Mosquito hexamerins: characterization during larval development

We report here the first examination of hexamerins expressed during mosquito larval development. Haemolymph proteins from fourth-instar larvae of six species representing the two major subfamilies of mosquitoes were characterized by immunoblotting using antisera to calliphorin, the major hexamerin of the blowfly, Calliphora vicina , or to LSP1 or LSP2, the two distinct hexamerins of Drosophila melanogaster . In each mosquito species the antisera demonstrated the presence of multiple abundant hexamerin polypeptides of 66–85 kDa in molecular weight. According to the subunit composition of native proteins, the larval hexamerins from both Aedes aegypti and Anopheles gambiae form heterohexamers. Furthermore, the two major Aedes hexamerin subunits (AaHex1 and AaHex2) are neither rich in aromatic amino acids nor methionine. cDNA clones encoding AaHex1 and AaHex2 were isolated and used to show that hexamerin mRNA is uniquely expressed in fourth-instar larvae of both A. aegypti and A. gambiae and disappears rapidly at the onset of pupal development.     

Aedes aegypti transducing densovirus pathogenesis and expression in Aedes aegypti and Anopheles gambiae larvae

Aedes aegypti densovirus (AeDNV) is a small DNA virus that has been developed into an expression and transducing vector for mosquitoes [Afanasiev et al. (1994) Exp Parasitol 79: 322-339; Afanasiev et al. (1999) Virology 257: 62-72; Carlson et al. (2000) Insect Transgenesis: Methods and Applications (Handler, A.M. & James, A.A., eds), pp. 139-159. CRC Press, Boca Raton]. Virions carrying a recombinant genome expressing the GFP gene were used to characterize the pathogenesis of the virus in 255 individual Aedes aegypti larvae. The anal papillae of the larvae were the primary site of infection confirming previous observations (Afanasiev etal., 1999; Allen-Muira et al. (1999) Virology 257: 54-61). GFP expression was observed in most cases to spread from the anal papillae to cells of the fat body, and subsequently to many other tissues including muscle fibers and nerves. Infected anal papillae were also observed to shrink, or melanize and subsequently fall off in a virus dependent manner. Three to four day-old larvae were less susceptible to viral infection and, if infected, were more likely to survive into adulthood, with 14% of them still expressing GFP as adults. Higher salt concentrations of 0.10-0.15 M inhibited viral infection. Anopheles gambiae larvae also showed infection of the anal papillae (17%) but subsequent viral dissemination did not occur. The persistence of the reporter gene expression into adulthood of Aedes aegypti indicates that transduction of mosquito larvae with recombinant AeDNV may be a means of introducing a gene of interest into a mosquito population for transient expression.     

Molecular characterization of the Aedes aegypti odorant receptor gene family

The olfactory-driven blood-feeding behaviour of female Aedes aegypti mosquitoes is the primary transmission mechanism by which the arboviruses causing dengue and yellow fevers affect over 40 million individuals worldwide. Bioinformatics analysis has been used to identify 131 putative odourant receptors from the A. aegypti genome that are likely to function in chemosensory perception in this mosquito. Comparison with the Anopheles gambiae olfactory subgenome demonstrates significant divergence of the odourant receptors that reflects a high degree of evolutionary activity potentially resulting from their critical roles during the mosquito life cycle. Expression analyses in the larval and adult olfactory chemosensory organs reveal that the ratio of odourant receptors to antennal glomeruli is not necessarily one to one in mosquitoes.     

Characterization of a carboxypeptidase A gene from the mosquito, Aedes aegypti

A gut-specific carboxypeptidase A gene (AeCPA) from the mosquito, Aedes aegypti, was cloned and characterized. The gene has an open reading frame that predicts a protein of 427 amino acids, 61% of which are identical to an Anopheles gambiae carboxypeptidase A sequence. AeCPA messenger RNA (mRNA) was not detected during larval and pupal development. In situ hybridization experiments indicated that AeCPA mRNA is expressed by posterior midgut epithelial cells. In sharp contrast to An. gambiae carboxypeptidase A gene expression, AeCPA mRNA accumulates to high levels only late ( approximately 16-24 h) after ingestion of a blood meal. The temporal profile of AeCPA gene induction is similar to that of Ae. aegypti late trypsin, suggesting the existence of common regulatory elements.     

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.     

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.     

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.     

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.     

