[show abstract][hide abstract] ABSTRACT: Innate immune signalling pathways are evolutionarily conserved between invertebrates and vertebrates. The analysis of NF-kappaB signalling in Drosophila has contributed important insights into how organisms respond to infection. Nevertheless, significant gaps remain in our understanding of how the activation of intracellular signalling elicits specific transcriptional programs. Here we report a genome-wide RNA interference survey for transcription factors that are required for Toll-dependent immune responses. In addition to the NF-kappaB homologs Dif, Dorsal and factors of the general transcription machinery, we identified Deformed Epidermal Autoregulatory Factor 1 (Deaf1) to be required for the expression of the Toll target gene Drosomycin in cultured cells and in Drosophila in vivo. We show that Deaf1 is required for the survival of flies after fungal, but not E. coli, infection. We determine that Deaf1 acts downstream of the NF-kappaB factors Dorsal and Dif. These results indicate that Deaf1 is an important contributor to innate immune responses in vivo.
Journal of Innate Immunity 01/2010; 2(2):181-94. · 4.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Proceedings of the National Academy of Sciences 08/2007; 104(27):11304-9. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Attempts over the last three decades to reconstruct the phylogenetic history of the Anopheles gambiae species complex have been important for developing better strategies to control malaria transmission.
We used fingerprint genotyping data from 414 field-collected female mosquitoes at 42 microsatellite loci to infer the evolutionary relationships of four species in the A. gambiae complex, the two major malaria vectors A. gambiae sensu stricto (A. gambiae s.s.) and A. arabiensis, as well as two minor vectors, A. merus and A. melas.
We identify six taxonomic units, including a clear separation of West and East Africa A. gambiae s.s. S molecular forms. We show that the phylogenetic relationships vary widely between different genomic regions, thus demonstrating the mosaic nature of the genome of these species. The two major malaria vectors are closely related and closer to A. merus than to A. melas at the genome-wide level, which is also true if only autosomes are considered. However, within the Xag inversion region of the X chromosome, the M and two S molecular forms are most similar to A. merus. Near the X centromere, outside the Xag region, the two S forms are highly dissimilar to the other taxa. Furthermore, our data suggest that the centromeric region of chromosome 3 is a strong discriminator between the major and minor malaria vectors.
Although further studies are needed to elucidate the basis of the phylogenetic variation among the different regions of the genome, the preponderance of sympatric admixtures among taxa strongly favor introgression of different genomic regions between species, rather than lineage sorting of ancestral polymorphism, as a possible mechanism.
PLoS ONE 02/2007; 2(11):e1249. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: A microarray containing approximately 20 000 expressed sequence tags (ESTs; 11 760 unique EST clusters) from the malaria vector, Anopheles gambiae, was used to monitor differences in global gene expression in two insecticide resistant and one susceptible strains. Statistical analysis identified 77 ESTs that were differentially transcribed among the three strains. These include the cytochrome P450 CYP314A1, over-transcribed in the DDT resistant ZAN/U strain, and many genes that belong to families not usually associated with insecticide resistance, such as peptidases, sodium/calcium exchangers and genes implicated in lipid and carbohydrate metabolism. Short-term (6 and 10 h) effects of exposure of the pyrethroid resistant RSP strain to permethrin were also detected. Several genes belonging to enzyme families already implicated in insecticide or xenobiotic detoxification were induced, including the carboxylesterase COEAE2F gene and members of the UDP-glucuronosyl transferase and nitrilase families.
[show abstract][hide abstract] ABSTRACT: 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).].
Genome Research 07/2005; 15(6):893-9. · 14.40 Impact Factor
[show abstract][hide abstract] ABSTRACT: Anopheles gambiae is the principal vector of malaria, a disease that afflicts more than 500 million people and causes more than 1 million deaths each year. Tenfold shotgun sequence coverage was obtained from the PEST strain of A. gambiae and assembled into scaffolds that span 278 million base pairs. A total of 91% of the genome was organized in 303 scaffolds; the largest scaffold was 23.1 million base pairs. There was substantial genetic variation within this strain, and the apparent existence of two haplotypes of approximately equal frequency ("dual haplotypes") in a substantial fraction of the genome likely reflects the outbred nature of the PEST strain. The sequence produced a conservative inference of more than 400,000 single-nucleotide polymorphisms that showed a markedly bimodal density distribution. Analysis of the genome sequence revealed strong evidence for about 14,000 protein-encoding transcripts. Prominent expansions in specific families of proteins likely involved in cell adhesion and immunity were noted. An expressed sequence tag analysis of genes regulated by blood feeding provided insights into the physiological adaptations of a hematophagous insect.
