Copyright 0 1989 by the Genetics Society of Amerlcd

Behavior and Cytogenetics offruitless in melanogaster: Different Courtship Defects Caused by Separate, Closely Linked Lesions

Donald A. Gailey and JeffreyC. Hall

Department of Biology, Brandeis University, Waltham, Massachusetts02254 Manuscript received September 22, 1988 Accepted for publication December 24, 1988

ABSTRACT Thefruitless (fru)courtship mutant was dissected into three defectsof male reproductive behavior, which were separable as to their genetic etiologies by applicationof existing and newly induced chromosomal aberrations.fru itself is a small inversion [In(3R)9OC; 9 1B] on genetic andcytological criteria. Uncovering the fru distal breakpoint with deletions usually led to males with two of thefru courtship abnormalities: no copulation attemptswith females (hence, behavioralsterility) and vigorous courtship among males, including the formation of “courtship chains.” However, certain genetic changes involving region 91B resulted in males who formed courtship chains but who mated with females. Uncovering the fru proximal breakpoint led to males that passively elicit inappropriately high levels of courtship. Thiselicitation property was separable genetically from thesterility and chain formation phenotypes and provisionally mapped to the interval 89F-90F, which includes the fru proximal breakpoint. Behavioral sterility and chaining werealso observed in males expressing certain abnormal genotypes, independent of thefru inversion. These included combinations ofdeficiencies, each with a breakpoint in 91B, and a transposon inserted in 91 B.

RECOPULATORY behaviors are sex-specific nervous system (CNS). Thus, a search for P components of reproduction. In Drosophila mel- that disrupt the male-specific behavior of attempted anogaster, males respond to females with a stereotypic copulation might not only reveal that are rele- set of actions, including following, production of a vant to, for example, male-specific musculature in the courtship song via wing vibration and curling of the abdomen (cf: LAWRENCEand JOHNSTON1984), but abdomen in order to initiate mating(reviews: HALLet that also lead to defects within the CNS. A mutant al. 1980; EWING1983; TOMPKINS1984; HALL1985). that could be in this category is fruitless (fru). A The responses of females to these male behaviors are cytogenetic analysis offru-associated courtship anom- less overt and undoubtedly involve nervous system- alies is the focus of this report. dependent phenomena (e.g., TOMPKINSand HALL fru is a homozygous-viable, male-sterile mutant that 1983). Females perceive male courtship cues that are was induced with X-rays (GILL1963a, b). The effects chemical (reviews: JALLON1984; TOMPKINS1984), of fru seem to be male specific, in that females homo- auditory (e.g., EWINC1977; HALL 1984, 1986), and zygous for fru show no obvious phenotypic or behav- visual (e.g., TOMPKINS1984; MARKOW1987) inna- ioral differences from wild type (HALL1978a). Males ture. Receptive females ultimately respond to the cues homozygous for fru, however, show at least three of conspecific males by slowing their locomotor move- obvious deviations fromnormal male courtship be- ments, and mating ensues (MARKOWand HANSON havior. (1)They do notcurl their abdomens femalesat 1981; TOMPKINSet al. 1982; GAILEY,LACAILLADE to attempt copulation, and are thereby behaviorally and HAIL 1986). sterile (HALL 1978a); to this extent fru could be an It should be possible in Drosophila to “dissect out” example of a controlling male-specific actions in genes pertinent to these behavioral dimorphisms. For courtship. (2) They court otherfru males and normal exanlple, mosaic analysis of precopulatory behavior males (HALL1978a); this could mean that fru is rele- has led to the identification of thoracic tissues which vant to the discrimination by malesof appropriate must be male for copulation attempts tobe performed courtshipobjects, since normal males rarely court by gynandromorphs (HOTTAand BENZER1976; HALL each other. (3) They stimulate normal males to court 1979), implying that this behavior is not generated or them; consequently, courtship-stimulating cues could controlled locally within the male abdomen per se, but emanate from fru males (HALL1978a), a hypothesis probably is subserved by male tissues in the central supported by the results of bioassay experiments in which solvent extracts of fru males stimulated court- Dedicated to R. W. Siege1 and his fruitful career as a ship (TOMPKINS,HALL and HALL1980). geneticist. In the current study we show that the fru pheno-

(kwt.ti<< 121: ii:i-78.~(April. 1989) 774 D. A. Gailey and J. C. Hall

TABLE 1

Chromosomal aberrations used in the cytogenetic localizationof thefruitless phenotypes

Marker Aberration genotype" Cvtology Origin Referenceb

Deficiencies Df(3R)PI4 X-ray LINDSLEYand ZIMM(1985); K. MOSES (personal communication) Df(3R)Cha"' X-ray LINDSLEYand ZIMM(1985); K. MOSES(personal communication) Df(3R)Cha"' 91B;91D X-ray LINDSLEYand ZIMM (1985) Df(3R)Cha"' 90F:91 F' y-ray LINDSLEYand ZIMM (1985); K. MOSES(personal communication) Df(3H)148.5-1 91B3;91D1 y-ray GORCZYCAand HALL(1984); W. M. GELBART (personal communication) Df(3R)DCI 90E1-2;91C3-7 ?-ray This report Df(3R)DG2 89E-F;91B1-2 y-ray This report Df(3R)DG3 89F;90F 7-y This report Df(3R)DG4 90E1-2;90F3-11 ?-ray This report Df(3R)DCS 9OE-F;9lE-F way This report Df(3R)glBXZ 90F8-11;91B1-2 X-ray K. MOSES(personal communication) Df(3R)glBXZO 90C7-8;91B1-2 X-ray K. Mosm (personal communication) Df(3R)gl+BX5 91B1-2;91D1-2 X-ray K. MOSES (personal communication) Df(3R)gl+BX6 90C9-10:90F2-11 X-ray K. MOSES(personal communication) Marked transposon P(w+)ARO-Z w+ inserted at 91B1-2 Transposon LEVIS, HAZELRICCand RUBIN (1985); K. Moses mobilization (personal communication) Duplication Dp(3R)DGd sr+ gl- 81-90A;90F-90A; y-ray This report 90A-100

