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Prophenoloxidase, a melanin-synthesizing enzyme, is considered to be an important arthropod immune protein. In mosquitoes, prophenoloxidase has been shown to be involved in refractory mechanisms against malaria parasites. In our study we used Anopheles gambiae, the most important human malaria vector, to characterize the first arthropod prophenoloxidase gene at the genomic level. The complete nucleotide sequence, including the immediate 5' flanking sequence (-855 bp) of the prophenoloxidase 1 gene, was determined. The gene spans 10 kb and is composed of five exons and four introns coding for a 2.5-kb mRNA. In the 5' flanking sequence, we found several putative regulatory motifs, two of which were identified as ecdysteroid regulatory elements. Electrophoretic mobility gel-shift assays and supershift assays demonstrated that the Aedes aegypti ecdysone receptor/Ultraspiracle nuclear receptor complex, and, seemingly, the endogenous Anopheles gambiae nuclear receptor complex, was able to bind one of the ecdysteroid response elements. Furthermore, 20-hydroxyecdysone stimulation was shown to up-regulate the transcription of the prophenoloxidase 1 gene in an A. gambiae cell line.

Chromosomal rearrangements abound in nature and can be studied in detail in organisms with polytene chromosomes. In Drosophila and in Anopheline mosquitoes most speciation processes seem to be associated with the establishment of chromosomal rearrangements, particularly of paracentric inversions. It is not known what triggers inversions in natural populations. In the laboratory inversions are commonly generated by X-rays, mutagens or after the activity of certain transposable elements (TEs). The Anopheles gambiae complex is comprised of six sibling species, each one characterized by the presence of fixed paracentric inversions on their chromosomes. Two of these, An. gambiae s.s. and An. arabiensis, are the most important vectors of human malaria and are structured into sub-populations, each carrying a characteristic set of polymorphic chromosomal inversions. We have cloned the breakpoints of the naturally occurring polymorphic inversion In(2R)d' of An. arabiensis. Analysis of the surrounding sequences demonstrated that adjacent to the distal breakpoint lies a transposable element that we called Odysseus. Characteristics of Odysseus' terminal region and its cytological distribution in different strains as well as within the same strain indicate that Odysseus is an actively transposing element. The presence of Odysseus at the junction of the naturally occurring inversion In(2R)d' suggests that the inversion may be the result of the TEs activity. Cytological evidence from Drosophila melanogaster has also implicated the hobo transposable element in the generation of certain Hawaiian endemic inversions. This picture supports the hypothesis of the important role of TEs in generating natural inversions.

Genomic DNA corresponding to the soluble guanylyl cyclase beta-subunit (GCSbeta) gene was cloned and sequenced from Anopheles gambiae. The sequence was 8103 bp long and presumably included the entire coding region. The deduced amino acid sequence was 71% and 62% similar to previously known Drosophila and vertebrate GCSbeta, while the C-terminus of A. gambiae GCSbeta was shorter. Because of the conserved characteristics in each functional domain, the high G+C% in the third codon positions compared to the introns, the lack of internal stop codons, and the fact that we identified the gene from a cDNA, we conclude that this A. gambiae gene is functional. This is the first detailed description of a guanylyl cyclase gene structure (e.g. intron-exon boundaries). Interestingly, within the fifth intron we found high similarity to the flanking regions of the Pegasus-27 transposable element and other noncoding regions of the A. gambiae genome.

Anopheles arabiensis, one of the two most potent malaria vectors of the gambiae complex, is characterized by the presence of chromosomal paracentric inversions. Elucidation of the nature and the dynamics of these inversions is of paramount importance for the understanding of the population genetics and evolutionary biology of this mosquito and of the impact on malaria epidemiology. We report here the cloning of the breakpoints of the naturally occurring polymorphic inversion 2Rd' of A. arabiensis. A cDNA clone that cytologically mapped on the proximal breakpoint was the starting material for the isolation of a cosmid clone that spanned the breakpoint. Analysis of the surrounding sequences demonstrated that adjacent to the distal breakpoint lies a repetitive element that exhibits distinct distribution in different A. arabiensis strains. Sequencing analysis of that area revealed elements characteristic of transposable element terminal repeats. We called this presumed transposable element Odysseus. The presence of Odysseus at the junction of the naturally occuring inversion 2Rd' suggests that the inversion may be the result of the transposable element's activity. Characteristics of Odysseus' terminal region as well as its cytological distribution in different strains may indicate a relatively recent activity of Odysseus.

