Haematophagous insects are frequently carriers of parasitic diseases, including malaria. The mosquito Anopheles gambiae is the major vector of malaria in sub-Saharan Africa and is thus responsible for thousands of deaths daily. Although the role of olfaction in A. gambiae host detection has been demonstrated, little is known about the combinations of ligands and odorant binding proteins (OBPs) that can produce specific odor-related responses in vivo. We identified a ligand, indole, for an A. gambiae odorant binding protein, AgamOBP1, modeled the interaction in silico and confirmed the interaction using biochemical assays. RNAi-mediated gene silencing coupled with electrophysiological analyses confirmed that AgamOBP1 binds indole in A. gambiae and that the antennal receptor cells do not respond to indole in the absence of AgamOBP1. This case represents the first documented instance of a specific A. gambiae OBP–ligand pairing combination, demonstrates the significance of OBPs in odor recognition, and can be expanded to the identification of other ligands for OBPs of Anopheles and other medically important insects.

Female Anopheles gambiae mosquitoes respond to odours emitted from humans in order to find a blood meal, while males are nectar feeders. This complex behaviour is controlled at several levels, but is probably initiated by the interaction of various molecules in the antennal sensilla. Important molecules in the early odour recognition events include odourant binding proteins (OBPs), which may be involved in odour molecule transport, odourant receptors (ORs) that are expressed in the chemosensory neurones and odour degrading enzymes (ODEs). To obtain a better understanding of the expression patterns of genes that may be involved in host odour reception in females, we generated a custom microarray to study their steady state mRNA levels in chemosensory tissues, antennae and palps. These results were supported by quantitative RT PCR. Our study detected several OBPs that are expressed at significantly higher levels in antennae and palps of females vs. males, while others showed the opposite expression pattern. Most OBPs are slightly down-regulated 24 h after blood feeding, but some, especially those with higher expression levels in males, are up-regulated in blood-fed females, suggesting a shift in blood-fed females from human host seeking to nectar feeding.

Sex-specific expression of transgenes in pest insects enables novel genetic control strategies, based either on genetic sexing or the spread of transgenes through the germ-line, to be developed and then tested for implementation. We describe the isolation of the beta tubulin genes from the yellow fever mosquito, Aedes aegypti, and the identification of the particular beta2 tubulin gene which has expression confined to the testes. We demonstrate that the beta2 tubulin promoter of Ae. aegypti can direct the expression of a DsRed genetic marker in the testes and show that labelled sperm can be detected in inseminated spermathecae. The applications for this technology in the genetic control of Ae. aegypti are discussed.

One of the functions of telomeres is to counteract the terminal nucleotide loss associated with DNA replication. While the vast majority of eukaryotic organisms maintain their chromosome ends via telomerase, an enzyme system that generates short, tandem repeats on the ends of chromosomes, other mechanisms such as the transposition of retrotransposons or recombination can also be used in some species. Chromosome end regression and extension were studied in a medically important mosquito, the malaria vector Anopheles gambiae, to determine how this dipteran insect maintains its chromosome ends. The insertion of a transgenic pUChsneo plasmid at the left end of chromosome 2 provided a unique marker for measuring the dynamics of the 2L telomere over a period of about 3 years. The terminal length was relatively uniform in the 1993 population with the chromosomes ending within the white gene sequence of the inserted transgene. Cloned terminal chromosome fragments did not end in short repeat sequences that could have been synthesized by telomerase. By late 1995, the chromosome ends had become heterogeneous: some had further shortened while other chromosomes had been elongated by regenerating part of the integrated pUChsneo plasmid. A model is presented for extension of the 2L chromosome by recombination between homologous 2L chromosome ends by using the partial plasmid duplication generated during its original integration. It is postulated that this mechanism is also important in wild-type telomere elongation.Telomeres are essential chromosomal structures whose functional integrity is linked to cell cycle progression. Considerable efforts are being made to understand telomere structure and to target elongation mechanisms in order to develop new means of proliferation control (12). Every eukaryotic organism must compensate for terminal loss of DNA from chromosome ends because DNA polymerases cannot completely replicate the ends of linear chromosomes. So far, evidence for three different terminal elongation mechanisms has been established. Most eukaryotes have a short, tandemly repeated DNA sequence motif on their chromosome ends. These telomeric tandem arrays are extended by a specific reverse transcriptase, telomerase, which carries an internal RNA template (12). Alternatively, Drosophila melanogaster elongates its telomeres by a very different mechanism that is based on transpositions of specific retrotransposons, HeT-A and TART, to chromosome termini (22). In addition, recombination of repetitive telomeric ends has been considered as a possible elongation mechanism (4, 10, 42). This pathway has been well documented in yeast where telomeres are extended by telomerase, but recombination can be used as an efficient bypass mechanism for elongation (19,20,30,39).Understanding the mechanism of telomere elongation in the malaria-transmitting mosquito Anopheles gambiae may be useful for developing new strategies for vector control. Therefore, we examined the dynamics of chromosome length variation at the 2L chromosome end. For...

