The white gene from the yellow fever mosquito, Aedes aegypti

We report the cloning and primary characterization of both cDNA and genomic fragments from the white gene of the yellow fever mosquito, Aedes aegypti . Comparisons of the conceptual translation product with white genes from four other species within the order Diptera show that the Ae. aegypti gene is most similar to the white gene of the mosquito vector of human malaria, Anopheles gambiae (86% identity and 92% similarity). The analysis of the primary sequence of genomic DNA at the 5'-end of the coding region revealed the presence of an intron that is also present in An. gambiae , but not in the vinegar fly, Drosophila melanogaster . The isolated clones of the Ae. aegypti white gene will enable the construction of a marker gene for use in the development of a germline transformation system for this species.     

Developmental variation in epidermal growth factor receptor size and localization in the malaria mosquito, Anopheles gambiae

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.     

Genomic and evolutionary analyses of Tango transposons in Aedes aegypti, Anopheles gambiae and other mosquito species

Tango is a transposon of the Tc1 family and was originally discovered in the African malaria mosquito, Anopheles gambiae. Here we report a systematic analysis of the genome sequence of the yellow fever mosquito, Aedes aegypti, which uncovered three distinct Tango transposons. We name the only An. gambiae Tango transposon AgTango1 and the three Ae. aegypti Tango elements AeTango1-3. Like AgTango1, AeTango1 and AeTango2 elements both have members that retain characteristics of autonomous elements such as intact open reading frames and terminal inverted repeats (TIRs). AeTango3 is a degenerate transposon with no full-length members. All full-length Tango transposons contain subterminal direct repeats within their TIRs. AgTango1 and AeTango1-3 form a single clade among other Tc1 transposons. Within this clade, AgTango1 and AeTango1 are closely related and share approximately 80% identity at the amino acid level, which exceeds the level of similarity of the majority of host genes in the two species. A survey of Tango in other mosquito species was carried out using degenerate PCR. Tango was isolated and sequenced in all members of the An. gambiae species complex, Aedes albopictus and Ochlerotatus atropalpus. Oc. atropalpus contains a rich diversity of Tango elements, while Tango elements in Ae. albopictus and the An. gambiae species complex all belong to Tango1. No Tango was detected in Culex pipiens quinquefasciatus, Anopheles stephensi, Anopheles dirus, Anopheles farauti or Anopheles albimanus using degenerate PCR. Bioinformatic searches of the Cx. p. quinquefasciatus (~10 x coverage) and An. stephensi (0.33 x coverage) databases also failed to uncover any Tango elements. Although other evolutionary scenarios cannot be ruled out, there are indications that Tango1 underwent horizontal transfer among divergent mosquito species.     

Transposable element (TE) display and rapid detection of TE insertion polymorphism in the Anopheles gambiae species complex

Transposable element (TE) display was shown to be a highly specific and reproducible method of detecting the insertion sites of TEs in individuals of the African malaria mosquito, Anopheles gambiae, and its sibling species, A. arabiensis. Relatively high levels of insertion polymorphism were observed during the TE display of several families of miniature inverted-repeat TEs (MITEs) that have variable copy numbers. The genomic locations of selected insertion sites were identified by matching the sequences of their corresponding bands in a TE display gel to specific regions of the draft A. gambiae genome assembly. We discuss different scenarios in which TE display will provide powerful dominant and co-dominant genetic markers to study the behaviour of TEs in A. gambiae populations and to illustrate the complex population genetics of this intriguing disease vector. We suggest that TE display can also provide tools for a phylogenetic analysis of the A. gambiae complex.     

cpbAg1 encodes an active carboxypeptidase B expressed in the midgut of Anopheles gambiae

We previously used differential display to identify several Anopheles gambiae genes, whose expression in the mosquito midgut was regulated upon ingestion of Plasmodium falciparum. Here, we report the characterization of one of these genes, cpbAg1, which codes for the first zinc-carboxypeptidase B identified in An. gambiae and in any insect. Expression of cpbAg1 in baculovirus gave rise to an active enzyme, and determination of the N-terminal amino acids confirmed that CPBAg1 contains a signal peptide and a pro-peptide, typical features of digestive zinc carboxypeptidases. cpbAg1 mRNA was mainly produced in the mosquito midgut, where it accumulated in unfed females and was rapidly down-regulated upon blood feeding. Annotation of the An. gambiae genome predicts twenty-three sequences coding for zinc-carboxypeptidases of which only two (cpbAg1 and cpbAg2) are expressed at a significant level in the mosquito midgut.     

