Volume 16, Issue 11, Pages 1154-1159 (June 2006) A Drosophila Protein Specific to Pheromone-Sensing Gustatory Hairs Delays Males' Copulation Attempts  Su K. Park, Kevin J. Mann, Heping Lin, Elena Starostina, Aaron Kolski-Andreaco, Claudio W. Pikielny  Current Biology  Volume 16, Issue 11, Pages 1154-1159 (June 2006) DOI: 10.1016/j.cub.2006.04.028 Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 1 CheB42a and Gr68a Are Expressed in the Same Stereotypic Subset of Gustatory Sensilla on the Front Legs of Males (A) GFP expression placed under indirect control of upstream CheB42a regulatory sequences via the Gal4/UAS system was visualized (green arrow) as described [8]. The cell bodies of both neurons and support cells that contribute to a gustatory sensillum are located proximal to the hair base (farther away from the tip of the hair) and beneath the cuticle [21] (see Figure 2A). Gustatory sensilla (arrowheads) are identified by their curved shaft, whereas mechanosensory sensilla (small arrows) have a straight shaft and a bract at their base. (B) Representative left front legs of males carrying, in addition to two copies of a UAS-GFP reporter transgene, one or both Gal4 driver transgenes, as indicated. For each leg, GFP fluorescence was superimposed on a bright-field image to allow orientation relative to morphological landmarks. Faithful reproduction of endogenous CheB42a expression with this CheB42a-Gal4 transgene was previously demonstrated by direct comparison with anti-CheB42a immunostaining [8]. Less directly, the reduced male response to female hydrocarbon pheromones as a result of Gr68a-Gal4-driven expression of either neurotoxins or double-stranded Gr68a RNA validates the use of Gr68a-Gal4-driven expression of GFP to identify the functional expression sites of the endogenous Gr68a gene [9]. (C) Schematic view of the surface of a male left front leg showing the position of 29 gustatory sensilla [17, 19, 20]. When the fly is at rest, the surface of the tarsus shown is directed toward the anterior of the fly, whereas the surface of the tibia shown is as seen from the side of the fly. GFP-expressing sensilla are indicated by green circles. TSn, tarsal segment n. Current Biology 2006 16, 1154-1159DOI: (10.1016/j.cub.2006.04.028) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 2 CheB42a Is Specifically Expressed in the Sheath Cells that Envelop Gr68a-Expressing Gustatory Neurons (A) Cartoon of a gustatory sensillum of male front legs that expresses CheB42a, PBPRP2, and Gr68a. Gustatory hairs on the legs of Drosophila have two fluid-filled cavities [21]. The inner lumen is the site of the earliest steps in gustatory-signal transduction because it contains the dendrites of gustatory neurons and communicates with the external environment through a terminal pore. Gr68a, a putative pheromone receptor, is expressed in at least some of the gustatory neurons within this subset of hairs [9], and CheB42a is expressed in the thecogen or sheath cells that surround Gr68a-expressing neurons (see below). Finally, PBPRP2 is expressed in trichogen and tormogen cells and secreted into the external lumen of the hair [21]. For simplicity, the mechanosensory neuron and two of the four gustatory neurons are not shown. (B) CheB42a is expressed in non-neuronal cells that envelop gustatory neurons. Frozen sections of male front legs were stained with anti-CheB42a (green, first panel) [8] and 22C10, a monoclonal antibody against a neuronal-specific surface marker (red, right panel) [22]. The two fluorescent signals are digitally superimposed in the central panel. (C) The gustatory neurons that express Gr68a, a putative pheromone receptor [9], are immediately adjacent to CheB42a-expressing cells and partially surrounded by their membranes. Increasing contrast and saturating signal in the cell body allowed visualization of the weaker CheB42a signal that surrounds the Gr68a-expressing neurons. CheB42a (green) and GFP were immunologically detected in animals that express GFP under control of Gr68a-Gal4. (D) Expression of CheB42a and PBPRP2 within the same gustatory sensilla occurs in different non-neuronal cells. In this image, the tip of the leg (distal) is at the top. Immunological detection of CheB42a and GFP was performed as in (C), in animals in which GFP is expressed under control of the pbprp2 promoter [21]. Immunohistochemical methods to detect specific proteins in frozen sections of male front legs were as described for CheB42a [8] as well as PBPRP2 and GFP [24]. Current Biology 2006 16, 1154-1159DOI: (10.1016/j.cub.2006.04.028) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 3 Female-Specific Cuticular Hydrocarbons Trigger Earlier and More Frequent Copulation Attempts from CheB42a Mutant Males (A) The response of males to w1118 virgin females was compared for control males (black bars), males homozygous for CheB42aΔ5-68 (white bars), and CheB42aΔ5-68 homozygous males that carry Tg2, a transgenic construct that encodes CheB42a but no other protein-coding gene (gray bars) [13]. An asterisk indicates p < 0.0000007; double asterisks indicate p < 0.002. (B) A single copy of alternative transgenes is the only genetic difference between the two types of males tested in this experiment; both are heterozygous for CheB42aΔ5-68 and Δ5-22, another deletion of the locus that removes not only CheB42a but also the neighboring ppk25 gene [13]. Control males (black bars) carry Tg1[13], a transgene that encodes both CheB42a and ppk25, whereas the transgene present in test males (white bars) is identical to Tg1, except for a point mutation that changes the initiating ATG of CheB42a to ATA and results in the absence of detectable CheB42a protein (data not shown). An asterisk indicates p < 0.02; double asterisks indicate p < 0.002. (C) The responses of CheB42aΔ5-68 and control males toward heat-shocked “hs-traF” females that carry the hs-Gal4 and UAS-traF transgenes and lack cuticular hydrocarbons [25] or, as controls, genetically identical, non-heat-shocked hs-traF female siblings were measured. An asterisk indicates p < 0.006; double asterisks indicate p < 0.008. (D) “Oeno-traF” males that carry the oenocyte-Gal4 and UAS-traF transgenes and display female-specific hydrocarbon pheromones on their cuticles [26] were used as sexual objects for CheB42aΔ5-68 and control males. An asterisk indicates p < 0.00038; double asterisks indicate p < 0.00067. For all panels, analysis of courtship behavior was as described previously [13], with the following modifications. Males of each genotype were placed in the presence of intact (not decapitated) sexual objects, and each couple's behavior was videotaped under normal overhead lights for 10 min. Blind analysis of each individual step in the courtship sequence was performed with Lifesong X (0.75) [32]. p values were derived by ANOVA. Current Biology 2006 16, 1154-1159DOI: (10.1016/j.cub.2006.04.028) Copyright © 2006 Elsevier Ltd Terms and Conditions