line in the natural vector Phlebotomus papatasi. The intensity of infection, parasite location and morphological forms were compared by microscopy. Parasite growth curves and IC50 values have been determined before and after the passage in Ph. papatasi. qPCR was used to assess the amplification rates of some antimony-resistance gene markers. In the digestive tract of sand flies, Sb(III)-resistant lines developed similar infection rates as the wild-type lines during the early-stage infections, but significant differences were observed during the late-stage of the infections. Thus, on day 7 p. i., resistant lines showed lower representation of heavy infections with colonization of the stomodeal valve and lower percentage of metacyclic promastigote forms in comparison to wild-type strains. Observed differences between both resistant lines suggest that the level of Sb(III)-resistance negatively correlates with the quality of the development in the vector. Nevertheless, both resistant lines developed mature infections with the presence of infective metacyclic forms in almost half of infected sandflies. The passage of parasites through the sand fly guts does not significantly influence their capacity to multiply in vitro. The IC50 values and molecular analysis of antimony-resistance genes showed that the resistant phenotype of Sb(III)-resistant parasites is maintained after passage through the sand fly. Sb(III)-resistant lines of L. major MON-25 were able to produce mature infections in Ph. papatasi suggesting a possible circulation in the field using this vector.

Evidence of a conserved mammalian immunosuppression mechanism in Lutzomyia longipalpis upon infection with Leishmania.

Sand flies (Diptera: Phlebotominae) belonging to the Lutzomyia genus transmit Leishmania infantum parasites. To understand the complex interaction between the vector and the parasite, we have been investigating the sand fly immune responses during the Leishmania infection. Our previous studies showed that genes involved in the IMD, Toll, and Jak-STAT immunity pathways are regulated upon Leishmania and bacterial challenges. Nevertheless, the parasite can thrive in the vectors' gut, indicating the existence of mechanisms capable of modulating the vector defenses, as was already seen in mammalian Leishmania infections. In this study, we investigated the expression of Lutzomyia longipalpis genes involved in regulating the Toll pathway under parasitic infection. Leishmania infantum infection upregulated the expression of two L. longipalpis genes coding for the putative repressors cactus and protein tyrosine phosphatase SHP. These findings suggest that the parasite can modulate the vectors' immune response. In mammalian infections, the Leishmania surface glycoprotein GP63 is one of the inducers of host immune depression, and one of the known effectors is SHP. In L. longipalpis we found a similar effect: a genetically modified strain of Leishmania amazonensis over-expressing the metalloprotease GP63 induced a higher expression of the sand fly SHP indicating that the L. longipalpis SHP and parasite GP63 increased expressions are connected. Immuno-stained microscopy of L. longipalpis LL5 embryonic cells cultured with Leishmania strains or parasite conditioned medium showed cells internalization of parasite GP63. A similar internalization of GP63 was observed in the sand fly gut tissue after feeding on parasites, parasite exosomes, or parasite conditioned medium, indicating that GP63 can travel through cells in vitro or in vivo. When the sand fly SHP gene was silenced by RNAi and females infected by L. infantum, parasite loads decreased in the early phase of infection as expected, although no significant differences were seen in late infections of the stomodeal valve. Our findings show the possible role of a pathway repressor involved in regulating the L. longipalpis immune response during Leishmania infections inside the insect. In addition, they point out a conserved immunosuppressive effect of GP63 between mammals and sand flies in the early stage of parasite infection.

Leishmania (L.) amazonensis LaLRR17 increases parasite entry in macrophage by a mechanism dependent on GRP78.

