Proc. Nati. Acad. Sci. USAVol. 84, pp. 8026-8030, November 1987Genetics

Sexual behavior: Its genetic control during development andadulthood in Drosophila melanogaster

(courtship behavior/tra-2 gene/central nervous system plasticity)

JOHN M. BELOTE* AND BRUCE S. BAKERtDepartment of Biology, University of California at San Diego, La Jolla, CA 92093

Communicated by E. B. Lewis, July 27, 1987

ABSTRACT Courtship behavior in Drosophila melanogas-ter males is an innate behavior pattern. Whether or not a fly willdisplay male courtship behavior is governed by the action of aset of regulatory genes that control all aspects of somatic sexualdifferentiation. The wild-type function of one of these regula-tory genes, transformer-2 (tra-2), is necessary for female sexualdifferentiation; in the absence oftra-2+ function XX individualsdifferentiate as males. A temperature-sensitive tra-2 allele hasbeen used to investigate, by means of temperature shifts, whenand how male courtship behavior is specified during develop-ment. The removal of tra-2s function in the adult (by a shift ofthe tra-2a mutant flies to the restrictive temperature) can leadto the appearance of male courtship behavior in flies thatotherwise would not display these behaviors. These experi-ments suggest that the regulatory hierarchy controlling sexualdifferentiation is functioning in the adult central nervoussystem. More importantly, these results suggest that the adultcentral nervous system has some functional plasticity withrespect to the innate behavioral pattern of male courtship andis maintained in a particular state of differentiation by theactive control of gene expression in the adult.

Relatively little is known about the manner in which theneural circuits and controls that underlie particular adultbehavioral patterns are programmed into the central nervoussystem (CNS) during development. Among the more elabo-rate behavioral patterns seen in Drosophila melanogaster isthe sequence of courtship behaviors males carry out thatculminate with copulation (1-3). When stimulated by aprospective mate, males exhibit a complex sequence ofcourtship behaviors; the most conspicuous are (1) orientingtowards and following the female, (2) extending and vibratinga wing to produce a courtship song, (3) licking the female'sgenitalia, (4) curling the abdomen in an attempt to copulate,and (5), if the female is receptive, copulation (ref. 4, see Fig.1). This sequential action pattern is not strictly followed,since males may break off the performance of one behaviorin the series and resume courtship with an earlier behavior.However, males generally do not proceed to later stages ofcourtship without having first carried out the earlier steps inthe sequence. Areas in the dorsal posterior brain as well asthe thoracic ganglion have been shown to be responsible fordifferent steps of this sequence (5-7). Males without priorexperience perform this sequence of behaviors with aplomb,indicating that it is largely genetically programmed [althoughit is modifiable by experience (8-11)], and hence the devel-opmental program necessary to establish male courtshipbehavior must be executed in the CNS prior to earlyadulthood.

In Drosophila the development of the CNS differs signif-icantly from that of almost all other tissues. Most tissues of

the adult are derived from imaginal cells whose lineagesseparate early in embryogenesis from those that produce thelarval tissues. During the pupal period the larval cellsdegenerate and are supplanted by the imaginal cells thatdifferentiate into the structures of the adult. In the CNS, onthe other hand, there is relatively little cell death, and boththe cells that constitute the larval CNS and cells producedduring the late larval and early pupal periods are used to makethe CNS of the adult (12-14). In a specific portion of thedorsal CNS, the mushroom bodies, where the transition oflarval CNS into the adult CNS has been carefully studied, thedifferentiated larval neurons are not incorporated into theadult CNS "as is." Rather, they undergo a massive reorga-nization: a large proportion of the projections of the larvalcells degenerate and the cell bodies send out new projectionsto participate in building the adult brain. In the mushroombodies this process of reorganization extends into adulthood(15). Since development of the CNS retains a cellular conti-nuity between the larval and adult nervous systems, theprogramming of the CNS for male courtship behavior by thesex determination regulatory hierarchy could, a priori, occurat any stage of development.

