Reproduction in higher animals requires the efficient and accurate display of innate mating behaviors. In Drosophila, male courtship consists of a stereotypic sequence of behaviors involving multiple sensory modalities, such as vision, audition, and chemosensation. For example, taste bristles located in the male forelegs and the labial palps are thought to recognize nonvolatile pheromones secreted by the female. A putative pheromone receptor, GR68a, is expressed in chemosensory neurons of about 20 male-specific gustatory bristles in the forelegs. Gr68a expression is dependent on the sex determination gene doublesex, which controls many aspects of sexual differentiation and is necessary for normal courtship behavior. Tetanus toxin-mediated inactivation of Gr68a-expressing neurons or transgene-mediated RNA interference of Gr68a RNA leads to a significant reduction in male courtship performance, suggesting that GR68a protein is an essential component of pheromone-driven courtship behavior in Drosophila (Bray, 2003).

Courtship behaviors are highly diversified, innate behaviors essential for propagation in higher animals. In general, courtship is composed of a series of behavioral displays controlled by the CNS, modulated by the endocrine system, and triggered only by highly specific, external stimuli that emanate from the mating object (Bray, 2003).

In wild flies, mating occurs near feeding sites to which they are attracted by long-range olfactory cues. Mate recognition, courtship, and mating are then mediated by visual, auditory, and pheromone signals and displayed in a stereotypic sequence of behaviors particularly well defined in the male: first, the male orients toward and follows a female (1), taps her abdomen with his forelegs (2), and proceeds to generate a 'courtship song' by rapid wing vibrations (3). He then licks the female's genitalia (4), curls his abdomen to attempt mounting (5), and eventually succeeds in mounting and copulation with the female (6). These steps entail visual (1) and chemosensory (2 and 4) recognition of female features by the male, auditory reception of the male courtship song by the female (3), and somatosensory agility of both sexes (3, 5, and 6). Even though this behavior is displayed in a stereotypical, sequential manner, a male generally executes each step multiple times before proceeding to the next. Females do not display a distinct courting behavior, but mated females actively reject a new potential mate by walking away, kicking with her hind legs, flicking of her wings, and extension of her ovipositor (Bray, 2003).

Efficient performance of courtship is the major determinant of mating success and can be quantified in single pair mating experiments by measuring mating latency (time from a first encounter between a male and a female until copulation), nonmating frequency, or the courtship index (CI = the percentage of time a male performs any of the first five courtship steps during a mating experiment). All these parameters have proved valuable for the quantification of male mating performance (Bray, 2003).

Mating behavior and sexual differentiation are regulated by a genetic cascade of splicing factors including Transformer (Tra) and Transformer2 (Tra2) that control the sex-specific, alternative splicing of mRNAs encoding the transcription factors Doublesex (Dsx) and Fruitless (Fru). Dsx^{m (male)}, Dsx^{f (female)}, and Fru^m control the expression of numerous male and female effector genes responsible for differentiation and maintenance of sexual identity. Several dsx-dependent effectors have been identified and are found to be expressed in endocrine tissues of the adult or in the genital disc during differentiation of adult structures. Fru^m and Dsx^m are required, but neither alone is sufficient, for wild-type male courtship behavior, because males lacking Fru^m but expressing Dsx^m (X/Y; fru¹/ fru¹) and intersexes which lack Dsx^m but express Fru^m [X/Y dsx¹/Df(dsx)] display severely reduced courtship behavior. Additionally, fru males court both males and females indiscriminately (Bray, 2003).

Several lines of evidence suggest that pheromone-elicited mate recognition is mediated mainly through the contact chemosensory system. For example, males tap the pheromone-coated, female abdomen and genitalia with their forelegs and labial palps, respectively, both of which are covered with taste bristles. The taste bristles on the forelegs are also implicated in a sex-specific function due to a quantitative difference in their number between males (50) and females (37). Finally, the chemical properties of the known female pheromones, which are nonvolatile, long-chain hydrocarbons, further support a role for contact chemosensory neurons/receptors in male courtship behavior (Bray, 2003).