a Presence (+), or absence (-) of normal marker gene function on aberration-bearing as inferred from crosses with 6% sr gl or a lethal allele of Cha (choline acetyltransferase). Absence of any marker designation(s) means that particular cross was not performed. Cytology on all was provided by K. MOSES,except for Df(3R)ChaM' and Df(3R)148.5-Z.The second through fifth deletions in this table were isolated as Cha- (cf: GORCZYCAand HALL 1984; P. L.MYERS and W. M. GELBART,unpublished data). The last four deletions were derived from a screen for deletions near or including gl (K. MOSES, unpublished data). Indicates cytological information supplied by K. MOSESthat differs slightly from the published breakpoints. Shows no visible aberration in the region ofgl at 91A (K. MOSES,personal communication); Df(jR)P14/Dp(3R)DG is viable and sr+ gl- in phenotype; Df(?R)DG3/Dp(3R)DCis viable and sr+ gl+ (no other crosses were performed). types do not all map to a single genetic locus but are maintained in the laboratory of J. C. HALLby mass transfer associated with a small inversion on the third chro- of unselected adults. Chromosomal aberrations used to map thefruitless phe- mosome. The two mutant behaviors that fru males notypes are listed in Table 1 and schematically presented in themselves display in their courtship co-map with one Figure 1. Markers used in mapping by recombination were breakpoint of this inversion; the mutant behavior that bithorax (b~~~~,3, 58.8), stripe (sr, 3, 62.0) and glass, (gl, 3, fru males elicit from normal males maps provisionally 63.1). to the other breakpoint. Balancer chromosomes (Bal) used were In(3LR)TM3 (=TM3), marked with the dominant Stubble (Sb); In(3LR)TM6 (=TM6),marked with the dominant mutation MATERIALSAND METHODS Ultrabithorax (UbxPl5);and In(3LR)TM6B (=TM6B; CRAY- MER 1984), markedwith the dominant mutationTubby (Tb), The maintenance of fly cultures and procedures of fly which imparts a distinctive body shape in both adults and handling for behavioral assays were as detailed in GAILEY, larvae andfacilitated selection ofhomozygousfru/fru larvae JACKSON and SIEGEL(1 982) and GAILEY, LACAILLADE and for salivary gland squashes. HALL (1986). Flies weregrown on a mediumcontaining P(w+)ARO-I is a transposon containing the white+ (w, I, cornmeal, molasses, agar, yeast and the mold-inhibitor Te- 1.5) gene, and was derived from mobilization of the tran- gosept . sposon P[(w,ry)AR]4-3 andreinsertion at the cytogenetic Fly stocks: Several of the genetic variants used in this region91B1-2 (LEVIS, HAZELRIGGand RUBIN 1985; K. analysis appear in LINDSLEYand GRELL (1 968),or LINDSLEY MOSES,personal communication). This transposonis main- and ZIMM (1 985); others arenewly isolated (Table 1). tained in the stock wl'", TM3/ARO-1, which was provided fruitless (fru, 3, 62.0 HALL 1978a)"derived from X-ray to us by K. MOSESand G. M. RUBIN. mutagenesis(GILL 1963a, b). fru/"ru males expressthe Screen for new aberrations in the region of sr and gl: fruitless syndrome of behavioraldefects, frulfru females Wild-type males from a Canton-S strain received 4.5 kR y- appear normal (HALL 1978a).Because of the male sterility, irradiationfrom a 137Cssource, delivered at the rate of fru stocks are kept with balancer chromosomes (see below). approximately 265 R/min. They were immediately crossed The starting stock for the current analysis was TMb/fru, to bx sr gl homozygous females. FI males displaying the sr “I fruitless Courtship I In

4” -”) EFABCDEFABCDEF 90 91 I E3 =a I kc fiu break “g’&

0 0 FIGURE2.-fru courtship chain beh;lvior. To the left are nine Df(3R)g1RX//frumales (see Table 1 and Figure 4 for details on this deletion). Arrows indicate instances of wing vibration. To the right are nine Df(3R)g/RXI/+ males. I. ship stimulation phenotype). One line was selected arbitrar- P (w+) ARO-I ily (a TM6/fru female-derived line) and was the source for FIGURE 1 .-Approximate breakpoint cytology of aberration- fru chromosomes in all further crosses. This stock is main- bearing chromosomes used in the mapping offru phenotypes (com- tained as TM3/fru. pilation of cytology in Table 1 and functional tests for complemen- Next, ten sublines of this TM3Ifru stock were established tation). Background shading categories are based on the CI values from ten individual pairs ofTM3/fru flies. These lines were generated by Aberration/fru males (ie., thefru courtship stimula- single-pair inbred for seven generations using TM3/fru sib- tion phenotype). White background, CI < 10; light gray back- lings. One line was homozygous lethal at the third genera- ground, 11 < CI < 24: dark gray background. CI > 24. The tion. When crossed to the stock carrying Df(3R)P14, the assessment off” behavioral sterility and courtship chain formation 0flf.u male segregants werefru in phenotype: thus, lethality in Abenationlfru males is denoted as follows: black-filled circle = was again not fru-associated. fru/fru males of the nine re- sterility and courtship chain formation; gray-filled circle = fertility maining lines weresterile and formed courtship chains. The and courtship chain formation; no circle = wild tvpe for these two viabilityof frulfru flies before and afterthe single-pair f” phenotypes. See Table 3 and text. inbreedings stayed roughly the same, ca. 25-40%. Crosses of these nine inbred lines with the Df(3R)P 14 stock revealed and/or gl phenotype(s) were individually mated to TM6/Sb similar viability for Df/fru flies. virgin females, and stocks were established (the TM6 chro- Moreover, in the course of mappingfru by recombination mosome contains the bx marker and allowed for easy selec- (see RESULTS and cf. HALL 197th) many potentially bx fru tion against the bx sr gl chromosome in bx SI gl/Aberration chromosomes were formed; two such recombinants were FI males). Aberrations were recovered in the approximate chosen arbitrarily and established as balanced, isochromo- frequency of 1/5000 males screened. soma1 stocks. Male homozygotes in each line were sterile Several new aberrations weresupplied by K. MOSES and formed courtship chains (not tested for the courtship (Table 1): these were X-ray induced in males carrying the stimulation phenotype). transposonP[(w,ry)A]2-1 inserted at 91C (HAZELRICG, These pedigrees reveal that fru is stable and is a consist- LEVISand RURIN1984) and isolated by monitoring for loss ently abnormal behavioral mutant. of w+ function (K. MOSES, personal communication). Behavioral assays: The following general tests were de- Cytology: Temporary mounts of chromosomal spreads vised to reveal three specificfru phenotypes: from the salivary glands of fru/+ and fru/fru larvae were Behavioral sterility: Males whose fertility was to be tested prepared with lacto-aceto-orcein (HUMASON1979) and in- were collected at eclosion (grouped not more than 10 per terpreted with the aid of the revised chromosome map of vial) and aged 5-6 days. Then they were placed individually LEFEVRE(1 976). in food vials each containing four wild-type virgin females, Preliminary crosses withfm: Preliminary tests with the aged 4-6 days. In somecases, vials were scanned at this TM6/fru starting stock revealed that fru homozygotes ap- point for occurrences of courtship. Otherwise, all vials were peared at a low frequency. fru/fru males courted but did scored for presence oflarval progeny 7 days later. Vials not copulate and formed courtship chains with each other: containing a dead male and no progeny were not counted. thus at least twofru phenotypes persisted in this stock (see Suchcases in all experiments were rare. The testes of methods for behavioral analyses, below). As a test for het- surviving males were dissected out in buffered saline solu- erogeneity of these phenotypes within this stock, 40 isochro- tion (IKEDA and KAPLAN 1972) and in every case contained mosomal fru lines were established by standard Bal chro- motile sperm. mosome technique (20 linesestablished from TM6/fru Formation of courtship chains: A detailed description of males, 20 from TM6/fru females). Homozygousfru males this fru behavior is given in RFSULTS (also see Figure 2). from each line were then tested for these twofru pheno- Males to be tested were collected and grouped at eclosion, types. One male-derived line and one female-derived line 10 per vial. On the seventh day, the behavior of males was proved to be homozygous lethal. Crosses of these two lines observed for a 0.5-hr period, usuallv several vials at a time. to a stock carrying Df(3R)P14 (see Table 1) yielded Df/fru The minimum criterion for scoring a vial of males as mutant males that expressed the sterility and courtship chain phe- for this behavior was the occurrence of at least one “court- notypes: thus the lethality in these lines is not fru-associated. ship chain” made up of at least 4 participating males (cf: In each of the remaining 38 linesfru/fru flies were viable, Figure 2). and males of this genotype displayed the fru phenotypes Courtshipstimulation: Homozygous fru males stimulate noted above (none of the 40 lines was tested for the court- wild-type males to court them; this fru phenotype is easiest 776 D. A. Gailey and J. C. Hall