In Drosophila melanogaster a single regulatory hierarchycontrols all facets of somatic sexual differentiation (see ref.16 for review), including the differentiation of those parts ofthe CNS responsible for sexual behavior. For example, thereare sex determination mutants [transformer (tra), transform-er-2 (tra-2)] that block female sexual differentiation in so-matic cells and cause chromosomally female (i.e., XX)individuals to develop somatically into phenotypic males inall aspects. This includes their external cuticular structures,their internal genital duct systems, the synthesis of severalsex-specific proteins (17-23), and their courtship behaviorpatterns (7, 23-25). These observations suggest that theactivities of the wild-type alleles of the tra and tra-2 loci arenecessary in chromosomally female individuals both to causefemale sexual differentiation and to repress male sexualdifferentiation. These two genes have no apparent effect onsomatic sexual differentiation in XY males. tra' and tra-2'are thought to carry out their dual functions in females bycontrolling the expression of the bifunctional doublesex (dsx)locus, which carries out opposite negative regulatory func-tions in the two sexes (16). In wild-type females dsx+ is underthe control of tra+ and tra-2+, and it functions to repress maledifferentiation; female differentiation, not being repressed,occurs. If either tra+ or tra-2' activity is missing in an XXfemale, then dsx+ is expressed as it normally would be in amale, in which its active function is to repress female sexualdifferentiation; male differentiation, not being repressed,occurs. Thus, loss-of-function mutations at either the tra or

Abbreviation: CNS, central nervous system.*Present address: Department of Biology, Syracuse University,Syracuse, NY 13244.tDepartment of Biological Sciences, Stanford University, Stanford,CA 94305.

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tra-2 locus cause XX individuals not to undergo femaledifferentiation but to follow instead the normal male devel-opmental pathway with respect to all somatic sexual char-acteristics thus far examined.

Temperature-sensitive tra-2 mutants (which behave asloss-of-function mutations at the restrictive temperature of290C, whereas they are essentially wild type when homozy-gous at the permissive temperature of 16'C) have been usedto show that this regulatory hierarchy functions throughoutthe late larval, pupal, and adult periods to determine and/ormaintain various aspects of sexual differentiation (23, 26). Inthe present study we use temperature shifts with one of thesetemperature-sensitive alleles (tra-21sI) to examine how malecourtship behavior is established and maintained by thefunctioning of this regulatory hierarchy.

MATERIALS AND METHODSFlies were raised on a cornmeal/molasses/agar mediumseeded with live yeast. Adults were collected under lightanesthetization with ether within 8 hr after eclosion (emer-gence from the pupal case) and placed singly in vialscontaining the above medium. All subsequent transfers weredone without ether anesthetization. Two days after eclosionflies were transferred to fresh vials and either kept at the sametemperature or shifted to the other temperature, at whichthey were maintained until being tested for courtship behav-ior. For all courtship assays, flies were transferred to freshvials and allowed to become acclimated to room temperature(220C) for at least ½ hr prior to testing. To reduce subjectivityin scoring the mating behaviors, shifted and unshifted flies aswell as a few Canton-S wild-type male controls were codedby one person while a second person recorded their courtshipbehaviors. The behaviors were observed in a plastic appa-ratus containing 10 cylindrical mating chambers (5). One flyto be tested was introduced into each of the 10 chamberstogether with one virgin Canton-S female that had beencollected at least 3 days earlier. Each pair was observed for30 min and courtship behavior was recorded. Flies werescored positive for following if they oriented themselvesbehind the female and closely followed her for more than 1 or2 sec at a time. Wing extension was scored positive if the flybeing tested extended its right and/or left wing while orient-ing towards the female. Tapping or licking was scored if thefly contacted the female repeatedly with its forelegs, or if itextended its proboscis and contacted the female's genitalia.Attempted copulation was scored if the fly curled its abdo-men and directed its genitalia towards the female. Copulationwas scored as positive if the male being tested engaged itspenis apparatus and the mating pair remained together forseveral minutes. In our courtship assays we did not differ-entiate between flies that courted vigorously and those thatdisplayed the various behaviors weakly or sporadically. Aftereach trial the mating chamber was washed, rinsed withethanol, and air dried.