Leishmaniases affect 12 million people worldwide. They are caused by Leishmania spp., protozoan parasites transmitted to mammals by female phlebotomine flies. During the life cycle, promastigote forms of the parasite live in the gut of infected sandflies and convert into amastigotes inside the vertebrate macrophages. The parasite evades macrophage's microbicidal responses due to virulence factors that affect parasite phagocytosis, survival and/or proliferation. The interaction between Leishmania and macrophage molecules is essential to phagocytosis and parasite survival. Proteins containing leucine-rich repeats (LRRs) are common in several organisms, and these motifs are usually involved in protein–protein interactions. We have identified the LRR17 gene, which encodes a protein with 6 LRR domains, in the genomes of several Leishmania species. We show here that promastigotes of Leishmania (L.) amazonensis overexpressing LaLRR17 are more infective in vitro. We produced recombinant LaLRR17 protein and identified macrophage 78 kDa glucose-regulated protein (GRP78) as a ligand for LaLRR17 employing affinity chromatography followed by mass spectrometry. We showed that GRP78 binds to LaLRR17 and that its blocking precludes the increase of infection conferred by LaLRR17. Our results are the first to report LRR17 gene and protein, and we hope they stimulate further studies on how this protein increases phagocytosis of Leishmania.

Infectiousness of Asymptomatic Meriones shawi, Reservoir Host of Leishmania major.

Leishmaniases are neglected diseases caused by protozoans of the genus Leishmania that threaten millions of people worldwide. Cutaneous leishmaniasis (CL) caused by L. major is a typical zoonosis transmitted by phlebotomine sand flies and maintained in rodent reservoirs. The female sand fly was assumed to become infected by feeding on the skin lesion of the host, and the relative contribution of asymptomatic individuals to disease transmission was unknown. In this study, we infected 32 Meriones shawi, North African reservoirs, with a natural dose of L. major obtained from the gut of infected sand flies. Skin manifestations appeared in 90% of the animals, and xenodiagnosis with the proven vector Phlebotomus papatasi showed transmissibility in 67% of the rodents, and 45% were repeatedly infectious to sand flies. Notably, the analysis of 113 xenodiagnostic trials with 2189 sand flies showed no significant difference in the transmissibility of animals in the asymptomatic and symptomatic periods; asymptomatic animals were infectious several weeks before the appearance of skin lesions and several months after their healing. These results clearly confirm that skin lesions are not a prerequisite for vector infection in CL and that asymptomatic animals are an essential source of L. major infection. These data are important for modeling the epidemiology of CL caused by L. major.

Molecular detection of Wolbachia and Bartonella as part of the microbiome of phlebotomine sand flies from Chiapas, Mexico

Phlebotomine sand flies are dipterans of relevance due to their role as vectors of several pathogens worldwide. Bacteria in the gut of sand flies possibly affect their vectorial capacity and competence to transmit parasites. A retrospective study was performed in sand fly specimens that had previously been collected in four localities of the state of Chiapas during the period 2009–2011 to detect Wolbachia and Bartonella and their possible coinfection with Leishmania. For the molecular detection of bacteria, we used primers and conditions that had previously been reported. A total of 531 sand fly specimens of 10 species were analyzed. Four Wolbachia strains were detected in five sand fly species, showing a prevalence of 8.6%. All the Wolbachia strains had previously been reported in other taxa. In one sand fly species, we also detected a new lineage of Bartonella evidenced by a phylogenetic analysis. No sand fly specimens showed coinfections of these bacteria and Leishmania. The bacteria found in the phlebotomine sand flies are possibly transmitted by plant-mediated horizontal transmission and during blood meal feeding.

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

Leishmania is the unicellular parasite transmitted by phlebotomine sand fly bite. It exists in two different forms; extracellular promastigotes, occurring in the gut of sand flies, and intracellular, round-shaped amastigotes residing mainly in vertebrate macrophages. As amastigotes originating from infected animals are often present in insufficient quality and quantity, two alternative types of amastigotes were introduced for laboratory experiments: axenic amastigotes and amastigotes from macrophages infected in vitro. Nevertheless, there is very little information about the degree of similarity/difference among these three types of amastigotes on proteomic level, whose comparison is crucial for assessing the suitability of using alternative types of amastigotes in experiments. In this study, L. mexicana amastigotes obtained from lesion of infected BALB/c mice were proteomically compared with alternatively cultivated amastigotes (axenic and macrophage-derived ones). Amastigotes of all three types were isolated, individually treated and analysed by LC-MS/MS proteomic analysis with quantification using TMT10-plex isobaric labeling. Significant differences were observed in the abundance of metabolic enzymes, virulence factors and proteins involved in translation and condensation of DNA. The most pronounced differences were observed between axenic amastigotes and lesion-derived amastigotes, macrophage-derived amastigotes were mostly intermediate between axenic and lesion-derived ones.