[show abstract][hide abstract] ABSTRACT: We have identified 242 Anopheles gambiae genes from 18 gene families implicated in innate immunity and have detected marked diversification relative to Drosophila melanogaster. Immune-related gene families involved in recognition, signal modulation, and effector systems show a marked deficit of orthologs and excessive gene expansions, possibly reflecting selection pressures from different pathogens encountered in these insects' very different life-styles. In contrast, the multifunctional Toll signal transduction pathway is substantially conserved, presumably because of counterselection for developmental stability. Representative expression profiles confirm that sequence diversification is accompanied by specific responses to different immune challenges. Alternative RNA splicing may also contribute to expansion of the immune repertoire.
[show abstract][hide abstract] ABSTRACT: Successful propagation of the malaria parasite Plasmodium falciparum within a susceptible mosquito vector is a prerequisite for the transmission of malaria. A field-based genetic analysis of the major human malaria vector, Anopheles gambiae, has revealed natural factors that reduce the transmission of P. falciparum. Differences in P. falciparum oocyst numbers between mosquito isofemale families fed on the same infected blood indicated a large genetic component affecting resistance to the parasite, and genome-wide scanning in pedigrees of wild mosquitoes detected segregating resistance alleles. The apparently high natural frequency of resistance alleles suggests that malaria parasites (or a similar pathogen) exert a significant selective pressure on vector populations.
[show abstract][hide abstract] ABSTRACT: Genome evolution entails changes in the DNA sequence of genes and intergenic regions, changes in gene numbers, and also changes in gene order along the chromosomes. Genes are reshuffled by chromosomal rearrangements such as deletions/insertions, inversions, translocations, and transpositions. Here we report a comparative study of genome organization in the main African malaria vector, Anopheles gambiae, relative to the recently determined sequence of the Drosophila melanogaster genome. The ancestral lines of these two dipteran insects are thought to have separated approximately 250 Myr, a long period that makes this genome comparison especially interesting. Sequence comparisons have identified 113 pairs of putative orthologs of the two species. Chromosomal mapping of orthologous genes reveals that each polytene chromosome arm has a homolog in the other species. Between 41% and 73% of the known orthologous genes remain linked in the respective homologous chromosomal arms, with the remainder translocated to various nonhomologous arms. Within homologous arms, gene order is extensively reshuffled, but a limited degree of conserved local synteny (microsynteny) can be recognized.
Genome Research 02/2002; 12(1):57-66. · 14.40 Impact Factor
[show abstract][hide abstract] ABSTRACT: The AGER gene encoding the epidermal growth factor receptor (EGFR) of the malaria mosquito Anopheles gambiae was cloned and sequenced. It represents a canonical member of this family of tyrosine kinase proteins exhibiting many similarities to orthologues from other species, both on the level of genomic organization and protein structure. The mRNA can be detected throughout development. Western analysis with an antibody raised against the extracellular domain of the mosquito protein suggests developmental variation in protein size and location that may be involved in the function of EGFR in the mosquito.
[show abstract][hide abstract] ABSTRACT: We have isolated an mRNA encoding a beta integrin subunit of the malaria mosquito Anopheles gambiae. Our analysis predicts a protein that is very similar to betaPS, the fruitfly orthologue. The gene is expressed during all developmental stages and it is found in all body parts, including the midgut. Finally, the expression of the gene does not seem to be modulated during blood meals, except for a substantial increase 48 h posthaematophagy, when digestion is nearly complete.
[show abstract][hide abstract] ABSTRACT: A recombinant Anopheles gambiae defensin peptide was used to define the antimicrobial activity spectrum against bacteria, filamentous fungi and yeast. Results showed that most of the Gram-positive bacterial species tested were sensitive to the recombinant peptide in a range of concentrations from 0.1 to 0.75 microM. No activity was detected against Gram-negative bacteria, with the exception of some E. coli strains. Growth inhibitory activity was detected against some species of filamentous fungi. Defensin was not active against yeast. The kinetics of bactericidal and fungicidal effects were determined for Micrococcus luteus and Neurospora crassa, respectively. Differential mass spectrometry analysis was used to demonstrate induction of defensin in the hemolymph of bacteria-infected adult female mosquitoes. Native peptide levels were quantitated in both hemolymph and midgut tissues. The polytene chromosome position of the defensin locus was mapped by in situ hybridization.
