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• W2138814742 abstract "All animals exhibit innate behaviors that are specified during their development. Drosophila melanogaster males (but not females) perform an elaborate and innate courtship ritual directed toward females (but not males). Male courtship requires products of the fruitless (fru) gene, which is spliced differently in males and females. We have generated alleles of fru that are constitutively spliced in either the male or the female mode. We show that male splicing is essential for male courtship behavior and sexual orientation. More importantly, male splicing is also sufficient to generate male behavior in otherwise normal females. These females direct their courtship toward other females (or males engineered to produce female pheromones). The splicing of a single neuronal gene thus specifies essentially all aspects of a complex innate behavior. All animals exhibit innate behaviors that are specified during their development. Drosophila melanogaster males (but not females) perform an elaborate and innate courtship ritual directed toward females (but not males). Male courtship requires products of the fruitless (fru) gene, which is spliced differently in males and females. We have generated alleles of fru that are constitutively spliced in either the male or the female mode. We show that male splicing is essential for male courtship behavior and sexual orientation. More importantly, male splicing is also sufficient to generate male behavior in otherwise normal females. These females direct their courtship toward other females (or males engineered to produce female pheromones). The splicing of a single neuronal gene thus specifies essentially all aspects of a complex innate behavior. Animals are born not only with their characteristic body plan and morphology, but also a set of innate behaviors, or instincts, that are manifested as stereotyped responses to environmental stimuli (Tinbergen, 1951Tinbergen N. The Study of Instinct. Clarendon Press, Oxford1951Google Scholar). Enormous progress has been made over the past several decades in elucidating the developmental processes that direct the formation of the body plan and its parts. In contrast, our understanding of how innate behaviors are specified is still rudimentary at best. It is not even clear whether the general principles gleaned from the study of morphological development also apply to the development of behavior. For example, body parts are often specified by “switch” or “selector” genes, the action of which is both necessary and sufficient to trigger the development of a complete anatomical structure (Garcia-Bellido, 1975Garcia-Bellido A. Genetic control of wing disc development in Drosophila.Ciba Found. Symp. 1975; 0: 161-182PubMed Google Scholar). Might instincts be specified in a similar way? Are there behavioral switch genes that create the potential for a complex innate behavior (Baker et al., 2001Baker B.S. Taylor B.J. Hall J.C. Are complex behaviors specified by dedicated regulatory genes? Reasoning from Drosophila.Cell. 2001; 105: 13-24Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar)? Or, at the other extreme, do instincts emerge diffusely from the combined actions of the vast number of genes that contribute to nervous system development and function, so that no single gene can be said to specify any particular behavior (Greenspan, 1995Greenspan R.J. Understanding the genetic construction of behavior.Sci. Am. 1995; 272: 72-78Crossref PubMed Scopus (39) Google Scholar)? If behavioral switch genes exist, then one place in which they are likely to be found is in the specification of sexual behaviors (Baker et al., 2001Baker B.S. Taylor B.J. Hall J.C. Are complex behaviors specified by dedicated regulatory genes? Reasoning from Drosophila.Cell. 2001; 105: 13-24Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Males and females generally have dramatically distinct and innate sexual behaviors. These behaviors are essential for their reproductive success, and so strong selective pressure is likely to have favored the evolution of genes that “hardwire” them into the brain. The initial steps of sexual differentiation have been well characterized for several model organisms, and genetic perturbations in these