Comparative analysis of the gut microbiota of sand fly vectors of zoonotic visceral leishmaniasis (ZVL) in Iran; host-environment interplay shapes diversity.

The development of Leishmania parasites within sand fly vectors occurs entirely in the insect gut lumen, in the presence of symbiotic and commensal bacteria. The impacts of host species and environment on the gut microbiome are currently poorly understood. We employed MiSeq sequencing of the V3-16S rRNA gene amplicons to characterize and compare the gut microbiota of field-collected populations of Phlebotomus kandelakii, P. perfiliewi, P. alexandri, and P. major, the primary or secondary vectors of zoonotic visceral leishmaniasis (ZVL) in three distinct regions of Iran where ZVL is endemic. In total, 160,550 quality-filtered reads of the V3 region yielded a total of 72 operational taxonomic units (OTUs), belonging to 23 phyla, 47 classes, 91 orders, 131 families, and 335 genera. More than 50% of the bacteria identified were Proteobacteria, followed by Firmicutes (22%), Deinococcus-Thermus (9%), Actinobacteria (6%), and Bacteroidetes (5%). The core microbiome was dominated by eight genera: Acinetobacter, Streptococcus, Enterococcus, Staphylococcus, Bacillus, Propionibacterium, Kocuria, and Corynebacterium. Wolbachia were found in P. alexandri and P. perfiliewi, while Asaia sp. was reported in P. perfiliewi. Substantial variations in the gut bacterial composition were found between geographically distinct populations of the same sand fly species, as well as between different species at the same location, suggesting that sand fly gut microbiota is shaped by both the host species and geographical location. Phlebotomus kandelakii and P. perfiliewi in the northwest, and P. alexandri in the south, the major ZVL vectors, harbor the highest bacterial diversity, suggesting a possible relationship between microbiome diversity and the capacity for parasite transmission. In addition, large numbers of gram-positive human or animal pathogens were found, suggesting that sand fly vectors of ZVL could pose a potential additional threat to livestock and humans in the region studied. The presence of Bacillus subtilis, Enterobacter cloacae, and Asaia sp suggests that these bacteria could be promising candidates for a paratransgenesis approach to the fight against Leishmaniasis.

Can floral nectars reduce transmission of Leishmania?

Insect-vectored Leishmania are responsible for loss of more disability-adjusted life years than any parasite besides malaria. Elucidation of the environmental factors that affect parasite transmission by vectors is essential to develop sustainable methods of parasite control that do not have off-target effects on beneficial insects or environmental health. Many phytochemicals that inhibit growth of sand fly-vectored Leishmania-which have been exhaustively studied in the search for phytochemical-based drugs-are abundant in nectars, which provide sugar-based meals to infected sand flies. In a quantitative meta-analysis, we compare inhibitory phytochemical concentrations for Leishmania to concentrations present in floral nectar and pollen. We show that nectar concentrations of several flowering plant species exceed those that inhibit growth of Leishmania cell cultures, suggesting an unexplored, landscape ecology-based approach to reduce Leishmania transmission. If nectar compounds are as effective against parasites in the sand fly gut as predicted from experiments in vitro, strategic planting of antiparasitic phytochemical-rich floral resources or phytochemically enriched baits could reduce Leishmania loads in vectors. Such interventions could provide an environmentally friendly complement to existing means of disease control.

Effects of anti-Leishmania compounds in the behavior of the sand fly vector Lutzomyia longipalpis.

