The malaria vector mosquito Anopheles gambiae expresses a suite of larval-specific defensin genes

cDNAs of Anopheles gambiae Defensin 2 ( AgDef2 ), Defensin 3 ( AgDef3 ) and Defensin 4 ( AgDef4 ), identified in the genome sequence, have been characterized and their expression profiles investigated. In contrast to both typical defensins and insect antimicrobial peptides generally, the newly identified defensins were not upregulated with acute-phase kinetics following immune challenge in insects or cell culture. However, mRNA abundance of AgDef2, AgDef3 and AgDef4 increased significantly during the larval stages. Promoter analysis of all three genes failed to identify putative immune response elements previously identified in other mosquito defensin genes. As previous studies failed to identify these larval-specific defensins, it seems likely that further antimicrobial peptide genes with nontypical expression profiles will be identified as more genome sequences become available.     

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.     

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.     

Molecular analysis of multiple cytochrome P450 genes from the malaria vector,Anopheles gambiae

Cytochrome P450s are a superfamily of haemoproteins, important in the metabolism of endogenous compounds and xenobiotics. As a first step to elucidating the role of this family in insecticide resistance in the malaria mosquito, Anopheles gambiae, we have cloned and mapped multiple P450 genes. Sixteen cDNAs encoding full-length P450s were cloned and physically mapped to the mosquito's polytene chromosomes. Fourteen of these encode putative CYP6 proteins and two encode P450s belonging to the CYP9 class. Eighteen new A. gambiae Cyp4 P450 genes were identified using degenerate PCR primers, cDNAs were detected for ten and in situ locations for thirteen members of this gene family.     

Optimization of the Gal4-UAS system in an Anopheles gambiae cell line

The development of the bipartite Gal4-UAS system in Anopheles gambiae would improve the functional characterization of genes in this important malaria vector. Towards this aim, we used Gal4 driver plasmids to successfully activate expression of the reporter gene, luciferase, from UAS responder plasmids when cotransfected into an An. gambiae cell line. To optimize Gal4-regulated gene expression in mosquitoes, we compared the efficiency of a series of alternative Gal4 transactivators to drive reporter gene expression from responder plasmids incorporating different numbers of tandemly arrayed Gal4 binding sites or upstream activation sequences (UAS). The results indicated that the native Gal4 is only weakly active in these cells. Modified forms of Gal4, including those carrying minimal VP16 activation domains, as well as a deleted form of Gal4, give up to 20-fold greater activity than the native protein, when used in conjunction with a responder plasmid having 14 UAS repeats. The identification of Gal4-UAS vectors that are efficiently expressed in a mosquito cell line should facilitate the transfer of this versatile expression system to An. gambiae, and potentially to other insects of medical importance.     

A muscle-specific actin gene from the Mediterranean fruit fly, Ceratitis capitata

A characterization of an actin gene isolated from the genome of the Mediterranean fruit fly, Ceratitis capitata , including the complete sequencing of the coding, 3' and 5' flanking regions of this gene and a partial cDNA was carried out. The partial cDNA was derived from the 3' untranslated region of the actin gene described here, and has been used to identify this gene uniquely. The DNA sequence data presented here, together with the pattern of expression exhibited by this gene during development, strongly support the interpretation that this is a muscle-specific actin gene. Peaks of expression are seen in tissues and during temporal phases of development where muscle differentiation is occurring. The derived protein sequence of the Medfly actin gene shows the highest degrees of similarity, 98.4 and 96.6% respectively, with the two muscle-specific actin genes 79B and 88F from D. melanogaster . The Medfly actin gene also has a single intervening sequence, and an intron is found at the same position in the 79B and 88F actin genes. In the coding region at the DNA level, 17.2 and 16.4% nucleotide differences, respectively, are observed between the Medfly actin gene and these same two D. melanogaster actin genes. The disparity between the amino acid and nucleotide comparisons can be explained, in part, by a high level of synonymous changes in the DNA sequence. In addition, despite the many similarities, codon usage appears to be very different between the actin genes of these species.     

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.     

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.     

Annotation and analysis of low-complexity protein families of Anopheles gambiae that are associated with cuticle

We have characterized four new families of homologous genes of the mosquito, Anopheles gambiae , all of which include members shown by previous work to be cuticular in nature. The CPLCG, CPLCW, CPLCP, and CPLCA families (where CPLC is 'cuticular protein of low complexity') encode proteins with a high proportion of low-complexity sequence. We have also annotated the An. gambiae Tweedle genes, a family of cuticular protein genes first described in Drosophila , and additional ungrouped An. gambiae cuticular proteins identified by proteomics. Our annotations reveal multiple gene-family expansions that are specific to Diptera or Culicidae. The CPLCG and CPLCW families occur within a large and dynamic tandem array on chromosome 3R that includes sets of concertedly evolving genes. Most gene families exhibit two or more different expression profiles during development.     

Short-hairpin RNA expressed from polymerase III promoters mediates RNA interference in mosquito cells

Putative U6snRNA polymerase III (PolIII) promoters were cloned from the Anopheles gambiae and Aedes aegypti genomes. The PolIII promoters were tested for their ability to express short-hairpin RNA (shRNA) targeted to firefly luciferase and to mediate RNA interference (RNAi) knockdown of a co-transfected luciferase reporter gene vector in AG-55 Anopheles gambiae and ATC-10 Aedes aegypti cells. Promoters capable of silencing expression of the co-transfected luciferase plasmid by up to 95% in AG-55 cells and up to 75% in ATC-10 cells were identified. RNase protection experiments allowed detection of the 19 nt luciferase short-interfering RNA (siRNA) in transfected cells. These findings indicate that mosquito U6snRNA gene promoters can be used for production of shRNA to induce the RNAi response in mosquito cells.     

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.