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.

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.

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.Anopheles gambiae mosquitoes are the principle vectors of Plasmodium falciparum, the parasite causing the most severe form of human malaria. However, within the A. gambiae complex mosquitoes may differ in their efficiency of transmitting malaria (1). Transmission requires completion of a complex sporogonic cycle, which takes place over 2 weeks within the mosquito and can be aborted by innate immune responses, such as the encapsulation of early oocysts soon after invasion of the midgut (2). Genes responsible for such refractory phenotypes would be candidates for inclusion in a vector control strategy based on genetically manipulated mosquitoes (3, 4).Vertebrate immune defense has been attributed to two general systems, innate and adaptive immunity, which are interconnected (5). In contrast, insect immune defense lacks the adaptive component and therefore an antibody-mediated immune response. Instead, it fully relies on innate immune mechanisms, such as the inducible synthesis of antimicrobial peptides, and the coagulation and melanization cascades (6, 7). The ongoing exploration of immune mechanisms in A. gambiae is hampered by the small size of this insect, limited knowledge of its genetics, and the absence of an efficient method for germline transformation. Insect cell lines were previously shown to exhibit immune properties, including the inducible synthesis of antibacterial peptides (8 -10). Therefore, we have taken the approach of establishing A. gambiae cell lines as an in vitro system to analyze the immune mechanisms of the mosquito. We have reported that one such line expresses in an inducible manner a panel of immune marker genes (11).In the course of establishing a larger variety of immuneresponsive A. gambiae cell lines, we identified some that secrete prophenoloxidase (PPO...

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.

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.