Leishmaniasis is an infectious parasitic disease caused by pathogens of the genus Leishmania transmitted through the bite of adult female sand flies. To reduce case numbers, it is necessary to combine different control approaches, especially those aimed at the sand fly vectors. Innovative forms of control with the use of attractive sugar baits explored the fact that adult sand flies need to feed on sugars of plant origin. Leishmania parasites develop in the gut of sand flies, interacting with the sugars in the diet of adults. Recent studies have shown that sugar baits containing plant-derived compounds can reduce sand fly survival, the number of parasites per gut, and the percentage of infected sand flies. Several synthetic compounds produced from naphthoquinones and pterocarpans have anti-parasitic activity on Leishmania amazonensis and/or Leishmania infantum in cell culture. This work aimed to assess the inclusion of these compounds in sugar baits for blocking transmission, targeting the development of the Leishmania parasite inside the sand fly vector. We evaluated the attractant or repellent properties of these compounds, as well as of the reference compound N,N'-diethyl-m-toluamide (DEET), in sugar baits. We also observed changes in feeding preference caused by these compounds, looking for anti-feeding or stimulation of ingestion. Pterocarpanquinone L4 and pentamidine showed attractant and repellent properties, respectively. Based on the effects in feeding preference and intake volume, pterocarpanquinone L6, and the pyrazole-derived compound P8 were chosen as the most promising compounds for the future development of anti-Leishmania sugar baits. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Sand Flies and Their Microbiota

Sand flies are a significant public health concern in many parts of the world where they are known to transmit agents of several zoonotic diseases to humans, such as leishmaniasis. Vector control remains a key component of many anti-leishmaniasis programs and probably will remain so until an effective vaccine becomes available. The sand fly gut microbiota has recently emerged as an encouraging field for the exploration of vector-based disease control. In particular, the gut microbiome was previously reported to either enhance or inhibit parasite activity depending on the species of bacteria and, thus, has the potential to alter vector competence. Here, we describe the technological advances that are currently expanding our understanding of microbiota composition in sand flies. The acquisition and composition of microbiomes are influenced by several abiotic and biotic factors, including host immunity, genetics, and the environment. Therefore, the microbiomes of sand flies can vary substantially between individuals, life stages, species, and over geographical space, and this variation likely contributes to differences in host phenotypes, highlighting opportunities for novel vector control strategies.

Advances in Understanding Leishmania Pathobiology: What Does RNA-Seq Tell Us?

Leishmaniasis is a vector-borne disease caused by a protozoa parasite from over 20 Leishmania species. The clinical manifestations and the outcome of the disease vary greatly. Global RNA sequencing (RNA-Seq) analyses emerged as a powerful technique to profile the changes in the transcriptome that occur in the Leishmania parasites and their infected host cells as the parasites progresses through their life cycle. Following the bite of a sandfly vector, Leishmania are transmitted to a mammalian host where neutrophils and macrophages are key cells mediating the interactions with the parasites and result in either the elimination the infection or contributing to its proliferation. This review focuses on RNA-Seq based transcriptomics analyses and summarizes the main findings derived from this technology. In doing so, we will highlight caveats in our understanding of the parasite’s pathobiology and suggest novel directions for research, including integrating more recent data highlighting the role of the bacterial members of the sandfly gut microbiota and the mammalian host skin microbiota in their potential role in influencing the quantitative and qualitative aspects of leishmaniasis pathology.

Remarkable diversity, new records and Leishmania detection in the sand fly fauna of an area of high endemicity for cutaneous leishmaniasis in Acre state, Brazilian Amazonian Forest

The species richness of Amazonian phlebotomines is considered to be one of the highest in the world. In the present study, we investigated the richness and diversity of phlebotomine fauna in Xapuri city, Acre state, Western Brazilian Amazonia, which is an area that is highly endemic for cutaneous leishmaniasis. Sand fly collections were performed monthly from August 2013 to July 2015 (288 h total of sampling effort) in intradomiciliary, peridomiciliary, and forested environments of two localities. Collected females were dissected, microscopically examined for flagellates in their guts, and preserved in ethanol. A total of 21,197 specimens comprising 14 genera and 57 species were collected, and the majority of these were Nyssomyia, Psychodopygus, and Trichophoromyia genera. Three new records of phlebotomine species for Acre are presented here, including Brumptomyia brumpti, Psathyromyia pradobarrientosi, and for the first time in Brazil, Th. omagua. In Xapuri, the phlebotomine fauna of different ecotopes was varied in regard to abundance, diversity, and frequency, and they included proven and permissive vectors of Leishmania spp. The fauna discovered in the forested areas (57 species) was richer and more diverse than was that (33 species) identified in the peri‑ and intra-domiciles. The identification of Leishmania subgenera that were present in sand fly guts according to SSU rRNA sequences revealed ten and three species harboring Leishmania of subgenera Viannia and Leishmania (most likely Leishmania amazonensis), respectively. The presence of Leishmania (Leishmania) in sand flies are reported here for the first time in Acre. The presence of L. (Viannia) spp. in Brumptomyia sp. and Lutzomyia sherlocki. and the occurrence of mixed infections with Leishmania of both subgenera in Ps. lainsoni have been reported for the first time in Brazil. Taken together, data from previous studies and from the present study highlight the remarkable complexity of phlebotomine fauna that is possibly due to the well-preserved Xapuri forested areas sustaining vital economic activities of plant extraction and ecological tourism. Our findings also provide new insights into the ongoing adaptation of Trichophoromyia and Psychodopygus species to human habitats.