[show abstract][hide abstract] ABSTRACT: Laminin is a major constituent of the basal lamina surrounding the midgut of the malaria vectors that has been implicated in the development of the Plasmodium oocyst. In this report we describe the cloning of the Anopheles gambiae gene encoding the laminin γ1 polypeptide and follow its expression during mosquito development. To further investigate the putative role of laminin in the transmission of the malaria parasite we studied the potential binding of the P25 surface protein of Plasmodium berghei using a yeast two-hybrid system. Heterodimer formation was observed and does not require any additional protein factors since purified fusion proteins can also bind each other in vitro. Laminin γ1 also interacts with the paralogue of P25, namely P28, albeit more weakly, possibly explaining why the two parasite proteins can substitute for each other in deletion mutants. This represents the first direct evidence for molecular interactions between a surface protein of the Plasmodium parasite with an Anopheles protein; the strong interplay between laminin γ1 and P25 suggests that this pair of proteins may function as a receptor/ligand complex regulating parasite development in the mosquito vector.
Molecular and Biochemical Parasitology 03/2001; · 2.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Anopheline mosquito species are obligatory vectors for human malaria, an infectious disease that affects hundreds of millions of people living in tropical and subtropical countries. The lack of a suitable gene transfer technology for these mosquitoes has hampered the molecular genetic analysis of their physiology, including the molecular interactions between the vector and the malaria parasite. Here we show that a transposon, based on the Minos element and bearing exogenous DNA, can integrate efficiently and stably into the germ line of the human malaria vector Anopheles stephensi, through a transposase-mediated process.
[show abstract][hide abstract] ABSTRACT: Parasites of the genus Plasmodium are transmitted to mammalian hosts by anopheline mosquitoes. Within the insect vector, parasite growth and development are potentially limited by antimicrobial defence molecules. Here, we describe the isolation of cDNA and genomic clones encoding a cecropin antibacterial peptide from the malaria vector mosquito Anopheles gambiae. The locus was mapped to polytene division 1C of the X chromosome. Cecropin RNA was induced by infection with bacteria and Plasmodium. RNA levels varied in different body parts of the adult mosquito. During development, cecropin expression was limited to the early pupal stage. The peptide was purified from both adult mosquitoes and cell culture supernatants. Anopheles gambiae synthetic cecropins displayed activity against Gram-negative and Gram-positive bacteria, filamentous fungi and yeasts.
[show abstract][hide abstract] ABSTRACT: The ability of the Minos transposable element to function as a transformation vector in anopheline mosquitoes was assessed. Two recently established
Anopheles gambiae cell lines were stably transformed by using marked Minos transposons in the presence of a helper plasmid expressing transposase. The markers were either the green fluorescent protein
or the hygromycin B phosphotransferase gene driven by the Drosophila Hsp70 promoter. Cloning and sequencing of the integration sites demonstrated that insertions in the cell genome occurred
through the action of Minos transposase. Furthermore, an interplasmid transposition assay established that Minos transposase is active in the cytoplasmic environment of Anopheles stephensi embryos: interplasmid transposition events isolated from injected preblastoderm embryos were identified as Minos transposase-mediated integrations, and no events were recorded in the absence of an active transposase. These results demonstrate
that Minos vectors are suitable candidates for germ-line transformation of anopheline mosquitoes.
Proceedings of the National Academy of Sciences 02/2000; 97(5):2157-2162. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cell lines from the malaria vector Anopheles gambiae have been established as a tool for the study of the mosquito innate immune system in vitro. Here, we describe the first continuous insect cell line that produces prophenoloxidase (PPO). This cell line (4a-3B) expresses constitutively six PPO genes, three of which are novel (PPO4, PPO5, and PPO6). The PPO genes show distinct temporal expression profiles in the intact mosquito, spanning stages from the embryo to the adult in an overlapping manner. Transient induction of larva-specific PPO genes in blood-fed adult females suggests that the developmental hormone 20-hydroxyecdysone may be involved in PPO gene regulation. Indeed, exposure of 4a-3B cells to 20-hydroxyecdysone in culture results in induction of those PPO genes that are mainly expressed in early developmental stages, and repression of PPO5, which is preferentially expressed at the adult stage. The cell line shows bacteria-induced immune transcripts that encode defensin and Gram-negative bacteria-binding protein, but no induction of PPO transcripts. This cell line most likely derives from a hemocyte lineage, and represents an appropriate in vitro model for the study of the humoral and cellular immune defenses of A. gambiae.
Journal of Biological Chemistry 05/1999; 274(17):11727-35. · 4.65 Impact Factor