Galpha encoding gene family of the malaria vector mosquito Anopheles gambiae: expression analysis and immunolocalization of AGalphaq and AGalphao in female antennae

Abstract

To initiate a comprehensive investigation of chemosensory signal transduction downstream of odorant receptors, we identified and characterized the complete set of genes that encode G-protein alpha subunits in the genome of the malaria vector mosquito An. gambiae. Data are provided on the tissue-specific expression patterns of 10 corresponding aga-transcripts in adult mosquitoes and pre-imago developmental stages. Specific immunoreactivity in chemosensory hairs of female antennae provides evidence in support of the participation of a subset of AGalphaq isoforms in olfactory signal transduction in this mosquito. In contrast, AGalphao is localized along the flagellar axon bundle but is absent from chemosensory sensilla, which suggests that this G-protein alpha subunit does not participate in olfactory signal transduction.

Fig. 1

Phylogeny of D. melanogaster G_α-proteins compared with that of An. gambiae homologs. Scale bar denotes 5% sequence-divergence. Protein sequences for An. gambiae were deduced from sequence information available through the mosquito genome project and at TIGR, which was combined with sequence information of RT-PCR products. For bootstrap analysis, alignment-gaps were excluded. The Saccharomyces cerevisiae G_α-protein of the pheromone response pathway, Gpa1p was included in bootstrap analysis as an outgroup. DmGq2 was not included because no protein corresponding to this mRNA has been detected (Scott et al., 1995).

Fig. 2

A: Genomic organization at the An. gambiae ago-locus: the protein coding region is distributed over more than 56 kbp; coding exons are displayed as bars and sizes of selected subfragments are indicated below the schematic. Exons 1N and 1A are respective parts of two transcripts, agon and agoa, that originate from alternative splicing. Exon 2 is encoded in a region of ∼5 kbp that was not sequenced during the course of the genome project. B: Structure of the An. gambiae agq-locus: three homologous pairs of exons D and D*, G and G*, as well as H and H*, respectively, are spliced alternatively as shown (transcripts agq1-4). Transcript structure is depicted for the regions analyzed by sequencing of RT-PCR products. Additionally, an RT-PCR product consistent with the expected complete agq2 ORF A–H* was cloned and sequenced. Hatched boxes: exons encoding translation-STOP.

Fig. 3

Expression analysis of agα transcripts in adult tissues (A) and preimago developmental stages as well as female adult tissues prior to and 20 hours after a blood meal (C) using RT-PCR. Target transcripts are denoted on the left margin. The rpS7 transcript was amplified as a control for cDNA integrity. All PCR products that originated from cDNA, as judged by size, were cloned and sequenced. Asterisks indicate sequenced RT-PCR products that may not be translated (see text) and PCR product sizes are denoted on the right, investigated tissues at the top margin. RNA was isolated from the following mosquito tissues in (A): m.ant., male antennae; m.p.p., male palps and proboscides; f.ant., female antennae; f.p.p., female palps and proboscides; heads, heads without appendages; bodies, thoraces and abdomens including wings, without legs; gen, genomic DNA control. C: h.+app., heads including appendages; th., thorax including wings and legs; abd., abdomens; bf., 20 hours post blood meal; e. larv., 1-week-old larvae; l.larv., 2-week-old larvae. Left and right-most lanes in (A) and left lane in (C): 100 bp-ladder (New England Biolabs, Beverly, MA). All RT-PCR results are shown for 34 PCR cycles. Note that ago and agq primer binding sites are flanking large introns and agi, agm, and agc primers target exon–exon boundaries, thus obviating amplification of genomic fragments. B: Relative expression levels of agα transcripts in female An. gambiae antennae, determined by real-time RT-PCR are described as copies/1,000 copies rpS7. Error bars represent 1 standard deviation (n = 3). Note that primers for real-time analysis were placed close to the 3′ end of the respective reading frames. Hence, agoa and agon could not be distinguished.

Fig. 4

Immunoblots of An. gambiae heads plus appendages. Each lane represents ∼0.5 head equivalents. Anti-Gq/11, anti-DGq1, and anti-Go sera label protein bands consistent with the expected molecular weights for AG_αq and AG_αo isoforms (arrowhead; unmodified AG_αq = 41.6 kDa, unmodified AG_αo = 40.4kDa), respectively. Anti-Gq/11 specific labeling is inhibited by preadsorption with its cognate peptide (q11P), which does not reduce anti-DGq1 immunoreactivity consistent with its specificity to AG_αq-isoforms 1 and 4. Anti-Go serum reactivity (arrowhead) is blocked by preincubation along with recombinant rat G_αo.

Fig. 5

Immunolocalization of Gα proteins in frozen sections of adult female An. gambiae heads. Anti-DGq1 serum-dependent labeling (A,D) is specific to the retina (R). Anti-Gq/11 dependent labeling (B,E) is strong in the retina of the compound eye and the lamina ganglionaris (L). More moderate anti-Gq/11 labeling was also observed in the neuropil of the medulla and central nervous system (CNS) and in adjacent neuronal cell bodies. Anti-Go labeling (C,F) was observed in the CNS neuropil and the fat body (FB), while neuronal cell bodies and the lamina ganglionaris were labeled to a lesser extent. No labeling was observed in the retina. Gα, magenta; neurons (anti-HRP (Sun and Salvaterra, 1995), green; DNA, blue; magenta/green overlap, white. Top row: composite images; bottom row: Gα-specific channel. Scale bars = 50 µm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 6

Anti-Gq/11 serum (magenta) labeling is associated with most or all trichodic (examples labeled with asterisk), grooved peg (examples labeled with arrowhead), and large coeloconic sensilla (open arrows), the flagellar axon bundle, and the base of sensilla chaetica (open arrowheads). A: Intermediate segments of antenna. B,C: Enhancement of panel A as indicated by asterisk. Note that anti-HRP serum predominantly labels neuronal somata and inner dendrites of trichodic ORNs, while anti-Gq/11-dependent labeling is confined to the sensory hair, which contains the outer dendritic region of ORNs. D,E: Anti-Gq/11 labeling in grooved peg sensilla is weaker and more diffuse than in trichodic sensilla. F,G: Labeling of large coeloconic sensilla is associated with the sensillum base. A,B,D,F: Composite images. C,E,G: Respective Anti-Gq/11 (magenta) specific channel. Neurons (anti-HRP/nervana), green; DNA, blue; magenta/green overlap, white. Stack sizes: (A) 4.1 µm (B,C) 0.51 µm (D,E) 0.51 µm (F,G) 3.6 µm. Note that the brightfield component is not confocal and thus represents the entire 15-µm section. Scale bars = 50 µm in A; 5 µm in B–G. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 7

Anti-DGq1 dependent labeling was not observed along the antenna (A,B). Anti-Go-specific labeling (C,D) is associated with the flagellar axon bundle and the base of sensilla chaetica (asterisk). In situ hybridization utilizing specific antisense RNA probe (E,F) identifies ago expression in neurons (arrowhead) that are distinguished by anti-HRP serum (E) (Sun and Salvaterra, 1995), while neurons in control sections hybridized to sense RNA probe remain unlabeled (data not shown). Gα, ago, magenta; neurons, green; DNA, blue; magenta/green overlap, white (note: overlap may not generate white color, if one signal is significantly stronger, within the dynamic range of detection). Stack sizes are (A,B) 4.2 µm (C,D) 5.1 µm (E,F) 2 µm. Scale bars = 20 µm in B,D; 5 µm in F. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]