RESULTSTo carry out temperature-shift experiments to examine howmale courtship behavior is controlled by tra-2 it was firstnecessary to identify an XX; tra-2 genotype that, whencarriers were reared throughout development at the restric-tive temperature (290C), resulted in a high proportion ofadults that displayed male courtship behavior. Preliminaryobservations on XX; tra-2t`J/tra-2f1s and XX; tra-2's2/tra-25s2flies raised at 290C revealed that, while such flies aremorphologically male, they often fail to display a sustainedpattern of courtship activity when tested. XX; tra/tra indi-viduals taken from long-established stocks also often dopoorly in male courtship tests, and this has been shown to be

due to modifiers in the background genotype of these stocks(J. Hall, personal communication). Thus, various combina-tions of tra-2sI or tra-2s2 with other alleles of tra-2 weretested to determine the genotype that gave the highestfrequency and extent of male courtship behavior when theflies were raised at 290C. In this way, XX; tra-2's1/Df(2R)trix(a chromosome deleted for the tra-2+ region; B.B., unpub-lished data) individuals were found to exhibit the mostreproducible male courtship when raised at 290C (C. Loer,personal communication). About 80% of these flies displayedthe following and wing-extension behaviors, with about 67%of them attempting copulation and a substantial numberachieving copulation (Fig. 1A). Flies of this same genotypewhen cultured at 16'C develop into morphologically inter-sexual individuals (unpublished observations), presumablybecause one dose of the tra-2's' gene, even at this normallypermissive temperature, does not provide enough wild-typefunction to allow normal female development. These inter-sexes have malelike pigmentation, intermediate or malelikesex combs, and rotated male genitalia that are often onlypartially developed. Such intersexes are not malelike withrespect to their sexual behavior: fewer than 5% of such fliesever displayed, even weakly, the wing-extension behavior,and none proceeded to the later steps in the courtshipsequence (Fig. 1B). Wild-type control males showed no effectof culture temperature on their courtship performance (datanot shown).

In the first series of temperature shifts, XX; tra2fsI/Df(2R)trix adults that had been cultured at 29TC until 2 daysafter eclosion were shifted to 16'C and maintained at thattemperature for 6-14 days. When these flies were tested theirbehavioral profiles were virtually indistinguishable fromthose of similar flies kept continuously at 290C for a compa-rable time (Fig. 1A). This result suggests that the CNS is setto direct the performance of male courtship by the adult atsome time prior to early adulthood.To define when this determination of the male courtship

behavior occurs, XX; tra-2'sJ/Df(2R)trix individuals wereshifted from 290C to 16'C during the pupal stage (Fig. 2) andbehavior of the resulting adults was assayed (Fig. 3). Indi-viduals down-shifted such that the last 2 days of their pupalperiod were at 16'C (Fig. 3B) exhibited as high a frequencyof male courtship behavior as individuals raised throughoutdevelopment at 290C (Fig. 3A). Conversely, individualsdown-shifted prior to 8 days before eclosion (about 40% ofthe way into the pupal period; Fig. 3 F and G) displayed thesame low levels of courtship as flies kept at 16'C throughouttheir development (Fig. 3H). Thus the inactivity of the tra-2'gene product during the embryonic, larval, and early pupalstages does not appreciably shift these flies' behavior towardmaleness. Moreover, pupae down-shifted at successivelylater times during the interval of 8 to 2 days before eclosionshow a progressive increase in both the frequency and extentof male behavior they display (Fig. 3 C-E). Taken together,the above results show that the temperature-sensitive periodfor the induction of male courtship behavior commencesabout 40% of the way through pupal development andterminates about 85% ofthe way through pupal development;the absence of wild-type tra-2 function during this time issufficient to determine male courtship (Fig. 2).Although the above temperature shifts show that develop-