sex-determination hierarchies can alter all aspects of the sexual phenotype—innate behaviors as well as gross anatomy. Several genes near the top of these sex-determination hierarchies thus qualify as developmental switch genes, but they cannot be considered specifically as behavioral switch genes. A switch gene for a sexual behavior should act to specify either male or female behavior, irrespective of the overall sexual phenotype of the animal. A candidate for such a gene is the fruitless (fru) gene of Drosophila, which is intimately linked to male sexual orientation and behavior (Baker et al., 2001Baker B.S. Taylor B.J. Hall J.C. Are complex behaviors specified by dedicated regulatory genes? Reasoning from Drosophila.Cell. 2001; 105: 13-24Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Male courtship in Drosophila is an elaborate ritual that involves multiple sensory inputs and complex motor outputs (Hall, 1994Hall J.C. The mating of a fly.Science. 1994; 264: 1702-1714Crossref PubMed Scopus (508) Google Scholar; see Movie S1 in the Supplemental Data available with this article online). It is largely a fixed-action pattern, in which the male orients toward and follows the female, taps her with his forelegs, sings a species-specific courtship song by extending and vibrating one wing, licks her genitalia, and finally curls his abdomen for copulation. If the female is sufficiently aroused and has not recently mated, she accepts his advances by slowing down and opening her vaginal plates to allow copulation. An obvious but nonetheless remarkable aspect of this behavior is that mature males court only females, never other males, whereas females do not court at all. Certain loss-of-function alleles of the fru gene disrupt both male courtship behavior and sexual orientation: performance of the courtship ritual is below par, and it is directed indiscriminately at either sex (Anand et al., 2001Anand A. Villella A. Ryner L.C. Carlo T. Goodwin S.F. Song H.J. Gailey D.A. Morales A. Hall J.C. Baker B.S. Taylor B.J. Molecular genetic dissection of the sex-specific and vital functions of the Drosophila melanogaster sex determination gene fruitless.Genetics. 2001; 158: 1569-1595PubMed Google Scholar, Ito et al., 1996Ito H. Fujitani K. Usui K. Shimizu-Nishikawa K. Tanaka S. Yamamoto D. Sexual orientation in Drosophila is altered by the satori mutation in the sex-determination gene fruitless that encodes a zinc finger protein with a BTB domain.Proc. Natl. Acad. Sci. USA. 1996; 93: 9687-9692Crossref PubMed Scopus (225) Google Scholar, Lee et al., 2001Lee G. Villella A. Taylor B.J. Hall J.C. New reproductive anomalies in fruitless-mutant Drosophila males: extreme lengthening of mating durations and infertility correlated with defective serotonergic innervation of reproductive organs.J. Neurobiol. 2001; 47: 121-149Crossref PubMed Scopus (72) Google Scholar, Ryner et al., 1996Ryner L.C. Goodwin S.F. Castrillon D.H. Anand A. Villella A. Baker B.S. Hall J.C. Taylor B.J. Wasserman S.A. Control of male sexual behavior and sexual orientation in Drosophila by the fruitless gene.Cell. 1996; 87: 1079-1089Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, Villella et al., 1997Villella A. Gailey D.A. Berwald B. Ohshima S. Barnes P.T. Hall J.C. Extended reproductive roles of the fruitless gene in Drosophila melanogaster revealed by behavioral analysis of new fru mutants.Genetics. 1997; 147: 1107-1130PubMed Google Scholar). Strong fru alleles completely block courtship behavior, but weaker fru alleles variously disrupt individual steps, with each step affected in some allelic combination (Anand et al., 2001Anand A. Villella A. Ryner L.C. Carlo T. Goodwin S.F. Song H.J. Gailey D.A. Morales A. Hall J.C. Baker B.S. Taylor B.J. Molecular genetic dissection of the sex-specific and vital functions of the Drosophila melanogaster sex determination gene fruitless.Genetics. 2001; 158: 1569-1595PubMed Google Scholar, Lee et al., 2001Lee G. Villella A. Taylor B.J. Hall J.C. New reproductive anomalies in fruitless-mutant Drosophila males: extreme lengthening of mating durations and infertility correlated with defective serotonergic innervation of reproductive organs.J. Neurobiol. 