SNP discovery and molecular evolution in Anopheles gambiae, with special emphasis on innate immune system

<p>Abstract</p> <p>Background</p> <p><it>Anopheles </it>innate immunity affects <it>Plasmodium </it>development and is a potential target of innovative malaria control strategies. The extent and distribution of nucleotide diversity in immunity genes might provide insights into the evolutionary forces that condition pathogen-vector interactions. The discovery of polymorphisms is an essential step towards association studies of susceptibility to infection.</p> <p>Results</p> <p>We sequenced coding fragments of 72 immune related genes in natural populations of <it>Anopheles gambiae </it>and of 37 randomly chosen genes to provide a background measure of genetic diversity across the genome. Mean nucleotide diversity (π) was 0.0092 in the <it>A. gambiae </it>S form, 0.0076 in the M form and 0.0064 in <it>A. arabiensis</it>. Within each species, no statistically significant differences in mean nucleotide diversity were detected between immune related and non immune related genes. Strong purifying selection was detected in genes of both categories, presumably reflecting strong functional constraints.</p> <p>Conclusion</p> <p>Our results suggest similar patterns and rates of molecular evolution in immune and non-immune genes in <it>A. gambiae</it>. The 3,214 Single Nucleotide Polymorphisms (SNPs) that we identified are the first large set of <it>Anopheles </it>SNPs from fresh, field-collected material and are relevant markers for future phenotype-association studies.</p

Λειτουργική γονιδιοματική και αναπτυξιακό μεταγράφωμα του κουνουπιού φορέα της ελονοσίας, Anopheles gambiae

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

AnoEST: Toward A. gambiae functional genomics

Here, we present an analysis of 215,634 EST and cDNA sequences of a major vector of human malaria Anopheles gambiae structured into the AnoEST database. The expressed sequences are grouped into clusters using genomic sequence as template and associated with inferred functional annotation, including the following: corresponding Ensembl gene prediction, putative orthologous genes in other species, homology to known proteins, protein domains, associated Gene Ontology terms, and corresponding classification into broad GO-slim functional groups. AnoEST is a vital resource for interpretation of expression profiles derived using recently developed A. gambiae cDNA microarrays. Using these cDNA microarrays, we have experimentally confirmed the expression of 7961 clusters during mosquito development. Of these, 3100 are not associated with currently predicted genes. Moreover, we found that clusters with confirmed expression are nonbiased with respect to the current gene annotation or homology to known proteins. Consequently, we expect that many as yet unconfirmed clusters are likely to be actual A. gambiae genes. [AnoEST is publicly available at http://komar.embl.de, and is also accessible as a Distributed Annotation Service (DAS).

Discovery of Plasmodium modulators by genome-wide analysis of circulating hemocytes in Anopheles gambiae

Insect hemocytes mediate important cellular immune responses including phagocytosis and encapsulation and also secrete immune factors such as opsonins, melanization factors, and antimicrobial peptides. However, the molecular composition of these important immune cells has not been elucidated in depth, because of their scarcity in the circulating hemolymph, their adhesion to multiple tissues and the lack of primary culture methods to produce sufficient material for a genome-wide analysis. In this study, we report a genome-wide molecular characterization of circulating hemocytes collected from the hemolymph of adult female Anopheles gambiae mosquitoes—the major mosquito vector of human malaria in subSaharan Africa. Their molecular profile identified 1,485 transcripts with enriched expression in these cells, and many of these genes belong to innate immune gene families. This hemocyte-specific transcriptome is compared to those of Drosophila melanogaster and two other mosquitoes, Aedes aegypti and Armigeres subalbatus. We report the identification of two genes as ubiquitous hemocyte markers and several others as hemocyte subpopulation markers. We assess, via an RNAi screen, the roles in development of Plasmodium berghei of 63 genes expressed in hemocytes and provide a molecular comparison of the transcriptome of these cells during malaria infection

SNP discovery and molecular evolution in , with special emphasis on innate immune system-0

(black line). Data from immune related genes and control genes are included. Abscise represents position of the genes along the genome. Chromosomes and centromeres are represented below. The asterisk shows the position of TEP1 gene.<p><b>Copyright information:</b></p><p>Taken from "SNP discovery and molecular evolution in , with special emphasis on innate immune system"</p><p>http://www.biomedcentral.com/1471-2164/9/227</p><p>BMC Genomics 2008;9():227-227.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2405807.</p><p></p

SNP discovery and molecular evolution in , with special emphasis on innate immune system-1

Estimates are shown in the top part of each graph and the corresponding P-values are shown below, in grey for /M form comparisons, white for /S form and in black for M form/S form. Data from immune related genes and control genes are included. Horizontal straight lines represent the significance threshold at P= 0.05, dashed lines: P = 0.05 after correction for multiple tests (Bonferroni sequential procedure). Missing data are indicated with a dot. Negative Fst values that are always not significant were represented as equal to zero. Highly significant P values (<p><b>Copyright information:</b></p><p>Taken from "SNP discovery and molecular evolution in , with special emphasis on innate immune system"</p><p>http://www.biomedcentral.com/1471-2164/9/227</p><p>BMC Genomics 2008;9():227-227.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2405807.</p><p></p

SNP discovery and molecular evolution in , with special emphasis on innate immune system-2

(black line). Data from immune related genes and control genes are included. Abscise represents position of the genes along the genome. Chromosomes and centromeres are represented below. The asterisk shows the position of TEP1 gene.<p><b>Copyright information:</b></p><p>Taken from "SNP discovery and molecular evolution in , with special emphasis on innate immune system"</p><p>http://www.biomedcentral.com/1471-2164/9/227</p><p>BMC Genomics 2008;9():227-227.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2405807.</p><p></p