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 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.     

Functional validation of the carbon dioxide receptor genes in Aedes aegypti mosquitoes using RNA interference

Carbon dioxide (CO(2)) is an important long-range chemosensory cue used by blood-feeding female mosquitoes to find their hosts. The CO(2) receptor in Drosophila melanogaster was previously determined to be a heterodimer comprised of two gustatory receptor (Gr) proteins, DmGr21a and DmGr63a. In the mosquito Aedes aegypti, two putative orthologous genes, AaGr1 and AaGr3, were identified in the genome database, along with an apparent paralogue of AaGr1, AaGr2. In this study, RNA interference (RNAi)-mediated gene knockdown of either AaGr1 or AaGr3 resulted in a loss of CO(2) sensitivity in both male and female mosquitoes, suggesting that these two proteins, like the Drosophila orthologues, function as a heterodimer. RNAi-mediated knockdown of AaGr2 expression had no impact on CO(2) reception. All three Gr genes were expressed in the maxillary palps of both Ae. aegypti and the West Nile virus vector mosquito, Culex pipiens quinquefasciatus. Interestingly, expression of the two CO(2) receptor genes was not equivalent in the two sexes and the implications of differential sex expression of the CO(2) receptor in different species are discussed. The functional identification of the CO(2) receptor in a mosquito could prove invaluable in the strategic design of compounds that disrupt the mosquito's ability to find hosts.     

Female-biased expression of odourant receptor genes in the adult antennae of the silkworm, Bombyx mori

Olfaction plays an important role in the life history of insects, including key behaviours such as host selection, oviposition and mate recognition. Odour perception by insects is primarily mediated by the large diverse family of odourant receptors (Ors) that are expressed on the dendrites of olfactory neurones housed within chemosensilla. However, few Or sequences have been identified from the Lepidoptera, an insect order that includes some of the most important pest species worldwide. We have identified 41 Or gene sequences from the silkworm (Bombyx mori) genome, more than double the number of published Or sequences from the Lepidoptera. Many silkworm Ors appear to be orthologs of the 17 published tobacco budworm (Heliothis virescens) Ors indicating that many Or lineages may be conserved within the Lepidoptera. The majority of the Or genes are expressed in adult female and male antennae (determined by quantitative real-time PCR analysis), supporting their probable roles in adult olfaction. Several Or genes are expressed at high levels in both male and female antennae, suggesting they mediate the perception of common host or conspecific volatiles important to both sexes. BmOrs 45-47 group together in the same phylogenetic branch and all three are expressed at moderate female-biased ratios, six to eight times higher in female compared to male moth antennae. Interestingly, BmOrs19 and 30 appear to be expressed predominantly in female antennae, opposite to that of the published silkworm pheromone receptors BmOrs 1 and 3 that are specific to male antennae. These results suggest that BmOr19 and 30 may detect odours critical to female behaviour, such as oviposition cues or male-produced courtship pheromones.     

Molecular characterization of arrestin family members in the malaria vector mosquito, Anopheles gambiae

Olfaction influences many insect behaviours including mate seeking and host selection. The molecular machinery underlying insect olfactory systems is a G protein-coupled receptor pathway that, in addition to activation, requires adaptation for olfactory sensitivity and discrimination. We have previously identified ARR1 (henceforth AgARR1), a sensory arrestin from the malaria vector mosquito Anopheles gambiae that has been postulated to modulate olfactory adaptation. This report describes three additional arrestin family members including ARR2 (henceforth AgARR2), which is similar to previously characterized insect sensory arrestins and is expressed at significantly higher levels in the antennae of male vs. female A. gambiae mosquitoes. This finding is consistent with the hypothesis that AgARR2 may be important for the regulation of olfactory-driven behaviours particular to male mosquitoes.     

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.     

The Aedes aegypti genome: a comparative perspective

The sequencing of the second mosquito genome, Aedes aegypti , in addition to Anopheles gambiae , is a major milestone that will drive molecular-level and genome-wide high-throughput studies of not only these but also other mosquito vectors of human pathogens. Here we overview the ancestry of the mosquito genes, list the major expansions of gene families that may relate to species adaptation processes, as exemplified by CYP9 cytochrome P450 genes, and discuss the conservation of chromosomal gene arrangements among the two mosquitoes and fruit fly. Many more invertebrate genomes are expected to be sequenced in the near future, including additional vectors of human pathogens (see http://www.vectorbase.org ), and further comparative analyses will become increasingly refined and informative, hopefully improving our understanding of the genetic basis of phenotypical differences among these species, their vectorial capacity, and ultimately leading to the development of novel disease control strategies.     

Microarray-based survey of a subset of putative olfactory genes in the mosquito Anopheles gambiae

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.     

cDNA cloning of biotransformation enzymes belonging to the cytochrome P450 family in the antennae of the noctuid moth Mamestra brassicae

The involvement of cytochrome P450 enzymes in olfaction was demonstrated in vertebrates some time ago. In insects these enzymes are well known for their role in insecticide resistance, but the involvement of P450 in pheromone degradation was only recently demonstrated. Using a PCR strategy, we have isolated two cDNAs from the antennae of the cabbage armyworm Mamestra brassicae - CYP4L4 and CYP4S4 - which encode microsomal P450s. CYP4S4 expression is restricted to the antennae, whereas CYP4L4 is also found in the proboscis and legs. Moreover, the two genes are strongly expressed in one type of sensory unit of the antennae - the sensilla trichodea - which are tuned to the detection of odourants. The putative function of the corresponding enzymes is discussed with regard to their respective expression patterns.