behavior cytogenetics of fruitless in drosophila ...that disrupt the male-specific behavior of...

Copyright 0 1989 by the Genetics Society of Amerlcd

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

Donald A. Gailey and Jeffrey C. Hall Department of Biology, Brandeis University, Waltham, Massachusetts 02254

Manuscript received September 2 2 , 1988 Accepted for publication December 24, 1988

ABSTRACT Thefruitless ( f r u ) courtship mutant was dissected into three defects of male reproductive behavior,

which were separable as to their genetic etiologies by application of existing and newly induced chromosomal aberrations. f r u itself is a small inversion [ I n ( 3 R ) 9OC; 9 1 B] on genetic and cytological criteria. Uncovering the fru distal breakpoint with deletions usually led to males with two of thefru courtship abnormalities: no copulation attempts with females (hence, behavioral sterility) 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 f r u proximal breakpoint led to males that passively elicit inappropriately high levels of courtship. This elicitation property was separable genetically from the sterility and chain formation phenotypes and provisionally mapped to the interval 89F-90F, which includes the f r u proximal breakpoint. Behavioral sterility and chaining were also observed in males expressing certain abnormal genotypes, independent of thefru inversion. These included combinations of deficiencies, each with a breakpoint in 91B, and a transposon inserted in 91 B.

P RECOPULATORY behaviors are sex-specific components of reproduction. In Drosophila mel-

anogaster, males respond to females with a stereotypic set of actions, including following, production of a courtship song via wing vibration and curling of the abdomen in order to initiate mating (reviews: HALL et al. 1980; EWING 1983; TOMPKINS 1984; HALL 1985). The responses of females to these male behaviors are less overt and undoubtedly involve nervous system- dependent phenomena (e.g., TOMPKINS and HALL 1983). Females perceive male courtship cues that are chemical (reviews: JALLON 1984; TOMPKINS 1984), auditory (e .g . , EWINC 1977; HALL 1984, 1986), and visual (e.g., TOMPKINS 1984; MARKOW 1987) in nature. Receptive females ultimately respond to the cues of conspecific males by slowing their locomotor move- ments, and mating ensues (MARKOW and HANSON 1981; TOMPKINS et al. 1982; GAILEY, LACAILLADE and HAIL 1986).

I t should be possible in Drosophila to “dissect out” genes pertinent to these behavioral dimorphisms. For exanlple, mosaic analysis of precopulatory behavior has led to the identification of thoracic tissues which must be male for copulation attempts to be performed b y gynandromorphs (HOTTA and BENZER 1976; HALL 1979), implying that this behavior is not generated or controlled locally within the male abdomen per se, but probably is subserved by male tissues in the central

Dedicated to R. W. Siege1 and his fruitful career as a geneticist.

(kwt.ti<< 121: i i : i - 7 8 . ~ (April. 1989)

nervous system (CNS). Thus, a search for mutations that disrupt the male-specific behavior of attempted copulation might not only reveal genes that are relevant to, for example, male-specific musculature in the abdomen (cf : LAWRENCE and JOHNSTON 1984), but that also lead to defects within the CNS. A mutant that could be in this category is fruitless ( f r u ) . A cytogenetic analysis offru-associated courtship anomalies is the focus of this report.

f ru is a homozygous-viable, male-sterile mutant that was induced with X-rays (GILL 1963a, b). The effects of f ru seem to be male specific, in that females homozygous for f r u show no obvious phenotypic or behavioral differences from wild type (HALL 1978a). Males homozygous for f ru , however, show at least three obvious deviations from normal male courtship behavior. (1) They do not curl their abdomens at females to attempt copulation, and are thereby behaviorally sterile (HALL 1978a); to this extent f ru could be an example of a gene controlling male-specific actions in courtship. (2) They court otherfru males and normal males (HALL 1978a); this could mean that f ru is relevant to the discrimination by males of appropriate courtship objects, since normal males rarely court each other. (3) They stimulate normal males to court them; consequently, courtship-stimulating cues could emanate from f r u males (HALL 1978a), a hypothesis supported by the results of bioassay experiments in which solvent extracts of f r u males stimulated courtship (TOMPKINS, HALL and HALL 1980).

In the current study we show that the f r u pheno-

774 D. A. Gailey and J. C. Hall

TABLE 1

Chromosomal aberrations used in the cytogenetic localization of thefruitless phenotypes

Aberration genotype" Cvtology Origin Referenceb Marker

Deficiencies Df(3R)P I4

Df(3R)Cha"'

Df(3R)Cha"' Df(3R)Cha"'

Df(3H)148.5-1

Df(3R)DC I Df(3R)DG2 Df(3R)DG3

Df(3R)DCS Df(3R)glBXZ Df(3R)glBXZO Df(3R)gl+BX5 Df(3R)gl+BX6

Marked transposon P(w+)ARO-Z

Df(3R)DG4

Duplication Dp(3R)DGd sr+ gl-

91B;91D 90F:91 F'

91B3;91D1

90E1-2;91C3-7 89E-F;91B1-2 89F;90F 90E1-2;90F3-11 9OE-F;9lE-F 90F8-11;91B1-2 90C7-8;91B1-2

90C9-10:90F2-11 91B1-2;91D1-2

w + inserted at 91B1-2

81-90A;90F-90A; 90A-100

X-ray

X-ray

X-ray y-ray

y-ray

?-ray y-ray 7-y ?-ray w a y X-ray X-ray X-ray X-ray

Transposon mobilization

y-ray

LINDSLEY and ZIMM (1985); K. MOSES (personal

LINDSLEY and ZIMM (1985); K . MOSES (personal

LINDSLEY and ZIMM (1 985) LINDSLEY and ZIMM (1985); K. MOSES (personal

GORCZYCA and HALL (1984); W. M. GELBART

This report This report This report This report This report K. MOSES (personal communication) K. Mosm (personal communication) K. MOSES (personal communication) K. MOSES (personal communication)

LEVIS, HAZELRICC and RUBIN (1 985); K. M o s e s

communication)

communication)

communication)

(personal communication)

(personal communication)

This report

a Presence (+), or absence (-) of normal marker gene function on aberration-bearing chromosome 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 chromosomes 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: GORCZYCA and 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. MOSES that differs slightly from the published breakpoints. Shows n o 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)DC is viable and sr+ gl+ (no other crosses were performed).

types do not all map to a single genetic locus but are associated with a small inversion on the third chromosome. The two mutant behaviors that f ru males themselves display in their courtship co-map with one breakpoint of this inversion; the mutant behavior that f ru males elicit from normal males maps provisionally to the other breakpoint.

MATERIALS AND METHODS

The maintenance of fly cultures and procedures of fly handling for behavioral assays were as detailed in GAILEY, JACKSON and SIEGEL (1 982) and GAILEY, LACAILLADE and HALL (1986). Flies were grown on a medium containing cornmeal, molasses, agar, yeast and the mold-inhibitor Te- gosept .

Fly stocks: Several of the genetic variants used in this analysis appear in LINDSLEY and GRELL (1 968), or LINDSLEY and ZIMM (1 985); others are newly isolated (Table 1).

fruitless ( f r u , 3, 62.0 HALL 1978a)"derived from X-ray mutagenesis (GILL 1963a, b). fru/"ru males express the fruitless syndrome of behavioral defects, f ru l f ru females appear normal (HALL 1978a). Because of the male sterility, f r u stocks are kept with balancer chromosomes (see below). The starting stock for the current analysis was TMb/fru,

maintained in the laboratory of J. C. HALL by mass transfer of unselected adults.

Chromosomal aberrations used to map thefruitless phenotypes are listed in Table 1 and schematically presented in Figure 1. Markers used in mapping by recombination were bithorax ( b ~ ~ ~ ~ , 3, 58.8), stripe (sr, 3, 62.0) and glass, (gl, 3, 63.1).

