identification of brain sites controlling …€¦laurie tompkins*.' and jeffrey c. hall**...

Copyright 0 1983 by the Genetics Society of America

IDENTIFICATION O F BRAIN SITES CONTROLLING FEMALE RECEPTIVITY IN MOSAICS OF DROSOPHILA MELANOGASTER

LAURIE TOMPKINS*.' AND JEFFREY C. HALL**

'Department of Biology, Brandeis University, Waltham, Massachusetts 02254, and

**Department of Biology, Temple University, Philadelphia, Pennsylvania 19122

Manuscript received July 19,1982 Revised copy accepted September 16,1982

ABSTRACT

We have identified cells in the brain of Drosophila melanogoster that are required to be of female genotype for receptivity to copulation with males. To do this, we determined experimental conditions in which female flies virtually always copulate, then measured the minimum amount of male courtship that is required to stimulate females to indicate their receptivity to copulation. We then observed gynandromorphs with female genitalia to determine whether the sex mosaics elicited at least the minimum amount of courtship and, if so, whether they copulated. By analyzing these gynandromorphs, in which the genotype of external and internal tissues could be ascertained, we were able to identify a group of cells in the dorsal anterior brain that, when bilaterally female, is necessary and sufficient for receptivity to copulation. This group of cells is anatomically distinct from those that are required to be of male genotype for the performance of courtship behaviors.

N Drosophila melanogaster, diplo-X individuals with a normal complement I of autosomes are female, whereas haplo-X individuals are male (BRIDGES 1925). Female embryos that lose one of their X chromosomes in some cells during development become sex mosaics (gynandromorphs). Since the sexual behaviors performed by normal male and female flies are entirely different (STURTEVANT 1915), the question of whether sex mosaics behave as males or as females is of interest. When the presence of male or female cells in specific tissues of gynandromorphs is correlated with the ability of the mosaics to perform a sexual behavior, it is possible to identify behavioral foci-parts of the fly whose sex determines the behavior.

A number of investigators have observed that some mosaics with female tissues can perform male courtship behaviors: orienting toward and following a female, tapping the female's abdomen, extending and vibrating a wing to produce the courtship song, licking the female's genitalia, and attempting copulation (see SPIETH 1952 and BASTOCK and MANNING 1955 for a more complete description of these behaviors). Several early studies (reviewed by PATTERSON and STONE 1938) showed that the presence of male genitalia in a

I To whom correspondence should be sent, Present address: Department of Biology, Temple University, Philadelphia, PA 19122.

Genetics 103 179-195 February, 1983.

180 L. TOMPKINS AND J . C. HALL

gynandromorph is neither necessary nor sufficient for the performance of courtship. More recently, HOTTA and BENZER (1976) identified a focus within the head that is required to be of male genotype for following and wing extension and showed that there must also be male tissue in the thorax for attempted copulation with a female. These studies were limited, however, in that they utilized externally marked mosaics in which it was not possible to determine the genotype of internal tissues. In such mosaics, the presence of an internal focus can only be inferred from mapping the focus relative to cells in the embryonic blastoderm that will subsequently give rise to external structures in the adult (HOTTA and BENZER 1972). Accordingly, to localize foci for courtship directly and with more precision, HALL (1977, 1979) and VON SCHILCHER and HALL (1979) observed the behavior of sex mosaics in which the genotype of cells in the central nervous system, as well as the cuticle, could be ascertained. These investigators identified foci in the dorsal posterior brain for tapping, following, wing extension and licking (HALL 1977, 1979) and foci in the thoracic ganglion for courtship song and attempted copulation with a female (HALL 1979; VON SCHILCHER and HALL 1979).

The sexual responses of females have received comparatively little attention, presumably because the behavior of females is more subtle than the courtship performed by males. However, the ability of externally marked sex mosaics to stimulate courtship and to copulate with males has been analyzed. Although the genotype of the genitalia is irrelevant to the ability of a gynandromorph to elicit courtship (PATTERSON and STONE 1938), female tissue within the abdomen is necessary for “sex appeal” (HALL 1977; NISSANI 1977; JALLON and HOTTA 1979).

For copulation with males, there is an obvious requirement for female genitalia. It has also been suggested that there are anterior foci that must be of female genotype for receptivity to copulation (HOTTA and BENZER 1976; NISSANI 1977; COOK 1978; W. A. HARRIS in ARNOLD and KANKEL 1981; SZABAD and FAJSZI 1982). However, identification of foci has been complicated by the fact that a gynandromorph will fail to copulate with a male if it is tested in conditions that are not optimal for copulation, if it elicits insufficient courtship or if it is unreceptive.

