Vector-borne diseases create considerable risks to human health. Leishmaniases are a group of neglected vector-borne diseases caused by protists parasites from the Leishmania genus. Approximately 350 million people worldwide are at risk of being infected with Leishmania, and clinical manifestations in humans vary from cutaneous to visceral types. Leishmania is transmitted to humans by hematophagous female sand flies (Diptera, Psychodidae, Phlebotominae). Here, we focus on Phlebotomus perniciosus, the natural vector of Leishmania infantum (the causative agent of visceral leishmaniasis) in the western region of the Mediterranean basin of Europe. Leishmania cycle occurs in the sand fly midgut, where parasites must overcome natural barriers such as high activity levels of midgut digestive proteases, competition with the native gut microbiota, and the activation of sand fly immune responses. Recently it was shown that insect immunity is under hormonal regulation; the ecdysone hormone binds to its receptor (EcR) and triggers the expression of IMD immune signalling pathway-related genes. In sand flies gut, the IMD pathway controls Leishmania infection, but ecdysone's role in sand fly immunity and microbiota has never been studied. The NEPHIMLEI project, in a pioneering way, proposes manipulating P.perniciosus neuroendocrine system to affect sand fly gut immunity and microbiota, intending the blockage of L. infantum development in the vector. Sand fly ecdysone function will be impaired by azadirachtin (an ecdysone inhibitor) oral treatment or by silencing the EcR gene by RNAi. Also, sand fly immunity will be enhanced by suppressing repressor genes from both ecdysone and IMD pathways (Eip75B and caspar, respectively) using the CRISPR/Cas9 system. The altered ecdysone signaling could change P. perniciosus IMD-related immune responses, affecting microbiota and inducing sand fly resistance against L. infantum.
The NEPHIMEI proposal will unveil the mechanisms by which ecdysone regulates the insect's immunity. However, as there is no study describing P. perniciosus immune signaling pathways, we will first investigate these genes' function in maintaining microbiota homeostasis and during L. infantum infection. Then, the neuroendocrine system's role in P. perniciosus immunity and parasite-insect-microbiota interaction will be explored. Based on evidence that ecdysone signaling amplifies Drosophila immune responses, hence affecting parasites' development, it is expected the knockout of the repressor gene Eip75B will make the ecdysone signaling pathway permanently activated. In this sense, immune genes that are positively regulated by ecdysone will be constantly transcribed. Therefore, effector molecules such as antimicrobial peptides will be circulating in the insect gut before parasite infection. This immune priming could affect parasite development in two ways: by directly affecting the effector molecules in parasite cells or by causing a microbiota dysregulation, which could also present a collateral effect in parasite survival. Using the CRISPR gene editing system we will construct a sand fly lineage containing a nonnaturally occurring gene deletion (knock out), which has the potential to interrupt leishmaniasis transmission by the vector effectively. Besides the scientific impact, the acknowledgment of new sand fly molecules that could interrupt the Leishmania cycle will also contribute to the development, together with potential stakeholders from the industry, of a solution to deliver a commercialized product to control leishmaniasis, which will have societal and economic impacts.
