We used bioinformatic approaches to identify a total of 276 G protein–coupled receptors (GPCRs) from the Anopheles gambiae genome. These include GPCRs that are likely to play roles in pathways affecting almost every aspect of the mosquito's life cycle. Seventy-nine candidate odorant receptors were characterized for tissue expression and, along with 76 putative gustatory receptors, for their molecular evolution relative to Drosophila melanogaster . Examples of lineage-specific gene expansions were observed as well as a single instance of unusually high sequence conservation.

The mosquito Anopheles gambiae is the major vector of malaria in sub-Saharan Africa. It locates its human hosts primarily through olfaction, but little is known about the molecular basis of this process. Here we functionally characterize the Anopheles gambiae odorant receptor (AgOr) repertoire. We identify receptors that respond strongly to components of human odour and that may act in the process of human recognition. Some of these receptors are narrowly tuned, and some salient odorants elicit strong responses from only one or a few receptors, suggesting a central role for specific transmission channels in human host-seeking behaviour. This analysis of the Anopheles gambiae receptors permits a comparison with the corresponding Drosophila melanogaster odorant receptor repertoire. We find that odorants are differentially encoded by the two species in ways consistent with their ecological needs. Our analysis of the Anopheles gambiae repertoire identifies receptors that may be useful targets for controlling the transmission of malaria.

INTRODUCTION: Control of mosquito vectors has historically proven to be an effective means of eliminating malaria. Human malaria is transmitted only by mosquitoes in the genus Anopheles , but not all species within the genus, or even all members of each vector species, are efficient malaria vectors. Variation in vectorial capacity for human malaria among Anopheles mosquito species is determined by many factors, including behavior, immunity, and life history. RATIONALE: This variation in vectorial capacity suggests an underlying genetic/genomic plasticity that results in variation of key traits determining vectorial capacity within the genus. Sequencing the genome of Anopheles gambiae , the most important malaria vector in sub-Saharan Africa, has offered numerous insights into how that species became highly specialized to live among and feed upon humans and how susceptibility to mosquito control strategies is determined. Until very recently, similar genomic resources have not existed for other anophelines, limiting comparisons to individual genes or sets of genomic markers with no genome-wide data to investigate attributes associated with vectorial capacity across the genus. RESULTS: We sequenced and assembled the genomes and transcriptomes of 16 anophelines from Africa, Asia, Europe, and Latin America, spanning ~100 million years of evolution and chosen to represent a range of evolutionary distances from An. gambiae , a variety of geographic locations and ecological conditions, and varying degrees of vectorial capacity. Genome assembly quality reflected DNA template quality and homozygosity. Despite variation in contiguity, the assemblies were remarkably complete and searches for arthropod-wide single-copy orthologs generally revealed few missing genes. Genome annotation supported with RNA sequencing transcriptomes yielded between 10,738 and 16,149 protein-coding genes for each species. Relative to Drosophila, the closest dipteran genus for which equivalent genomic resources exist, Anopheles exhibits a dynamic genomic evolutionary profile. Comparative analyses show a fivefold faster rate of gene gain and loss, elevated gene shuffling on the X chromosome, and more intron losses in Anopheles . Some determinants of vectorial capacity, such as chemosensory genes, do not show elevated turnover but instead diversify through protein-sequence changes. We also document evidence of variation in important reproductive phenotypes, genes controlling immunity to Plasmodium malaria parasites and other microbes, genes encoding cuticular and salivary proteins, and genes conferring metabolic insecticide resistance. This dynamism of anopheline genes and genomes may contribute to their flexible capacity to take advantage of new ecological niches, including adapting to humans as primary hosts. CONCLUSIONS: Anopheline mosquitoes exhibit a molecular evolutionary profile very distinct from Drosophila , and their genomes harbor strong evidence of functional variation in traits that determine vectorial capacity. These 16 new reference genome assemblies provide a foundation for hypothesis generation and testing to further our understanding of the diverse biological traits that determine vectorial capacity. Geography, vector status, and molecular phylogeny of the 16 newly sequenced anopheline mosquitoes and selected other dipterans.: The maximum likelihood molecular phylogeny of all sequenced anophelines and two mosquito outgroups was constructed from the aligned protein sequences of 1085 single-copy orthologs. Shapes between branch termini and species names indicate vector status and are colored according to geographic ranges depicted on the map.

A systematic functional analysis across much of the conventional Anopheles gambiae odorant receptor (AgOR) repertoire was carried out in Xenopus oocytes using two-electrode, voltage-clamp electrophysiology. The resulting data indicate that each AgOR manifests a distinct odor-response profile and tuning breadth. The large diversity of tuning responses ranges from AgORs that are responsive to a single or small number of odorants (specialists) to more broadly tuned receptors (generalists). Several AgORs were identified that respond robustly to a range of human volatiles that may play a critical role in anopheline host selection. AgOR responses were analyzed further by constructing a multidimensional odor space representing the relationships between odorants and AgOR responses. Within this space, the distance between odorants is related to both chemical class and concentration and may correlate with olfactory discrimination. This study provides a comprehensive overview of olfactory coding mechanisms of An. gambiae that ultimately may aid in fostering the design and development of olfactory-based strategies for reducing the transmission of malaria and other mosquito-borne diseases.

The period gene (per) is required for Drosophila melanogaster to manifest circadian (congruent to 24 hr) rhythms. We report here that per protein (PER) undergoes daily oscillations in apparent molecular mass as well as abundance. The mobility changes are largely or exclusively due to multiple phosphorylation events. The temporal profile of the classic short-period form of PER (PERS) is altered in a manner consistent with the mutant strain's behavioral phenotype. As changes in abundance and phosphorylation persist under constant environmental conditions, they reflect or contribute to a free-running rhythm. We suggest that the phosphorylation status of PER is an important determinant in the Drosophila clock's time-keeping mechanism.