TABLE 2 soma1 stock (see MATERIALS AND METHODS),and, as Behavioral phenotypes offruitless males compared with wild controls, wild-typemales or males from across be- type and the fml+ heterozygote tween fru and wild type. All +/+ and fru/+ males initiated copulation within 30 min. All frulfru males Fertility Chain Courtship stimulus courted within 30 min and the preliminary compo- (knutype” fraction’ behavior‘ (CI SEM)d nents of male courtship (cJ BASTOCKand MANNING f../f.. 0/100 10/10 28 +- 3* 1955)appeared superficiallynormal (although fru 16 k 2* frul+ 100/100 0/10 males generate a defective courtship song; seeDISCUS- +/+ 100/100 0/10 4t I* SION). However, not onefru male initiated copulation a “+” indicates a fru+-bearingchromosome from a Canton-S stock. orattempted to initiate copulation by curlingthe Males (5-6 days old) wereplaced individually in food vials abdomen.With regard to sterility, fru is asimple tont;tining 4 wild-type virgin females and then scored for larval recessive mutation (Table 2, second line). progct~y7 days later. Testes of all males yielding no progeny were dissected out in buffer, and found to contain motile sperm. The frulfru males court other frulfru males and wild-type denominatorrepresents the total number of males tested;the males: This conclusion was reached by HALL (1978a) nunlcrator equals the number of males yielding progeny. after observing the courtship behaviorof many male Males were collected and grouped upon eclosion IO/food vial. On rhc 7th day, vials were observed for a 0.5-hr period, and were trios involving several genotypic combinations. Here scored as positive for fru chain behavior only if there occurred at we have analyzed only the courtship among frulfru least one courtship chain of four or more males. The denominator males. When in groups, they spend much of the time represents thetotal nunlber of vials containing males of the specified gelwtype so observed, and the numerator the number of vials in courting each otherin lines-which we call “courtship which fru courtship behavior occurred. chains” (Figure 2). This is an easily recognized court- ” (:I (courtshipindex) is the mean percentage of time in an ship interaction unique tofrulfru males in groups. observation period that 5-day-old wild-type males spent courting 5- d;ly-old males of the specified genotype. A comparison of chain behavior in fru/fru, fru/+, * n = 50 observations per genotype; the three mean CI values and wild-type males is in Table 2 (“Chain behavior”). differ significantly at the P < 0.001 level, t-test matrix. Only frulfru males were ever observed to display such measured in pairings of wild-type test males and anesthe- behavior. We estimated that these chains occurred for tizedfrulfru males (HALL!978a). We observed and quan- about 50% of the observation periods,with a chain of tified this kind of courtship as in GAILEY, LACAILLADEand 4-10 males often snaking about the inner surface of HALL(1986). In brief, observation chambers were formed the food vial; many instances of wing vibration were by covering the depressions of porcelain well plates with microscope slides. To test a male forfru-like elicitation of observed. Wild-type and fru/+ males never displayed courtship, he was ether anesthetized and then placed in a this behavior, tending to disperse themselves evenly depression. Positioning a glass slide to make a small opening about the vial; instances of courtship behavior were in the chamber, a wild-type males was carefully aspirated in, limited to pairs of males and were infrequent as well the slide moved to close the chamber, and the observation as brief. Hence, this aspect of the fru phenotype is begun. Any courtship that occurred was measured with respect to Courtship Index (CI) determinations (e.g., also recessive. GAILEY,LACAILLADE and HALL1986). frulfru males stimulate wild-type males to court them Statistical analysis: When it was deemed appropriate (see even when the mutant males are anesthetized or have had REsu1.n) data wereanalyzed by a t-test matrix (BMDP their heads and thoraces removed: This seemed to sug- Statistical Software, Los Angeles, California 90024). gest that fru/fru males contain a chemical courtship- stimulating cue (HALL 1978a; TOMPKINS,HALL and RESULTS HALL 1980). Here,we assessed the courtship perform- Abnormalities of male courtship performed and ance of individual wild-type males that were paired elicited by the fruitless mutant: The results in Table with ether-immobilized flies ofvarying genotype, 2 summarize components of thefru phenotype. These quantified as CI (e.g., GAILEY, LACAILLADE and HALL behavioraldefects were first reported by HALL 1986). Numerousreports in theliterature indicate (1 978a), and the current data arefully consistent with that 5-day-oldwild-type males stimulatevery little thoseearlier findings. Details of the fr-u syndrome courtship; yet when newly emerged, males stimulate follow. as much courtship as virgin females (review: TOMP- fru/fru males are behaviorallysterile: Theycourt KINS 1989). females with persistence, yet they do not curl their As expected, we found that 5-day-old frulfru males abdomens for attemptsat copulation; they never pro- stimulated wild-type males to perform the same kind duce progeny when kept with virgin females for an of vigorouscourtship that virgin females or newly extendedperiod, even though their reproductive emerged malesinduce (Table 2, “Courtshipstimu- tracts appear normal upon dissection and the testes lus”). The wild-type males often courtedfrulfru males seem to have normal numbers of motile sperm (HALL vigorously, with frequent bouts ofwing vibration and 197th).This result hasbeen confirmed (Table 2, attempts at copulation;this is evidenced by the mean “Fertility fraction”) with males from a fru isochromo- CI of 28%. Thus, in a hypothetical 8-min observation fruitless Courtship 777

". ". . period, a wild-type male would court a frulfru male for about 2 min. By comparison, anesthetized wild- type nules stimulated much less courtship, with short 900-F bouts ofwing vibration, and only rare attempts at copulation. With a mean CI of 496, a wild-type male would court another wild-type male for about 20 sec 4 I of ;I hypothetical 8 min observation period. There is a dominant effect of the fru chromosome on this aspect of the fru phenotype; fru/+ males stimulated wild-type males to display a level of courtship (Table 2) intermediate to, and significantly different from (P < 0.001, t-test matrix), that elicited by frulfru and wild type. The fruitless phenotypes are associated with an 918' 91A inversion: The phenotypes described above were 4 found in meiotic mapping crosses to be inseparable A fronl the marker sr (3, 62.0) and were uncovered by Df(3R)P14 (HALL 1978a). Since this deficiency also uncovers ST, these results are mutually consistent and indicated that the fru phenotypes map within the cytological interval 90C2-D 1 to 9 1 A2-3 [but see Table 1 of the current report for a new placement of the Df(3K)P14distal breakpoint to 91B1-2 (K. MOW$, personal con~munication)]. Making use of sr and the flanking markers b~~~~(3, 58.8) and gl (3, 63.1), we found that recombination of the fru chromosonle within the sr gl interval was completely suppressed(O/10,038, us. 47/3 143 in con- trols) and that recombination in the 6x sr interval was reduced (277/10,038, us. 104/3 143), suggesting the presence of ;I chromosomal aberration. The salivary gland chromosonles of fru/+ larvae showed that the fru chromosome contains an apparent inversion, with B 91A breakpoints at 9OC and 9 1 B (Figure 3A). The cyto- genetic interval 90D to 9 1 B forms a distinctive band- FIGLIRF.J.-l'olytenc cIIrotIIosoIIw 1)rcpara~iomol.frtr-l~e;~ritlg ing pattern,and in the fru/fru homozygote appears third itIst;tr I;~rv;~e.A, fru/+. Labels indicate the 9OC-9 11% interval intact-but inverted (Figure 3B). The two inversion of 3K. The upper chrolnatitl shows the wild-tvpe hand secpence; lower cllromatid carriesfru and implies the inverted b;lnd sequencc breakpoints on thefru chromosome flank the sr and ;IS indicated. H,fru/'ru. p indicates the position of thcfru prosinlal gl loci, which,by other cytological and genetic criteria, brcakpoint: d indicates the position of thefrzr distal breakpoint. are within 90E-F and 9 1 A 1-3, respectively (K. MOSES, personal communication; cf: COSTELLOand WYMAN located at or near eitherfru breakpoint and be uncov- 1986). ered by this deletion. It is plausible that the pheno- Note that the following terminologywill be adopted types associated with this genetic variant stem inde- from this point on: at its first mention an aberration pendently from two or more damaged loci; thus the will be listed by complete name, e.g., Df(3R)P14.It fru behavioral abnormalities could map differentially will subsequentlybe referred to by an abbreviated to onefru breakpoint or the other but "co-map" when name, in this case, PI4 (cf: Table 1, Figure 2). fru is heterozygous with P14. The collection of chro- The Fact that all threefru phenotypes mapped very mosomal aberrations in Figure 1 partitions the P14- close to sr and were uncovered by PI4 led HALL defined interval and allowed us to test whether fru (197th) to the provisionalconclusion that fru is a phenotypes map specifically to different, but nearby single-gene variant. The current finding that thefru regions of the third chromosome. phenotypes are associated with an inversion calls this Deficiency mapping of the fruitless phenotypes: supposition into question. Since the inversion break- We mapped the threefru phenotypes by assessing the points of thefru chromosome and the breakpoints of behavior of Aberrationlfru males. Aberration/+ PI4 roughly coincide, it is possible that a damaged males served as controls. The results were that fru gene relevant to a particularfru phenotype could be sterility and courtship chain behavior map at region 778 D. A. Gailey and J. C. Hall