ment following the male pathway during the pupal period issufficient to produce a male behavioral pattern in the adult,they do not establish that this is a necessary condition forproducing male behavior. For example, it could be that amale behavioral pattern can be induced at any time during orafter the midpupal period by the inactivation of the tra-2'function (e.g., that wild-type tra-2 function is requiredcontinuously during these stages to repress male courtshipbehavior). Alternatively, it could be that the CNS is pro-

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FIG. 1. Courtship behavior profiles of XX; tra-21s) bw/Df(2R)trix flies. (A) Flies were raised at 290C to adulthood and then either (l) keptfor 6-10 days after eclosion at 290C (solid bars; n = 95) or (it) at 2 days after eclosion at 290C shifted to 16WC for 6-14 days (shaded bars; n =114). (B) Flies were raised at 16WC to adulthood and then either (i) kept for 6-10 days after eclosion at 16WC (solid bars; n = 100) or (ii) at 2 daysafter eclosion at 16WC shifted to 290C for 6-10 days (shaded bars; n = 96). Horizontal axes represent the maximum stage of courtship displayedby each individual: 0, no male courtship behavior observed; 1, orienting toward and following the female; 2, wing extension and vibration; 3,licking and/or tapping the female's genitalia; 4, attempted copulation; and 5, copulation. A fly carrying out any given step in courtship hadinvariably carried out all prior steps in the courtship sequence. Vertical axes represent the percentage of individuals in each of these classes.The illustration of courtship behaviors is from Hall (27).

grammed irreversibly during the midpupal period to be eithermale or not-male (i.e., that the CNS displays plasticity withrespect to adult behavior only during the midpupal period).By carrying out the reciprocal temperature shift (from 16TC

to 29TC) on adult flies one can distinguish between thesepossibilities. For these experiments, XX; tra-2's/Dft2R)trixflies were raised and kept at 16TC until 2 days after eclosion.As seen in Fig. 1B, few of these flies, when kept 16TC forseveral additional days and then tested, carried out even thefirst steps of male courtship behavior. However, a strikinglydifferent result was obtained when, as 2-day-old adults, suchflies were instead shifted to 29TC, maintained at that temper-ature for 6-10 days, and then tested for male courtship: asubstantial proportion of them displayed male courtshipbehavior (Fig. 1B). About 45% ofthese flies displayed at leastthe first steps in courtship (orienting toward and following thefemale) and about 33% of them progressed at least to wingextension. Of the latter flies 27% progressed to licking thefemale's genitalia and about half of these in turn attempted

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copulation. The failure of any of these flies to achievecopulation may not be meaningful, since these flies do nothave normal genitalia, and so copulation may not have beenpossible for mechanical reasons.While these flies showed an increased frequency and

extent of male courtship behavior, many of the shifted fliesdid not display any male behavior. There are three possibleexplanations for why some flies did not display male behav-ior. The first is based on the finding that flies of the abovegenotype shifted to 290C at 2 days after eclosion and testedfor male courtship at only 2-4 days after the shift did notshow any significant male behavior (data not shown). Thisimplies that the switch to male sexual behavior takes some-where between 4 and 10 days at 290C to occur. Thus it maybe that some of the flies that failed to exhibit male behaviorafter 6-10 days at 290C had not been at 290C for a sufficienttime period to bring about a behavioral change. Alternatively,the fact that these flies were raised at 16'C during the pupaland early adult period may have led to some, but not all, of

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FIG. 2. Protocol for, and summary of, shifts from 290C to 160C during the pupal period. Cultures containing larvae and pupae of randomages were shifted from 290C to 16'C and emerging adults were collected every 2 days. The ages of individuals at the time of the temperatureshift are taken as the time it took them after the shift to reach eclosion at 160C. Times are in days prior to eclosion at the indicated temperature;the pupal period lasts approximately 3.5 days at 290C and 14 days at 16'C.