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.

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BackgroundMosquito eggshells show remarkable diversity in physical properties and structure consistent with adaptations to the wide variety of environments exploited by these insects. We applied proteomic, transcriptomic, and hybridization in situ techniques to identify gene products and pathways that participate in the assembly of the Aedes aegypti eggshell. Aedes aegypti population density is low during cold and dry seasons and increases immediately after rainfall. The survival of embryos through unfavorable periods is a key factor in the persistence of their populations. The work described here supports integrated vector control approaches that target eggshell formation and result in Ae. aegypti drought-intolerant phenotypes for public health initiatives directed to reduce mosquito-borne diseases.ResultsA total of 130 proteins were identified from the combined mass spectrometric analyses of eggshell preparations.ConclusionsClassification of proteins according to their known and putative functions revealed the complexity of the eggshell structure. Three novel Ae. aegypti vitelline membrane proteins were discovered. Odorant-binding and cysteine-rich proteins that may be structural components of the eggshell were identified. Enzymes with peroxidase, laccase and phenoloxidase activities also were identified, and their likely involvements in cross-linking reactions that stabilize the eggshell structure are discussed.

Telomeres are generally considered heterochromatic. On the basis of DNA composition, the telomeric region of Drosophila melanogaster contains two distinct subdomains: a subtelomeric region of repetitive DNA, termed TAS, and a terminal array of retrotransposons, which perform the elongation function instead of telomerase. We have identified several P-element insertions into this retrotransposon array and compared expression levels of transgenes with similar integrations into TAS and euchromatic regions. In contrast to insertions in TAS, which are silenced, reporter genes in the terminal HeT-A, TAHRE, or TART retroelements did not exhibit repressed expression in comparison with the same transgene construct in euchromatin. These data, in combination with cytological studies, provide evidence that the subtelomeric TAS region exhibits features resembling heterochromatin, while the terminal retrotransposon array exhibits euchromatic characteristics. D NA sequences at the ends of eukaryotic chromosomes are the products of a telomere elongation process. In most eukaryotes, these sequences are simple repeating units that are synthesized by telomerase, but in Drosophila melanogaster they are tandem head-to-tail arrays of three non-long terminal repeat retrotransposons, HeT-A, TAHRE, and TART ( Despite these differences, a common feature of eukaryotic chromosomes is a region of complex repeats located adjacent to the terminal sequences. These complex repeats are referred to as subtelomeric regions, or telomere-associated sequences (TAS), and differ in sequence, structure, and length among species and among telomeres within an individual (Pryde et al. 1997). The repetitive nature and the high density of transposable elements in these subtelomeric regions (Mefford and Trask 2002) are reminiscent of heterochromatin. In D. melanogaster, TAS consist of several kilobases of complex repeats, which exhibit similarities between the different chromosome ends. Sequences of the 2L and X TAS regions have been described in detail (Karpen and Spradling 1992;Walter et al. 1995), and in situ hybridizations to polytene chromosomes showed that 2L TAS share homology with 3L TAS, while X TAS share homology with 2R and 3R TAS. The 2L TAS appear to be 15 kb in length and composed of relatively simple