TABLE 3 Cytogenetic localization of thefruitless phenotypes

Fertility fraction” Chain behavior” Chain fraction” Fertility Courtship ~tirnulus“~~ (CI * SEM) Aberration AberrationlfruAberrationlfruAberration/+AberrationAberrationlfruAberration/+Aberration/+

P(w+)ARO-f 48/50 50/50 6/10 3+1 7fl Df(?R)DG2 0/50 41/50 3/5 48 f 6 21 f4 Df(?R)DG? 39/50 39/50 0/5 41 f5 13+3 Df(3R)P14 0/50 50/50 5/5 24 f 4 11 f3 Df(?R)glEXf 0 0/50 50/50 5/5 12f 3 4fl Df(3R)gl+EX6 50/50 50/50 0/5 15f 2 4fl Df(3R)DGI 0/50 50/50 5/5 19% 3 16f4 Df(3R)DG4 47/50 46/50 0/5 15f3 6+2 Df(3R)glEXf 0/50 50/50 5/5 321 4+1 Df(3R)DG5 0/50 50/50 5/5 4fl 3fl Df(3R)Cha” 0/50 50/50 5/5 521 4f1 Df(3R)Cha” 0/50 50/50 5/5 6f2 3fl Df(3R)ChaM5 0/50 50/50 5/5 3&1 4fl Df(3R)gl+EX5 50/50 50/50 4/5 521 2fl Df(3R)lI8.5-1 50/50 50/50 0/5 321 2fl Dp(3R)DG 48/50 50/50 0/5 9f2 21 f4

a See footnotes b, c, and d, Table 2. * n = 20 observations per genotype.