Coadaptation of isoacceptor tRNA genes and codon usage bias for translation efficiency in Aedes aegypti and Anopheles gambiae

The transfer RNAs (tRNAs) are essential components of translational machinery. We determined that tRNA isoacceptors (tRNAs with different anticodons but incorporating the same amino acid in protein synthesis) show differential copy number abundance, genomic distribution patterns and sequence evolution between Aedes aegypti and Anopheles gambiae mosquitoes. The tRNA-Ala genes are present in unusually high copy number in the Ae. aegypti genome but not in An. gambiae. Many of the tRNA-Ala genes of Ae. aegypti are flanked by a highly conserved sequence that is not observed in An. gambiae. The relative abundance of tRNA isoacceptor genes is correlated with preferred (or optimal) and nonpreferred (or rare) codons for ～2-4% of the predicted protein coding genes in both species. The majority (～74-85%) of these genes are related to pathways involved with translation, energy metabolism and carbohydrate metabolism. Our results suggest that these genes and the related pathways may be under translational selection in these mosquitoes.     

A cluster of four D7-related genes is expressed in the salivary glands of the African malaria vector Anopheles gambiae

Four genes expressed in the Anopheles gambiae adult female salivary glands and similar in sequence to the Aedes aegypti D7 gene were identified. The genes, called D7-related (D7r), are included in a single cluster encompassing approximately six kilobases on chromosome arm 3R. The deduced proteins contain secretory signals and they are probably injected by the mosquito into the host with the saliva during blood feeding. The region of similarity to D7 encompasses the carboxy-terminal part of the Ae. aegypti protein and the different An. gambiae D7r show a degree of similarity to each other, varying from 53% to 73%. The weak but significant similarity to members of a wide family of insect proteins, including odourant- and pheromone-binding proteins, raises the possibility that the D7r-encoded proteins may bind and/or carry small hydrophobic ligands.     

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.     

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.     

Development of a new Sindbis virus transducing system and its characterization in three Culicine mosquitoes and two Lepidopteran species

Alphavirus transducing systems (ATSs) are alphavirus-based tools for expressing genes in insects. Here we describe an ATS (5'dsMRE16ic) based entirely on Sindbis MRE16 virus. GFP expression was used to characterize alimentary tract infections and dissemination in three Culicine and two Lepidopteran species. Following per os infection, 5'dsMRE16ic-EGFP efficiently infected Aedes aegypti and Culex tritaeniorhynchus, but not Culex pipiens pipiens. Ae. aegypti clearly showed accumulation of green fluorescent protein (GFP) in the posterior midgut and foregut/midgut junction within 2-3 days postinfection. Following parenteral infection of larvae, Bombyx mori had extensive GFP expression in larvae and adults, but Manduca sexta larvae were mostly resistant. 5'dsMRE16ic should be a valuable tool for gene expression in several important insect species that are otherwise difficult to manipulate genetically.     

The D7 family of salivary proteins in blood sucking diptera

The D7 subfamily of salivary proteins is widespread in blood sucking Diptera and belongs to the superfamily of pheromone/odourant binding proteins. Although D7 proteins are among the most abundant salivary proteins in adult female mosquitoes and sand flies, their role in blood feeding remains elusive. In the present work we report the sequence of seventeen novel D7 proteins, and propose an evolutionary scenario for the appearance of the several forms of this protein, based on a total of twenty-one sequences from Culex quinquefasciatus, Aedes aegypti, Anopheles gambiae, An. arabiensis, An. stephensi, An. darlingi mosquitoes and Lutzomyia longipalpis and Phlebotomus papatasi sand flies.     

Characterization of two globin genes from the malaria mosquito Anopheles gambiae: divergent origin of nematoceran haemoglobins

The chironomid midges are the only insects that harbour true haemoglobin in their haemolymph. Here we report the identification of haemoglobin genes in two other nematoceran species. Two paralogous haemoglobin genes (glob1 and glob2) from the malaria mosquito Anopheles gambiae were cloned and sequenced. Furthermore, we identified two orthologous haemoglobin genes in the yellow fever mosquito Aedes aegypti. All four haemoglobins were predicted to be intracellular proteins, with the amino acids required for heme- and oxygen-binding being conserved. In situ-hybridization studies showed that glob1 and glob2 expression in An. gambiae is mainly associated with the tracheal system. This pattern resembles that of other insect intracellular globins. We also observed expression of glob2 in visceral muscles. Phylogenetic analyses showed that the globins of the mosquitoes and the Chironomidae are not orthologous. The chironomid haemoglobins share a recent common origin with the brachyceran glob1 proteins. The mosquito glob1 and glob2 proteins, which separated by gene duplication around 170 million years ago, form a distinct clade of more ancient evolutionary origin within the insects. The glob1 genes have introns in the ancestral globin positions B12.2 and G7.0. An additional intron was observed in Ae. aegypti glob1 helix position E18.0, providing evidence for a recent intron gain event. Both mosquito glob2 genes have lost the B12.2 intron. This pattern must be interpreted in terms of dynamic intron gain and loss events in the globin gene lineage.