RESUMO
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.
Assuntos
Anopheles/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genoma de Inseto/genética , Hemócitos/metabolismo , Aedes/genética , Animais , Drosophila melanogaster/genética , Feminino , Marcadores Genéticos , Malária , Plasmodium , RNA Mensageiro/análiseRESUMO
BACKGROUND: Anopheles innate immunity affects Plasmodium 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. RESULTS: We sequenced coding fragments of 72 immune related genes in natural populations of Anopheles gambiae and of 37 randomly chosen genes to provide a background measure of genetic diversity across the genome. Mean nucleotide diversity (pi) was 0.0092 in the A. gambiae S form, 0.0076 in the M form and 0.0064 in A. arabiensis. 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. CONCLUSION: Our results suggest similar patterns and rates of molecular evolution in immune and non-immune genes in A. gambiae. The 3,214 Single Nucleotide Polymorphisms (SNPs) that we identified are the first large set of Anopheles SNPs from fresh, field-collected material and are relevant markers for future phenotype-association studies.
Assuntos
Anopheles/genética , Anopheles/imunologia , Evolução Molecular , Imunidade Inata/genética , Polimorfismo de Nucleotídeo Único , Animais , Genes de Insetos , Variação Genética , Humanos , Insetos Vetores/genética , Insetos Vetores/imunologia , Insetos Vetores/parasitologia , Malária Falciparum/prevenção & controle , Malária Falciparum/transmissão , Plasmodium falciparum/imunologia , Plasmodium falciparum/patogenicidade , Seleção Genética , Especificidade da EspécieRESUMO
The African mosquito Anopheles gambiae is the major vector of human malaria. We report a genome-wide survey of mosquito gene expression profiles clustered temporally into developmental programs and spatially into adult tissue-specific patterns. Global expression analysis shows that genes that belong to related functional categories or that encode the same or functionally linked protein domains are associated with characteristic developmental programs or tissue patterns. Comparative analysis of our data together with data published from Drosophila melanogaster reveal an overall strong and positive correlation of developmental expression between orthologous genes. The degree of correlation varies, depending on association of orthologs with certain developmental programs or functional groups. Interestingly, the similarity of gene expression is not correlated with the coding sequence similarity of orthologs, indicating that expression profiles and coding sequences evolve independently. In addition to providing a comprehensive view of temporal and spatial gene expression during the A. gambiae life cycle, this large-scale comparative transcriptomic analysis has detected important evolutionary features of insect transcriptomes.
Assuntos
Anopheles/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Estágios do Ciclo de Vida/genética , RNA Mensageiro/genética , Animais , Anopheles/crescimento & desenvolvimento , Anopheles/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Feminino , Insetos Vetores/genética , Estágios do Ciclo de Vida/fisiologia , Malária/parasitologia , Masculino , Camundongos , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
Mosquitoes are vectors of parasitic and viral diseases of immense importance for public health. The acquisition of the genome sequence of the yellow fever and Dengue vector, Aedes aegypti (Aa), has enabled a comparative phylogenomic analysis of the insect immune repertoire: in Aa, the malaria vector Anopheles gambiae (Ag), and the fruit fly Drosophila melanogaster (Dm). Analysis of immune signaling pathways and response modules reveals both conservative and rapidly evolving features associated with different functional gene categories and particular aspects of immune reactions. These dynamics reflect in part continuous readjustment between accommodation and rejection of pathogens and suggest how innate immunity may have evolved.
Assuntos
Aedes/genética , Anopheles/genética , Evolução Molecular , Imunidade Inata/genética , Insetos Vetores/genética , Aedes/imunologia , Animais , Anopheles/imunologia , Peptídeos Catiônicos Antimicrobianos/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Drosophila melanogaster/genética , Drosophila melanogaster/imunologia , Genes de Insetos , Proteínas de Insetos/genética , Proteínas de Insetos/fisiologia , Insetos Vetores/imunologia , Malária/transmissão , Melaninas/metabolismo , Família Multigênica , Transdução de Sinais , Especificidade da EspécieRESUMO
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).].