9 1 B (Table 3), the site of the distal breakpoint on the ARO-llfru males showed normal fertility,they formed fru chromosome (Figure 3). A genetic factor respon- courtship chains when in groups (Table3). These two sible forfru courtship stimulatingcapacity clearly does genotypes representthe only two in Table 3 that not map to this distal site (Table 3) but may be located functionally “uncouple” fru sterility and courtship within a cytogenetic interval that includes the proxi- chain behavior. Such results provide evidence in sup- mal fru inversion breakpoint at region 9OC. Pertinent port of the existence of a ‘Ifru chain factor,” which details of these results follow. may not be the selfsame element as that which causes fru sterility maps at or near the 91A-B boundary: The fru sterility. The reciprocal separation of these two Df (3R)gl+BXSchromosome complementsfru sterility, fru phenotypes-behaviorally sterile males which do in that all gl+BXS/fru males tested were fertile (Table not form courtship chains-was not observed for any 3). This chromosome supplies sufficientfru+ function genotypic combination. to give wild-type fertility and places a hypothetical Df(?R)148.5-l/fru males did not formchains (Table “fru sterility factor” to theleft of the gl+BX5 chromo- 3). This observation places the putative “fru chain some’s lefthand(proximal) breakpoint at 91B1-2. factor” at a site proximal to the lefthand breakpoint Df(3R)DGJ uncovers the lefthand fru breakpoint at of this deficiency chromosome. 9OC but not the righthand breakpoint at 91B. DG?/ fru courtship stimulation factor does not map to the fru males are as fertile as their DC3/+ counterparts 91A-Bboundary: We first had to considerthat the (Table 3), and this deficiency serves as a control to deficiency mapping offru courtshipstimulation would indicate that no separate, or interacting, “fru sterility be complicated by the fact that fru/+ heterozygotes factor” maps tothe lefthand fru breakpoint. [We elicit an intermediate level of courtship, i.e., between attribute theapparent partial sterility in DG?/fru the high level stimulated byfrulfru homozygotes and males togeneral debilitation as a result of severe the low level by wild-type controls (Table 2). Thus, in aneuploidy;compare fertility fractions of DG?/fru, a single-factor model for thisfru phenotype, we pre- DC3/+, and Df(3R)DG2/+ males (Table 3).] All re- dicted that Df/fru males in which the deficiency fails maining deficiencies with one breakpoint at the 9 1A- to uncover the fru defect would generate an inter- B boundary, when heterozygous with the fru chro- mediate CI as the baseline control level of courtship mosome, resulted in complete male sterility (Figure 1, (similar to fru/+, Table 2); and that a 0flf.u combi- Table 3). nation in which the deficiency does uncover thefru fru courtship chain behavior maps to the 9IA-B bound- defect would generate a high CI (similar tofrulfru, ary: Application of the deletions to the localization of Table 2). But this prediction was not met: The “Court- this phenotype led to conclusions similar to those from ship stimulus” column in Table 3 (“Aberrationlfru”) the sterility mapping crosses. There were, however, reveals a range of CIS from very low [e.g., CI = 3% important exceptions. Although gl+BXS/fru and for Df(?R)glBXl/fru], to very high (e.g., CI = 48% fruitless Courtship 779 for DG2/fru). As a visual aid, the shading ofdeficien- point itself reduces or removes the expression of a ciesin Figure 1 indicates the CI range inwhich a hypothetical “jru courtship stimulation factor,” then particular “Aberrationlfru” genotypefell (CJ:Table 3; these two Of/+ combinations could be the equivalent notethat Of/+ controls are discussed as a group, of fru/+, with similarly reduced fru+ function from below). The white background bars indicate the CI this locus and resultant courtship-stimulating pheno- range 0- lo%, light gray 1 1-24%, and dark gray types. >24%. Two Df/fru combinations elicited more court- This same argument can be applied to athird ship than did frulfru males, and seven 0flf.u combi- genotype, P14/+; this was the deletion first used to nations were courted only at a wild-type-likelevel uncover fru-associated courtshipstimulation (HALL (compare Tables 2and 3). Nevertheless, the following 1978a). The lefthand breakpoint in P14, which maps points are reasonably clear. within region 9OC, could reduce or remove the DGB/fru and DG3/fru males stimulated the highest expression of the fru stimulation locus on the PI4 levels of courtship, roughly twice that associated with chromosome and also result in intermediate courtship the next highest combination, Plllfru. Both of these stimulation when heterozygous with wild type. DG deficiencies uncover the proximal fru breakpoint The fourth genotype, Dp(3R)DG/+, produces flies (Figure 1). The DG3 chromosome does not uncover that arehyperploid (+/+/+) for the90A-90F interval. the distalfru breakpoint;this provides strongevidence Males of this genotype stimulated afrulfru-like level that fru courtship stimulation does notexclusively map of courtship; conversely, DpDGlfrumales (+/+/fru to thesame region asfru sterility plus courtship chain for the 90A-90F interval) were like wild type in that behavior. That is, stimulation maps within the region they elicited almost no courtship (Table 3). In terms defined by the breakpoints ofDG3, which did not offru+ function at the hypothetical stimulation locus, uncover fru sterility or chain behavior (Table 3). DpDG/fru males are expected to be wild-type for this Of the Df/fru genotypes that yielded intermediate character if fru is severely hypomorphic. DpDC/+ CI values (light gray bars,Figure l), three of the males maybe hyperploid for thestimulation locus and deficiencies have proximal breakpoints that map near thus have an elevatedlevel offru+ function. Deviation the fru proximal breakpoint.P14/fru males stimulated from the wild-type level of expression at this locus- the highest CI of the intermediate group(CI = 24%), whetheran increase (e.g., DpDC/+), or adecrease a value similar to the CI stimulated by fru/fru males (e.g.,DG3/+, or DG3/fru)-results in males that elicit (CI = 28%; compare Tables2 and 3). inappropriately high levels of courtship. Eight deficiencies used in this analysis containa The final genotype of this group, which yielded a breakpointat, or completely remove,region 91B CI > lo%, is Df(3R)DGI/+. This deletion does not (near thefru distal breakpoint) and leave intact chro- uncover the leftl-iandfi~~breakpoint (Figure 1); the mosomal material at 9OC (at thefru proximal break- high stimulation CI (Table 3) was unexpected (note point). Seven of these eight deficiencies, when heter- also that the same value was determined for DGl/fru ozygous with fru, produced males that unexpectedly as for DGI/+). stimulateda low, wild-type level of courtship (see Effects of a transposon at 91B1-2:The P(w+)ARO-I Figure 1, deficiencies designated by open bars). The factor is a w+-marked transposon inserted at 91B1-2 combination ARO-l/fru also falls within this group (CI (Table 1). The balanced stock of this variant contains = 3%; Table 3). The obvious common feature of this only the single insert at 91B1-2 (K. MOSES, personal group is that none of these genotypes leads to the communication); the chromosome bearing ARO-I was level of courtship expected for fru/fru; this again is semilethal leading to viability levels that were only ca. strong evidence that a putativefru courtshipstimulat- 5% of normal. By extracting potentially recombinant ing factor does not mapto thedistal fru breakpoint at chromosomes from ARO-I/ARO-I mothers, eight iso- region 9 1B. chromosomal sublines were established in which the Aberration/+ controls may define a region of dos- viability was increased to about 20%; one line was age sensitivity for the fru courtship stimulation phe- arbitrarily chosen for assessment of fru phenotypes notype. Five of these control combinations led to CI and subsequent crosses involving ARO-I. values >lo%, whereas the remainingcombinations ARO-I/ARO-I males from this line appear normal gave low levels of stimulation, close to that of wild- in size and mobility when compared with their balan- type males (Table 2 vs. Table 3, “Aberration/+”). cer-bearing siblings. When observed in groups their Two of these genotypesare DG2/+ and DG3/+, which behavior was striking: ARO-I/ARO-I males exhibited remove region 9OC, the site of the lefthand fru break- fru chain behavior (line 1, Table 4) that qualitatively point. Such males are hemizygous forthe normal appeared as robust as that of fru/fru males. ARO-I/ geneticmaterial at 9OC and elicit anintermediate ARO-Imales were not, however, completely sterile level of courtship, roughly the sameas that forfru/+ (line 1, Table 4). To determine whether this semister- (compare Tables 2 and 3). If this proximal fru break- ility was fru-like andthus behavioral, 20 ARO-I/ 780 D. A. Gailey and J. C. Hall

TABLE 4 By comparison, ARO-I/+ males never formed court- ftuitless phenotypes analyzed in males expressing deletion and ship chains and were fully fertile (Table 3). transposon genotypes Taken together, the preceding results are further evidence that fru sterility and chain behavior are Courtship stimulus associated with the fru distal breakpoint. On the other (CI k SEM)~,‘ Viability FertilityChain __ hand, ARO-IIARO-1, ARO-I/+, and ARO-l/fru males (;?notype (%)” fractionb behavior’ <6-hr male .!-day male stimulate very little courtship in wild-type males(com- AKO-Z/~4RO-l 19 11/50 10/10 43 + 8 8 & 1 pare“Courtship Stimulus,” line 1, Tables 3 and 4, Cha “’/P I 4 60 0125 515 50 f 8 2 & 1 with Table 2) and serve as evidence that fru courtship O~ha’”7/D(;2d 42 0125 315 37f 9 5 & 1 stimulation is not associated with the fru distal break- (:ha“’S/ARO-l 83 0125 415 29 & 7 3 & 1 point. gl+UX5/P I4 83 25/25 015 34 f 9 4 f 1 Deficiency and transposon combinations that re- gl+13X5/DC2d 27 0125 215 30 _t 5 4 f 1 sult in sterility and courtship chain behavior: Of the gl+BX5/ARO-I 25/25 75 015 31 f 6 3 + 1 seven deletions that contain a breakpoint at the 91A- 148.5-l/P14 25/25 25 015 34+6 2 f 1 B boundary, five failed to complement bothfru steril- 14N.5-l/DC2d 75 0125 11.5 36 6 4 1 f + ity and chain behavior (Figure 1) andthus could 14K.5-l/ARO-I 86 25/25 015 47 + 7 5 f 1 remove or damage the fru 91B factor. If this locus is Expressed as the fraction “observed/expected (X100)” of prog- nonvital, males heterozygous for two deficiencies, or eny flies bearing both specified aberrations (ie., “genotype”) from the generalized cross: DfllBal X Df2/Bal.More than 500 progeny a deficiency and ARO-1, would survive and be fru were scored from each cross. mutants. This in is fact thephenotype of See footnotes b. c, and d, Table 2. l‘he amount of courtship wild-type males displayed with either Df (?R)ChaM’/P14 and C~U“~/ARO-Imales: They newly emerged or 5-day-old males of the specified aberration gen- were behaviorally sterile and formed courtship chains oty e 1z - 10 observations each group. while obviously not carrying the fru inversion (Table ’ill cLosses involving the Df(3R)DG2 chromosome yielded flies which were small and sluggish; the assessment of these genotypes 4). as causingfru phenotypes is thus tenuous (see text). The results of testing the remaining deletionltran- sposon combinations for viability and fru phenotypes ARO-1 males were placed individually with virgin fe- were extensive and complex. A review of the seven males and scanned for a l-hr period for occurrences deficiencies andtheir interactions with fru should of courtship behavior: 10 males courted persistently increase the comprehensibility of the behavioral and but did not curl their abdomensto attempt copulation; viability data to be described in this section. The DG2, 6 males failed to court; 4 males copulated and were P14, Df(?R)glBXlO, glBXl, and ChaM5deletions un- the only males which produced progeny after all 20 cover both fru sterility and courtship chain behavior; had been kept with females for a week. gl+BX5 uncovers only the fru courtship chain pheno- These results and observations imply that ARO-1 is type; 148.5-1 uncovers neither fru phenotype (Figure hypomorphic for fru+ function(s) at 91B, leading to 1, and see above, Table 3). incomplete penetrance offru-like sterility. The follow- Behavioral analyses of malesfrom crosses that com- ing results further support such a notion. glBXl is a plemented for viability are in Table 4 and are sche- small deficiency which uncovers both fru sterility and matically summarized in Figure 4. Also see Figure 5, chain behavior (Table 3).The glBXl/ARO-1 genotype which is a model of the functional placement of break- is poorly viable (5%)but superficially indistinguishable points at 9 1 B based on the results of all complemen- from ARO-1IARO-1 in appearance. Two groups of 10 tation tests-both for viability, and fru sterility plus glBXl/ARO-1 males showed frequent instances of chain behavior. chain behavior in a 30-min observation period. Four- As alreadymentioned, P14/ChaM5 is not only a teen of these 20 males courted females with no ob- viable combination, but males of this genotype are served curling in a l-hr period. All 20 males were still also behaviorally sterile and court each other vigor- alive after a week with females; none produced prog- ously in courtship chains (Figure 4, Table 4). We eny although all malescontained motile sperm in their conclude that each of these deficiencies removes the testes. This genotype is the first of several we shall fru 9 1B factor, or damages it by chromosomal break- present in which males fully express fru sterility and age within the locus (Figure 5). chain behavior in the absence of the fru inversion. The combination P14/gl+BX5 also allows for viabil- Heterozygous ARO-l/fru males also perform char- ity, but males of this genotype are behaviorally wild acteristic fru chain behavior, although notas intensely type (Figure 4, Table 4), suggesting that this pair of as either frulfru or ARO-l/ARO-1 males (four of 10 breakpoints does not overlap (Figure 5), or at least ARO-l/fru groups performed no chain behavior in a that they do not each remove or completely inactivate 30-nlin observation period; line 1, Table 3); but ARO-1/ fru+ function(s). fru rnales are essentially normal in fertility (Table 3). glCBX5 could define the distal limit of a putative fruitless Courtship 78 1

