Periodic Reporting for period 1 - NEPHIMLEI (Neuroendocrine regulation of Phlebotomus perniciosus immunity and microbiota: fighting Leishmania infection)
Okres sprawozdawczy: 2023-09-01 do 2025-08-31
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.
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.
Technical Activities
Experimental Design:
Developed a robust protocol to disrupt the neuroendocrine system of Phlebotomus perniciosus using azadirachtin (Aza), a plant-derived ecdysone synthesis inhibitor.
Designed feeding regimens for larvae and adult females incorporating azadirachtin and azadirachtin with exogenous ecdysone to evaluate their effects on development and immune gene expression.
Gene Expression Analysis:
Identified key genes involved in ecdysone signaling (Eip74EF, Eip75B, Serpent, EcR) and antimicrobial peptide (AMP) production (Attacin, Defensin).
Used quantitative PCR (qPCR) to monitor the expression of these genes in larvae and adult females after azadirachtin treatment.
Developmental Monitoring:
Assessed the molting process in larvae from L1 to L2 and monitored adult emergence in azadirachtin-treated and control groups.
Immunological Assessments:
Investigated how azadirachtin-induced suppression of ecdysone signaling affected AMP production and basal immune competence in P. perniciosus.
Scientific Achievments
Disruption of Larval Development:
Demonstrated that azadirachtin treatment inhibited larval molting, with only 10% of treated L1 larvae molting to L2 compared to 95% in controls.
No adult emergence was observed in azadirachtin-treated groups, indicating a profound developmental block.
Regulation of Immune and Developmental Genes:
Showed significant suppression of Eip74EF and Serpent gene expression in azadirachtin-treated larvae.
Suppression was partially reversed by exogenous ecdysone supplementation, confirming the dependency of these genes on ecdysone signaling.
Stage-Specific Effects:
Highlighted differential impacts of azadirachtin on EcR expression between larvae and adult females, suggesting distinct hormonal compensatory mechanisms across developmental stages.
In adult females, azadirachtin suppressed EcR, Eip74EF, Eip75B, and Serpent expression at 1 day after feeding (DAF), with partial recovery by 7 DAF.
Immune Suppression:
Revealed that azadirachtin inhibited AMP production (Attacin, Defensin) by downregulating their regulators (Eip74EF, Eip75B, and Serpent).
Established the role of ecdysone signaling in maintaining immune competence in both larvae and adult females.
Neuroendocrine-Immune Crosstalk:
Provided evidence of the interplay between ecdysone signaling and immunity in P. perniciosus.
Confirmed that exogenous ecdysone supplementation counteracts azadirachtin’s immunosuppressive effects, restoring AMP expression.
Novel Insights into Vector Biology:
Proposed that suppression of immune genes during molting could render P. perniciosus more susceptible to infections, including Leishmania.
Identified Serpent and Eip74EF as potential biomarkers and targets for manipulating sand fly immunity.
Experimental Design:
Developed a robust protocol to disrupt the neuroendocrine system of Phlebotomus perniciosus using azadirachtin (Aza), a plant-derived ecdysone synthesis inhibitor.
Designed feeding regimens for larvae and adult females incorporating azadirachtin and azadirachtin with exogenous ecdysone to evaluate their effects on development and immune gene expression.
Gene Expression Analysis:
Identified key genes involved in ecdysone signaling (Eip74EF, Eip75B, Serpent, EcR) and antimicrobial peptide (AMP) production (Attacin, Defensin).
Used quantitative PCR (qPCR) to monitor the expression of these genes in larvae and adult females after azadirachtin treatment.
Developmental Monitoring:
Assessed the molting process in larvae from L1 to L2 and monitored adult emergence in azadirachtin-treated and control groups.
Immunological Assessments:
Investigated how azadirachtin-induced suppression of ecdysone signaling affected AMP production and basal immune competence in P. perniciosus.
Scientific Achievments
Disruption of Larval Development:
Demonstrated that azadirachtin treatment inhibited larval molting, with only 10% of treated L1 larvae molting to L2 compared to 95% in controls.
No adult emergence was observed in azadirachtin-treated groups, indicating a profound developmental block.
Regulation of Immune and Developmental Genes:
Showed significant suppression of Eip74EF and Serpent gene expression in azadirachtin-treated larvae.
Suppression was partially reversed by exogenous ecdysone supplementation, confirming the dependency of these genes on ecdysone signaling.
Stage-Specific Effects:
Highlighted differential impacts of azadirachtin on EcR expression between larvae and adult females, suggesting distinct hormonal compensatory mechanisms across developmental stages.
In adult females, azadirachtin suppressed EcR, Eip74EF, Eip75B, and Serpent expression at 1 day after feeding (DAF), with partial recovery by 7 DAF.