Taste bristles in the labial palps and legs are composed of two to four gustatory receptor neurons (GRNs). A family of about 70 G protein-coupled receptor (GPCR) genes has been identifed, members of which are expressed in small subsets of GRNs in all known taste organs including the labial palps and the forelegs (Clyne, 2000; Dunipace, 2001; Scott, 2001). Upon analysis of about one quarter of the ">Gustatory receptor genes, the expression and function of Gr68a, a Gr gene expressed in chemosensory neurons of about ten male-specific taste bristles in the foreleg, is described. It is proposed that GR68a recognizes a female pheromone(s) involved in the second step of the courtship display, which is essential for efficient execution of the entire courtship sequence and timely mating (Bray, 2003).

Thus, a set of about 20 neurons associated with male-specific taste bristles in the forelegs of Drosophila is crucially involved in pheromone recognition during male courtship behavior. These bristles are molecularly characterized by the expression of the proposed taste receptor GR68a. RNA-mediated repression of this gene shows that Gr68a is in fact directly involved in recognition of a female pheromone, providing a precedent for a sex-specific pheromone receptor with a defined function in courtship behavior (Bray, 2003).

In principle, courtship behaviors serve two purposes: to attract the attention of a mating partner and to identify the sex and mating status of a con-specific animal. The complex sequence of behaviors of male flies combines both these purposes and is critical in guiding the male in a coordinated fashion through the entire courtship ritual culminating in successful copulation. The perception of female pheromones during the second and fourth step of the sequence are crucial events of courtship and must be integrated with other sensory input, including visual cues (female-specific coloration of the abdomen) and behavioral responses of the female toward the male during the entire courtship (Bray, 2003).

The functional characterization of the Gr68a-expressing neurons associated with male-specific taste bristles of the forelegs provides an opportunity to dissect the male courtship behavior. When Gr68a-expressing neurons were functionally inactivated by coexpressing TNT, males show a significant reduction in courtship activity toward females or males with a female pheromone profile, but no increase of courtship toward other males. Therefore, these neurons mediate a stimulatory response of an attractive, female pheromone as opposed to a repressive response of an inhibitory male pheromone. The specific role for these neurons is associated with the tapping step during courtship (step 2), in which the male directly contacts the pheromone-coated abdomen of the female with the tarsi of his forelegs. By quantitatively analyzing individual courtship steps, males lacking functional Gr68a-expressing neurons were shown to stall during the second step. Moreover, the modest increase in initiation/orientation (step 1) suggests that these males 'start over' more often with the courtship sequence than males with intact Gr68a-expressing neurons. Knowing the identity of these neurons and the specific phenotype associated with their inactivation should provide future opportunities to address more complex questions pertinent to this intriguing behavior. For example, how is the pheromone input in step 2 integrated with visual information received during step 1 and additional pheromone input received in step 4? And how does this input affect the motorneuron output so strikingly displayed in steps 3 and 5? At least a partial answer to these questions will require the identification of the first- and second-order target neurons of the Gr68a-expressing sensory neurons, which eventually should become feasible using axonal, synaptic, and trans-synaptic marker proteins expressed under the control of the Gr68a promoter (Bray, 2003).

The courtship phenotype associated with inactivating Gr68a-expressing neurons is likely to be mediated by the GR68a receptor itself. Males in which GR68a expression is suppressed by RNAi have an increase in mating latency, fraction of nonmaters, and reduced courtship intensity, as was observed in males in which the Gr68a-expressing neurons were inactivated altogether. Moreover, the detailed courtship analysis reveals that these males also stall during the same step in the courtship sequence, with almost identical severity as males with inactivated Gr68a-expressing neurons, arguing for a major role of this receptor in recognition of a female pheromone. It is quite possible that Gr68a is the only Gr gene that is expressed in these neurons. Reported expression studies of about ten Gr genes (Dunipace, 2001; Scott, 2001) and ongoing studies of an additional ten Gr genes (N. Thorne and H. A., unpublished data cited in Bray, 2003) indicate that most Gr genes are expressed in distinct sets of gustatory neurons, and expression of different Gr genes are to a large extent nonoverlapping. In any case, even if Gr68a-expressing neurons coexpress another Gr gene, its transcripts are unlikely to be affected by the expression of ds_Gr68a RNA, because nucleotide sequence similarity between Gr68a and any other Gr gene is far too low to allow RNAi to occur (Bray, 2003).