Identification of odorant-binding proteins of the yellow fever mosquito Aedes aegypti: genome annotation and comparative analyses

The yellow fever mosquito Aedes aegypti is an important human health pest which vectors yellow fever and dengue viruses. Olfaction plays a crucial role in its attraction to hosts and although the molecular basis of this is not well understood it is likely that odorant-binding proteins (OBPs) are involved in the first step of molecular recognition. Based on the OBPs of Drosophila melanogaster and Anopheles gambiae we have defined sequence motifs based on OBP conserved cysteine and developed an algorithm which has allowed us to identify 66 genes encoding putative OBPs from the genome sequence and expressed sequence tags (ESTs) of Ae. aegypti. We have also identified 11 new OBP genes for An. gambiae. We have examined all of the corresponding peptide sequences for the properties of OBPs. The predicted molecular weights fall within the expected range but the predicted isoeletric points are spread over a wider range than found previously. Comparative analyses of the 66 OBP sequences of Ae. aegypti with other dipteran species reveal some mosquito-specific genes as well as conserved homologues. The genomic organisation of Ae. aegypti OBPs suggests that a rapid expansion of OBPs has occurred, probably by gene duplication. The analyses of OBP-containing regions for microsynteny indicate a very high synteny between Ae. aegypti and An. gambiae.     

Infection patterns of o'nyong nyong virus in the malaria-transmitting mosquito, Anopheles gambiae

Arthropod-borne alphaviruses transmitted by mosquitoes almost exclusively use culicines; however, the alphavirus o'nyong-nyong (ONNV) has the unusual characteristic of being transmitted primarily by anopheline mosquitoes. This unusual attribute makes ONNV a valuable tool in the characterization of mosquito determinants of infection as well as a useful expression system in Anopheles species. We developed a series of recombinant alphaviruses, based upon the genome of ONNV, designed for the expression of heterologous genes. The backbone genome is a full-length infectious cDNA clone of ONNV from which wild-type virus can be rescued. Additional constructs are variants of the primary clone and contain the complete genome plus a duplicated subgenomic promoter element with a multiple cloning site for insertion of heterologous genes. We inserted a green fluorescent protein (GFP) gene downstream of this promoter and used it to characterize infection and dissemination patterns of ONNV within An. gambiae mosquitoes. These experiments allowed us to identify atypical sites of initial infection and dissemination patterns in this mosquito species not frequently observed in comparable culicine infections. The utility of these ONNVs for studies in anopheline mosquitoes includes the potential for identification of vector infection determinants and to serve as tools for antimalaria studies. Viruses that can express a heterologous gene in a vector and rapidly and efficiently infect numerous tissues in An. gambiae mosquitoes will be a valuable asset in parasite-mosquito interaction and interference research.     

The rate of terminal nucleotide loss from a telomere of the mosquito Anopheles gambiae

Using a single copy pUChsneo transgene insertion at the Anopheles gambiae 2L telomere, this chromosome end was monitored by genomic Southern blots for forty-four mosquito generations. During this time, the chromosome end lost terminal nucleotides at an apparently constant rate of 55 bp/generation, which can be accounted for by incomplete DNA replication and does not imply exonuclease activity. No telomere elongation events were detected, suggesting that a previously described gene conversion event at this transgene does not occur very frequently. Moreover, no evidence for elongation by transposable elements was found, as described in Drosophila melanogaster. These results are consistent with the proposal that gene conversion between complex terminal satellite repeats that are present at natural telomeres, represents the major telomere elongation mechanism in A. gambiae. Such recombination events between repetitive sequences would occur more frequently than between the single copy pUChsneo transgene on the 2L homologues.     

