Background: Microbial ecology of phlebotomine sand flies is not well understood although bacteria likely play an important role in the sand fly biology and vector capacity for Leishmania parasites. In this study, we assessed the significance of the microbial community of rabbit feces in oviposition and larval development of Lutzomyia longipalpis as well as bacterial colonization of the gut of freshly emerged flies. Methods: Sterile (by autoclaving) and non-sterile (control) rabbit feces were used in the two-choice assay to determine their oviposition attractiveness to sand fly females. Bacteria were identified by amplification and sequencing of the 16S rRNA gene with universal eubacterial primers. Sterile, control (non-sterile), and sterilized and inoculated rabbit feces were used to assess the significance of bacteria in L. longipalpis development. Newly emerged adult flies were surface-sterilized and screened for the bacterial population size and diversity by the culturing approach. The digestive tract of L4 sterile and control larvae was incubated with Phalloidin to visualize muscle tissues and DAPI to visualize nuclei. Results: Two-choice behavioural assays revealed a great preference of L. longipalpis to lay eggs on rabbit feces with an active complex bacterial community (control) (85.8 % of eggs) in comparison to that of sterile (autoclaved) rabbit feces (14.2 %). Bioassays demonstrated that L. longipalpis larvae can develop in sterile rabbit feces although development time to adult stage was greatly extended (47 days) and survival of larvae was significantly lower (77.8 %) compared to that of larvae developing in the control rabbit feces (32 days and 91.7 %). Larval survival on sterilized rabbit feces inoculated with the individual bacterial isolates originating from this substrate varied greatly depending on a bacterial strain. Rhizobium radiobacter supported larval development to adult stage into the greatest extent (39 days, 88.0 %) in contrast to that of Bacillus spp. (76 days, 36.0 %). From the complex natural bacterial community of rabbit feces, R. radiobacter survived pupation and colonized the newly emerged females most successfully (82.6 % of all bacteria cultured); however, only 25 % of females were positive for bacteria in the digestive tract upon emergence. Immunohistochemistry did not reveal any obvious differences in anatomy of the digestive tract between control and axenic larvae. Conclusions: The bacterial community in the sand fly larval habitat affects oviposition and larval development although bacteria are not essential for successful development of L. longipalpis. Different bacteria contribute to larval development to various degrees and some, e.g. Rhizobium radiobacter, survive pupation and colonize the digestive tract of newly emerged females. With the establishment of the axenic rearing system, this study opens new venues to study the effect of bacteria on the gut epithelial immunity and vector competence of sand flies for Leishmania parasites with a goal to develop paratransgenic approaches for Leishmania control.

Background: In sandflies, the blood meal is responsible for the induction of several physiologic processes that culminate in egg development and maturation. During blood feeding, infected sandflies are also able to transmit the parasite Leishmania to a suitable host. Many blood-induced molecules play significant roles during Leishmania development in the sandfly midgut, including parasite killing within the endoperitrophic space. In this work, we randomly sequenced transcripts from three distinct high quality full-length female Phlebotomus papatasi midgut-specific cDNA libraries from sugar-fed, blood-fed and Leishmania major-infected sandflies. Furthermore, we compared the transcript expression profiles from the three different cDNA libraries by customized bioinformatics analysis and validated these findings by semi-quantitative PCR and real-time PCR. Results: Transcriptome analysis of 4010 cDNA clones resulted in the identification of the most abundant P. papatasi midgut-specific transcripts. The identified molecules included those with putative roles in digestion and peritrophic matrix formation, among others. Moreover, we identified sandfly midgut transcripts that are expressed only after a blood meal, such as microvilli associated-like protein (PpMVP1, PpMVP2 and PpMVP3), a peritrophin (PpPer1), trypsin 4 (PpTryp4), chymotrypsin PpChym2, and two unknown proteins. Of interest, many of these overabundant transcripts such as PpChym2, PpMVP1, PpMVP2, PpPer1 and PpPer2 were of lower abundance when the sandfly was given a blood meal in the presence of L. major. Conclusion: This tissue-specific transcriptome analysis provides a comprehensive look at the repertoire of transcripts present in the midgut of the sandfly P. papatasi. Furthermore, the customized bioinformatic analysis allowed us to compare and identify the overall transcript abundance from sugar-fed, blood-fed and Leishmania-infected sandflies. The suggested upregulation of specific transcripts in a blood-fed cDNA library were validated by real-time PCR, suggesting that this customized bioinformatic analysis is a powerful and accurate tool useful in analysing expression profiles from different cDNA libraries. Additionally, the findings presented in this work suggest that the Leishmania parasite is modulating key enzymes or proteins in the gut of the sandfly that may be beneficial for its establishment and survival.