Balancer chromosomes (Bal) used were In(3LR)TM3 (=TM3), marked with the dominant mutation Stubble ( S b ) ; In(3LR)TM6 (=TM6), marked with the dominant mutation Ultrabithorax (UbxPl5); and In(3LR)TM6B (=TM6B; CRAY- MER 1984), marked with the dominant mutation Tubby (Tb) , which imparts a distinctive body shape in both adults and larvae and facilitated selection of homozygousfru/fru larvae for salivary gland squashes.

P(w+)ARO-I is a transposon containing the white+ (w, I, 1.5) gene, and was derived from mobilization of the transposon P[(w,ry)AR]4-3 and reinsertion at the cytogenetic region 91B1-2 (LEVIS, HAZELRIGG and RUBIN 1985; K. MOSES, personal communication). This transposon is maintained in the stock wl'", TM3/ARO-1, which was provided to us by K. MOSES and G. M . RUBIN.

Screen for new aberrations in the region of sr and gl: Wild-type males from a Canton-S strain received 4.5 kR y- irradiation from a 137Cs source, delivered at the rate of approximately 265 R/min. They were immediately crossed to bx sr gl homozygous females. FI males displaying the sr

fruitless Courtship “I

I In

4” -”)

E F A B C D E F A B C D E F

90 91 I kc

E3 = a I break fiu “g’&

0 0

I. P (w+) A R O - I

FIGURE 1 .-Approximate breakpoint cytology of aberration- bearing chromosomes used in the mapping offru phenotypes (com- pilation of cytology in Table 1 and functional tests for complementation). Background shading categories are based on the CI values generated by Aberration/fru males ( i e . , thefru courtship stimulation phenotype). White background, CI < 10; light gray background, 1 1 < CI < 24: dark gray background. CI > 24. The assessment off” behavioral sterility and courtship chain formation in Abenationlfru males is denoted as follows: black-filled circle = sterility and courtship chain formation; gray-filled circle = fertility and courtship chain formation; no circle = wild tvpe for these two f” phenotypes. See Table 3 and text.

and/or gl phenotype(s) were individually mated to TM6/Sb virgin females, and stocks were established (the TM6 chromosome contains the bx marker and allowed for easy selection against the bx sr gl chromosome in bx S I gl/Aberration FI males). Aberrations were recovered in the approximate frequency of 1/5000 males screened.

Several new aberrations were supplied by K . MOSES (Table 1): these were X-ray induced in males carrying the transposon P[(w,ry)A]2-1 inserted at 91C (HAZELRICG, LEVIS and RURIN 1984) and isolated by monitoring for loss of w+ function (K. MOSES, personal communication).

Cytology: Temporary mounts of chromosomal spreads from the salivary glands of fru/+ and fru/fru larvae were prepared with lacto-aceto-orcein (HUMASON 1979) and in- terpreted with the aid of the revised chromosome map of LEFEVRE (1 976).

Preliminary crosses withfm: Preliminary tests with the TM6/fru starting stock revealed that fru homozygotes appeared at a low frequency. fru/fru males courted but did not copulate and formed courtship chains with each other: thus at least twofru phenotypes persisted in this stock (see methods for behavioral analyses, below). As a test for het- erogeneity of these phenotypes within this stock, 40 isochro- mosomal fru lines were established by standard Bal chromosome technique (20 lines established from TM6/fru males, 20 from TM6/fru females). Homozygousfru males from each line were then tested for these twofru phenotypes. One male-derived line and one female-derived line proved to be homozygous lethal. Crosses of these two lines to a stock carrying Df(3R)P14 (see Table 1) yielded Df/fru males that expressed the sterility and courtship chain phenotypes: thus the lethality in these lines is not fru-associated. In each of the remaining 38 linesfru/fru flies were viable, and males of this genotype displayed the fru phenotypes noted above (none of the 40 lines was tested for the court-

FIGURE 2.-fru courtship chain beh;lvior. T o the left are nine Df(3R)g1RX//fru males (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.

ship stimulation phenotype). One line was selected arbitrarily (a TM6/fru female-derived line) and was the source for fru chromosomes in all further crosses. This stock is maintained as TM3/fru.

Next, ten sublines of this TM3Ifru stock were established from ten individual pairs of TM3/fru flies. These lines were single-pair inbred for seven generations using TM3/fru siblings. One line was homozygous lethal at the third generation. When crossed to the stock carrying Df(3R)P14, the 0flf.u male segregants werefru in phenotype: thus, lethality was again not fru-associated. fru/fru males of the nine remaining lines were sterile and formed courtship chains. The viability of frulfru flies before and after the single-pair inbreedings stayed roughly the same, ca. 25-40%. Crosses of these nine inbred lines with the Df(3R)P 14 stock revealed similar viability for Df/fru flies.

Moreover, in the course of mappingfru by recombination (see RESULTS and cf. HALL 197th) many potentially bx fru chromosomes were formed; two such recombinants were chosen arbitrarily and established as balanced, isochromo- soma1 stocks. Male homozygotes in each line were sterile and formed courtship chains (not tested for the courtship stimulation phenotype).

These pedigrees reveal that fru is stable and is a consistently abnormal behavioral mutant.

Behavioral assays: The following general tests were de- vised to reveal three specificfru phenotypes:

Behavioral sterility: Males whose fertility was to be tested were collected at eclosion (grouped not more than 10 per vial) and aged 5-6 days. Then they were placed individually in food vials each containing four wild-type virgin females, aged 4-6 days. In some cases, vials were scanned at this point for occurrences of courtship. Otherwise, all vials were scored for presence of larval progeny 7 days later. Vials containing a dead male and no progeny were not counted. Such cases in all experiments were rare. The testes of surviving males were dissected out in buffered saline solution (IKEDA and KAPLAN 1972) and in every case contained motile sperm.

Formation of courtship chains: A detailed description of this fru behavior is given in RFSULTS (also see Figure 2). Males to be tested were collected and grouped at eclosion, 10 per vial. On the seventh day, the behavior of males was observed for a 0.5-hr period, usuallv several vials at a time. The minimum criterion for scoring a vial of males as mutant for this behavior was the occurrence of at least one “courtship chain” made up of at least 4 participating males (cf: Figure 2).

Courtship stimulation: Homozygous fru males stimulate wild-type males to court them; this fru phenotype is easiest


TABLE 2

Behavioral phenotypes offruitless males compared with wild type and the fml+ heterozygote

Fertility (knutype” fraction’ behavior‘

Chain Courtship stimulus (CI SEM)d f../f.. 0/100 10/10 28 +- 3*

f ru l+ 100/100 0/10 16 k 2* +/+ 100/100 0/10 4 t I *

a “+” indicates a fru+-bearing chromosome from a Canton-S stock.

Males (5-6 days old) were placed individually in food vials tont;tining 4 wild-type virgin females and then scored for larval progct~y 7 days later. Testes of all males yielding no progeny were dissected out in buffer, and found to contain motile sperm. The denominator represents the total number of males tested; the nunlcrator equals the number of males yielding progeny.

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 scored a s positive for f r u chain behavior only if there occurred at least one courtship chain of four or more males. The denominator represents the total nunlber of vials containing males of the specified gelwtype so observed, and the numerator the number of vials i n which f ru courtship behavior occurred.

” (:I (courtship index) is the mean percentage of time in an observation period that 5-day-old wild-type males spent courting 5- d;ly-old males of the specified genotype.

* n = 50 observations per genotype; the three mean CI values differ significantly at the P < 0.001 level, t-test matrix.

measured in pairings of wild-type test males and anesthe- tizedfrulfru males (HALL !978a). We observed and quantified this kind of courtship as in GAILEY, LACAILLADE and HALL (1986). In brief, observation chambers were formed by covering the depressions of porcelain well plates with microscope slides. To test a male forfru-like elicitation of courtship, he was ether anesthetized and then placed in a depression. Positioning a glass slide to make a small opening i n the chamber, a wild-type males was carefully aspirated in, the slide moved to close the chamber, and the observation begun. Any courtship that occurred was measured with respect to Courtship Index (CI) determinations (e .g . , GAILEY, LACAILLADE and HALL 1986).

Statistical analysis: When it was deemed appropriate (see REsu1.n) data were analyzed by a t-test matrix (BMDP Statistical Software, Los Angeles, California 90024).