The Diversity of Midgut Bacteria among Wild-Caught Phlebotomus argentipes (Psychodidae: Phlebotominae), the Vector of Leishmaniasis in Sri Lanka.

Phlebotomus argentipes is the main suspected vector for leishmaniasis in Sri Lanka. Investigations on the presence of aerobic bacteria in the gut of sand flies which evidence a potential approach to control leishmaniasis transmission through a paratransgenic strategy are still not available for the local sand fly populations. Field-caught unfed female sand flies collected from three selected Medical Officer of Health (MOH) areas (Polpithigama, Maho, and Galgamuwa) in Kurunegala District, Sri Lanka from August to December 2018 were used. Prokaryotic 16S ribosomal RNA partial gene was amplified and sequenced. Morphological identification revealed the presence of only one sand fly species, P. argentipes (n = 1,969). A total of 20 organisms belonging to two phyla (Proteobactericea and Furmicutes) were detected within the gut microbial community of the studied sand fly specimens. This study documents the first-ever observation of Rhizobium sp. in the midgut of P. argentipes. The presence of Bacillus megaterium, which is considered as a nonpathogenic bacterium with potential use for paratransgenic manipulation of P. argentipes suggest that it may be used as a delivery vehicle to block the vectorial transmission of Leishmania parasites. In addition, Serratia marcescens may be used as a potential candidate to block the parasite development in sand fly vectors since it has evidenced antileishmanial activities in previous investigations. Hence, further studies are required to gain full insight into the potential use of this bacterium in the control of Leishmania parasites through paratransgenesis.

Bacteria composition and diversity in the gut of sand fly: impact on Leishmania and sand fly development

Sand flies (Diptera: Psychodidae: Phlebotominae) are the only proven vectors of leishmaniasis. Leishmania parasites develop and differentiate in the sand fly gut where they reside alongside the vector’s gut bacteria. Gut bacteria are important to sand fly’s nutrition, oviposition, and defence against pathogens. In vectors, gut bacteria also influence vector competence. However, the link between specific bacterial taxa and sand fly vector susceptibility is poorly documented. In this review, we investigated current published data on bacterial communities in sand flies and provide a better understanding of how gut bacteria influence sand fly-Leishmania interactions. Understanding sand fly-bacteria-Leishmania interactions will provide new targets in vector control and Leishmania-transmission blocking approaches.

Development of Leishmania orientalis in the sand fly Lutzomyia longipalpis (Diptera: Psychodidae) and the biting midge Culicoides soronensis (Diptera: Ceratopogonidae)

Leishmania (Mundinia) orientalis is a newly described species causing human leishmaniasis in Thailand whose natural vector is unknown. L. orientalis infections in sand flies and/or biting midges under laboratory conditions have not been previously investigated. In this study, the development of L. orientalis in two experimental vectors, Lutzomyia longipalpis sand flies and Culicoides sonorensis biting midges was investigated for the first time using light microscopy, scanning electron microscopy, and histological examination. The results showed that L. orientalis was unable to establish infection in Lu. longipalpis. No parasites were found in the sand fly gut 4 days post-infected blood meal (PIBM). In contrast, the parasite successfully established infection in C. sonorensis. The parasites differentiated from amastigotes to procyclic promastigotes in the abdominal midgut (AMG) on day 1 PIBM. On day 2 PIBM, nectomonad promastigotes were observed in the AMG and migrated to the thoracic midgut (TMG). Leptomonad promastigotes appeared at the TMG on day 3 PIBM. Clusters of leptomonad promastigotes and metacyclic promastigotes colonized around the stomodeal valve with the accumulation of a promastigote secretory gel-like material from day 3 PIBM onwards. Haptomonad-like promastigotes were observed from day 5 PIBM, and the proportion of metacyclic promastigotes reached 23% on day 7 PIBM. The results suggest that biting midges or other sand fly genera or species might be vectors of L. orientalis.

