Functional genomics and developmental transcriptomics of the mosquito malaria vector, Anopheles gambiae

Abstract

The mosquito Anopheles gambiae is the major vector of human malaria in Africa. Malaria is caused by parasites of the genus Plasmodium which undergo a complex developmental cycle inside the mosquito. These parasites are then transmitted to humans by infectious bites and cause the clinical manifestations of the disease. Although mosquitoes were identified as vectors of the disease several decades ago, little knowledge was available, especially at the molecular level. This situation was improved the last two decades largely due to increased interest by researchers, which led to the publication of the A. gambiae genome, alongside the Plasmodium falciparum genome and the development of specific tools for functional genomic studies. The present study concentrates in post-genomic research of A. gambiae and describes efforts in several fields. In the field of bioinformatics, we used the genome information to position all publicly available expressed sequences (ESTs and cDNAs) to construct expressed contigs. This information was presented in a newly developed database, AnoEST and was supplemented with functional annotation information, which is valuable for the analysis of microarray experiments. In addition, this study provided evidence for the existence of several expressed sequences that have been missed by the automatic gene prediction pipeline of Ensembl. In the field of transcriptomics, we constructed a new microarray platform, MMC1 that encompasses 20,000 ESTs from A. gambiae. We used this platform to monitor global gene expression in nine different time periods of the lifecycle of Anopheles and four different tissues of the adult mosquito. Our analysis identified developmental programmes and tissue-specific patterns and showed that genes which belong to related functional categories, or that encode the same or functionally linked protein domains are clustered together. Comparative analysis of our data together with published data from Drosophila melanogaster, which diverged from Anopheles some 250 million years ago, revealed high correlation of developmental expression between orthologous genes. The degree of gene expression similarity is not correlated with the degree of coding sequence similarity, implying uncoupled evolution of gene expression profiles and coding sequences. This is the first large-scale comparative transcriptomic analysis in insects which detected important evolutionary features of insect transcriptomes. In the field of functional genomics, we present a comprehensive functional survey of leucine rich repeat immune gene 1, LRIM1, and its relation to Anopheles innate immune responses. We showed that LRIM1 is involved in responses against pathogenic bacteria and argue that the response is dependent on bacterial species and bacterial concentration. Finally, we demonstrated involvement of LRIM1 in the killing and melanisation of the Plasmodium berghei malaria parasites and showed evidence recruitment and localisation in close proximity to the malaria parasites. Thus, the multifaceted analysis presented in this thesis aims to highlight different aspects of A. gambiae research: bioinformatic and transcriptional studies that promote knowledge in mosquito basic biology and fuctional analyses that aim to identify important factors of the mosquito immune system. Our integrated approach in the study of A. gambiae may prove useful towards effective future vector control strategies against the malaria parasite.