InteractiveFly: GeneBrief

quick-to-court: Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Sexual behaviors are critical to the reproduction of most animal species, yet remarkably little is known about the molecular mechanisms that drive them. Neural elements associated with sexual behaviors have been defined in a number of systems, but in few species have the molecular elements underlying these behaviors been explored. One organism that exhibits a rich repertoire of sexual behaviors and offers a powerful means of investigating their genetic and molecular underpinnings is Drosophila (Gaines, 2000).

Male flies show stereotyped courtship behaviors toward virgin females. The male orients toward the female, follows her, taps her abdomen with his foreleg, extends and vibrates his wing, licks her genitalia, and then mounts her and initiates copulation. A number of mutants showing abnormalities in courtship behavior have been identified, many of which show reduced levels of courtship behavior. Many of these mutants exhibit sensory defects, defects in the generation of the courtship song, and/or reduced motor activity (Gaines, 2000).

quick-to-court (qtc) is a gene whose mutations are unique, in that they cause augmentation of male courtship behavior toward both females and other males. The gene was identified by an enhancer trap insertion, which was isolated in a screen of 6400 enhancer trap lines for lines showing expression of a lacZ reporter gene in the olfactory system. This particular insertion is distinguished by its sexually dimorphic expression in the Drosophila antenna, exhibiting stronger staining in the male antenna than in the female antenna. Further investigation showed that the staining is due to the insertion of a P-element enhancer trap in cytogenetic region 25C of the second chromosome. The staining pattern is not specific to the antenna, but was also detected in a small number of cells in the adult brain, where it was expressed at comparable levels in males and females. Expression was also observed in association with the larval olfactory organ, the antenno-maxillary complex. qtc encodes a protein with predicted coiled-coil domains (Gaines, 2000).

Striking abnormalities are observed in a P(lacZ) enhancer trap line, subsequently designated as qtc¹. Sexually mature qtc¹ males perform much more courtship in response to each other than do control Canton-S-5 (CS) males. Specifically, mature qtc¹ males yield a male-male courtship index (CI: defined as the percentage of the observation period during which any courtship behaviors, i.e., orientation, following, tapping, wing vibration, licking and attempted copulation are observed) of 42 ± 5, indicating that courtship behaviors were observed on average during 42% of a 10-min observation period, whereas CS control males have a CI of only 4 ± 1. CS was chosen as a control because the sexual behavior of CS flies has been thoroughly characterized; however, since qtc¹ is not in a CS genetic background, a series of experiments were performed to determine whether the phenotype is in fact due to the P-element insertion in this line (Gaines, 2000).

The behavior of qtc¹ males first was compared to that of males from 6276, another line isolated from the same enhancer trap screen as the original isolate of qtc¹. Males from the 6276 line perform very low levels of male-male courtship (CI = 2 ± 0), similar to the levels in CS. The responses of males from four stocks derived by excision of the P-element insertion were then compared: three stocks in which the P element had excised precisely (qtc^r1, qtc^r2, and qtc^r3) and one in which the P element had excised imprecisely, creating a deletion of flanking DNA (qtc^Ex1). Males from the precise excision stocks (qtc^r1 and qtc^r2) show low levels of male-male courtship, similar to CS (low levels of male-male courtship also were observed in qtc^r3, for which fewer data have been collected). In comparison, males from the imprecise excision stock (qtc^Ex1) have high levels of male-male courtship. Since these excision lines were generated in parallel and are expected to have similar genetic backgrounds, these results indicate that the elevated levels of male-male courtship in the qtc¹ stock result from the insertion of the P element (Gaines, 2000).

qtc¹ males' courtship of each other also was qualitatively different from that of CS controls. In all pairs of CS males observed (10/10), one or both males performed at least one of the 'early' behaviors (orientation, tapping, and following), but in only half of the pairs observed did one or both males vibrate their wings to produce a courtship song, and none of the males licked or attempted copulation. By contrast, in all pairs of qtc¹ males observed, one or both males exhibited wing vibration. In half of the pairs observed, at least one of the qtc¹ males licked the other male's genitalia, and one qtc¹ male attempted to copulate with the other male in the observation chamber. Some aspects of qtc¹ males' responses to each other were normal. When groups of qtc¹ males were observed in a Petri dish, the males did not exhibit the chaining behavior shown by certain other mutants. Moreover, like CS males, all of the qtc¹ males that were observed in chambers or in Petri dishes ran away from the males that courted them. Thus, qtc¹ males' responses to male courtship appears normal (Gaines, 2000).