A transdisciplinary consideration of sand flies & leishmaniasis

A transdisciplinary consideration of sand flies & leishmaniasis

Coinfection with Leishmania major and Staphylococcus aureus enhances the pathologic responses to both microbes through a pathway involving IL-17A.

Cutaneous leishmaniasis (CL) is a parasitic disease causing chronic, ulcerating skin lesions. Most humans infected with the causative Leishmania protozoa are asymptomatic. Leishmania spp. are usually introduced by sand flies into the dermis of mammalian hosts in the presence of bacteria from either the host skin, sand fly gut or both. We hypothesized that bacteria at the dermal inoculation site of Leishmania major will influence the severity of infection that ensues. A C57BL/6 mouse ear model of single or coinfection with Leishmania major, Staphylococcus aureus, or both showed that single pathogen infections caused localized lesions that peaked after 2–3 days for S. aureus and 3 weeks for L. major infection, but that coinfection produced lesions that were two-fold larger than single infection throughout 4 weeks after coinfection. Coinfection increased S. aureus burdens over 7 days, whereas L. major burdens (3, 7, 28 days) were the same in singly and coinfected ears. Inflammatory lesions throughout the first 4 weeks of coinfection had more neutrophils than did singly infected lesions, and the recruited neutrophils from early (day 1) lesions had similar phagocytic and NADPH oxidase capacities. However, most neutrophils were apoptotic, and transcription of immunomodulatory genes that promote efferocytosis was not upregulated, suggesting that the increased numbers of neutrophils may, in part, reflect defective clearance and resolution of the inflammatory response. In addition, the presence of more IL-17A-producing γδ and non-γδ T cells in early lesions (1–7 days), and L. major antigen-responsive Th17 cells after 28 days of coinfection, with a corresponding increase in IL-1β, may recruit more naïve neutrophils into the inflammatory site. Neutralization studies suggest that IL-17A contributed to an enhanced inflammatory response, whereas IL-1β has an important role in controlling bacterial replication. Taken together, these data suggest that coinfection of L. major infection with S. aureus exacerbates disease, both by promoting more inflammation and neutrophil recruitment and by increasing neutrophil apoptosis and delaying resolution of the inflammatory response. These data illustrate the profound impact that coinfecting microorganisms can exert on inflammatory lesion pathology and host adaptive immune responses.

Functional genomics in sand fly-derived Leishmania promastigotes.

BackgroundLeishmania development in the sand fly gut leads to highly infective forms called metacyclic promastigotes. This process can be routinely mimicked in culture. Gene expression–profiling studies by transcriptome analysis have been performed with the aim of studying promastigote forms in the sand fly gut, as well as differences between sand fly–and culture-derived promastigotes.FindingsTranscriptome analysis has revealed the crucial role of the microenvironment in parasite development within the sand fly gut because substantial differences and moderate correlation between the transcriptomes of cultured and sand fly–derived promastigotes have been found. Sand fly–derived metacyclics are more infective than metacyclics in culture. Therefore, some caution should be exercised when using cultured promastigotes, depending on the experimental design. The most remarkable examples are the hydrophilic acidic surface protein/small endoplasmic reticulum protein (HASP/SHERP) cluster, the glycoprotein 63 (gp63), and autophagy genes, which are up-regulated in sand fly–derived promastigotes compared with cultured promastigotes. Because HASP/SHERP genes are up-regulated in nectomonad and metacyclic promastigotes in the sand fly, the encoded proteins are not metacyclic specific. Metacyclic promastigotes are distinguished by morphology and high infectivity. Isolating them from the sand fly gut is not exempt from technical difficulty, because other promastigote forms remain in the gut even 15 days after infection. Leishmania major procyclic promastigotes within the sand fly gut up-regulate genes involved in cell cycle regulation and glucose catabolism, whereas metacyclics increase transcript levels of fatty acid biosynthesis and ATP-coupled proton transport genes. Most parasite's signal transduction pathways remain uncharacterized. Future elucidation may improve understanding of parasite development, particularly signaling molecule-encoding genes in sand fly versus culture and between promastigote forms in the sand fly gut.ConclusionsTranscriptome analysis has been demonstrated to be technically efficacious to study differential gene expression in sand fly gut promastigote forms. Transcript and protein levels are not well correlated in these organisms (approximately 25% quantitative coincidences), especially under stress situations and at differentiation processes. However, transcript and protein levels behave similarly in approximately 60% of cases from a qualitative point of view (increase, decrease, or no variation). Changes in translational efficiency observed in other trypanosomatids strongly suggest that the differences are due to translational regulation and regulation of the steady-state protein levels. The lack of low-input sample strategies does not allow translatome and proteome analysis of sand fly–derived promastigotes so far.