RESULTS

Abnormalities of male courtship performed and elicited by the fruitless mutant: T h e results in Table 2 summarize components of the f r u phenotype. These behavioral defects were first reported by HALL ( 1 978a), and the current data are fully consistent with those earlier findings. Details of the fr-u syndrome follow.

f ru / f ru males are behaviorally sterile: They court females with persistence, yet they do not curl their abdomens for attempts at copulation; they never pro- duce progeny when kept with virgin females for an extended period, even though their reproductive tracts appear normal upon dissection and the testes seem to have normal numbers of motile sperm (HALL 197th). This result has been confirmed (Table 2, “Fertility fraction”) with males from a f r u isochromo-

soma1 stock (see MATERIALS AND METHODS), and, as controls, wild-type males o r males from a cross between f r u and wild type. All +/+ and fru/+ males initiated copulation within 30 min. All f ru l f ru males courted within 30 min and the preliminary components of male courtship (cJ BASTOCK and MANNING 1955) appeared superficially normal (although f r u males generate a defective courtship song; see DISCUS- SION). However, not one f r u male initiated copulation or attempted to initiate copulation by curling the abdomen. With regard to sterility, f r u is a simple recessive mutation (Table 2, second line).

f ru l f ru males court other f ru l f ru males and wild-type males: This conclusion was reached by HALL (1978a) after observing the courtship behavior of many male trios involving several genotypic combinations. Here we have analyzed only the courtship among f ru l f ru males. When in groups, they spend much of the time courting each other in lines-which we call “courtship chains” (Figure 2). This is an easily recognized courtship interaction unique to f ru l f ru males in groups.

A comparison of chain behavior in f ru / f ru , f ru /+ , and wild-type males is in Table 2 (“Chain behavior”). Only f ru l f ru males were ever observed to display such behavior. We estimated that these chains occurred for about 50% of the observation periods, with a chain of 4-10 males often snaking about the inner surface of the food vial; many instances of wing vibration were observed. Wild-type and f ru /+ males never displayed this behavior, tending to disperse themselves evenly about the vial; instances of courtship behavior were limited to pairs of males and were infrequent as well as brief. Hence, this aspect of the f r u phenotype is also recessive.

frulfru males stimulate wild-type males to court them even when the mutant males are anesthetized or have had their heads and thoraces removed: This seemed to suggest that f ru / f ru males contain a chemical courtship- stimulating cue (HALL 1978a; TOMPKINS, HALL and HALL 1980). Here, we assessed the courtship perform- ance of individual wild-type males that were paired with ether-immobilized flies of varying genotype, quantified as CI (e.g., GAILEY, LACAILLADE and HALL 1986). Numerous reports in the literature indicate that 5-day-old wild-type males stimulate very little courtship; yet when newly emerged, males stimulate as much courtship as virgin females (review: TOMP- KINS 1989).

As expected, we found that 5-day-old f ru l f ru males stimulated wild-type males to perform the same kind of vigorous courtship that virgin females o r newly emerged males induce (Table 2, “Courtship stimulus”). T h e wild-type males often courted f ru l f ru males vigorously, with frequent bouts of wing vibration and attempts at copulation; this is evidenced by the mean 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 m i n . By comparison, anesthetized wild- type nules stimulated much less courtship, with short bouts of wing 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 of ;I hypothetical 8 m i n 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 inversion: The phenotypes described above were found i n meiotic mapping crosses to be inseparable 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)P14 distal 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 f r u chromosonle within the sr gl interval was completely suppressed (O/10,038, us. 47/3 143 in controls) 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 breakpoints at 9OC and 9 1 B (Figure 3A). The cytogenetic interval 90D to 9 1 B forms a distinctive band- ing pattern, and in the fru/fru homozygote appears intact-but inverted (Figure 3B). The two inversion breakpoints on thefru chromosome flank the sr and gl loci, which, by other cytological and genetic criteria, are within 90E-F and 9 1 A 1-3, respectively (K. MOSES, personal communication; cf: COSTELLO and WYMAN 1986).

Note that the following terminology w i l l be adopted from this point on: at its first mention an aberration will be listed by complete name, e.g., Df(3R)P14. I t w i l l subsequently be referred to by an abbreviated name, in this case, PI4 (cf: Table 1 , Figure 2).

The Fact that all threefru phenotypes mapped very close to sr and were uncovered by PI4 led HALL (197th) to the provisional conclusion that fru is a single-gene variant. The current finding that thefru phenotypes are associated with an inversion calls this supposition into question. Since the inversion breakpoints of thefru chromosome and the breakpoints of P I 4 roughly coincide, it is possible that a damaged gene relevant to a particularfru phenotype could be

". .

I

900-F

4

918 ' 91A 4

A

B 91A FIGLIRF. J.-l'olytenc cIIrotIIosoIIw 1)rcpara~iom ol.frtr-l~e;~ritlg

third itIst;tr I;~rv;~e. A, fru/+. Labels indicate the 9OC-9 11% interval of 3K. The upper chrolnatitl shows the wild-tvpe hand secpence; lower cllromatid carriesfru and implies the inverted b;lnd sequencc ;IS indicated. H, fru/'ru. p indicates the position of thcfru prosinlal brcakpoint: d indicates the position of thefrzr distal breakpoint.

located at or near eitherfru breakpoint and be uncovered by this deletion. I t is plausible that the phenotypes associated with this genetic variant stem independently from two or more damaged loci; thus the fru behavioral abnormalities could map differentially to onefru breakpoint or the other but "co-map" when fru is heterozygous with P14. The collection of chromosomal aberrations in Figure 1 partitions the P14- defined interval and allowed us to test whether fru phenotypes map specifically to different, but nearby regions of the third chromosome.

Deficiency mapping of the fruitless phenotypes: We mapped the threefru phenotypes by assessing the behavior of Aberrationlfru males. Aberration/+ males served as controls. The results were that f ru sterility and courtship chain behavior map at region


TABLE 3

Cytogenetic localization of thefruitless phenotypes

Fertility fraction” Chain behavior” Courtship ~ t i rnu lus“~~ (CI * SEM)

Aberration Aberrationlfru Aberration/+ Aberrationlfru Aberration/+ Aberrationlfru Aberration/+

P(w+)ARO-f Df(?R)DG2 Df(?R)DG? Df(3R)P 14 Df(?R)glEXf 0 Df(3R)gl+EX6 Df(3R)DGI Df(3R)DG4 Df(3R)glEXf Df(3R)DG5 Df(3R)Cha” Df(3R)Cha” Df(3R)ChaM5 Df(3R)gl+EX5 Df(3R)lI8.5-1 Dp(3R)DG

48/50 0/50 39/50 0/50 0/50 50/50 0/50 47/50 0/50 0/50 0/50 0/50 0/50 50/50 50/50 48/50

50/50 41/50 39/50 50/50 50/50 50/50 50/50 46/50 50/50 50/50 50/50 50/50 50/50 50/50 50/50 50/50

6/10 3/5 0/5 5/5 5/5 0/5 5/5 0/5 5/5 5/5 5/5 5/5 5/5 4/5 0/5 0/5

3+1 48 f 6 41 f 5 24 f 4 12f 3 15f 2 19% 3 15f3 3 2 1 4 f l 5 2 1 6 f 2 3&1 5 2 1 3 2 1 9 f 2

7 f l 21 f 4 13+3 1 1 f 3 4 f l 4 f l 16f4 6 + 2 4 + 1 3 f l 4 f 1 3 f l 4 f l 2 f l 2 f l 21 f 4

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 f r u chromosome (Figure 3). A genetic factor respon- sible forfru courtship stimulating capacity clearly does not map to this distal site (Table 3) but may be located within a cytogenetic interval that includes the proximal f r u inversion breakpoint at region 9OC. Pertinent details of these results follow.

