Lutzomyia longipalpis is the most important vector of visceral leishmaniasis in Brazil. When female sandflies feed on blood, a peritrophic matrix (PM) is formed around the blood bolus. The PM is secreted by midgut cells and composed of proteins, glycoproteins and chitin microfibrils. The PM functions as both a physical barrier against pathogens present in the food bolus and blood meal digestion regulator. Previous studies of mosquitoes and sandflies have shown that the absence of a PM, resulting from adding an exogenous chitinase to the blood meal, accelerates digestion. In the present study, we analysed biological factors associated with the presence of a PM in L. longipalpis females. Insects fed blood containing chitinase (BCC) accelerated egg-laying relative to a control group fed blood without chitinase. However, in the BCC-fed insects, the number of females that died without laying eggs was higher and the number of eggs laid per female was lower. The eggs in both groups were viable and generated adults. Based on these data, we suggest that the absence of a PM accelerates nutrient acquisition, which results in premature egg production and oviposition; however, the absence of a PM reduces the total number of eggs laid per female. Reduced fecundity in the absence of a PM may be due to inefficient nutrient conversion or the loss of the protective role of the PM

Phlebotomine sand flies (Diptera, Psychodidae) belonging to the Lutzomyia genus transmit zoonoses in the New World. Lutzomyia longipalpis is the main vector of Leishmania infantum, which is the causative agent of visceral leishmaniasis in Brazil. To identify key molecular aspects involved in the interaction between vector and pathogens and contribute to developing disease transmission controls, we investigated the sand fly innate immunity mediated by the Janus kinase/signal transducer and activator of transcription (Jak-STAT) pathway in response to L. infantum infection. We used two study models: L. longipalpis LL5 embryonic cells co-cultured with L. infantum and sand fly females artificially infected with the parasite. We used qPCR to follow the L. longipalpis gene expression of molecules involved in the Jak-STAT pathway. Also, we modulated the Jak-STAT mediated immune response to understand its role in Leishmania parasite infection. For that, we used RNAi to silence the pathway regulators, protein inhibitor of activated STATs (PIAS) in LL5 cells, and STAT in adult females. In addition, the pathway suppression effect on parasite development within the vector was assessed by light microscopy in late-phase infection. The silencing of the repressor PIAS in LL5 cells led to a moderate increase in a protein tyrosine phosphatase 61F (PTP61F) expression. It suggests a compensatory regulation between these two repressors. L. infantum co-culture with LL5 cells upregulated repressors PIAS, suppressor of cytokine signaling (SOCS), and PTP61F. It also downmodulated virus-induced RNA-1 (VIR-1), a pathway effector, indicating that the parasite could repress the Jak-STAT pathway in LL5 cells. In Leishmania-infected L. longipalpis females, STAT and the antimicrobial peptide attacin were downregulated on the third day post-infection, suggesting a correlation that favors the parasite survival at the end of blood digestion in the sand fly. The antibiotic treatment of infected females showed that the reduction of gut bacteria had little effect on the Jak-STAT pathway regulation. STAT gene silencing mediated by RNAi reduced the expression of inducible nitric oxide synthase (iNOS) and favored Leishmania growth in sand flies on the first day post-infection. These results indicate that STAT participated in the iNOS regulation with subsequent effect on parasite survival.

Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the kinetoplastid parasites of the genus Leishmania, the causative agents of devastating diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. No approved efficacious vaccine exists for leishmaniasis and available therapeutic drugs are either toxic and/or expensive, or the parasites are becoming resistant to the more recently developed drugs. Therefore, sand fly and/or reservoir control are currently the most effective strategies to break transmission. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two geographically widespread and important sand fly vector species: Phlebotomus papatasi, a vector of Leishmania parasites that cause cutaneous leishmaniasis, (distributed in Europe, the Middle East and North Africa) and Lutzomyia longipalpis, a vector of Leishmania parasites that cause visceral leishmaniasis (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites.

Introduction: Production of different antimicrobial peptides (AMPs) is one of the insect’s prominent defense strategies, regulated mainly by Toll and immune deficiency (IMD) humoral pathways. Here we focused mainly on two AMPs of Phlebotomus papatasi, vector of Leishmania major parasites, their association with the relish transcription factor and the effective participation on Leishmania infection.Methods and results: We further characterized the role of previously described gut-specific P. papatasi defensin (PpDef1) and identified the second defensin (PpDef2) expressed in various sand fly tissues. Using the RNAi-mediated gene silencing, we report that the silencing of PpDef1 gene or simultaneous silencing of both defensin genes (PpDef1 and PpDef2) resulted in increased parasite levels in the sand fly (detectable by PCR) and higher sand fly mortality. In addition, we knocked down relish, the sole transcription factor of the IMD pathway, to evaluate the association of the IMD pathway with AMPs expression in P. papatasi. We demonstrated that the relish gene knockdown reduced the expression of PpDef2 and attacin, another AMP abundantly expressed in the sand fly body.Conclusions: Altogether, our experiments show the importance of defensins in the sand fly response toward L. major and the role of the IMD pathway in regulating AMPs in P. papatasi.