Aerobic midgut microbiota of sand fly vectors of zoonotic visceral leishmaniasis from northern Iran, a step toward finding potential paratransgenic candidates

BackgroundLeishmaniasis is caused by Leishmania parasites and is transmitted to humans through the bite of infected sand flies. Development of Leishmania to infective metacyclic promastigotes occurs within the sand fly gut where the gut microbiota influences development of the parasite. Paratransgenesis is a new control method in which symbiotic bacteria are isolated, transformed and reintroduced into the gut through their diet to express anti-parasitic molecules. In the present study, the midgut microbiota of three sand fly species from a steppe and a mountainous region of northern Iran, where zoonotic visceral leishmaniasis (ZVL) is endemic, was investigated.MethodsBriefly, adult female sand flies was collected during summer 2015 and, after dissection, the bacterial composition of the guts were analyzed using a culture-dependent method. Bacterial DNA from purified colonies was extracted to amplify the 16S rRNA gene which was then sequenced.ResultsThree ZVL sand fly vectors including Phlebotomus major, P. kandelakii and P. halepensis were found in the highlighted regions. In total, 39 distinct aerobic bacterial species were found in the sand fly midguts. The sand fly microbiota was dominated by Proteobacteria (56.4%) and Firmicutes (43.6%). Bacterial richness was significantly higher in the steppe region than in the mountainous region (32 vs 7 species). Phlebotomus kandelakii, the most important ZVL vector in the study area, had the highest bacterial richness among the three species. Bacillus subtilis and Pantoea agglomerans were isolated from the guts of the sand flies; these are already used for the paratransgenesis of sand flies and mosquitoes, respectively.ConclusionsThe existence of B. subtilis and P. agglomerans in the ZVL vectors and other sand fly species studied so far suggests that these two bacterial species are potential candidates for paratransgenic approach to prevent ZVL transmission. Further research needs to test the possible relationship between the gut microbiome richness and the vector competence of the ZVL vectors.

An Insight into the Constitutive Proteome Throughout Leishmania donovani Promastigote Growth and Differentiation.

Anthroponotic visceral leishmaniasis is a life-threatening disease caused by Leishmania donovani (Kinetoplastida: Trypanosomatidae) in East Africa and the Indian subcontinent. Unlike promastigote growth and differentiation in the sand fly gut or in axenic culture, L. donovani promastigote-into-amastigote development has been studied by high-throughput gene expression profiling. In this study, we have identified abundant constitutive proteins in axenically cultured promastigotes by two-dimension electrophoresis and matrix-assisted laser desorption-ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry. Most proteins involved in the trypanothione-based redox antioxidant system are expressed constitutively throughout axenic L. donovani promastigote growth and differentiation (tryparedoxin, trypanothione peroxidase, generic peroxidoxin, iron-superoxide dismutase, and elongation factor 1β). These findings are in agreement with previous data on other Old World species (i.e., L. major and L. infantum), whereas New World species (i.e., L. amazonensis and L. pifanoi) and Crithidia fasciculata show different expression patterns.