f r u sterility maps at or near the 91A-B boundary: The Df (3R)gl+BXS chromosome complements f r u sterility, i n that all gl+BXS/fru males tested were fertile (Table 3). This chromosome supplies sufficientfru+ function to give wild-type fertility and places a hypothetical “fru sterility factor” to the left of the gl+BX5 chromosome’s lefthand (proximal) breakpoint at 91B1-2. Df(3R)DGJ uncovers the lefthand f r u breakpoint at 9OC but not the righthand breakpoint at 91B. DG?/ f r u males are as fertile as their DC3/+ counterparts (Table 3), and this deficiency serves as a control to indicate that no separate, or interacting, “fru sterility factor” maps to the lefthand f ru breakpoint. [We attribute the apparent partial sterility in DG?/fru males to general debilitation as a result of severe aneuploidy; compare fertility fractions of DG?/fru, DC3/+, and Df(3R)DG2/+ males (Table 3).] All remaining deficiencies with one breakpoint at the 9 1 A- B boundary, when heterozygous with the f r u chromosome, resulted in complete male sterility (Figure 1, Table 3) .

f r u courtship chain behavior maps to the 9IA-B boundary: Application of the deletions to the localization of this phenotype led to conclusions similar to those from the sterility mapping crosses. There were, however, important exceptions. Although gl+BXS/fru and

ARO-llfru males showed normal fertility, they formed courtship chains when in groups (Table 3). These two genotypes represent the only two in Table 3 that functionally “uncouple” f r u sterility and courtship chain behavior. Such results provide evidence in support of the existence of a ‘Ifru chain factor,” which may not be the selfsame element as that which causes f ru sterility. The reciprocal separation of these two f r u phenotypes-behaviorally sterile males which do not form courtship chains-was not observed for any genotypic combination.

Df(?R)148.5-l/fru males did not form chains (Table 3). This observation places the putative “fru chain factor” at a site proximal to the lefthand breakpoint of this deficiency chromosome.

f r u courtship stimulation factor does not map to the 91A-B boundary: We first had to consider that the deficiency mapping offru courtship stimulation would be complicated by the fact that f ru /+ heterozygotes elicit an intermediate level of courtship, i.e., between the high level stimulated byfrulfru homozygotes and the low level by wild-type controls (Table 2). Thus, in a single-factor model for thisfru phenotype, we predicted that Df/fru males in which the deficiency fails to uncover the f r u defect would generate an intermediate CI as the baseline control level of courtship (similar to fru/+, Table 2); and that a 0 f l f . u combination in which the deficiency does uncover thefru defect would generate a high CI (similar tofrulfru, Table 2). But this prediction was not met: The “Court- ship stimulus” column in Table 3 (“Aberrationlfru”) reveals a range of CIS from very low [e.g., CI = 3% for Df(?R)glBXl/fru], to very high (e.g., CI = 48%


for DG2/fru). As a visual aid, the shading of deficiencies in Figure 1 indicates the CI range in which a particular “Aberrationlfru” genotype fell (CJ: Table 3; note that Of/+ controls are discussed as a group, below). The white background bars indicate the CI range 0- lo%, light gray 1 1 -24%, and dark gray >24%. Two Df/fru combinations elicited more courtship than did frulfru males, and seven 0flf.u combinations were courted only at a wild-type-like level (compare Tables 2 and 3). Nevertheless, the following points are reasonably clear.

DGB/fru and DG3/fru males stimulated the highest levels of courtship, roughly twice that associated with the next highest combination, Plllfru. Both of these DG deficiencies uncover the proximal fru breakpoint (Figure 1). The DG3 chromosome does not uncover the distal fru breakpoint; this provides strong evidence that fru courtship stimulation does not map exclusively to the same region as fru sterility plus courtship chain behavior. That is, stimulation maps within the region defined by the breakpoints of DG3, which did not uncover fru sterility or chain behavior (Table 3).

Of the Df/fru genotypes that yielded intermediate CI values (light gray bars, Figure l), three of the deficiencies have proximal breakpoints that map near the fru proximal breakpoint. P14/fru males stimulated the highest CI of the intermediate group (CI = 24%), a value similar to the CI stimulated by fru/fru males (CI = 28%; compare Tables 2 and 3).

Eight deficiencies used in this analysis contain a breakpoint at, or completely remove, region 91B (near thefru distal breakpoint) and leave intact chromosomal material at 9OC (at thefru proximal breakpoint). Seven of these eight deficiencies, when heterozygous with f ru , produced males that unexpectedly stimulated a low, wild-type level of courtship (see Figure 1, deficiencies designated by open bars). The combination ARO-l/fru also falls within this group (CI = 3%; Table 3). The obvious common feature of this group is that none of these genotypes leads to the level of courtship expected for fru/fru; this again is strong evidence that a putativefru courtship stimulating factor does not map to the distal fru breakpoint at region 9 1 B.

Aberration/+ controls may define a region of dosage sensitivity for the fru courtship stimulation phenotype. Five of these control combinations led to CI values >lo%, whereas the remaining combinations gave low levels of stimulation, close to that of wild- type males (Table 2 vs. Table 3, “Aberration/+”). T w o of these genotypes are DG2/+ and DG3/+, which remove region 9OC, the site of the lefthand fru breakpoint. Such males are hemizygous for the normal genetic material at 9OC and elicit an intermediate level of courtship, roughly the same as that forfru/+ (compare Tables 2 and 3). If this proximal fru break-

point itself reduces or removes the expression of a hypothetical “jru courtship stimulation factor,” then these two Of/+ combinations could be the equivalent of fru/+, with similarly reduced fru+ function from this locus and resultant courtship-stimulating phenotypes.

This same argument can be applied to a third genotype, P14/+; this was the deletion first used to uncover fru-associated courtship stimulation (HALL 1978a). The lefthand breakpoint in P14, which maps within region 9OC, could reduce or remove the expression of the fru stimulation locus on the PI4 chromosome and also result in intermediate courtship stimulation when heterozygous with wild type.

The fourth genotype, Dp(3R)DG/+, produces flies that are hyperploid (+/+/+) for the 90A-90F interval. Males of this genotype stimulated afrulfru-like level of courtship; conversely, DpDGlfru males (+/+/fru for the 90A-90F interval) were like wild type in that they elicited almost no courtship (Table 3). In terms offru+ function at the hypothetical stimulation locus, DpDG/fru males are expected to be wild-type for this character if fru is severely hypomorphic. DpDC/+ males may be hyperploid for the stimulation locus and thus have an elevated level of fru+ function. Deviation from the wild-type level of expression at this locus- whether an increase (e .g . , DpDC/+), or a decrease (e .g . , DG3/+, or DG3/fru)-results in males that elicit inappropriately high levels of courtship.

The final genotype of this group, which yielded a CI > lo%, is Df(3R)DGI/+. This deletion does not uncover the leftl-iandfi~~ breakpoint (Figure 1); the high stimulation CI (Table 3) was unexpected (note also that the same value was determined for DGl/fru as for DGI/+).

Effects of a transposon at 91B1-2: The P(w+)ARO-I factor is a w+-marked transposon inserted at 91 B1-2 (Table 1). The balanced stock of this variant contains only the single insert at 91B1-2 (K. MOSES, personal communication); the chromosome bearing ARO-I was semilethal leading to viability levels that were only ca. 5 % of normal. By extracting potentially recombinant chromosomes from ARO-I/ARO-I mothers, eight iso- chromosomal sublines were established in which the viability was increased to about 20%; one line was arbitrarily chosen for assessment of fru phenotypes and subsequent crosses involving ARO-I.