Immune Suppression:
Revealed that azadirachtin inhibited AMP production (Attacin, Defensin) by downregulating their regulators (Eip74EF, Eip75B, and Serpent).
Established the role of ecdysone signaling in maintaining immune competence in both larvae and adult females.
Neuroendocrine-Immune Crosstalk:
Provided evidence of the interplay between ecdysone signaling and immunity in P. perniciosus.
Confirmed that exogenous ecdysone supplementation counteracts azadirachtin’s immunosuppressive effects, restoring AMP expression.
Novel Insights into Vector Biology:
Proposed that suppression of immune genes during molting could render P. perniciosus more susceptible to infections, including Leishmania.
Identified Serpent and Eip74EF as potential biomarkers and targets for manipulating sand fly immunity.
The NEPHIMLEI project has generated significant findings that provide new insights into the neuroendocrine regulation of immunity in Phlebotomus perniciosus, a key vector of Leishmania infantum.
Background and Key Results
The project investigated the role of ecdysone, a hormone critical to insect immunity and development, using azadirachtin (Aza) to disrupt ecdysone synthesis in P. perniciosus. The key findings include:
Developmental Impact: Aza treatment inhibited the molting process in larvae, with only 10% of treated L1 larvae molting to L2, compared to 95% in controls.
Immune Gene Suppression: Aza suppressed ecdysone signaling and downregulated genes linked to immunity, including antimicrobial peptides (AMPs) Attacin and Defensin in both larvae and adult females.
Role of Ecdysone: The addition of ecdysone alongside Aza reversed these effects, restoring normal gene expression and demonstrating the direct role of ecdysone in regulating development and immunity.
Conclusions
The project highlights the essential role of ecdysone in coordinating the development and immune defenses in P. perniciosus. These findings suggest that manipulating ecdysone signaling could potentially interfere with Leishmania infection by modulating the sand fly immune response, offering a novel vector control strategy.
Potential Impacts and Next Steps
The NEPHIMLEI results will provide a foundation for innovative approaches to control leishmaniasis by targeting sand fly immunity. These findings have broad implications:
Further Research and Demonstration:
Explore the interaction between disrupted immunity and Leishmania survival in sand flies.
Conduct field studies to validate the applicability of hormonal manipulation under natural conditions.
Commercialization and Stakeholder Engagement:
Engage stakeholders like EntoGenex Europe and Magtech to develop scalable, market-ready solutions for pest and disease control.
Leverage the expertise of CPPT and Charles University Innovations Prague to facilitate intellectual property protection and technology transfer.
Regulatory and Market Frameworks:
Align the development of ecdysone-based strategies with regulatory standards for insecticides and pest control solutions and advocate for international collaborations to promote sustainable, environmentally friendly pest control methods.
The combination of scientific innovation, industrial interest, and regulatory alignment positions the NEPHIMLEI project to make a significant contribution to controlling vector-borne diseases and addressing critical challenges in public health and pest management.
Background and Key Results
The project investigated the role of ecdysone, a hormone critical to insect immunity and development, using azadirachtin (Aza) to disrupt ecdysone synthesis in P. perniciosus. The key findings include:
Developmental Impact: Aza treatment inhibited the molting process in larvae, with only 10% of treated L1 larvae molting to L2, compared to 95% in controls.
Immune Gene Suppression: Aza suppressed ecdysone signaling and downregulated genes linked to immunity, including antimicrobial peptides (AMPs) Attacin and Defensin in both larvae and adult females.
Role of Ecdysone: The addition of ecdysone alongside Aza reversed these effects, restoring normal gene expression and demonstrating the direct role of ecdysone in regulating development and immunity.
Conclusions
The project highlights the essential role of ecdysone in coordinating the development and immune defenses in P. perniciosus. These findings suggest that manipulating ecdysone signaling could potentially interfere with Leishmania infection by modulating the sand fly immune response, offering a novel vector control strategy.
Potential Impacts and Next Steps
The NEPHIMLEI results will provide a foundation for innovative approaches to control leishmaniasis by targeting sand fly immunity. These findings have broad implications:
Further Research and Demonstration:
Explore the interaction between disrupted immunity and Leishmania survival in sand flies.
Conduct field studies to validate the applicability of hormonal manipulation under natural conditions.
Commercialization and Stakeholder Engagement:
Engage stakeholders like EntoGenex Europe and Magtech to develop scalable, market-ready solutions for pest and disease control.
Leverage the expertise of CPPT and Charles University Innovations Prague to facilitate intellectual property protection and technology transfer.
Regulatory and Market Frameworks:
Align the development of ecdysone-based strategies with regulatory standards for insecticides and pest control solutions and advocate for international collaborations to promote sustainable, environmentally friendly pest control methods.
The combination of scientific innovation, industrial interest, and regulatory alignment positions the NEPHIMLEI project to make a significant contribution to controlling vector-borne diseases and addressing critical challenges in public health and pest management.