ChaMS gl+BXS 148.5-1 AROl jiu 9lA 91B 91c

P14

DG2

gZBxl0

glBXl

AR 0-1

l~A \ AROl fru FIGURE5.-Functional positions of fru-associated breakpoints. llle locations of theselesions are predicted by theoutcome of crosses sunlnlarizecl in Figure 4. “lethal” indicates a predicted vital locus to the right offru: “spmilefhal” indicates ;I predicted locus to = no courtship chains: fertile + the left offru which affects viability. An arrow that passes through = courtship chains & a locusindicates a loss of functionfor that locus on the named F = fde: F- = semt-fde chromosonle. An arrowstopping “within“ a locusimplies hvpo- S = sterile morphic expression [e.g., the lethal factor and Df(3R)I48.5-I].lhe (7) = equivocal display of chain gray arrow (bottom) suggestsa spreading effect associated with the formation behavior ARO-I insertion (see text). FIGURI.:4.--Sunlnl;1ry of complementationcrosses involving phenotypic assessment of viable combinations with chronlosomes with breakpoints at or near 9 1 B (cf: Table 4, andsee DG2. Although DG2 is a very large deficiency, it was text). like PI4 in that viable “double-deficiency” flies were produced whenit was heterozygous with either Cha”’, “frusterility factor,”in that gl+BX5lfru males are fully gl+BX5, or 148.5-1 (Figure 4). Whereas combinations fertile vet form courtship chains (Figure 1, Table 3). with PI4 led to vigorous,normal-looking flies, the This genotypeis special in that it is the only deficiency three viable genotypes involvingDG2 caused adults to which uncoupled thefru sterility and chain behavior phenotypes. The gl+BX5 chromosome mightbe inter- emerge late and be small and lethargic. These debili- preted asweakly expressing, or hypomorphic for,fru+ tationscould easily obscurethe assessment of fru function(s). The relevant breakpoint of this deletion phenotypes, and thus a question mark is attached to is represented as impinging on thefru locus from the each of the genotypesin Figure 4. However, males of right (Figure 5), i.e., from a location distal to thefru each DG2-bearing combination were sterile and met lesion in 9 1B. In combinationwith a chromosome that the minimalbehaviorally mutant criterion of four presumablyremoves fru+function(s) such as DG2, males courting in a chain in at least one trial (Table gl+BX5 would provide insufficient fru+ function(s) to 4). The inferred removal by DG2 of most, if not all rescue either courtship abnormality. Thisis consistent fru+ function(s) at 91 B leads to its functional break- with the observations that DG2/gl+BX5 males are ster- point placement ofDG2 in Figure 5. ile and form chains (Figure4, Table 4), but thatP14/ Any combination of either glBXl or glBXI0 with gl+BX5 males are behaviorally wild type (see above either Chd”, gl+BX5, or 148.5-1 led to lethality (Fig- and Figure 5). This latter combination leads to the ure 4). This suggests that glBXl and glBXl0 remove supposition that PI4 is also weakly hypomorphic (and not onlyfru+ function(s) but also inactivateor remove impinges on the fru locus from the left; Figure 5), ahypothetical vital locuslying to the right of fru providing a higher level offru+ function(s) than DG2. which is also inactivated by Cha“’, gl+BX5, and 148.5- 148.5-1 seems to set the righthand boundary of the I (Figure 5). fru 91B factor, in that 148.5-l/P14 males are com- In a series of further crosses involving ARO-I, this pletely wild type in behavior (Table 4, Figure 4). This chromosome produced the same “complementation does not, however, rule out the possibility that 148.5-1 pattern” with ChaM5, gl+BX5,and 148.5-1 as did PI4 is also weakly hypomorphic for fru+ function(s) and (Figure 4, Table 4). ARO-I/ChaM5and P14/Ch~“~ that this genotype, in terms offru+ expression, could males were essentially indistinguishableon all criteria: be the functionalwild-type equivalent of the P14/ they were completely sterile, and, by general visual gl+BX5 genotype. This possibility is suggested by the inspection,both were vigorous in chainformation. 782 D. A. Gailey and J. C. Hall