ARO-I/ARO-I males from this line appear normal in size and mobility when compared with their balancer-bearing siblings. When observed in groups their behavior was striking: ARO-I/ARO-I males exhibited fru chain behavior (line 1, Table 4) that qualitatively appeared as robust as that of fru/fru males. ARO-I/ ARO-I males were not, however, completely sterile (line 1, Table 4). T o determine whether this semister- ility was fru-like and thus behavioral, 20 ARO-I/


TABLE 4

ftuitless phenotypes analyzed in males expressing deletion and transposon genotypes

Courtship stimulus (CI k S E M ) ~ , ‘

(;?notype (%)” fractionb behavior’ <6-hr male .!-day male Viability Fertility Chain __

AKO-Z/~4RO-l 19 11/50 10/10 43 + 8 8 & 1

Cha “ ’ / P I 4 60 0125 515 50 f 8 2 & 1 O~ha’”7/D(;2d 42 0125 315 3 7 f 9 5 & 1 (:ha“’S/ARO-l 83 0125 415 29 & 7 3 & 1

gl+UX5/P I4 83 25/25 015 34 f 9 4 f 1 gl+13X5/DC2d 27 0125 215 30 _t 5 4 f 1 gl+BX5/ARO-I 75 25/25 015 31 f 6 3 + 1

148.5- l /P14 25 25/25 015 3 4 + 6 2 f 1 14N.5-l/DC2d 75 0125 11.5 36 f 6 4 + 1 14K.5-l/ARO-I 86 25/25 015 47 + 7 5 f 1

Expressed as the fraction “observed/expected (X100)” of progeny flies bearing both specified aberrations (ie., “genotype”) from the generalized cross: Df l lBa l X Df2/Bal. More than 500 progeny were scored from each cross.

See footnotes b. c, and d , Table 2. l‘he amount of courtship wild-type males displayed with either

newly emerged or 5-day-old males of the specified aberration gen- o t y e 1z - 10 observations each group. ’ill cLosses involving the Df(3R)DG2 chromosome yielded flies

which were small and sluggish; the assessment of these genotypes a s causingfru phenotypes is thus tenuous (see text).

ARO-1 males were placed individually with virgin females and scanned for a l-hr period for occurrences of courtship behavior: 10 males courted persistently but did not curl their abdomens to attempt copulation; 6 males failed to court; 4 males copulated and were the only males which produced progeny after all 20 had been kept with females for a week.

These results and observations imply that ARO-1 is hypomorphic for fru+ function(s) at 91B, leading to incomplete penetrance offru-like sterility. The following results further support such a notion. glBXl is a small deficiency which uncovers both f r u sterility and chain behavior (Table 3). The glBXl/ARO-1 genotype is poorly viable (5%) but superficially indistinguishable from ARO-1IARO-1 in appearance. Two groups of 10 glBXl/ARO-1 males showed frequent instances of chain behavior in a 30-min observation period. Four- teen of these 20 males courted females with no observed curling in a l-hr period. All 20 males were still alive after a week with females; none produced progeny although all males contained motile sperm in their testes. This genotype is the first of several we shall present in which males fully express f r u sterility and chain behavior in the absence of the f r u inversion.

Heterozygous ARO-l/fru males also perform char- acteristic f r u chain behavior, although not as intensely a s either frul fru or ARO-l/ARO-1 males (four of 10 ARO-l/fru groups performed no chain behavior in a 30-nlin observation period; line 1 , Table 3); but ARO-1/ f ru rnales are essentially normal in fertility (Table 3).

By comparison, ARO-I/+ males never formed courtship chains and were fully fertile (Table 3).

Taken together, the preceding results are further evidence that f r u sterility and chain behavior are associated with the f r u distal breakpoint. On the other hand, ARO-IIARO-1, ARO-I/+, and ARO-l/fru males stimulate very little courtship in wild-type males (compare “Courtship Stimulus,” line 1, Tables 3 and 4, with Table 2) and serve as evidence that f r u courtship stimulation is not associated with the f r u distal breakpoint.

Deficiency and transposon combinations that result in sterility and courtship chain behavior: Of the seven deletions that contain a breakpoint at the 91A- B boundary, five failed to complement both f r u steril- i ty and chain behavior (Figure 1) and thus could remove or damage the f r u 91B factor. If this locus is nonvital, males heterozygous for two deficiencies, or a deficiency and ARO-1, would survive and be f r u mutants. This is in fact the phenotype of Df (?R)ChaM’/P14 and C ~ U “ ~ / A R O - I males: They were behaviorally sterile and formed courtship chains while obviously not carrying the f r u inversion (Table

The results of testing the remaining deletionltran- sposon combinations for viability and f r u phenotypes were extensive and complex. A review of the seven deficiencies and their interactions with f r u should increase the comprehensibility of the behavioral and viability data to be described in this section. The DG2, P14, Df(?R)glBXlO, glBXl, and ChaM5 deletions uncover both f r u sterility and courtship chain behavior; gl+BX5 uncovers only the f r u courtship chain phenotype; 148.5-1 uncovers neither f r u phenotype (Figure 1, and see above, Table 3).

Behavioral analyses of males from crosses that com- plemented for viability are in Table 4 and are schematically summarized in Figure 4. Also see Figure 5, which is a model of the functional placement of breakpoints at 9 1 B based on the results of all complementation tests-both for viability, and f r u sterility plus chain behavior.

As already mentioned, P14/ChaM5 is not only a viable combination, but males of this genotype are also behaviorally sterile and court each other vigorously in courtship chains (Figure 4, Table 4). We conclude that each of these deficiencies removes the f r u 9 1 B factor, or damages it by chromosomal break- age within the locus (Figure 5).

The combination P14/gl+BX5 also allows for viability, but males of this genotype are behaviorally wild type (Figure 4, Table 4), suggesting that this pair of breakpoints does not overlap (Figure 5) , or at least that they do not each remove or completely inactivate fru+ function(s).

glCBX5 could define the distal limit of a putative

4).

fruitless Courtship 78 1

9lA 91B 91c

P14

DG2

gZBxl0

glBXl

AR 0- 1

ChaMS gl+BXS 148.5-1 AROl jiu

fru

+ = no courtship chains: fertile & = courtship chains

F = f d e : F- = semt-fde S = sterile (7) = equivocal display of chain

formation behavior

FIGURI.: 4.--Sunlnl;1ry o f complementation crosses involving chronlosomes with breakpoints at or near 9 1 B (cf: Table 4, and see text).

“fru sterility factor,” in that gl+BX5lfru males are fully fertile vet form courtship chains (Figure 1, Table 3). This genotype is special in that it is the only deficiency which uncoupled thefru sterility and chain behavior phenotypes. T h e gl+BX5 chromosome might be inter- preted as weakly expressing, or hypomorphic for,fru+ function(s). T h e relevant breakpoint of this deletion is represented as impinging on thefru locus from the right (Figure 5), i . e . , from a location distal to thefru lesion in 9 1 B. In combination with a chromosome that presumably removes fru+function(s) such as DG2, gl+BX5 would provide insufficient fru+ function(s) to rescue either courtship abnormality. This is consistent with the observations that DG2/gl+BX5 males are sterile and form chains (Figure 4, Table 4), but that P14/ gl+BX5 males are behaviorally wild type (see above and Figure 5) . This latter combination leads to the supposition that P I 4 is also weakly hypomorphic (and impinges on the f ru locus from the left; Figure 5), providing a higher level offru+ function(s) than DG2.

148.5-1 seems to set the righthand boundary of the f ru 91B factor, in that 148.5- l /P14 males are completely wild type in behavior (Table 4, Figure 4). This does not, however, rule out the possibility that 148.5-1 is also weakly hypomorphic for fru+ function(s) and that this genotype, in terms offru+ expression, could be the functional wild-type equivalent of the P14/ gl+BX5 genotype. This possibility is suggested by the

A l~ \

AROl

FIGURE 5.-Functional positions of fru-associated breakpoints. l l l e locations of these lesions are predicted by the outcome of crosses sunlnlarizecl in Figure 4. “lethal” indicates a predicted vital locus to the right offru: “spmilefhal” indicates ;I predicted locus to the left offru which affects viability. An arrow that passes through a locus indicates a loss o f function for that locus on the named chromosonle. An arrow stopping “within“ a locus implies hvpo- morphic expression [e.g., the lethal factor and Df(3R)I48.5-I]. l h e gray arrow (bottom) suggests a spreading effect associated with the ARO-I insertion (see text).

phenotypic assessment of viable combinations with DG2. Although DG2 is a very large deficiency, it was like P I 4 in that viable “double-deficiency” flies were produced when it was heterozygous with either Cha”’, gl+BX5, or 148.5-1 (Figure 4). Whereas combinations with P I 4 led to vigorous, normal-looking flies, the three viable genotypes involving DG2 caused adults to emerge late and be small and lethargic. These debili- tations could easily obscure the assessment of f ru phenotypes, and thus a question mark is attached to each of the genotypes in Figure 4. However, males of each DG2-bearing combination were sterile and met the minimal behaviorally mutant criterion of four males courting in a chain in at least one trial (Table 4). T h e inferred removal by DG2 of most, if not all fru+ function(s) at 91 B leads to its functional breakpoint placement of DG2 in Figure 5.