Gr68a is the first putative, sex-specific, pheromone receptor gene with a defined function in courtship behavior. A gene cluster containing 16 putative pheromone receptors (V1Rs) expressed in the vomeronasal organ was recently reported to be required for normal mating behavior of mice (Del Punta, 2002). Female mice homozygous for this multigene knockout show reduced aggression toward invaders, and homozygous male mice show reduced sexual aggression toward both sexes. However, none of the V1Rs included in this deletion were reported to be sex specific, and it remains to be investigated whether specific behavioral phenotypes can be associated with individual V1R genes (Bray, 2003).

The 70 Drosophila Gr genes, which are distantly related to the olfactory receptor (Or) genes, encode a diverse family of G protein-coupled receptors that share between 15% and 80% sequence similarity. No other candidate chemosensory receptors have emerged from the complete genome sequence of Drosophila, suggesting that the GR proteins might accommodate the detection of all nonvolatile substrates to which Drosophila is responsive. In mammals, distinct groups of nonvolatile compounds are recognized by unrelated G protein-coupled receptors, encoded by four distinct gene families that are expressed in neurons of the vomeronasal organ or in taste cells of the tongue. For example, the taste cells in the tongue express two classes of receptors, the T1Rs and T2Rs; the T1Rs were shown to detect sweet-tasting substrates such as various sugars and many L-amino acids, whereas the much more numerous T2Rs appear to recognize the large spectrum of compounds perceived as bitter tasting to humans (Bray, 2003 and references therein).

The only known substrate for a GR protein, GR5a, is trehalose, which is an important food source for Drosophila melanogaster. The number of biologically relevant sugars is fairly small compared to the number of Gr genes, and hence, members of this protein family are likely to recognize other classes of substrates. It is suggested that GR68a is a receptor that detects a pheromone(s) of Drosophila melanogaster females, possibly a long-chain hydrocarbon such as the female-specific 7,11 heptacosadiene and 7,11 nonacosadiene, both of which have been strongly implicated in eliciting male courtship behavior (Coyne, 1995; Ferveur, 1996). Thus, it is proposed that the GR protein family recognizes a whole spectrum of nonvolatile, complex substrates (sugars, amino acids, alkaloids and bitter-tasting compounds, hydrocarbons, and possible other pheromones, etc.) to which Drosophila responds. For example, different sugars might bind to receptors of the GR5a subfamily, which include seven additional receptors encoded by Gr61a and Gr64a-f (members of a subfamily were defined by sharing at least 34% sequence similarity [Dunipace, 2001]). Similarly, proteins encoded by the Gr68a subfamily -- Gr2a, Gr32a and Gr39a.a-39a.d -- might interact with different pheromone components. Expression of Gr32a has been analyzed and found to be restricted to the labelum and the distal tip of the forelegs of both sexes (Scott, 2001). Preliminary experiments indicate that simultaneous inactivation of Gr68a- and Gr32a-expressing neurons by TNT compounds the courtship defect in males, resulting in about 80% nonmaters and almost 20 min latency time, suggesting that this receptor might function in the fourth step of the courtship sequence. Further analysis of Gr32a and the other members of the Gr68a subfamily using RNAi should reveal the specific roles, if any, that these genes have during male mating behavior (Bray, 2003 and references therein).

Finally, amino acids and various classes of bitter-tasting substrates might be recognized by receptors encoded by yet other Gr subfamilies. It is even conceivable that some GR proteins detect volatile molecules, as a few Gr genes were found to be expressed in olfactory neurons both in the larvae and the adult (Dunipace, 2001; Scott, 2001). Thus, the highly diverse GR proteins are likely to mediate a multitude of strikingly different behaviors including courtship and mating, feeding behavior, and avoidance behavior elicited both by soluble and volatile compounds (Bray, 2003 and references therein).

GENE STRUCTURE

cDNA clone length - 1170

Exons - 1

PROTEIN STRUCTURE

Amino Acids - 389

Structural Domains

See SRS@EMBL-EBI for information on GR68a structure.

date revised: 20 March 2004

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