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FIG. 3. Results of tests of males' courtship behavior in XX; tra-2fsI/Dfl2R)trix individuals shifted from 290C to 160C during the pupal period.Axes are as in Fig. 1. For temperature-shift protocol see Fig. 2. The ages (as days prior to eclosion at 16'C) of the temperature-shifted flies areas follows: B, 0-2 days; C, 2-4 days; D, 4-6 days; E, 6-8 days; F, 8-10 days; G, >10 days. Controls: A, 290C unshifted; H, 16'C unshifted.

them being irreversibly committed to nonmale behavior.Finally, it should be noted that under some circumstancesfemale Drosophila display tracking behavior towards otherfemales that is very similar to the orientation and wing-extension steps of male courtship (28, 29). Thus it is possiblethat it is such female tracking behavior that was induced bythese adult temperature shifts. This seems unlikely to us,since 8 of the 96 flies shifted from 16WC to 290C as adultscarried courtship through to behaviors (licking female geni-talia and attempted copulation) that are not observed inencounters of wild-type females with one another (28, 29).The substantial time needed to bring about a behavioral

change in these adults almost certainly does not reflect thetime needed to inactivate the tra-2 product at 290C, sinceprevious work has shown that the tra-21sI product is inacti-vated in the adult fatbody within a few hours at 29TC (13).Thus the time required to change adult behavior probablyreflects a temporal requirement for the differentiation ofaspects of the CNS necessary for male courtship behavior.

DISCUSSIONThe temperature-sensitive period during pupation when malecourtship behavior is induced at 290C is after the time whenmost, if not all, cell divisions in the nervous system have beencompleted and overlaps the time during metamorphosis whenthe fly's CNS undergoes extensive reorganization (13-15).This is also the time when the peripheral nervous system(PNS) of the adult is differentiating. However, studies ofgynandromorphs (male-female mosaics) have shown that allsteps in male courtship through attempted copulation aredependent solely on the sex of the CNS and are independentof the sex of the PNS (5-7, 30, 31). Thus our data imply that,during the middle of the pupal period, the differentiation ofthose aspects of the CNS responsible for male-specificcourtship occurs. The absence of tra-2' function during thistime in a female is sufficient to establish the male behavioralprogram.That male courtship behavior can also be induced by a shift

to the restrictive temperature during adulthood demonstratesthat, in XX; tra-2tsl/Df(2)trix individuals, wild-type tra-2'function is necessary in adults to prevent the development ofmale courtship behavior. Previous work with temperature-sensitive tra-2 mutants showed that tra-2' function is alsonecessary in adults to regulate, at the RNA level, theexpression of the three yolk protein genes (22). In the case ofthe yolk protein genes, as we found here with respect to male

courtship behavior, a shift of XX; tra2ts/tra-2fs adults fromthe permissive to the restrictive temperature led to the malepattern of gene expression.

In the case of the yolk protein genes, temperature shiftswere carried out with tra-21s homozygous individuals, whichdeveloped to adulthood as morphologically normal females,and thus the active control of yolk protein gene expressionseen in these individuals almost certainly reflects a processthat is occurring in normal females. However, in the exper-iments reported here we used (for technical reasons de-scribed above) tra-2's' hemizygous individuals, which de-velop to adulthood at the permissive temperature as mor-phologically intersexual individuals. Thus, although it is clearthat a shift of such adults to the restrictive temperature elicitsmale courtship behavior, we cannot be sure that this degreeof plasticity is present in the CNS of wild-type adult females.It could be that this amount of developmental plasticity isunique to the genotype studied here. For example, it ispossible that in XX; tra-2's/Df(2)trix individuals reared at16°C, but not in normal females, some of the steps indifferentiation necessary for male courtship behavior occurduring the pupal period and the adult temperature sensitiveperiod reflects a plasticity in only a part of this process.