Any combination of either glBXl or glBXI0 with either Chd”, gl+BX5, or 148.5-1 led to lethality (Fig- ure 4). This suggests that glBXl and glBXl0 remove not onlyfru+ function(s) but also inactivate or remove a hypothetical vital locus lying to the right of fru which is also inactivated by Cha“’, gl+BX5, and 148.5- I (Figure 5).

In a series of further crosses involving ARO-I, this chromosome produced the same “complementation pattern” with ChaM5, gl+BX5, and 148.5-1 as did P I 4 (Figure 4, Table 4). ARO-I/ChaM5 and P 1 4 / C h ~ “ ~ males were essentially indistinguishable on all criteria: they were completely sterile, and, by general visual inspection, both were vigorous in chain formation.


Because its function in these crosses is analogous to PI4, ARO-I, too, could be hypomorphic for fru+ function(s) (see Figure 5, and also previous section on ARO-I). An important distinction is that P14/fru males are completely sterile whereas ARO-llfru males are virtually always fertile (Figure 4); both genotypes result in males that form courtship chains (Figure 4). Comparing these genotypes, ARO-I could supply suf- ficiently more fru+ function(s) to complement sterility.

Of the crosses involving ARO-I and deficiencies that extend proximally from 91B, only ARO-l/glBXI is a viable combination. Although viability is poor (5%), males of this genotype are behaviorally sterile and form courtship chains (Figure 4, and a previous section). It was observed in the culture vials that many ARO-l/P14 and ARO-l/glBXI flies developed fully, but died in the pupal case. Five pharate adult males of each genotype were removed from their pupal cases while still living; all died within 24 hr. T o account for this, we propose that a locus involved in viability is placed to the left of the fru 91B factor, and that expression at this locus is diminished through some sort of spreading effect from the site of ARO-I insertion (Figure 5). This insertion cannot impinge on the hypothetical vital locus placed to the right of fru, since flies carrying ARO-l-heterozygous with ChaM5, glfBX5, or 148.5-I-had normal viability (Table 3).

In summary, the eventual phenotype of a viable deficiency combination, or a transposon/deficiency combination, might be viewed as its relative sum of f~u’ functionts). The following hierarchy of expression is suggested for individual chromosomes: transposon and proximally extending deficiencies, ARO-I > P14 > DG2; distally extending deficiencies, 148.5-1 > gl+BX5 > Cha’”5. In none of these viable combinations were fru sterility and fru chain behavior functionally separated.

Deficiency/transposon combinations and courtship stimulation: It is evident from the previous sections that two of the f m mutant behaviors-behavioral sterility and chain formation-are not merely peculiar phenomena specific to the f ru inversion. These phenotypes are reproduced in the males of certain combinations of ARO-I and/or deficiencies with lesions in region 91B. The results in Table 4 (“C.ourtship stimulus,” 5-day male) were that none of these genotypes stimulated visibly more courtship than wild-type; no CI value was >lo%. These results are consistent with the observation that ARO-I/ ARO-I males stimulate very little courtship (Table 4), and reinforce the conclusion that the fm courtship stimulation factor does not map to region 91B.

As newly emerged males, however, all double-deletion and ARO-I/Df males stimulated high levels of courtship (Table 4, “Courtship stimulus,” <6-hr male),

as do newly emerged wild-type males (see, for example, JALLON and HOTTA 1979).

DISCUSSION

fru, a 9OC factor us. a 91B factor?fruitless is a small inversion with breakpoints at 9OC and 91B. This has allowed us to test whether three phenotypes associated with this mutant of reproductive behavior map differentially to one fru breakpoint or the other. Results of these mapping experiments indicated that the fru phenotypes do not have the same genetic etiology. Behavioral analysis for many combinations of thefru chromosome with deficiencies that partition the 9OC- 91B interval showed consistently that f ru sterility, courtship chain behavior, and thefru distal breakpoint co-map to region 91B. fru courtship stimulation provisionally maps proximally in region 89F-90F, as defined by the breakpoints of the DG3 deletion. Thefru proximal breakpoint is within this interval, and thus could be the factor causing inappropriately high levels of courtship elicitation.

Such an etiology for a mutant with “multiple phenotypes” has been previously documented. The ocelliless (oc, I , 23.1) variant was induced with X-rays and both of its phenotypes-female sterility and absence of simple eyes-map by recombination to the same location (LINDSLEY and GRELL 1968). In a manner analogous to fru, oc is associated with a small inversion (SPRADLINC and MAHOWALD 1981). Its distal breakpoint (located at 7F1-2) is 1-3 kb upstream from two chorion protein structural genes and disrupts their expression; the 7F material now moved to SA ( i e . , near the proximal breakpoint) results in overexpres- sion of sequences at 8A in oc follicle cells (SPRADLING and MAHOWALD 1981). In this case, the female sterility phenotype at least has a simpler etiology, which can be explained solely by the drastic reduction in chorion proteins.

Certain key examples reinforce the separability of fru-related courtship anomalies. (1) ChaM5/ARO-I males stimulate virtually no courtship and are thus wild type for thisfru phenotype; they also contain two doses of third chromosome material at 9OC (89F-90F inclusive). However, these males are completely sterile and form courtship chains; their mutant behavior, the site of ARO-1 insertion, and the lefthand ChaM5 breakpoint are located in region 91B. (2) The “reciprocal uncoupling” of the fru phenotypes is demonstrated by males of the genotypes DG3/+ and DpDG/+. These males have an altered dosage of genetic material that includes region 9OC, and they stimulated abnormally high amounts of courtship; they contain the normal dosage of material in region 91B, are fertile, and do not form courtship chains. On the strength of breakpoint cytology, fru sterility and courtship chain behavior map at 91B1-2. Courtship stimulation is less well-


defined by deficiency breakpoints but seems to map to the region 89F-90F. Because thefru breakpoints map very near or within these regions we restate the general proposal introduced above: thefru syndrome is a collection of phenotypes induced independently at the two breakpoints. f r u courtship stimulation is attributed to disruption of a locus at 9OC; sterility and courtship chain behavior are the result of disruption of a locus-or loci-at 9 1 B.

HALL (1978a) noted aberrant wing extension behavior infrulfru males. This has been further inves- tigated in the analysis of courtship wing vibrations by males in which one or the other f ru breakpoint was uncovered. f ru , and more specifically the distal f ru breakpoint, was found to cause substantially longer than normal silence intervals between pulses of tone (WHEELER et al. 1989). Thus,fru involves yet another element of defective courtship behavior. fm terminology: Our findings also suggest the in-

troduction of a new and perhaps more precise “fruitless lexicon.” Since it contains an inversion, we desig- nate the original fru-containing chromosome as Zn(3R)fru. The designation ‘yruitless” was reported by HALL (1978a)“with permission from K. S. GILL- to supplant the pejorative designation “fruity” (GILL 1963a, b). We think thatfruitless ( f r u ) is a meaningful designation for the “91B factor”; we propose to re- serve this name for the gene (or genes) which reside at this locus, whose mutant expression results (result) in male behavioral sterility and the formation of male courtship chains. The courtship stimulation phenotype, however, appears to have an independent genetic etiology, although the phenotype is in a sense related to “fruitless courtship,” in that one male is courting another. The details of this stimulation phenotype appear so different from the other phenotypes that we feel the elicitation factor, presumably in 9OC, should at present be referred to with the phenotypic designation “fruitless stimulation,” without, however, invoking a formal designation for the specifically relevant locus (if any).