2001; 47: 121-149Crossref PubMed Scopus (72) Google Scholar). This suggests that fru is required for every step of the courtship ritual, not just for a single critical step. For all of these fru alleles, female morphology and behavior appear normal. Of the many genes known to be involved in male courtship behavior (Billeter et al., 2002Billeter J.C. Goodwin S.F. O'Dell K.M. Genes mediating sex-specific behaviors in Drosophila.Adv. Genet. 2002; 47: 87-116Crossref PubMed Google Scholar), fru is unique in that it is sex-specifically spliced (Ito et al., 1996Ito H. Fujitani K. Usui K. Shimizu-Nishikawa K. Tanaka S. Yamamoto D. Sexual orientation in Drosophila is altered by the satori mutation in the sex-determination gene fruitless that encodes a zinc finger protein with a BTB domain.Proc. Natl. Acad. Sci. USA. 1996; 93: 9687-9692Crossref PubMed Scopus (225) Google Scholar, Ryner et al., 1996Ryner L.C. Goodwin S.F. Castrillon D.H. Anand A. Villella A. Baker B.S. Hall J.C. Taylor B.J. Wasserman S.A. Control of male sexual behavior and sexual orientation in Drosophila by the fruitless gene.Cell. 1996; 87: 1079-1089Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). Alternative splicing at both the 5′ and 3′ ends of the fru locus generates a complex set of transcripts, all of which encode BTB domain-containing zinc finger proteins. Most of these transcripts are not sex specific, but those initiated from the most distal (P1) promoter are spliced differently in males and females. The alleles of fru that affect male courtship are all associated with chromosomal insertions, deletions, or rearrangements that specifically disrupt these sex-specific P1 transcripts (Anand et al., 2001Anand A. Villella A. Ryner L.C. Carlo T. Goodwin S.F. Song H.J. Gailey D.A. Morales A. Hall J.C. Baker B.S. Taylor B.J. Molecular genetic dissection of the sex-specific and vital functions of the Drosophila melanogaster sex determination gene fruitless.Genetics. 2001; 158: 1569-1595PubMed Google Scholar, Goodwin et al., 2000Goodwin S.F. Taylor B.J. Villella A. Foss M. Ryner L.C. Baker B.S. Hall J.C. Aberrant splicing and altered spatial expression patterns in fruitless mutants of Drosophila melanogaster.Genetics. 2000; 154: 725-745PubMed Google Scholar). This has led to the hypothesis that the male-specific splicing of the fru P1 transcripts specifies male courtship behavior and sexual orientation (Baker et al., 2001Baker B.S. Taylor B.J. Hall J.C. Are complex behaviors specified by dedicated regulatory genes? Reasoning from Drosophila.Cell. 2001; 105: 13-24Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). This is the hypothesis we test here. We used gene targeting by homologous recombination to generate alleles of fru that are constitutively spliced in either the male or female mode. Forcing female splicing in the male results in a loss of male courtship behavior and orientation, confirming that male-specific splicing of fru is indeed essential for male behavior. More dramatically, females in which fru is spliced in the male mode behave as if they were males: they court other females. Thus, male-specific splicing of fru is both necessary and sufficient to specify male courtship behavior and sexual orientation. A complex innate behavior is thus specified by the action of a single gene, demonstrating that behavioral switch genes do indeed exist and identifying fru as one such gene. The fru locus spans approximately 130 kb, and includes at least four promoters (P1–P4; Figure 1A ; Ito et al., 1996Ito H. Fujitani K. Usui K. Shimizu-Nishikawa K. Tanaka S. Yamamoto D. Sexual orientation in Drosophila is altered by the satori mutation in the sex-determination gene fruitless that encodes a zinc finger protein with a BTB domain.Proc. Natl. Acad. Sci. USA. 1996; 93: 9687-9692Crossref PubMed Scopus (225) Google Scholar, Ryner et al., 1996Ryner L.C. Goodwin S.F. Castrillon D.H. Anand A. Villella A. Baker B.S. Hall J.C. Taylor B.J. Wasserman S.A. Control of male sexual behavior and sexual orientation in Drosophila by the fruitless gene.Cell. 1996; 87: 1079-1089Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). Transcripts from the P2–P4 promoters are not sex-specifically spliced and encode a set of common Fru isoforms that have essential functions in the development of both sexes (Figure 1B; Anand et al., 2001Anand A. Villella A. Ryner L.C. Carlo T. Goodwin S.F. Song H.J. Gailey D.A. Morales A. Hall J.C. Baker B.S. Taylor B.J. Molecular genetic dissection of the sex-specific and vital functions of the Drosophila melanogaster sex determination gene fruitless.Genetics. 