It was possible that pairs of qtc¹ males performed more male-male courtship, in comparison to pairs of CS controls, because the qtc¹ mutation affected males' propensity to court other males. Alternatively, the qtc¹ mutation could have affected males' ability to elicit courtship from other males. To distinguish between these two possibilities, a series of behavioral experiments were performed in which one qtc¹ male was paired with one CS male and each male's courtship index was determined individually. In addition, which of the two males initiated courtship first was noted. The qtc¹ males perform high levels of male-male courtship in response to the CS males, while the CS males perform little or no courtship in response to the qtc¹ males. Moreover, in all nine of the pairs in which both males performed at least some courtship (in one pair the CS male did not perform any courtship), the qtc¹ male initiated courtship first. The simplest interpretation of these results is that the qtc¹ mutation affects mature males' performance of male-male courtship but has no discernible effect on their ability to elicit male courtship. This conclusion is supported by analysis of cuticular hydrocarbons extracted from qtc¹ and CS males. This analysis revealed no differences in levels of 7-tricosene and 7-pentacosene, which are abundant hydrocarbons on the cuticles of mature males that have courtship-inhibiting and courtship-stimulating activity in bioassays, respectively. Nor do qtc¹ males synthesize detectable quantities of 7,11-heptacosadiene, a female-specific courtship-stimulating pheromone (Gaines, 2000).

A second phenotype was observed when qtc¹ males were paired with CS virgin females: qtc¹ males initiate courtship of females more quickly than do control CS males. To determine whether this phenotype is due to the P-element insertion, males of line 6276 were tested with CS females. 6276 males have significantly longer courtship latencies than qtc¹ males. Males homozygous for the imprecise excision (qtc^Ex1) initiate courtship more rapidly than those homozygous for precise excisions (qtc^r1 and qtc^r2). Accordingly, it is concluded that the rapid initiation of courtship in response to females most likely arises from the P-element insertion in the qtc¹ stock. Because the foregoing analysis indicates that the same mutation is responsible for the elevated levels of male-male courtship displayed by qtc¹ males, the gene that is mutated was designated as quick-to-court (qtc), based on the finding that mutant males are quick to initiate courtship of both virgin females and mature males (Gaines, 2000).

The qtc phenotype is highly specific. No morphological, sensory, motor, or general behavioral defects have been detected. Many aspects of sexual behavior are normal in qtc mutants. Mature qtc¹ males perform the full repertoire of courtship behaviors, show both normal levels of courtship and normal copulation latencies toward virgin CS females. qtc¹ females are normal in all courtship behaviors examined. qtc¹ thus defines two classes of sexual behaviors: those that are affected by qtc¹ and those that are not. These results suggest that some sexual behaviors are controlled by a genetic pathway that depends on qtc, and other behaviors are controlled by one or more distinct pathways (Gaines, 2000).

The male-male and male-female courtship phenotypes of qtc are related, in the sense that both may be considered as an increase in sexual activity. One interpretation of these phenotypes is that qtc males show an increased tendency to court: when paired with a female, they begin courting quickly; when paired with a male, they court despite the presence of inhibitory cues associated with the male partner. If this interpretation is correct, qtc may define a novel molecular component required for the control of male sexual drive. Since males expressing qtc mutations show elevated levels of courtship compared to controls, it is tempting to speculate that the qtc mutations affect neural circuitry that, in normal males, inhibits courtship (Gaines, 2000).