Because its function in these crosses is analogous to as do newly emerged wild-type males (see, for exam- PI4, ARO-I, too, could be hypomorphic forfru+ func- ple, JALLONand HOTTA1979). tion(s) (see Figure 5, and also previous section on ARO-I). An important distinction is that P14/fru DISCUSSION males are completely sterile whereas ARO-llfru males are virtually always fertile (Figure 4); both genotypes fru, a 9OC factor us. a 91B factor?fruitless is a small result in males that form courtship chains (Figure 4). inversion with breakpoints at 9OC and 91B. This has Comparing these genotypes, ARO-I could supply suf- allowed us to test whether three phenotypes associated with this mutant of reproductive behavior map differ- ficiently more fru+ function(s)to complement sterility. entially to one frubreakpoint or the other. Results of Of thecrosses involving ARO-I and deficiencies that thesemapping experiments indicated thatthe fru extend proximally from 91B, only ARO-l/glBXI is a phenotypes do not have the same genetic etiology. viable combination. Although viability is poor (5%), Behavioral analysis for many combinations of thefru males of this genotype are behaviorally sterile and chromosome with deficiencies that partition the 9OC- form courtship chains (Figure 4, and a previous sec- 91B interval showed consistently that fru sterility, tion). It was observed in the culture vials that many courtship chain behavior, and thefru distal breakpoint ARO-l/P14 and ARO-l/glBXI flies developed fully, co-map to region 91B. fru courtship stimulation pro- but died in the pupal case. Five pharate adult males visionally maps proximally in region 89F-90F, as de- of each genotype were removed from theirpupal cases fined by the breakpoints of the DG3 deletion. Thefru while still living; all died within 24 hr. To account for proximal breakpoint is within this interval, and thus this, we propose that a locus involved in viability is could be the factor causing inappropriately high levels placed tothe left of thefru 91B factor, and that of courtship elicitation. expression at this locus is diminished through some Such an etiology for a mutant with “multiple phe- sort of spreading effect from the site of ARO-I inser- notypes” has been previously documented. The ocel- tion (Figure 5). This insertion cannot impinge on the liless (oc, I, 23.1) variant was induced with X-rays and hypothetical vital locusplaced to the rightof fru, since both of its phenotypes-female sterility and absence flies carrying ARO-l-heterozygous with ChaM5, of simple eyes-map by recombination to the same glfBX5, or 148.5-I-had normal viability (Table 3). location (LINDSLEYand GRELL1968). Ina manner In summary,the eventual phenotype of a viable analogous to fru, oc is associated with a small inversion deficiency combination, or a transposon/deficiency (SPRADLINCand MAHOWALD1981). Its distal break- combination, might be viewed as its relative sum of point (located at 7F1-2) is 1-3 kb upstream from two f~u’ functionts). The following hierarchy of expres- chorion structural genes and disrupts their sion is suggested for individual chromosomes: tran- expression; the 7F material now moved to SA (ie., sposon and proximally extending deficiencies, ARO-I near the proximal breakpoint) results in overexpres- > P14 > DG2; distally extending deficiencies, 148.5-1 sion of sequences at 8A in oc follicle cells (SPRADLING > gl+BX5 > Cha’”5. In none of these viable combina- and MAHOWALD1981). In this case, the female steril- tions were fru sterility and fru chain behavior func- ity phenotype at least has a simpler etiology, which can be explained solely by the drastic reduction in tionally separated. chorion . Deficiency/transposon combinations and court- Certain key examples reinforce the separability of ship stimulation: It is evidentfrom the previous fru-relatedcourtship anomalies. (1) ChaM5/ARO-I sections that two of the fm mutant behaviors-behav- males stimulate virtually no courtship and are thus ioral sterility and chain formation-are not merely wild type for thisfru phenotype; they also contain two peculiar phenomena specific tothe fru inversion. doses of third chromosome material at 9OC (89F-90F Thesephenotypes arereproduced in the males of inclusive). However, these males are completely sterile certain combinations of ARO-I and/or deficiencies and form courtship chains; their mutant behavior, the withlesions in region 91B. The results in Table 4 site of ARO-1 insertion, and thelefthand ChaM5break- (“C.ourtship stimulus,” 5-day male) were that none of point are located in region 91B. (2) The “reciprocal these genotypes stimulated visibly morecourtship uncoupling” of the fru phenotypesis demonstrated by than wild-type; no CI value was >lo%. These results males of the genotypes DG3/+ and DpDG/+. These are consistent with the observation that ARO-I/ males have an altered dosage of genetic material that ARO-I males stimulate very little courtship (Table 4), includes region 9OC, and they stimulated abnormally and reinforce the conclusion that the fm courtship high amounts of courtship; they contain the normal stimulation factor does not map to region 91B. dosage of material in region 91B, are fertile, and do As newly emerged males, however, all double-dele- not form courtship chains. On the strength of break- tion and ARO-I/Df males stimulated high levelsof point cytology,fru sterility and courtship chain behav- courtship (Table4, “Courtship stimulus,” <6-hrmale), ior map at 91B1-2. Courtship stimulation is less well- fruitless Courtship fruitless 783 defined by deficiency breakpoints but seems to map J.-M. JALLON, unpublished observations). In normal to the region 89F-90F. Because thefru breakpoints D. melanogaster males this compound is an abundant map very near or within these regions we restate the (ANTONYand JALLON1982), and possibly courtship general proposal introduced above: thefru syndrome inhibitory (SCOTT1986), cuticular pheromone. Gen- is a collection of phenotypes induced independently erally, the enzymatic basis of pheromone production at the two breakpoints. fru courtship stimulation is and regulation in this species is poorly understood, attributed todisruption of a locus at 9OC; sterility and and its genetic regulation seems very complex (SCOTT courtship chain behavior are the result of disruption and RICHMOND1988). However, it could be useful to of a locus-or loci-at 9 1B. look into the putatively abnormal biochemistry asso- HALL(1978a) noted aberrant wing extension be- ciated with “fruitless stimulation” and attempt to de- havior infrulfru males. This has been further inves- termine how it might lead to a pheromonal change of tigated in the analysis of courtship wing vibrations by this kind. males in which one or the other fru breakpoint was fm and the 91B factor-one gene or more? The uncovered. fru, and more specifically the distal fru answer to this cannot be discerned from the results breakpoint, was found to cause substantially longer we presenthere. fru sterility and chain formation than normal silence intervals between pulses of tone almost always go together (Figures 1 and4). But males (WHEELERet al. 1989). Thus,fru involves yet another of two genotypes--gl+BX5/Zn(3R)fru and ARO-11 element of defective courtship behavior. Zn(3R)fru”show only the courtship chain phenotype; fm terminology: Our findings also suggest the in- they are fertile (Figure 4). On the other hand, fru troduction of a new and perhaps moreprecise “fruit- sterility was always coincident with courtship chain less lexicon.” Since it contains an inversion, we desig- behavior. These observations do not yet allow a deci- natethe original fru-containing chromosome as sion on whetherfru is one gene or two. Zn(3R)fru. The designation ‘yruitless” was reported As a working model, we propose that P14and ChaM5 by HALL(1978a)“with permission from K. S. GILL- both contain a breakpoint in fru. Whether heterozy- to supplant the pejorative designation “fruity” (GILL gous with Zn(3R)fru or each other, these deletions led 1963a, b). We think thatfruitless (fru)is a meaningful to both 91B-associated mutant phenotypes (Figure4). designation for the “91B factor”; we propose to re- This was also the case with the ChaM5/ARO-lcombi- serve this name for the gene (or genes) which reside nation (Figure 4). In this context the demonstration at this locus, whose mutant expression results (result) of 91Bphenotypes in double-deletion males is an in male behavioral sterility and theformation of male important new observation: It leads to the prediction courtship chains. The courtship stimulation pheno- that both deletions are amorphic for 9 1 fru+B func- type, however, appears to have an independent ge- tion, which certainly would be the case if their break- netic etiology, although the phenotype is in a sense pointsoverlap within fru. Since the resultant91B related to “fruitless courtship,” in that one male is phenotypes are thesame as in Zn(3R)fru homozygotes, courting another. The details of this stimulation phe- the distal inversion breakpoint may also be anamorph notype appear so different from the otherphenotypes (as opposed to thetype of genotypic change associated that we feel the elicitation factor, presumably in 9OC, with one of the ocelliless inversion breakpoints, see should at present be referred to with the phenotypic above). A corollary is that fru will turn out to be a designation “fruitless stimulation,” without, however, simple, single gene, as opposed tothe phenotypic invoking a formal designation for the specifically rel- abnormalities being somehow caused by the gratui- evant locus (if any). tous neomorphic juxtapositionof two usually separate The 9OC factor: This factor remainspoorly defined regions within chromosome 3. genetically; a future analysis of its mutant phenotype If frulfru, or ananalogous DflDf case, is truly must include new deficiencies and duplications that amorphic, fru would be a non-vital gene that influ- partition the 89F-90F interval. In addition, we have, ences behavior-ofwhich thereare several in D. in collaboration with J.-M. JALLON,begun to analyze melanoguster (see Discussion in KULKARNIand HALL the pheromone profiles of Zn(3R)fru-bearing flies, in 1987). Consistent with this notion is the observation an attempt touncover a specific chemical correlate to that Zn(3R)fru has never led to lethality when heter- the mutant courtshipstimulation (6TOMPKINS, HALL ozygous with any deficiency. In this respect,fru could and HALL1980). Our preliminary experiments reveal be like per (SMITHand KONOPKA 1981) and dunce neither missing nor novel compounds in Zn(3R)frul (BYERS,DAVIS and KICER 1981);these genes have Zn(3R)fi-u males, either when newly emerged, or 5 been deleted by the appropriate Df/Df combinations, days post-emergence. We have consistently seen, how- yielding adults which have good viability but exhibit ever, a reduction inlevels of amale-predominant severe behavioral defects. compound,7-tricosene, inZn(3R)fru homozygous Ultimately, a molecular analysis could lead to sev- males and in DG3/Zn(3R)fru males (D. A. GAILEYand eral pieces of new information aboutfru. This should 784 D. A. Gailey and J. C. Hall