Isolation of cDNA clones encoding putative odourant binding proteins from the antennae of the malaria-transmitting mosquito, Anopheles gambiae

One way of controlling disease transmission by blood-feeding mosquitoes is to reduce the frequency of insect-host interaction, thus reducing the probability of parasite transmission and re-infection. A better understanding of the olfactory processes responsible for allowing mosquitoes to identify human hosts is required in order to develop methods that will interfere with host seeking. We have therefore initiated a molecular approach to isolate and characterize the genes and their products that are involved in the olfactory recognition pathway of the mosquito Anopheles gambiae, which is the main malaria vector in sub-Saharan Africa. We report here the isolation and preliminary characterization of several cDNAs from male and female A. gambiae antennal libraries that encode putative odourant binding proteins. Their conceptual translation products show extensive sequence similarity to known insect odourant binding proteins (OBPs)/pheromone binding proteins (PBPs), especially to those of D. melanogaster. The A. gambiae OBPs described here are expressed in the antennae of both genders, and some of the A. gambiae OBP genes are well conserved in other disease-transmitting mosquito species, such as Aedes aegypti and Culex quinquefasciatus.     

Cloning and analysis of a cecropin gene from the malaria vector mosquito, Anopheles gambiae

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.     

Gnare: Automated System For High-Throughput Genome Analysis With Grid Computational Backend

Recent progress in genomics and experimental biology has brought exponential growth of the biological information available for computational analysis in public genomics databases. However, applying the potentially enormous scientific value of this information to the understanding of biological systems requires computing and data storage technology of an unprecedented scale. The Grid, with its aggregated and distributed computational and storage infrastructure, offers an ideal platform for high-throughput bioinformatics analysis. To leverage this we have developed the Genome Analysis Research Environment (GNARE)--a scalable computational system for the high-throughput analysis of genomes, which provides an integrated database and computational backend for data-driven bioinformatics applications. GNARE efficiently automates the major steps of genome analysis including acquisition of data from multiple genomic databases; data analysis by a diverse set of bioinformatics tools; and storage of results and annotations. High-throughput computations in GNARE are performed using distributed heterogeneous Grid computing resources such as Grid2003, TeraGrid, and the DOE Science Grid. Multi-step genome analysis workflows involving massive data processing, the use of application-specific tools and algorithms and updating of an integrated database to provide interactive web access to results are all expressed and controlled by a "virtual data" model which transparently maps computational workflows to distributed Grid resources. This paper describes how Grid technologies such as Globus, Condor, and the Gryphyn Virtual Data System were applied in the development of GNARE. It focuses on our approach to Grid resource allocation and to the use of GNARE as a computational framework for the development of bioinformatics applications.     

Analysis of the Anopheles gambiae genome using RAPD markers

RAPD analysis technique is used as a rapid and reliable tool for genome analysis in the malaria vector Anopheles gambiae. Using more than eighty different commercially available primers we identified more than sixty different DNA segments that were differentially amplified in different strains of An. gambiae s.s. and An. arabiensis. An estimate of the cytogenetic position of these markers is provided by their hybridization to divisional dot-blot filters. Potentially useful RAPD markers can be cytogenetically mapped with more precision by in situ hybridization and, as they segregate as dominant markers in a Mendelian fashion, they can also be genetically mapped relative to other genes or rearrangements. Finally, we identified markers for their potential use in the identification of different mosquito strains.     

Differential expression of the detoxification genes in the different life stages of the malaria vector Anopheles gambiae

The diverse habitats and diets encountered during the life cycle of an Anopheles mosquito have necessitated the development of extensive families of detoxification enzymes. Expansion of the three detoxification enzyme families (cytochrome P450s, carboxylesterases and glutathione transfereases), has occurred in mosquitoes compared with Drosophila, however, very little is known regarding the developmental expression of theses genes. Using a custom made microarray we determined the expression profile of the detoxification genes in adults, larvae and pupae of the malaria vector A. gambiae. The expression of approximately one quarter of these genes was developmentally regulated. The expression profile of each of these genes and the information this data provides on putative functions of the mosquito detoxification enzymes is discussed.     