While several Drosophila genes have been associated with high levels of male-male courtship, qtc is unique among them in that its wild-type function appears to be required for maintaining low levels of male-male sexual behavior, but not for the ability to execute the various courtship behaviors. The qtc phenotype shows a high degree of specificity in this sense. When fruitless (fru) males are placed in pairs, they show elevated levels of courtship behavior; when placed in groups, they form courtship chains, in which individual males court the fly ahead and are courted by the fly behind. fru encodes a zinc finger transcription factor that defines a branch of the sex determination hierarchy. qtc is different from fru in that qtc males carry out the full repertoire of courtship behaviors and are fertile; males expressing most fru alleles generate an abnormal courtship song, do not bend their abdomen into a copulating position, and are sterile. For most fru alleles, males show courtship toward females as well as males, but their courtship latency has not yet been measured. Males mutant for the recently described dissatisfaction (dsf) mutation court and attempt copulation with both males and females, but are slow to copulate, at least in part because of abnormal abdominal curling. dsf is different from qtc and fru in that dsf affects female, as well as male, courtship. dsf females resist copulation and fail to lay eggs. The abnormalities in female egg-laying and male abdominal curling are correlated with defects in motor neuron innervation of abdominal muscles. Flies in which the white (w) gene is ectopically expressed have also been shown to exhibit high levels of male-male courtship. When pairs of male flies that carry a mini-w gene under the control of the hsp70 heat-shock promoter are heat-shocked, they vigorously court each other; groups of heat-shocked males form courtship chains, lariats, and rings. The w gene encodes a polypeptide that is believed to play a role in transmembrane transport. Male-male courtship has not been observed in deletion or other loss-of-function mutants of w, and it is difficult to draw conclusions about the normal role of the w⁺ gene from these ectopic expression studies. Elevated levels of male-male courtship have also been observed in males exhibiting ectopic expression of the feminizing gene transformer in portions of the antennal lobes or mushroom bodies, structures implicated in olfactory processing (Gaines, 2000 and references therein).

The qtc gene encodes a predicted protein with coiled-coil domains, and the expression pattern includes the olfactory organs, the CNS, and the male reproductive tract. Many proteins with such coiled-coil domains form dimers as a result of hydrophobic interactions between two alpha-helices that wrap around each other. Some coiled-coil proteins, such as myosin, contain many heptad repeats; others contain few. Among the latter are several proteins containing leucine zippers, such as the yeast protein GCN4, or zinc fingers, such as the mouse protein Rpt-1. Some of these proteins contain DNA binding domains and function as transcription factors (Gaines, 2000 and references therein).

The qtc expression pattern is much broader than that of lacZ in the qtc¹ enhancer trap line. qtc¹ was identified in a screen for lines showing lacZ expression in the olfactory organs of adults and larvae, but little expression elsewhere. The qtc¹ enhancer trap line shows sexually dimorphic lacZ staining in a subregion of the third antennal segment and very limited staining in the CNS. The qtc transcript is distributed extensively in the antenna and the CNS and in some tissues outside the nervous system, e.g., in the male reproductive tract. Moreover, qtc expression in the antenna shows no evidence of sexual dimorphism in quantitative RNase protection experiments. There is ample precedent for P lacZ insertions whose expression patterns vary from those of the genes in which they are inserted. It is unlikely that the lacZ pattern of the qtc enhancer trap line reflects precisely the expression of a gene other than qtc. The P lacZ insertion is located within a small intron of qtc, an intron whose sequence does not indicate the presence of any internal genes. There is a gene that lies ~260 bp distal to the 3' end of qtc, but it encodes a predicted ribosomal protein and is ubiquitously expressed. No genes lying upstream from the 5' end of qtc have been identified, but the 5' end of qtc lies far (>18 kb) from the 5' end of the lacZ reporter gene. One possible interpretation of the discrepancy between the qtc and lacZ patterns is that qtc expression is controlled by multiple regulatory elements, one of which lies adjacent to the lacZ insertion in the third intron and drives sexually dimorphic expression in a group of antennal cells, conceivably in cells that receive pheromonal input (Gaines, 2000).

In summary, qtc encodes a novel protein that is required for the normal regulation of male sexual behavior. The molecular genetic underpinnings of sexual drive are poorly understood, and the identification of new components affecting this process provides new avenues for investigation. Further genetic analysis of qtc, including mosaic analysis or analysis of GAL4-driven qtc misexpression, may determine whether specific parts of the nervous system or reproductive tract require qtc function for normal sexual behavior. Such analysis may also determine whether the male-male and male-female courtship abnormalities have distinct anatomical foci. Finally, further molecular and genetic analysis of qtc may be useful in determining its molecular function and in identifying the pathway through which it exerts its role in the regulation of sexual behavior (Gaines, 2000).

