Organization of olfactory centres in the malaria mosquito Anopheles gambiae

Abstract

Mosquitoes are vectors for multiple infectious human diseases and use a variety of sensory cues (olfactory, temperature, humidity and visual) to locate a human host. A comprehensive understanding of the circuitry underlying sensory signalling in the mosquito brain is lacking. Here we used the Q-system of binary gene expression to develop transgenic lines of Anopheles gambiae in which olfactory receptor neurons expressing the odorant receptor co-receptor (Orco) gene are labelled with GFP. These neurons project from the antennae and maxillary palps to the antennal lobe (AL) and from the labella on the proboscis to the suboesophageal zone (SEZ), suggesting integration of olfactory and gustatory signals occurs in this brain region. We present detailed anatomical maps of olfactory innervations in the AL and the SEZ, identifying glomeruli that may respond to human body odours or carbon dioxide. Our results pave the way for anatomical and functional neurogenetic studies of sensory processing in mosquitoes.

Figure 1. Orco-QF2 expression in olfactory neurons in adult.

(a,b) Membrane-targeted GFP expression in adult female (a, top row) and male (b, top row) animals. Bottom row shows images of female (a) and male (b) QUAS-mCD8:GFP controls. GFP expression is visible in antennae (arrows), maxillary palps (white arrowheads) and proboscis (open arrowheads) of transgenic animals (top row), but not in QUAS-mCD8:GFP controls (bottom row). Transgenic animals also express fluorescent DsRed and/or CFP markers in the eye, which results in residual fluorescence in the GFP channel. Genotype: Orco-QF2, QUAS-mCD8:GFP. Scale bars, (a,b) left panels, 1 mm; (a,b) right panels, 200 μm. (c,d) Confocal images of GFP expression in the antennae (c,d, top rows), maxillary palps (c,d, middle rows) and proboscis (c,d, bottom rows) of female (c) and male (d) adult animals. Images in c,d left panels were acquired in RFP (autofluorescence) and GFP channels, which are shown separately for clarity. (c,d) Right panels are high-magnification images showing the cell bodies of labelled neurons. Scale bars, (c,d) left panels, 200 μm; (c,d) right panels, 20 μm.

Figure 2. Orco-QF2 drives GFP reporter expression in adult Orco+ neurons.

Antennae (top row), maxillary palps (middle row) and proboscis (bottom row) of adult female (left) and male (right) mosquitoes were immunostained with anti-CD8 (blue) and anti-DmOrco (red) antibodies. Images show immunofluorescence of CD8 and Orco, as well as genetically driven GFP (green). Same cell bodies are labelled in all three channels. White dashed line marks borders of the imaged tissue. All images were acquired at the same magnification. Genotype: Orco-QF2, QUAS-mCD8:GFP. Scale bar, 20 μm.

Figure 3. Orco+ olfactory neurons target two sensory brain regions.

(a) Orco+ receptor neurons send projections to the AL and SEZ of the brain, as detected in Orco-QF2, QUAS-mCD8:GFP animals by anti-GFP antibody labelling. Left column shows an overall image of the brain (scale bar, 100 μm). Middle and right columns show the AL and SEZ at higher magnifications (scale bar, 20 μm). Confocal sections are maximum z-projections. Arrowhead points to commissure. Inset shows commissure at higher magnification. (b) Shown are brains of male and female Orco-QF2, QUAS-mCD8:GFP mosquitoes in which the right antennae had been ablated (as schematized in the cartoon) 5 days before brain dissections. The ALs retain ipsilateral Orco+ innervation from the unablated left antennae and bilateral innervation from the intact maxillary palps. Inset shows commissure at higher magnification. Scale bars, 20 μm (c–e). Antennae (c), maxillary palps (d) or proboscis (e) of Orco-QF2, QUAS-mCD8:GFP mosquitoes were cut to ∼1/3 of their length (as depicted in the cartoons) and backfilled by neurobiotin that is incorporated into the membranes of severed neurons. The neurobiotin labelling (blue) of the brains, together with the GFP labelling (green), establishes the origin of Orco+ receptor neurons (antennal, maxillary palp or proboscis) that innervate the AL and SEZ. Backfills originating from the antenna innervated only the ipsilateral AL. Backfills originating from the maxillary palps innerved both ipsilateral and contralateral ALs. Backfills originating from the proboscis innervated only the SEZ brain region. The numbers indicate the distance in micrometres from the most anterior confocal section. Dashed white lines outline the AL/SEZ. Solid white lines outline the area labelled by the neurobiotin backfill (blue signal). Scale bars, 20 μm.

Figure 4. Reconstruction of the mosquito AL and SEZ based on Orco expression and olfactory tissue of origin.

(a) Confocal Z-stacks of female (left) and male (right) ALs, shown at the same magnification (most anterior—top row, most posterior—bottom row). Glomeruli were outlined manually, and GFP and neurobiotin backfill signals were used to assign each glomerulus to one of five groups: Orco+ antennal glomeruli (purple), Orco− antennal glomeruli (green), Orco+ maxillary palp glomeruli (blue), Orco− maxillary palp glomeruli (yellow) and glomeruli that were not labelled by backfills (grey). Glomeruli were numbered starting at the most anterior section. Scale bars, 20 μm. Also see Supplementary Table 5. (b) A. gambiae and D. melanogaster ALs show similarities in the arrangement of glomeruli targeted by Orco+ (purple) and Orco− neurons. Orco− neurons originating from the mosquito antennae are likely to be IR-expressing neurons (IR, green) and Orco− neurons from the maxillary palps are likely to be GR-expressing neurons (GR, yellow). The Drosophila AL is reprinted with permission from ref. with minor modifications. (c) Confocal Z-stacks of female (left) and male (right) SEZ, shown at the same magnification (most anterior, top row; most posterior, bottom row). Glomerular-like structures were outlined manually using the GFP signal. Glomerular structures were numbered starting at the most anterior section. Scale bars, 20 μm. (d) Three-dimensional modelling of olfactory neuron targeting in the SEZ. The Orco-QF2-labelled nerve bundle from the labella is shown for reference.