Periodic Reporting for period 1 - ReMVeC (How has the rapid scale up of malaria control in Africa impacted vector competence?)
Berichtszeitraum: 2023-05-01 bis 2025-10-31
Malaria remains one of the biggest causes of morbidity and mortality across the world, with 263 million cases and 597 000 deaths in 2023, with the latter primarily in Arican children under 5. The primary method for controlling malaria is through the use of insecticide-based vector control tools, targeting the adult female Anopheles mosquito, thereby preventing infection. Indeed, together, the use of insecticide treated bed nets and indoor residual spraying accounted for over 80% of all cases averted between 2000 and 2015. The remaining 20% was due to the use of curative drugs, usually in the form of artemisinin combination therapies, the front-line control for malaria. Unsurprisingly, the intense selection pressure on both the mosquito and parasite has led to the evolution of resistance to both insecticides and drugs, and, alarmingly, there has been a recent emergence and spread of artemisinin partial resistance in East Africa. Despite the importance of both insecticides and drugs in malaria control, we have little understanding of the impact of insecticide resistance or exposure on the development of the malaria parasite within the mosquito and thus transmission. Similarly, the changes in the parasite fitness due to drug resistance are well understood at blood stage but the competitive changes within the mosquito host remain largely uncharacterised. Within this project, these two key questions will be addressed using infectious feeds with the human malaria parasite P. falciparum and a combination of tools including phenotyping, molecular biology, CRISPR-Cas9, imaging, field work and multi-omics. Together, this project will aid in understanding the impacts of these two important phenotypes and shed light on the underlying mechanisms resulting in the changes observed through the following questions:
1. How is Plasmodium falciparum development in the mosquito affected by insecticide use?
2. Do drug resistance mutations in Plasmodium impact fitness during development within the Anopheles host?
3. What is the genetic interplay between parasite infection and insecticide response?
1. How is Plasmodium falciparum development in the mosquito affected by insecticide use?
2. Do drug resistance mutations in Plasmodium impact fitness during development within the Anopheles host?
3. What is the genetic interplay between parasite infection and insecticide response?
Aim 1: So far within this aim we have performed infectious feeds and have complete datasets for the differences in vector competence between An. gambiae, An. coluzzii and An. arabiensis in paired insecticide resistant (IR) and insecticide susceptible population. We have shown an impact of IR on parasite development and demonstrated clear differences in vector competence between species, regardless of IR status. We have data on oocyst prevalence, intensity and size, as well as melanisation and rupture rate. We have further got sporozoite counts every two days from day 8 to day 18 to assess changes on the extrinsic incubation period of the parasite. Additionally, we have optimised insecticide treated bed net exposure alongside an infectious blood meal, allowing sufficient sub-lethal exposure whilst ensuring high enough survival to complete the parasite development with enough mosquitoes.
Aim 2: Here we have established a work flow to analyse within-mosquito competition assays for drug resistant vs wild type parasites for both oocyst and sporozoite stage, allowing determination of DNA ratio after a feed of 1:1 mutant to wildtype. We have further developed a strategy to determine changes at each mosquito stage, including activated gametocyte, retort, ookinete, oocyst and sporozoite stage using a mixture of phenotyping, qPCR and imaging techniques. So far, in collaboration with Radboud Medical Centre, we have analysed a number of mutations involved in artemisinin partial resistance both in single infections and in competition with wild type.
Aim 3: We have produced bulk RNAseq data for a large time course covering all significant events during the parasite developmental cycle. The datasets include An. gambiae mosquitoes under the following conditions: P. falciparum infected, heat inactivated, bed net exposed every 4 days and unexposed mosquitoes. So far, the data has been analysed using a range of bioinformatics tools aimed at discerning patterns and overlaps in the time course data sets. We have identified a number of interesting transcripts for downstream validation in the second phase of the ERC grant.
Aim 2: Here we have established a work flow to analyse within-mosquito competition assays for drug resistant vs wild type parasites for both oocyst and sporozoite stage, allowing determination of DNA ratio after a feed of 1:1 mutant to wildtype. We have further developed a strategy to determine changes at each mosquito stage, including activated gametocyte, retort, ookinete, oocyst and sporozoite stage using a mixture of phenotyping, qPCR and imaging techniques. So far, in collaboration with Radboud Medical Centre, we have analysed a number of mutations involved in artemisinin partial resistance both in single infections and in competition with wild type.
Aim 3: We have produced bulk RNAseq data for a large time course covering all significant events during the parasite developmental cycle. The datasets include An. gambiae mosquitoes under the following conditions: P. falciparum infected, heat inactivated, bed net exposed every 4 days and unexposed mosquitoes. So far, the data has been analysed using a range of bioinformatics tools aimed at discerning patterns and overlaps in the time course data sets. We have identified a number of interesting transcripts for downstream validation in the second phase of the ERC grant.
Aim 1: To my knowledge, this is the first study to use paired insecticide resistant and susceptible mosquitoes across multiple species and compare, using dissections every 2 days, the parasite development time between species and phenotypes. We have shown clear differences between these that go beyond the current state of the art.
Aim 2: Again, to my knowledge, this is the first study to utilise CRISPR-Cas9 to integrate African mutations into African parasite backgrounds and perform fitness studies within the mosquito stage, both in single infections and in multiple infections. The pipeline set up can analyse these data within 44 days, which represents a fast experimental time for P. falciparum infections. As above, we have shown clear differences that extend beyond current state of the art.
Aim 3: The RNAseq data developed here represents a unique resource, pairing four experimental groups enabling control of blood feeding and aging/circadian rhythm whilst exploring the overlap between response to P. falciparum development over the complete life cycle and insecticide exposure every 4 days. The dataset shows unexpected responses to different stages of the parasite life cycle and response to insecticide treated bed nets.
Aim 2: Again, to my knowledge, this is the first study to utilise CRISPR-Cas9 to integrate African mutations into African parasite backgrounds and perform fitness studies within the mosquito stage, both in single infections and in multiple infections. The pipeline set up can analyse these data within 44 days, which represents a fast experimental time for P. falciparum infections. As above, we have shown clear differences that extend beyond current state of the art.
Aim 3: The RNAseq data developed here represents a unique resource, pairing four experimental groups enabling control of blood feeding and aging/circadian rhythm whilst exploring the overlap between response to P. falciparum development over the complete life cycle and insecticide exposure every 4 days. The dataset shows unexpected responses to different stages of the parasite life cycle and response to insecticide treated bed nets.