2001; 158: 1569-1595PubMed Google Scholar, Ryner et al., 1996Ryner L.C. Goodwin S.F. Castrillon D.H. Anand A. Villella A. Baker B.S. Hall J.C. Taylor B.J. Wasserman S.A. Control of male sexual behavior and sexual orientation in Drosophila by the fruitless gene.Cell. 1996; 87: 1079-1089Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). Transcripts initiated from the distal P1 promoter include the S exon, which is sex-specifically spliced under the control of the sex-determination factors Tra and Tra-2 (Heinrichs et al., 1998Heinrichs V. Ryner L.C. Baker B.S. Regulation of sex-specific selection of fruitless 5′ splice sites by transformer and transformer-2.Mol. Cell. Biol. 1998; 18: 450-458Crossref PubMed Google Scholar, Ito et al., 1996Ito H. Fujitani K. Usui K. Shimizu-Nishikawa K. Tanaka S. Yamamoto D. Sexual orientation in Drosophila is altered by the satori mutation in the sex-determination gene fruitless that encodes a zinc finger protein with a BTB domain.Proc. Natl. Acad. Sci. USA. 1996; 93: 9687-9692Crossref PubMed Scopus (225) Google Scholar, Ryner et al., 1996Ryner L.C. Goodwin S.F. Castrillon D.H. Anand A. Villella A. Baker B.S. Hall J.C. Taylor B.J. Wasserman S.A. Control of male sexual behavior and sexual orientation in Drosophila by the fruitless gene.Cell. 1996; 87: 1079-1089Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). In males, Tra is absent and the S exon is spliced at its default male-specific donor site. This results in an in-frame fusion to the exons common to all fru transcripts, adding a 101 amino acid N-terminal extension that is unique to these male-specific FruM isoforms. In females, Tra binds to fru P1 pre-mRNAs to promote splicing at a more 3′ donor site (Heinrichs et al., 1998Heinrichs V. Ryner L.C. Baker B.S. Regulation of sex-specific selection of fruitless 5′ splice sites by transformer and transformer-2.Mol. Cell. Biol. 1998; 18: 450-458Crossref PubMed Google Scholar) and to block translation of these transcripts (Usui-Aoki et al., 2000Usui-Aoki K. Ito H. Ui-Tei K. Takahashi K. Lukacsovich T. Awano W. Nakata H. Piao Z.F. Nilsson E.E. Tomida J. Yamamoto D. Formation of the male-specific muscle in female Drosophila by ectopic fruitless expression.Nat. Cell Biol. 2000; 2: 500-506Crossref PubMed Scopus (114) Google Scholar). Both mechanisms ensure that no full-length FruM proteins are produced in females. We generated four alleles of fru by gene targeting: fruF, an allele that should prevent male-specific splicing; fruM and fruΔtra, both of which should force male splicing; and fruC, a control allele in which splicing should be unchanged (Figures 1C and S1). In fruF, point mutations introduced at the male splice donor site of the S exon should abolish splicing at this site but not alter the coding potential of the unspliced transcripts. In fruM, the entire 1601 bp female-specific part of the S exon is deleted, while fruΔtra contains a 261 bp deletion that just eliminates the Tra binding sites. In fruC, the only sequence modification is the insertion of an FRT site in the intron following the S exon. This FRT insertion is a footprint of the targeting procedure and is also present in the fruF, fruM, and fruΔtra alleles. We established several independent lines for each of these four alleles and verified them by molecular, histological, and behavioral analyses. Independent derivatives of the same allele were indistinguishable in all of these assays. A single line for each allele was then backcrossed to the wild-type Canton S strain for four generations prior to more extensive behavioral tests. For all molecular, histological, and behavioral data presented here, unless otherwise stated, the fruC, fruF, fruM, and fruΔtra alleles were examined in trans to fru4-40. The fru4-40 allele results from a deletion of at least 70 kb that removes all P1 and P2 transcripts and is genetically null for the fru behavioral phenotypes (Anand et al., 2001Anand A. Villella A. Ryner L.C. Carlo T. Goodwin S.F. Song H.J. Gailey D.A. Morales A. Hall J.C. Baker B.S. Taylor B.J. Molecular genetic dissection of the sex-specific and vital functions of the Drosophila melanogaster sex determination gene fruitless.Genetics. 