Molecular analysis of the serine/threonine kinase Akt and its expression in the mosquito Aedes aegypti

A key component of the insulin-signalling pathway, the protein kinase Akt, was identified and cloned as a cDNA from ovaries of the mosquito Aedes aegypti. An ortholog gene was found in the Anopheles gambiae genome database, and like other Akts, both mosquito Akts possess pleckstrin homology domains for membrane binding and a serine/threonine kinase domain. When Ae. aegypti ovaries were treated with bovine insulin in vitro, a putative Akt was threonine-phosphorylated, as expected for Akts. AaegAKT was only expressed in embryos for the first 6 h after oviposition and in ovaries before and during a gonotrophic cycle.     

Microsatellite DNA and isozyme variability in a West African population of Anopheles gambiae

Microsatellites are defined as tracts of tandemly repeated short DNA sequences. Polymorphisms in this class of DNA are currently being used to generate a genetic map of the mosquito Anopheles gambiae. In the present study we explore the potential of microsatellites as a tool for studying the genetic structure of natural populations of this malaria vector. Genetic polymorphism at twenty enzyme coding gene loci and eleven microsatellite DNA loci was surveyed in a population of An. gambiae from Mali, West Africa. All of the microsatellite loci surveyed were polymorphic, as compared to 40% of the isozyme loci. The mean heterozygosity for the isozyme loci was only 0.097 (±0.0035), but for the microsatellite loci it was 0.732 (±0.060). The pattern of variability was very different between isozymes and microsatellites. Typically, at an isozyme locus a single allele occurred at a frequency ≥0.75, whereas at microsatellite loci the most common allele had a frequency <0.50. We conclude that micro-satellites provide a rich source of genetic polymorphisms for the study of the population genetics of An. gambiae and are in many ways superior to isozymes for this purpose. We discuss the potential for utilizing genetically mapped microsatellite loci to explore the effect of chromosomal inversions on the distribution of genetic polymorphisms in An. gambiae.     

Leveraging genomic databases: from an Aedes albopictus mosquito cell line to the malaria vector Anopheles gambiae via the Drosophila genome project

An important justification for genome sequencing efforts is the anticipation that data from model organisms will provide a framework for the more rapid analysis of other, less studied genomes. In this investigation, we sequenced an internal region of 25 amino acids from a 52 kDa protein that was differentially expressed in 20-hydroxyecdysone-treated Aedes albopictus cells in culture. Within the GenBank non-mouse and non-human expressed sequence tag (EST) database, this "Aedes peptide" uncovered a putative homology to hypothetical translation products from Anopheles gambiae, Caenorhabditis elegans and Drosophila melanogaster. The hypothetical translation product from D. melanogaster, which included 462 amino acids, uncovered five expressed sequence tags (ESTs) from the malaria vector, Anopheles gambiae. When the Anopheles ESTs were aligned against the hypothetical Drosophila protein, we found that in aggregate they covered 324 amino acids, with gaps measuring 19, 30, and 87 amino acids. To approximate the complete amino acid sequence, gaps between translation products from Anopheles ESTs were replaced with corresponding amino acids from Drosophila to arrive at a calculated mass of 51 104 and a pI of 5.84 for the mosquito protein, consistent with the position of the Ae. albopictus protein on two-dimensional polyacrylamide gels. Finally, tandem mass spectrometry of a tryptic digest of the 52 kDa Ae. albopictus protein revealed 33 peptides with masses within 1 Dalton of those predicted from an in silico digestion of the reconstructed Anophleles protein. In addition to providing the first direct evidence that a hypothetical protein in Drosophila is in fact translated, this analysis provides a general approach for maximizing recovery, from existing databases, of information that can facilitate prioritization of efforts among several candidate proteins.