The 9OC factor: This factor remains poorly defined genetically; a future analysis of its mutant phenotype must include new deficiencies and duplications that partition the 89F-90F interval. In addition, we have, in collaboration with J.-M. JALLON, begun to analyze the pheromone profiles of Zn(3R)fru-bearing flies, in an attempt to uncover a specific chemical correlate to the mutant courtship stimulation (6 TOMPKINS, HALL and HALL 1980). Our preliminary experiments reveal neither missing nor novel compounds in Zn(3R)frul Zn(3R)fi-u males, either when newly emerged, or 5 days post-emergence. We have consistently seen, however, a reduction in levels of a male-predominant compound, 7-tricosene, in Zn(3R)fru homozygous males and in DG3/Zn(3R)fru males (D. A. GAILEY and

J.-M. JALLON, unpublished observations). In normal D. melanogaster males this compound is an abundant (ANTONY and JALLON 1982), and possibly courtship inhibitory (SCOTT 1986), cuticular pheromone. Gen- erally, the enzymatic basis of pheromone production and regulation in this species is poorly understood, and its genetic regulation seems very complex (SCOTT and RICHMOND 1988). However, it could be useful to look into the putatively abnormal biochemistry associated with “fruitless stimulation” and attempt to determine how it might lead to a pheromonal change of this kind. fm and the 91B factor-one gene or more? The

answer to this cannot be discerned from the results we present here. f ru sterility and chain formation almost always go together (Figures 1 and 4). But males of two genotypes--gl+BX5/Zn(3R)fru and ARO-11 Zn(3R)fru”show only the courtship chain phenotype; they are fertile (Figure 4). On the other hand, f ru sterility was always coincident with courtship chain behavior. These observations do not yet allow a deci- sion on whetherfru is one gene or two.

As a working model, we propose that P14 and ChaM5 both contain a breakpoint in fru. Whether heterozygous with Zn(3R)fru or each other, these deletions led to both 91B-associated mutant phenotypes (Figure 4). This was also the case with the ChaM5/ARO-l combination (Figure 4). In this context the demonstration of 91B phenotypes in double-deletion males is an important new observation: It leads to the prediction that both deletions are amorphic for 9 1 B fru+ function, which certainly would be the case if their breakpoints overlap within fru. Since the resultant 91B phenotypes are the same as in Zn(3R)fru homozygotes, the distal inversion breakpoint may also be an amorph (as opposed to the type of genotypic change associated with one of the ocelliless inversion breakpoints, see above). A corollary is that f ru will turn out to be a simple, single gene, as opposed to the phenotypic abnormalities being somehow caused by the gratui- tous neomorphic juxtaposition of two usually separate regions within chromosome 3.

If frulfru, or an analogous DflDf case, is truly amorphic, f ru would be a non-vital gene that influences behavior-of which there are several in D. melanoguster (see Discussion in KULKARNI and HALL 1987). Consistent with this notion is the observation that Zn(3R)fru has never led to lethality when heterozygous with any deficiency. In this respect,fru could be like per (SMITH and KONOPKA 1981) and dunce (BYERS, DAVIS and KICER 1981); these genes have been deleted by the appropriate Df/Df combinations, yielding adults which have good viability but exhibit severe behavioral defects.

Ultimately, a molecular analysis could lead to several 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 transposon is very near to (possibly at) the 91Bfru locus. In fact, K. MOSES and G. M. RUBIN (personal corn- munication) are already investigating cloned material involving this element in their efforts to isolate and manipulate theglass gene (cf: Table 1). If this material also leads to the cloning and molecular identification offru, the following sorts of questions can be asked experimentally: Is DNA in fact missing from the putatively overlapping-Dfcombinations that lead to sterility and chain behavior (cf : molecular proofs of the per- genotypes, BARCIELLO and YOUNG 1984; REDDY et al. 1984)? Will one “simple transcription unit” in 91 B turn out to encode the function that influences these features of normal Drosophila behavior, whereby males do not court other males but vigorously court and readily mate with females?

Since the ARO-1 transposon seems near f ru (c f : Table 3 and 4), valuable information about the genetic organization offru might be obtained by mobilizing this transposon and selecting for imprecise excisions (ROBERTSON et al. 1988) which could remove third chromosome material near the insertion site. Such small deletions could potentially give further information on the genetic organization of f ru and the predicted nearby vital and “semilethal” loci (Figure

That f ru males do not curl their abdomens in attempts at copulation implies that there is a blockade in the anatomical/physiological “pathway” underlying this behavior. This f ru defect could be muscle- or nerve-related (c f : LAWRENCE and JOHNSTON 1986). Either of these kinds of tissue etiologies might apply also to the production offru males’ aberrant courtship song (WHEELER et al. 1989). This singing defect, however, might have a thoracic focus, with the curling inability hypothetically having an abdominal one. The tissue affected such that f ru males court each other could be neural and in the fly’s head.

Mosaic analysis (reviews: HALL 1978b, 1984) could yield clues about the anatomical sites involved in these different behavioral phenotypes. Since f ru is autosomal, generation offru//fru+ mosaics would be another set of experiments facilitated by the cloning of this gene. As exemplified by GAILEY et al. (1987), transformation involving afru’ DNA fragment into a Ring- X chromosome would permit the production of high frequencies of diplo-X//haplo-X mosaics involving this autosomal locus. Problems of sexual dimorphisms would be avoided in fru//fru+ mosaics by turning them into pseudomales with the transformer mutation (4 KULKARNI and HALL 1987; KULKARNI, STEINLAUF and HALL 1988).

5).

We are most grateful to K. MOSES and G. M. RUBIN for donating several of the crucial chromosomal aberrations used in this study. K. MOSES a l s o provided breakpoint data on his and our newly

induced aberrations. We thanL him, a s well, for helpful discussions. We appreciate technical assistance fi-on) B. ORIEL, hl. DOURSOUN- IAN, K. DESFOSSES and A. ARONSKY; and co~ntllents 011 the I I I ; I I I ~ ~ -

script from 1’. MERRIIL, K. KFNDAHI. and S . RORINOW. Frllis ~vork was supported bv ;I grant fronl the National Institute o f Healtll (GM-21473) and a Neurobiology Training grant (T32 NS07292) from the National Institute of Neurological and Collr~llunicative Disorders and Stroke.

L I T E R A T U R E C I T E D

ANTONY, C. , and J.”. JALLON, 1982 The chemical t,asis for- sex recognition i n Drosophila melanogaster. J. Insect Physiol. 28: 873-880.

BARGIELLO, T. A,. and M. W . YOUNG, 1984 Molec~~l;~r genetics o f a biological clock i n Drosophila. Proc. Natl. Acad. Sci. USA 81: 2 142-2 146.

BASTOCK, M., and A. MANNING, 1955 Courtship of Drosophila melanogaster. Behaviour 8: 85-1 I I.

BYERS, D., R. L. DAVIS and J. A. KIGER. I981 Ikfect in cyclic- AMP pllosphodiesterase due to the dunce mutation of learning i n Drosophila melanogaster. Nature 289: 79-8 1 .

COSTELLO, W. J., and R. Y. WYMAN, 1986 Developrllent of a11 indirect flight muscle in a muscle-specific mutant o f Drosophiln melanogaster. Dev. Biol. 118: 247-258.

CRAYMER, I . . , 1984 Keport. Drosophila Inform. Serv. 60: 234. EWINC., A . W., 1977 Colllmunicatiorr i n Iliptera, 11;). 403-417 i n

How Animals Communicate, edited by 1’. A . SEREOK. Indiana University Press, Bloonrington.

EWING, A. W , , 1983 Functional aspects of Drosophila courtship. Biol. Rev. 58: 275-292.

GAILEY, D. A. F. R. JACKSON and K. W. SIEGEI., 1982 Male courtship i n Drosophila: the conditioned response to inlmlture males and i t s genetic control. Genetics 102: 771-782.

GAILEY, D. A. , R. C. LACAILLADE: and J . <:. HALL, 1986 Cl~e~nosensory elements of courtship i n nornral a d mutant, ol~actioll-deficient Drosophila melanogaster. Behav. <;e- net. 16: 375-405.