2001; 158: 1569-1595PubMed Google Scholar). Thus, any fru P1 product or activity detected in these assays can be assigned to the engineered fruC, fruF, fruM, or fruΔtra allele. PCR amplification and DNA sequencing of the entire 18 kb targeted region confirmed the predicted structure of the fru locus in each of the four alleles, and RT-PCR experiments confirmed that the predicted transcripts are indeed generated (Figure 1C; splicing at the female donor site is variable in fruF males, presumably because no Tra is present to promote use of the normal female splice site.) FruM proteins could also be detected in the brains of fruC males, and fruM and fruΔtra flies of either sex, both in adults (Figure 1D) and 48 hr pupae. The distribution of FruM appears identical in each case and also matches the reported expression of FruM in wild-type males (Lee et al., 2000Lee G. Foss M. Goodwin S.F. Carlo T. Taylor B.J. Hall J.C. Spatial, temporal, and sexually dimorphic expression patterns of the fruitless gene in the Drosophila central nervous system.J. Neurobiol. 2000; 43: 404-426Crossref PubMed Scopus (164) Google Scholar). We conclude that the modifications we have introduced into the fru locus eliminate the sex differences in FruM expression but do not alter its distribution. The sex determination hierarchy in Drosophila bifurcates downstream of Tra. Like fru, the doublesex (dsx) gene is also differentially spliced under the control of Tra and produces either male (DsxM) or female (DsxF) isoforms of a DM-domain transcription factor (Burtis and Baker, 1989Burtis K.C. Baker B.S. Drosophila doublesex gene controls somatic sexual differentiation by producing alternatively spliced mRNAs encoding related sex-specific polypeptides.Cell. 1989; 56: 997-1010Abstract Full Text PDF PubMed Scopus (472) Google Scholar, Erdman and Burtis, 1993Erdman S.E. Burtis K.C. The Drosophila doublesex proteins share a novel zinc finger related DNA binding domain.EMBO J. 1993; 12: 527-535Crossref PubMed Scopus (191) Google Scholar, Hoshijima et al., 1991Hoshijima K. Inoue K. Higuchi I. Sakamoto H. Shimura Y. Control of doublesex alternative splicing by transformer and transformer-2 in Drosophila.Science. 1991; 252: 833-836Crossref PubMed Scopus (215) Google Scholar). These Dsx proteins direct male or female morphological development, respectively, but have little influence on sexual behavior: males that lack DsxM still court, albeit at reduced levels (Villella and Hall, 1996Villella A. Hall J.C. Courtship anomalies caused by doublesex mutations in Drosophila melanogaster.Genetics. 1996; 143: 331-344Crossref PubMed Google Scholar), whereas females that produce DsxM resemble normal males but do not court (Taylor et al., 1994Taylor B.J. Villella A. Ryner L.C. Baker B.S. Hall J.C. Behavioral and neurobiological implications of sex-determining factors in Drosophila.Dev. Genet. 1994; 15: 275-296Crossref PubMed Scopus (95) Google Scholar). This has led to the notion that dsx regulates gross sexual anatomy, while fru regulates sexual behavior (Taylor et al., 1994Taylor B.J. Villella A. Ryner L.C. Baker B.S. Hall J.C. Behavioral and neurobiological implications of sex-determining factors in Drosophila.Dev. Genet. 1994; 15: 275-296Crossref PubMed Scopus (95) Google Scholar). Consistent with this view, our fru P1 splice mutations do not generally alter external or internal sexual anatomy: fruF males have the normal male anatomy, whereas fruM and fruΔtra females are anatomically normal females. The one exception is that fruM and fruΔtra females have a male-specific muscle of Lawrence (MoL), and fruF males lack this muscle (Figure S2). The MoL is a dorsal abdominal muscle that uniquely depends on fru rather than dsx for its sexual differentiation (Gailey et al., 1991Gailey D.A. Taylor B.J. Hall J.C. Elements of the fruitless locus regulate development of the muscle of Lawrence, a male-specific structure in the abdomen of Drosophila melanogaster adults.Development. 1991; 113: 879-890PubMed Google Scholar, Lawrence and Johnston, 1984Lawrence P.A. Johnston P. The genetic specification of pattern in a Drosophila muscle.Cell. 1984; 36: 775-782Abstract Full Text PDF PubMed Scopus (46) Google Scholar, Taylor, 1992Taylor B.J. Differentiation of a male-specific muscle in Drosophila melanogaster does not require the sex-determining genes doublesex or intersex.Genetics. 