DEVELOPMENTAL BIOLOGY

The qtc enhancer trap line was originally identified by virtue of its reporter gene expression in the olfactory system, with limited expression also detected in the brain. To determine whether the qtc gene is expressed in the olfactory system and brain, Northern blots and in situ hybridizations to RNA were performed in tissue sections (Gaines, 2000).

A 2.4-kb transcript in heads and antennae is observed to hybridize to a qtc probe. The transcript is present in both males and females, in comparable amounts. Quantitative RNase protection experiments reveal no differences between male and female antennae in qtc transcript levels. In another experiment, the 2.4-kb transcript was also observed in bodies from which heads had been removed and in embryos, although the intensity of hybridization was less than that to head RNA. Following long exposure, an additional band of 4.4 kb can be observed in the head RNA track (Gaines, 2000).

The qtc mutations were found to affect the 2.4-kb transcript. Specifically, the P-element insertion in qtc¹, which introduces ~14 kb of DNA into the third intron of the qtc gene, greatly reduces the intensity of labeling and affects the band pattern; the same alterations are observed for the parental insertion line qtc¹ b pr. The qtc^Ex1 deletion mutation, generated by imprecise excision of the P-element insertion, also reduces the intensity of labeling and alters the band pattern. The changes in band pattern presumably reflect changes in the length of the transcription unit, the deletion or addition of splice junctions, and stability of the mRNAs: rapid degradation of other disrupted transcripts by mRNA surveillance pathways has been demonstrated. In contrast, wild-type levels of the adjacent L37a gene expression are observed in both qtc¹ insertion lines and in the deletion mutant line qtc^Ex1 (Gaines, 2000).

Localization of the qtc transcript was confirmed and extended by in situ hybridization to RNA in tissue sections. A qtc antisense probe hybridizes to sections of the antenna and the maxillary palp, another olfactory organ of the fly head. Within the antenna, expression is seen in both the second and third segments and is concentrated near the cuticle, where the cell bodies of sensory neurons are located. Expression in the maxillary palp also appears near the cuticle. These hybridization results show that the gene is expressed in two olfactory organs: the third antennal segment and the maxillary palp. However, they also show that expression is not limited to olfactory cells, since the second antennal segment contains no olfactory sensilla (Gaines, 2000).

Abundant expression is also seen in the CNS within the head, consistent with the strong hybridization to head RNA in Northern blots. There are high levels of expression in the visual system: expression is seen in the retina and the optic lobe. Uniform expression is also seen in the cortex of the brain, where the cell bodies of brain neuropil are located (Gaines, 2000).

The probe also hybridizes to sections from the thorax and abdomens of males and females. In both sexes, expression is seen in the ventral ganglion within the thorax. In males, expression is seen near the tip of the abdomen, closely associated with the ejaculatory bulb and testis. In females, expression is also observed in the distal end of the abdomen, likely associated with the reproductive system (Gaines, 2000).

EFFECTS OF MUTATION

Several aspects of the sexual behavior of qtc males and females are normal, in comparison to CS males and females. Mature qtc¹ males perform normal levels of courtship toward CS virgin females during the observation period: the CI of qtc¹ males paired with CS females was 75 ± 3, compared with 78 ± 3 for CS males paired with CS females. Moreover, mutant males performed 'advanced' courtship behaviors toward CS females: qtc¹ males showed wing vibration in all of 20 cases, and in most cases they showed licking of female genitalia and curling of the abdomen to attempt copulation. Moreover, qtc¹ and CS males that began to mate with females during the 10-min observation period had copulation latencies that were not significantly different. qtc¹ females elicit as much courtship from CS males as do CS females. Immature qtc¹ males elicite the same level of courtship from CS males as do immature CS males. qtc¹ females are as likely to mate with CS males (65%) during the observation period as are CS females (67%), and the mutant females' copulation latencies are normal. During courtship tests of qtc¹ males with CS females, qtc¹ males are observed to perform the full repertoire of wild-type courtship behaviors (Gaines, 2000).

In addition, sensory function of qtc¹ appears normal in limited testing of visual and olfactory physiology by electroretinogram and electroantennogram recordings and in tests of both adult and larval olfactory behavior. Detailed examination of qtc mutants did not reveal any general behavioral defects in such activities as walking or grooming (Gaines, 2000).