The Leishmania developmental life cycle within its sand fly vector occurs exclusively in the lumen of the insect’s digestive tract in the presence of symbiotic bacteria. The composition of the gut microbiota and the factors that influence its composition are currently poorly understood. A set of factors, including the host and its environment, may influence this composition. It has been demonstrated that the insect gut microbiota influences the development of several human pathogens, such as Plasmodium falciparum. For sand flies and Leishmania, understanding the interactions between the parasite and the microbial environment of the vector midgut can provide new tools to control Leishmania transmission. The midguts of female Phlebotomus perniciosus from laboratory colonies or from the field were collected during the months of July, September and October 2011 and dissected. The midguts were analyzed by culture-dependent and culture-independent methods. A total of 441 and 115 cultivable isolates were assigned to 30 and 11 phylotypes from field-collected and colonized P. perniciosus, respectively. Analysis of monthly variations in microbiota composition shows a species diversity decline in October, which is to the end of the Leishmania infantum transmission period. In parallel, a compilation and a meta-analysis of all available data concerning the microbiota of two Psychodidae genera, namely Phlebotomus and Lutzomyia, was performed and compared to P. perniciosus, data obtained herein. This integrated analysis did not reveal any substantial divergences between Old and New world sand flies with regards to the midgut bacterial phyla and genera diversity. But clearly, most bacterial species (>76%) are sparsely distributed between Phlebotominae species. Our results pinpoint the need for a more exhaustive understanding of the bacterial richness and abundance at the species level in Phlebotominae sand flies in order to capture the role of midgut bacteria during Leishmania development and transmission. The occurrence of Bacillus subtilis in P. perniciosus and at least two other sand fly species studied so far suggests that this bacterial species is a potential candidate for paratransgenic or biolological approaches for the control of sand fly populations in order to prevent Leishmania transmission. The use of conventional microbiological methods gave us the opportunity to investigate the richness of symbiotic bacteria that inhabit the gut of P. perniciosus during its main period of activity. Our results were subsequently analyzed in the framework of what has been done on sand flies microbiota in order to validate our results and to address the question of the definition of the core bacterial microbiota of sand flies. A meta-analysis on the respective gut microbiota of Old and New World sand flies shows that the majority of bacterial species is observed only in one host whereas less than 8% are shared by more than two hosts. Our results pinpoint the need for a more exhaustive understanding of the microbiota composition and dynamic in phlebotominae, with the aim to implement new biological approaches for the control of sand fly populations in order to prevent Leishmania transmission.

Insect-borne diseases are responsible for severe mortality and morbidity worldwide. As control of insect vector populations relies primarily on the use of insecticides, the emergence of insecticide resistance as well to unintended consequences of insecticide use pose significant challenges to their continued application. Novel approaches to reduce pathogen transmission by disease vectors are been attempted, including transmission-blocking vaccines (TBVs) thought to be a feasible strategy to reduce pathogen burden in endemic areas. TBVs aim at preventing the transmission of pathogens from infected to uninfected vertebrate host by targeting molecule(s) expressed on the surface of pathogens during their developmental phase within the insect vector or by targeting molecules expressed by the vectors. For pathogen-based molecules, the majority of the TBV candidates selected as well as most of the data available regarding the effectiveness of this approach come from studies using malaria parasites. However, TBV candidates also have been identified from midgut tissues of mosquitoes and sand flies. In spite of the successes achieved in the potential application of TBVs against insect-borne diseases, many significant barriers remain. In this review, many of the TBV strategies against insect-borne pathogens and their respective ramification with regards to the immune response of the vertebrate host are discussed.