1992; 132: 179-191PubMed Google Scholar), evidently requiring fru in the motor neurons that innervate it rather than the muscle itself (Currie and Bate, 1995Currie D.A. Bate M. Innervation is essential for the development and differentiation of a sex-specific adult muscle in Drosophila melanogaster.Development. 1995; 121: 2549-2557PubMed Google Scholar). It is not involved in courtship behavior, but may facilitate the termination of copulation (Lee et al., 2001Lee G. Villella A. Taylor B.J. Hall J.C. New reproductive anomalies in fruitless-mutant Drosophila males: extreme lengthening of mating durations and infertility correlated with defective serotonergic innervation of reproductive organs.J. Neurobiol. 2001; 47: 121-149Crossref PubMed Scopus (72) Google Scholar). We also verified that correct splicing of fru, rather than dsx, is essential in the male nervous system for male courtship behavior. Ectopic expression of tra in all postmitotic neurons in males normally leads to a dramatic reduction in courtship vigor (elav-GAL4/UAS-tra; Kido and Ito, 2002Kido A. Ito K. Mushroom bodies are not required for courtship behavior by normal and sexually mosaic Drosophila.J. Neurobiol. 2002; 52: 302-311Crossref PubMed Scopus (39) Google Scholar). In these males, fru, dsx, and any other tra targets are presumably spliced in the female mode within the nervous system. However, restoring male splicing of fru alone, by introducing either a fruM or fruΔtra allele, is sufficient to restore normal courtship levels (Figure S3). Having generated alleles of fru that force either male or female splicing and confirmed that they do not affect general sexual anatomy, we were now in a position to test the hypothesis that fru splicing specifies sexual behavior (Ito et al., 1996Ito H. Fujitani K. Usui K. Shimizu-Nishikawa K. Tanaka S. Yamamoto D. Sexual orientation in Drosophila is altered by the satori mutation in the sex-determination gene fruitless that encodes a zinc finger protein with a BTB domain.Proc. Natl. Acad. Sci. USA. 1996; 93: 9687-9692Crossref PubMed Scopus (225) Google Scholar, Ryner et al., 1996Ryner L.C. Goodwin S.F. Castrillon D.H. Anand A. Villella A. Baker B.S. Hall J.C. Taylor B.J. Wasserman S.A. Control of male sexual behavior and sexual orientation in Drosophila by the fruitless gene.Cell. 1996; 87: 1079-1089Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). First, we asked whether male behavior requires male splicing. If so, fruF males should display little or no courtship, and, if they have any residual courtship activity at all, it should be directed at males as well as females. fruM and fruΔtra males should behave normally. We used courtship, fertility, and chaining assays to test these predictions (Figure 2). In male-female courtship assays, a test male is paired with a wild-type virgin female in a 10 mm observation chamber, and the percentage of time the male courts the female during the first 8 min or until copulation, is recorded as his courtship index (CI). In these assays, wild-type, fruC, fruM, and fruΔtra males are all avid courters (CI > 70%; Figure 2A and Movie S1). In contrast, fruF males, like males carrying the classic P-induced fru alleles fru3 and fru4, barely court at all (CI < 5%). We also tested male courtship in competitive mating assays, in which a wild-type virgin female is placed in a chamber with two males—a test male and a wild-type (fru+) competitor. The trio is then observed for up to 1 hr to record which of the two males succeeds in copulating with the female (Figure 2B). In these assays, fruF males always lost out to the fru+ control males, whereas fruC, fruM, and fruΔtra males were all at least as successful as their fru+ competitors. Consistent with the loss of courtship behavior, fruF males are also completely sterile (0% fertility; n = 196), whereas fru+, fruC, fruM, and fruΔtra males are all fully fertile (>99%; n = 230, 140, 110, and 131, respectively). Together, these data establish that male-specific splicing of fru P1 transcripts is indeed essential for male courtship behavior. To test for sexual orientation, we first performed courtship assays in which single fru test males were paired