During the last larval instar, dipteran insects synthesize two hexamerins rich in aromatic residues, typified by the larval serum proteins 1 and 2 (LSP-1 and LSP-2) of Drosophila melanogaster. We report here the characterization of a complete cDNA sequence encoding a LSP-1-like protein from a lower dipteran insect, the malaria mosquito Anopheles gambiae. The cDNA encodes the subunit of a homohexamer, A. gambiae hexamerin-1.1 (AgHex-1.1), which is a major pupal protein but only a minor constituent of late larval hemolymph. AgHex-1.1 is moderately rich in methionine (3.9%) and particularly rich in aromatic residues (21% Phe+Tyr). Cytogenetic analysis reveals AgHex-1.1 to be encoded by a single-copy gene localized to division 22F within the proximal 2La inversion breakpoint of chromosome 2 of A. gambiae. The AgHex-1.1 transcript is first detected in fourth-instar larvae (L4) and disappears abruptly in early pupae. In situ hybridization shows accumulation of the transcript uniquely in the larval fat body. AgHex-1.1 mRNA is re-expressed in male and female adults at about 10% of the L4 level, with no effect of bloodfeeding in females. The potential roles of AgHex-1.1 in Anopheles development and reproductive maturation are discussed.

The African Anopheles gambiae complex of six sibling species has many polymorphic and fixed paracentric inversions detectable in polytene chromosomes. These have been used to infer phylogenetic relationships as classically done with Drosophila. Two species, A. gambiae and A. merus, were thought to be sister taxa based on a shared X inversion designated Xag. Recent DNA data have conflicted with this phylogenetic inference as they have supported a sister taxa relationship of A. gambiae and A. arabiensis. A possible explanation is that the Xag is not monophyletic. Here we present data from a gene (soluble guanylate cyclase) within the Xag that strongly supports the monophyly of the Xag. We conjecture that introgression may be occurring between the widely sympatric species A. gambiae and A. arabiensis and that the previous DNA phylogenies have been detecting the introgression. Evidently, introgression is not uniform across the genome, and species-specific regions, like the X-chromosome inversions, do not introgress probably due to selective elimination in hybrids and backcrosses.

Random cDNA clones, cosmid clones and RAPD polymorphic fragments have been localized by in situ hybridization to the ovarian nurse cell polytene chromosomes of the malaria vector Anopheles gambiae. We thus established 85 molecular markers for 110 sites within the whole A. gambiae polytene chromosome complement. The cDNA clones analyzed were isolated at random, and their exact localizations were determined by in situ hybridization. For 15 of the cDNA clones, a partial nucleotide sequence has been obtained; for nine of them sequence searches in the GenBank database revealed high degrees of similarity with published sequences. The cosmid clones analyzed were obtained as the result of screening with a few of the aforementioned cDNA clones of particular interest, or taken from a small set of randomly isolated cosmid clones. The RAPD clones are polymorphic fragments, potentially diagnostic for the various chromosomal forms of A. gambiae that are currently being analyzed.

The epidemiology of malaria in Africa is complicated by the fact that its principal vector, the mosquito Anopheles gambiae, constitutes a complex of six sibling species. Each species is characterized by a unique array of paracentric inversions, as deduced by karyotypic analysis. In addition, most of the species carry a number of polymorphic inversions. In order to develop an understanding of the evolutionary histories of different parts of the genome, we compared the genetic variation of areas inside and outside inversions in two distinct inversion karyotypes of A. gambiae. Thirty-five cDNA clones were mapped on the five arms of the A. gambiae chromosomes with divisional probes. Sixteen of these clones, localized both inside and outside inversions of chromosome 2, were used as probes in order to determine the nucleotide diversity of different parts of the genome in the two inversion karyotypes. We observed that the sequence diversity inside the inversion is more than three-fold lower than in areas outside the inversion and that the degree of divergence increases gradually at loci at increasing distance from the inversion. To interpret the data we present a selectionist and a stochastic model, both of which point to a relatively recent origin of the studied inversion and may suggest differences between the evolutionary history of inversions in Anopheles and Drosophila species.

We introduce here a simple approach for rapidly determining restriction maps for a number of regions of a genome; this involves "anchoring" a map with a rare restriction site (in this case the seldom-cutting EagI) followed by partial digestion of a frequent-cutting enzyme (e.g., Sau 3A). We applied this technology to five species of the Anopheles gambiae complex. In a single Southern blot we obtained about a 15-kb restriction map each for the mtDNA, rRNA gene, and a scnDNA region for each of five species. Phylogenetic analyses of these regions yield trees at odds with the more traditional chromosome inversion-based trees. The value of the approach for systematic purposes is the ease with which several large, independent regions of the genome can be quickly assayed for molecular variation.