GAILEY, D. A , , D. L. BORDNE, A. M. VALL~S, J. C. HAIL and K. WHITE, 1987 Construction of an unstable ring-X ch1~0111oSo111e hearing the autosomal dopa decal-boxylase gene i n Drosophila melanogaster and analysis of Ddc mosaics. Genetics 115: 305- 31 1 .

GILL, K. S., 1963a A mutation causing abnort~lal mating behavior. Drosophila Inform. Serv. 38: 33.

GILL, K. S., 1963h A mutation causing abnormal courtship and mating behavior in males of Ilrosophila melanogaster (abstract).

GORCZYCA, M., and J. C. HAIL, 1984 Identification of a cholit~- ergic synapse in the giant fiber pathway of Drosophila using conditional mutations ofacetylcholine synthesis. J. Neurogenet. 1: 289-3 13.

HALL, J. (;., 197821 Courtship among males due to a male-sterile mutation in Drosophila melanogaster. Behav. Genet. 8: 12.5- 141.

HALL, J. C., 1978b Behavioral analysis in Drosophila nwsaics, pp. 259-305 in Genetic Mosaics and Cell Dz;fferentiation, edited by W. J. GEHRING. Springer-Verlag. Berlin.

HALL,]. C., 1979 Control of male reproductive hehaviot- by the central nervous system o f Drosophila: dissection o f a courtship pathway by genetic mosaics. Genetics 92: 437-457.

HAI.I., J. C . , 1984 (:omplex brain and behavioml lu!?rtlons dis- rupted by nlutations i n Ihosophila. Dev. Genet. 4: 3.55-378.

HAIL, J . C . , 1985 Genetic analysis of behavior in insects. pp. 287- 383 in Comprehensive Insect Physiology Biochemistry and Phar- macology, Vol. 9, edited by G. A. KERKUT and 1.. I . GII.BER’1’.

Pergamon, Oxford.

A t n . Zool. 3: 507.


HALL, J. C., 1986 Learning and rhythms in courting, mutant Drosophila. Trends Neurosci. 9: 414-4 18

HAIL, J. C . , R. UT. SIEGEL, L. TOMPKINS and C. P. KYRIACOU, 1980 Neurogenetics of courtship in Drosophila. Stadler Ge- net. Symp. 12: 43-82.

HAZELRIGG, T , , R. L . E v I s ~ ~ ~ G. M. RURIN, 1984 Transformation of white locus DNA in Drosophila: dosage compensation, zeste intct-action. ; ~ n d position effects. Cell 36: 469-481.

HOTTA, Y. , ; ~ n d S. BENZER, 1976 Courtship in Drosophila mosaics: sex-specific foci for sequential action patterns. Proc. Natl. Acad. Sri. USA 73: 41.54-4158.

HUMASON, G. L>. , 1959 Aceto-orcein, alternate solution, p. 474 in Animal Tissue Techniques, Ed. 4. W. H. Freeman, San Francisco.

I K E u A . I(., and W'. D. KAPLAN, 1972 Neurophysiological tech- rliqucs i n Drosophzla. Am. Zool. 14: 1055-1066.

JAI.I.oN,,J.-M.. 1984 .4 few chemical words exchanged by Drosoph- ila during courtship and mating. Behav. Genet. 14: 441-478.

JAI.I.oN,,].-M., and Y. HOTTA, 1979 Genetic and behavioral stud- ics of female sex appeal i n Drosoph'ila. Behav. Genet. 9: 257- 255.

KULKARNI, S. J., and J. C. HALL, 1987 Behavioral and cytogenetic atmlysis o f thr cacophony courtship song nlutant and interacting genetic variants in Drosophila melanogaster. Genetics 115: 46 1- 47.5.

I(UI.KARN1, S. ,J., A. F. STEINLAUF and J. C. HALL, 1988 The dissonance mutant of courtship song in Drosophila melanogaster: isolation, behavior and cytogenetics. Genetics 118: 267-285.

I.AWRF.NCE, P. A , , and P. JOHNSTON, 1984 The genetic specifica- tion of pattern i n a Drosophila muscle. Cell 36: 775-782.

I.AWRKNCE, P. A,. and P. JOHNSTON, 1986 The muscle pattern of a segment of Drosophila n~ay be determined by neurons and not b y contrihuting myoblasts. Cell 45: 505-5 13.

I.LFEVRE. G . , 1976 The polytene chromosomes, pp. 61-63 in The Genctirs and Biology of Drosophila. Vol. la , edited by M. ASH- R ~ T R N E R and E. NOVITSKI. Academic Press, London.

I,E.VIS, K., 7'. HAZELRIGG and G. M. RURIN, 1985 Effects of genomic position on the expression of transduced copies of the white gene of Drosophila Science 229: 558-56 I .

I.INI)SI.F.Y. 1). Id., and E. H. GRELL, 1968 Genetic Variations of Drosophila melanogaster. Carnegie Inst. Wash. Publ. 627.

Inform. Sew. 62: 221-222. IJNDSLEY, 1). L., and C;. %I", 1985 Rearrangements. Drosophila

M A K K O W . -1. A , , 1987 Behavioral and sensory basis of courtship

success in Drosophila. Proc. Natl. Acad. Sci. USA 84: 6200- 6204.

MARKOW, T. A,. and S. J. HANSON, 1981 Multivariate analysis of Drosophila courtship. Proc. Natl. Acad. Sci. USA 78: 430-434.

REDDY, P., W. A. ZEHRING, D. A. WHEELER, V. PIRROTTA, C. HADFIELD, J. C. HALL and M. ROSBASH, 1984 Molecular analysis of the period locus in Drosophila rnelanogaster and identification of a transcript involved in biological rhythms. Cell 38: 701-710.

ROBERTSON, H. M., C. R. PRESTON, R. W. PHILLIS, D. M. JOHNSON- SCHLITZ, W. K. BENZ and W. R. ENGELS, 1988 A stable genomic source of P element transposase in Drosophila melanogaster. Genetics 118: 461-470.

Scolr, D., 1986 Sexual mimicry regulates the attractiveness of mated Drosophila melanogaster females. Proc. Natl. Acad. Sci. USA 83: 8429-8433.

Sco-rT, D., and R. C. RICHMOND, 1988 A genetic analysis of male- predominant pheromones in Drosophila melanogaster. Genetics 1 1 9 639-646.

SMITH, R., and R. J. KONOPKA, 1981 Circadian clock phenotypes of chromosomal aberrations with a breakpoint at the per locus. Mol. Gen. Genet. 183: 243-251.

SPRADLING, A. C., and A. P. MAHOWALD, 1981 A chromosome inversion alters the pattern of specific DNA replication in Drosophila follicle cells. Cell 27: 203-209.

TOMPKINS, Id., 1984 Genetic analysis of sex appeal in Drosophila. Behav. Genet. 14: 41 1-440.

TOMPKINS, L., 1989 Homosexual courtship in Drosophila, in MBL Lectures in Biology: Perspectives in Neural Systems and Behavior, edited by T. J. CAREW and D. KELLEY. Alan R. Liss, New York (in press).

TOMPKINS, L., and J. C. HALL, 1983 Identification of brain sites controlling female receptivity in mosaics of Drosophila melanogaster. Genetics 103: 179-195.

TOMPKINS, L., J . C. HALL and L. M. HALL, 1980 Courtship stimulating volatile compounds from normal and mutant Dro- sophila. J. Insect Physiol. 26: 689-697.

TOMPKINS, L., A. GROSS, J. C. HALL, D. A. GAILEY and R. W. SIEGEL, 1982 The role of female movement in the sexual behavior of Drosophila melanogaster. Behav. Genet. 13: 565- 578.

WHEELER, D. A . , S. J. KULKARNI, D. A. GAILEY and J. C. HALL, 1989 Spectral analysis of courtship songs in behavioral mutants o f Drosophila melanogaster. Behav. Genet. (in press).

Communicating editor: W. M. GELRART

behavior cytogenetics of fruitless in drosophila ...that disrupt the male-specific behavior of...

Documents