Genes involved in sex pheromone discrimination in Drosophila melanogaster and their background-dependent effect

Mate choice is based on the comparison of the sensory quality of potential mating partners, and sex pheromones play an important role in this process. In Drosophila melanogaster, contact pheromones differ between male and female in their content and in their effects on male courtship, both inhibitory and stimulatory. To investigate the genetic basis of sex pheromone discrimination, males showing either a higher or lower ability to discriminate sex pheromones over were experimentally selected 20 generations. This experimental selection was carried out in parallel on two different genetic backgrounds: wild-type and desat1 mutant, in which parental males showed high and low sex pheromone discrimination ability respectively. Male perception of male and female pheromones was separately affected during the process of selection. A comparison of transcriptomic activity between high and low discrimination lines revealed genes not only that varied according to the starting genetic background, but varied reciprocally. Mutants in two of these genes, Shaker and quick-to-court, were capable of producing similar effects on discrimination on their own, in some instances mimicking the selected lines, in others not. This suggests that discrimination of sex pheromones depends on genes whose activity is sensitive to genetic context and provides a rare, genetically defined example of the phenomenon known as "allele flips," in which interactions have reciprocal effects on different genetic backgrounds (Houot, 2012).

EVOLUTIONARY HOMOLOGS

Sex specific molecular responses of quick-to-court protein in Indian malarial vector Anopheles culicifacies: conflict of mating versus blood feeding behaviour

Understanding the molecular basis of mosquito behavioural complexity plays a central role in designing novel molecular tools to fight against their vector-borne diseases. Although the olfactory system plays an important role in guiding and managing many behavioural responses including feeding and mating, but the sex-specific regulation of olfactory responses remain poorly investigated. From an ongoing transcriptomic data annotation of olfactory tissue of blood fed adult female An. culicifacies mosquitoes, this study has identified a 383 bp long unique transcript encoding a Drosophila homolog of the quick-to-court protein. Previously this was shown to regulate courtship behaviour in adult male Drosophila. A comprehensive in silico analysis of the quick-to-court (qtc) gene of An. culicifacies (Ac-qtc) predicts a 1536 bp single copy gene encoding 511 amino acid protein, having a high degree of conservation with other insect homologs. The age-dependent increased expression of putative Ac-qtc correlated with the maturation of the olfactory system, necessary to meet the sex-specific conflicting demand of mating (mate finding) versus host-seeking behavioural responses. Sixteen to eighteen hours of starvation did not alter Ac-qtc expression in both sexes, however, blood feeding significantly modulated its response in the adult female mosquitoes, confirming that it may not be involved in sugar feeding associated behavioural regulation. Finally, a dual behavioural and molecular assay indicated that natural dysregulation of Ac-qtc in the late evening might promote the mating events for successful insemination. It is hypothesized that Ac-qtc may play a unique role to regulate the sex-specific conflicting demand of mosquito courtship behaviour versus blood feeding behaviour in the adult female mosquitoes. Further elucidation of this molecular mechanism may provide further information to evaluate Ac-qtc as a key molecular target for mosquito-borne disease management (Das De, 2017).

REFERENCES

Search PubMed for articles about Drosophila quick-to-court

Das De, T., Sharma, P., Rawal, C., Kumari, S., Tavetiya, S., Yadav, J., Hasija, Y. and Dixit, R. (2017). Sex specific molecular responses of quick-to-court protein in Indian malarial vector Anopheles culicifacies: conflict of mating versus blood feeding behaviour. Heliyon 3(7): e00361. PubMed ID: 28765838

Gaines, P., Tompkins, L., Woodard, C. T. and Carlson, J. R. (2000). quick-to-court, a Drosophila mutant with elevated levels of sexual behavior, is defective in a predicted coiled-coil protein. Genetics 154(4): 1627-1637. 10747058

Houot, B., Fraichard, S., Greenspan, R. J. and Ferveur, J. F. (2012). Genes involved in sex pheromone discrimination in Drosophila melanogaster and their background-dependent effect. PLoS One 7(1): e30799. PubMed ID: 22292044

date revised: 10 August 2018

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