Figure 1.
General organization of a trichoid sensillum hair.
Two olfactory sensory neurons (OSNs, red) project their dendrites into the sensillum lymph (blue). The cell bodies of the OSNs are surrounded by three support cells, two of which express “classic” odorant binding proteins (OBPs, dark green) and secret them into the sensillum lymph.
Figure 2.
Gene expression of “classic” OBPs and the “Plus-C” OBP48 in the antenna of A. gambiae females.
WM-FISH using AgOBP-specific DIG-labeled antisense RNA probes. Cells bearing AgOBP transcripts have been visualized by red fluorescence. The same (9th) flagellomere from different animals is shown. Each AgOBP is expressed in either a high, moderate or low number of cells distributed in the antennal segment. Scale bars: 20 µm.
Figure 3.
Expression of AgOBP pairs in different cells of the female antenna.
Two-color WM-FISH using combinations of differentially labeled antisense RNAs and detection systems visualizing expression of the two AgOBPs by green and red fluorescence, respectively. No co-labeling of single cells is visible indicating expression of the pairs in different cell populations. This labeling pattern was independent of a more proximal or distal position (upper row) of the antennal segment. The number of each antennal segment is indicated. Scale bars: 20 µm.
Figure 4.
Co-expression of AgOBP pairs in subpopulation of antennal cells.
Two-color WM-FISH with female antennae and visualization of cells bearing distinct AgOBP transcripts by green and red fluorescence, respectively. Labeling of the same cells by both AgOBP probes is shown for six pairings. In the case of strong hybridization signals co-labeling appears as yellow color in the overlay (left) of the red and green fluorescence channels (right). For the pair AgOBP7/AgOBP48, which displayed rather weak signals co-labeled cells are marked by arrow heads. The separated fluorescence channels (right) are shown in reduced size. Numbers indicate the position of the antennal segment. Scale bars: 20 µm.
Figure 5.
Direct vicinity of AgOR2- and AgOBP-expressing cells.
Spatial organization and relative localization of cells transcribing AgOR2 and AgOBP1 (A) and AgOBP4 (B) on female antenna. Double WM-FISH using differentially labeled antisense RNA probes, with visualization of AgOR2 by red and AgOBPs by green fluorescence (Boxed areas are shown at higher magnification on the right). Scale bars: 20 µm.
Figure 6.
Immunolocalization of OBPs in the antenna of female A. gambiae.
Whole mount preparations were probed with antisera specific for AgOBP1 (A and B) or AgOBP4 (C). Immunoreactivity was visualized by an Alexa488 secondary antibody. (A) Staining by AgOBP1 antibodies in consecutive antennal segments (6 to 8). A high number of AgOBP-expressing cells is visible and can be assigned to sensilla trichodea. (B) Higher magnification of the area boxed in A. AgOBP1 immunostaining is found within the sensilla hairs as well as in supporting cells below the sensilla. (C) Staining of AgOBP4-expressing cells and sensi lla in antennal segment 10. The antennal stretch shown in (A) was assembled from three single pictures. Scale bars: 20 µm.
Figure 7.
Co-localization of AgOBP and AgOR expression in single sensilla of female antenna.
Combination of WM-FIHC using AgOBP-specific antibodies and WM-FISH employing AgOR-specific DIG-labeled antisense probes. (A) AgOBP1 and AgOR2, (B) AgOBP4 and AgOR2, (C) AgOBP1 and AgOR1. OBP-immunostaining (green) is found in direct association with a red labeled OR-transcribing cell (left pictures) and can be followed into a distinct sensillum (boxed areas shown at higher magnification on the right). Scale bars: 20 µm (left).