A highly conserved candidate chemoreceptor expressed in both olfactory and gustatory tissues in the malaria vector Anopheles gambiae

Abstract

Anopheles gambiae is a highly anthropophilic mosquito responsible for the majority of malaria transmission in Africa. The biting and host preference behavior of this disease vector is largely influenced by its sense of smell, which is presumably facilitated by G protein-coupled receptor signaling [Takken, W. & Knols, B. (1999) Annu. Rev. Entomol. 44, 131-157]. Because of the importance of host preference to the mosquitoes' ability to transmit disease, we have initiated studies intended to elucidate the molecular mechanisms underlying olfaction in An. gambiae. In the course of these studies, we have identified a number of genes potentially involved in signal transduction, including a family of candidate odorant receptors. One of these receptors, encoded by GPRor7 (hereafter referred to as AgOr7), is remarkably similar to an odorant receptor that is expressed broadly in olfactory tissues and has been identified in Drosophila melanogaster and other insects [Krieger, J., Klink, O., Mohl, C., Raming, K. & Breer, H. (2003) J. Comp. Physiol. A 189, 519-526; Vosshall, L. B., Amrein, H., Morozov, P. S., Rzhetsky, A. & Axel, R. (1999) Cell 96, 725-736]. We have observed AgOr7 expression in olfactory and gustatory tissues in adult An. gambiae and during several stages of the mosquitoes' development. Within the female adult peripheral chemosensory system, antiserum against the AgOR7 polypeptide labels most sensilla of the antenna and maxillary palp as well as a subset of proboscis sensilla. Furthermore, AgOR7 antiserum labeling is observed within the larval antenna and maxillary palpus. These results are consistent with a role for AgOr7 in both olfaction and gustation in An. gambiae and raise the possibility that AgOr7 orthologs may also be of general importance to both modalities of chemosensation in other insects.

Fig. 1.

(A) Alignment of AgOR7 ortholog peptides, displayed in the single-letter amino acid code. Identical residues are boxed and shaded. Transmembrane regions (TMs) I-VII are indicated with black bars. Previous placement of TMVII (4) is indicated with a gray line. Black dotted line shows the location of the peptide antigen used in antiserum production. Threonine (T) and tyrosine (Y) residues that are sites of potential phosphorylation are enclosed in heavy black boxes. For a list of genes and accession numbers see Methods. (B) Exon and intron structures of the AgOr7 and DOr83b genes. Numbers within boxes indicate exon sizes in amino acids. Intron lengths (not drawn to scale) in base pairs are shown below each line. Asterisks highlight conserved splice sites. Heavy black line shows the position of the An. gambiae BAC from which the AgOr7 gene was isolated. (C) Microsynteny within the genomic regions surrounding AgOr7 and DOr83b. Gene names and their relative positions are listed above and below lines, respectively. Arrowheads indicate directions of transcription, and like arrowheads are apparent orthologs: gray ≈50% identical, black and white ≈80% identical at the amino acid level.

Fig. 2.

Expression of the AgOr7 gene in An. gambiae by RT-PCR. Lanes are as follows: gen, genomic DNA control; em, embryos; el, early-stage larvae; ll, late-stage larvae; pup, pupae; bod, bodies; leg, legs; ant, antennae; mp, maxillary palps; prb, proboscises; neg, no template. Numbers indicate expected sizes of genomic and cDNA products in base pairs (bp).

Fig. 3.

Localization of the AgOR7 protein in An. gambiae female antenna and maxillary palp. Magenta is anti-AgOR7 marked with Cy3-labeled secondary antibody. Green is anti-horseradish peroxidase conjugated with FITC. (A) SEM of the 10th flagellar segment. SC, sensilla chaetica; ST, sensilla trichodica; GP, grooved peg. (Scale bar is 25 μm.) (B) Antenna AgOR7 labeling in flagellar segments 2-9. JO, Johnston's organ. (Scale bar is 100 μm.) (C) AgOR7 labeling in the trichodic sensilla of a single distal antennal segment. (Scale bar is 25 μm.) (D) AgOR7 labeling is absent from grooved peg sensilla. (Scale bar is ≈4 μm.) (E) SEM of a palp segment showing nonsensory scales and capitate peg (CP) sensilla. (Scale bar is 40 μm.) (F) AgOR7 labeling in a capitate peg of the third palpal segment. (Scale bar is 40 μm.)

Fig. 4.

Localization of the AgOR7 protein in An. gambiae female proboscis. Magenta is anti-AgOR7 marked with Cy3-labeled secondary antibody. Green is anti-horseradish peroxidase conjugated with FITC. (A) SEM of the labellum. (Inset) A higher-magnification SEM of the sensillum type that is labeled by anti-AgOR7 in B. (Inset scale bar is 5 μm.) (B) AgOR7 labeling in a distinct sensillar type (arrows) in one labellar lobe of the proboscis. (Scale bars are 50 μm.)