with wild-type males rather than females. Male-male courtship is low for all genotypes. However, fruF males, like fru3 and fru4 males, court other males somewhat more actively than do any of the control males (fru+, fruC, fruM or fruΔtra; Figure 2C). Comparing courtship levels in these single-pair assays is more difficult for male-male assays than for male-female assays, as courtship levels are generally much lower. A more reliable way to test for male-male courtship is to monitor chaining behavior in groups of males. If groups of fru mutant males are left on food plates for several hours or days, they begin to form courtship chains in which each male courts the one ahead of him (Hall, 1978Hall J.C. Courtship among males due to a male-sterile mutation in Drosophila melanogaster.Behav. Genet. 1978; 8: 125-141Crossref PubMed Scopus (97) Google Scholar). It is not clear how this chaining behavior relates to normal courtship, and it probably involves environmental and social stimuli that are absent in the single-pair assays. Nevertheless, it is a robust male-male courtship behavior displayed by classical fru mutants but not wild-type males and can be readily quantified by a chaining index (ChI, the percentage of time three or more males form a chain during a 10 min observation period). Using this assay, we observed dramatically elevated levels of male-male courtship amongst fruF males (ChI = 63%, p < 0.0001; Figure 2D and Movie S2) compared to fru+, fruC, fruM, or fruΔtra males (ChI < 1%). We conclude that male-specific fru splicing not only promotes male-female courtship, it also inhibits male-male courtship. Intriguingly, in the competition assays, fruM and fruΔtra males had a slight but significant edge over their fru+ competitors, winning 71% (n = 41, p = 0.0002) and 61% (n = 62, p = 0.01) of assays, respectively (Figure 2B). This prompted us to compare individual courtship steps performed by fruC, fruM, and fruΔtra males in single-pair assays with wild-type virgin females (Table 1). Qualitatively, courtship performed by fruM and fruΔtra males is indistinguishable from courtship by fruC males. However, fruM and fruΔtra males initiate courtship more rapidly than fruC males (p < 0.01 for both genotypes). Once courtship is initiated, fruM and fruΔtra males spend as much time as fruC males performing each of the other steps (tapping, wing extension, licking, and attempted copulation; p > 0.05 for each step). Although we have not been able to detect any differences by molecular or histological means (Figure 1), it is possible that forced male splicing results in slightly elevated levels of FruM proteins in at least some cells in fruM and fruΔtra males. A tentative inference from this result is therefore that FruM is not only essential for male courtship behavior but may also contribute quantitatively to its initiation.Table 1Comparison of Courtship by fruC Males and fruM and fruΔtra Males and FemalesfruC MalefruM MalefruΔtra MalefruM FemalefruΔtra Femalen1310112115All courtship steps (CI)72.94 ± 3.4189.75 ± 2.8781.86 ± 4.2342.40 ± 3.58**45.76 ± 3.55*Courtship latency (s)50.00 ± 15.236.90 ± 2.64*10.27 ± 3.82*48.86 ± 10.5741.73 ± 7.72Tapping (s)51.23 ± 10.2873.00 ± 8.9556.09 ± 6.7986.67 ± 9.49*147.20 ± 18.02***Wing extension (s)128.77 ± 16.66181.30 ± 19.63153.36 ± 22.08105.57 ± 16.1486.20 ± 12.87Licking3.77 ± 0.765.60 ± 0.629.09 ± 3.030.62 ± 0.22*0.27 ± 0.12*Attempted copulation1.54 ± 0.292.40 ± 0.653.36 ± 1.060 ± 0**0 ± 0**Courtship assays for fruC males and fruM and fruΔtra males and females paired with wild-type virgin females were recorded at higher magnification to monitor individual courtship steps. Values are mean ± SEM. For tapping and wing extension, the total time engaged in these steps was recorded; for licking and attempted copulation, it was the total number of events. *p < 0.01; **p < 0.001; ***p < 0.0001 compared to fruC males (Kruskal-Wallis ANOVA test). p > 0." @default.
• W2138814742 created "2016-06-24" @default.
• W2138814742 creator A5039360877 @default.
• W2138814742 creator A5073254434 @default.
• W2138814742 date "2005-06-01" @default.
• W2138814742 modified "2023-10-10" @default.
• W2138814742 title "fruitless Splicing Specifies Male Courtship Behavior in Drosophila" @default.
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