be possible in the near future, since the ARO-1 tran- induced aberrations. We thanL him, as well, for helpful discussions. sposon is very near to (possibly at) the 91Bfru locus. We appreciate technical assistance fi-on) B. ORIEL,hl. DOURSOUN- In fact, K. MOSES and G. M. RUBIN(personal corn- IAN, K. DESFOSSESand A. ARONSKY; and co~ntllents011 the III;III~~- script from 1’. MERRIIL, K. KFNDAHI. and S. RORINOW.Frllis ~vork munication) are already investigating cloned material was supported bv ;I grant fronl the National Institute of Healtll involving this element in their efforts to isolate and (GM-21473) and a Neurobiology Training grant (T32 NS07292) manipulate theglass gene(cf: Table 1). If this material from theNational Institute of Neurological and Collr~llunicative also leads to the cloning and molecular identification Disorders and Stroke. offru, the following sorts of questions can be asked experimentally: Is DNA in fact missing from the pu- LITERATURECITED tatively overlapping-Dfcombinations that lead to ste- rility and chain behavior (cf: molecular proofs of the ANTONY,C., and J.”. JALLON, 1982 The chemical t,asis for- sex per- genotypes, BARCIELLOand YOUNG 1984; REDDY recognition in Drosophilamelanogaster. J. Insect Physiol. 28: et al. 1984)? Will one “simple transcription unit” in 873-880. BARGIELLO,T. A,. and M. W.YOUNG, 1984 Molec~~l;~rgenetics 91 B turn out to encode the function that influences ofa biological clock in Drosophila. Proc. Natl. Acad. Sci. USA these features of normal Drosophila behavior, 81: 2 142-2 146. whereby males do not court other males but vigor- BASTOCK,M., and A. MANNING,1955 Courtship of Drosophila ously court and readily mate with females? melanogaster. Behaviour 8: 85-1 I I. Since the ARO-1 transposon seems near fru (cf: BYERS,D., R. L. DAVISand J. A. KIGER. I981Ikfect in cyclic- AMP pllosphodiesterase due to the dunce mutation of learning Table 3 and 4),valuable information about thegenetic in . Nature 289: 79-8 1. organization offru might be obtained by mobilizing COSTELLO,W. J.,and R. Y. WYMAN,1986 Developrllent of a11 this transposon and selecting for imprecise excisions indirect flight muscle in a muscle-specific mutant of Drosophiln (ROBERTSONet al. 1988) which could remove third melanogaster. Dev. Biol. 118: 247-258. chromosome material nearthe insertion site. Such CRAYMER,I.., 1984Keport. Drosophila Inform. Serv. 60: 234. EWINC.,A. W., 1977 Colllmunicatiorr in Iliptera, 11;). 403-417 in small deletions could potentially give further infor- How AnimalsCommunicate, edited by 1’. A. SEREOK.Indiana mation on the genetic organization of fru and the University Press, Bloonrington. predictednearby vital and “semilethal” loci (Figure EWING,A. W,, 1983 Functionalaspects of Drosophila courtship. 5). Biol. Rev. 58: 275-292. That fru males do not curl their abdomens in at- GAILEY,D. A. F. R. JACKSON and K. W. SIEGEI., 1982 Male courtship in Drosophila: the conditioned response to inlmlture tempts at copulation implies that there is a blockade males and its genetic control. Genetics 102: 771-782. in the anatomical/physiological “pathway” underlying GAILEY, D. A., R. C. LACAILLADE:and J. <:. HALL, this behavior. This fru defect could be muscle- or 1986Cl~e~nosensory elements of courtship in nornral ad nerve-related (cf: LAWRENCEand JOHNSTON 1986). mutant, ol~actioll-deficient Drosophila melanogaster. Behav. <;e- Either of these kinds of tissue etiologies might apply net. 16: 375-405. GAILEY,D. A,, D. L. BORDNE,A. M. VALL~S, J.C. HAIL and K. also to the productionoffru males’ aberrant courtship WHITE, 1987 Construction of an unstable ring-X ch1~0111oSo111e song(WHEELER et al. 1989). This singing defect, hearing the autosomal dopa decal-boxylase gene in Drosophila however, might have a thoracicfocus, with the curling melanogaster and analysis of Ddc mosaics. Genetics 115: 305- inability hypothetically having an abdominal one. The 31 1. GILL,K. S., 1963a A mutation causing abnort~lalmating behavior. tissue affected such that fru males court each other Drosophila Inform. Serv. 38: 33. could be neural and in the fly’s head. GILL,K. S., 1963h A mutation causing abnormalcourtship and Mosaic analysis (reviews: HALL 1978b, 1984)could mating behavior in males of Ilrosophila melanogaster (abstract). yield clues about theanatomical sites involved in these Atn. Zool. 3: 507. different behavioral phenotypes. Since fru is autoso- GORCZYCA,M., and J. C. HAIL, 1984 Identification of a cholit~- ergic synapse in the giant fiber pathway of Drosophila using mal, generation offru//fru+ mosaics would be another conditional mutations ofacetylcholinesynthesis. J. Neurogenet. set of experiments facilitated by the cloning of this 1: 289-3 13. gene. As exemplified by GAILEYet al. (1987), trans- HALL, J.(;., 197821 Courtship among males due to a male-sterile formation involving afru’ DNA fragment into aRing- mutation in Drosophilamelanogaster. Behav. Genet. 8: 12.5- X chromosome would permit the production of high 141. HALL,J. C., 1978b Behavioral analysis in Drosophila nwsaics, pp. frequencies of diplo-X//haplo-X mosaics involving this 259-305 in Genetic Mosaics and Cell Dz;fferentiation, edited by autosomal locus. Problems of sexual dimorphisms W. J. GEHRING. Springer-Verlag.Berlin. would be avoided in fru//fru+mosaics by turning them HALL,]. 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