Regardless of which of these possibilities is correct, theseexperiments have strong implications for how behavioralpatterns are established and maintained in the CNS. Thesimple fact that a shift of an adult to the restrictive temper-ature can elicit male courtship behavior very strongly sug-gests that the regulatory hierarchy controlling sex determi-nation is functioning in the CNS of wild-type adults. That is,it seems unlikely that these regulatory genes would beexpressed in XX; tra-2fsI/Df(2)trix adults and not in wild-typeindividuals. If these regulatory genes are indeed functioningin the wild-type adult CNS, this would imply that thedifferentiated state of the adult CNS is not immutably fixedat some earlier stage ofdevelopment. Instead it would appearthat the adult Drosophila CNS has, at least in part, afunctional plasticity with respect to the innate action patternof male courtship behavior and is maintained in a particularstate of differentiation by the active control of gene expres-sion.The adult Drosophila CNS has at least some structural

plasticity in a sexually dimorphic region of the brain-themushroom bodies (15). The number of Kenyon cell axonfibers in the mushroom bodies is about 10% higher in femalesthan it is in males. This sexual dimorphism in the Drosophilabrain may be analogous to the occurrence of marked physical

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differences in brain regions of other invertebrates (e.g., refs.32-34) and many, perhaps all, vertebrates (35), where thesedifferences have been implicated in such sexually dimorphicbehaviors as courtship in song birds (e.g., ref. 36) andcopulatory behavior in rats (e.g., refs. 35 and 37). The sexualdimorphism in the Drosophila mushroom body structure hasnot been causally connected to differences in sexual behav-ior, but the mushroom bodies do appear to be involved in theassimilation or processing of olfactory information (14),which is known to be important in Drosophila sexual behav-ior (38, 39). Moreover, fate-mapping studies have implicatedthe dorsal posterior region of the brain (where the somata ofthe mushroom bodies are located) in the control ofsome stepsin male courtship behavior (6). In light of the results present-ed here-i.e., an adult temperature-sensitive period for theeffect of tra-2tsJ on courtship behavior-it is intriguing that inwild type the sexually dimorphic mushroom bodies continueto differentiate new Kenyon cell axons in the caudal pedunclefor several days after eclosion.More generally, we would like to comment on the impli-

cations of our finding of a functional plasticity in the adultwith respect to male courtship behavior for the widely heldview that invertebrate nervous systems are relatively "hardwired." In its simplest form "hard wiring" is taken to meanthat the properties of an invertebrate nervous system are laiddown by a set of machinery that functions during theestablishment ofthe system but, once the nervous system hasdifferentiated, the machinery that designed it is removed andthe properties ofthe system are then largely those dictated bythe previously established pattern of transmitters, connec-tions, and receptors. What our results show is that one of theimportant pieces of machinery (the genes regulating sexdetermination) that determine a major innate property of theDrosophila CNS (sexual courtship behavior) does not justfunction during development to establish this behavior pat-tern and then get turned off, but rather seems to actcontinuously, even when the mature (adult) system is func-tioning, to maintain its properties. Thus the hard wiring of thefly's nervous system with respect to one important kind ofinnate behavior, and by analogy perhaps others as well,appears to require the continuous active control of geneexpression and does not simply reflect the previously con-structed and fixed structural and physiological properties ofthe cells.

We thank R. Malloy for excellent technical assistance and A. T. C.Carpenter, B. Chase, C. Goodman, J. Hall, M. Heisenberg, and B.Taylor for their comments on the manuscript. This research wassupported by National Science Foundation Grant PCM 8202812 andNational Institutes of Health Grant GM 23345.

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