We attempted to localize the putative anterior foci for receptivity by doing the following experiments. First, we identified conditions in which most female flies copulate, then measured the amount of courtship stimulated by females before copulation. From these data, we determined the minimum amount of courtship that was necessary to ensure that a male would repeatedly attempt copulation and a female would virtually always respond to one attempt by opening her vaginal plates. We then observed the sexual behavior of gynandromorphs with female genitalia in which the distribution of male and female tissue in the central nervous system could be ascertained. Finally, the distribution of female tissue in the few gynandromorphs that stimulated at least the minimum amount of courtship was correlated with their receptivity, using an analytical procedure for mosaic analysis which permits identification of foci for a behavior in a nonrandomly selected population of gynandromorphs. Our

FEMALE RECEPTIVITY OF MOSAICS 181

results provide evidence for a pair of discrete, submissive foci in the brain that are required to be of female genotype for a fly to be receptive to copulation with a male.

MATERIALS AND METHODS

For a description of most of the mutations mentioned in this section, see LINDSLEY and GRELL (1968).

Stocks: Sex mosaics were generated by crossing virgin y w; Acph-1"" females to (CO Acph-1 bv)+ y cv.y'/Y; pal; ca Acph-I"" males. This cross is virtually identical with the mosaic-producing scheme described in KANKEL and HALL (1976). However, the paternal X chromosome used in these experiments bears a translocation of the tip of the right arm of the third chromosome onto the distal tip (HARRIS 1977) and a proximal duplication of the distal tip of the X. This chromosome was constructed by crossing (ca Acph-1 bv)' y cv V' f+/y cv v f-y'; ca females to ca males, then selecting a recombinant (ca Acph-1 bv)' y cv v+ Fey'; ca male, that expressed only the cross- veinless mutation, from among the progeny. In addition, the paternal third chromosomes in the mosaic-producing stock were made homozygous for claret so that retention of the (ca Acph-1 bv)' translocation on the X chromosome could be ascertained by observing that the males had wild-type eye color.

Expression of the paternal loss (pal) mutation in the male parents resulted in loss of the paternal X chromosome (BAKER 1975) in 0.6-0.8% of the initially diplo-X progeny in these experiments. The resulting sex mosaics, which were of the genotype (ca Acph-1 bv)+ y cv. y'/y w; ca Acph-l""//O/ y w; ca Acph-l"", were recognized as flies with patches of wild-type brown-colored (female) and yellow (male) cuticle and, in some cases, wild-type red (female) and white (male) eye tissue. In addition, female tissue in the central nervous system had acid phosphatase activity, due to the presence of a wild-type allele of the Acph-1 gene on the paternal X chromosome. Male tissue in the central nervous system, in contrast, lacked acid phosphatase activity, since the mosaics were homozygous for Acph-l"", a null allele (BELL and MACINTYRE 1973).

One set of controls for these mosaics was generated by crossing (ca Acph-1 bv)' y cv. y'/Y; Cy/ pal; ca Acph-1"" males to virgin y w; Acph-1"" females, then selecting female progeny with normal (Cy') wings. These flies ("duplication-bearing females") were of the same genotype as female tissues in gynandromorphs. In addition, as a control for the possibility that the markers used to identify male tissue in gynandromorphs influenced the behaviors under investigation, y w; Acph- In" ("yellow white") females were obtained from the stock that provided the female parents of the mosaics.

Males with which the mosaics and the control females were tested were obtained from a wild- type Canton-S stock, initiated from a single male-female pair in 1976 and subsequently maintained in mass culture (TOMPKINS, HALL and HALL 1980).

Behavioral assays: All stocks were raised in bottles on a standard cornmeal-molasses medium at 25', on a 12:12 1ight:dark cycle, with the onset of light at 8 a.m. All behavioral assays were performed between 2 and 6 pm., a part of the day during which males court females vigorously.

Gynandromorphs with female genitalia, control females, and males were collected as young adults (1-6 h posteclosion), separated by sex under light ether anesthesia, then maintained individually in vials at 25'. Four days after collection, one Canton-S male and either a gynandromorph or a female were aspirated without anesthesia into a cylindrical plexiglass chamber (volume ca. 0.2 cm3; HALL 1977). After 1-2 min for acclimation to the chamber, the pair of flies was observed at 1OX magnification until they copulated or for 1 hr, whichever occurred first. During the observation period, the courtship index (C.I.), the percentage of time during which the male performed any of the courtship behaviors (TOMPKINS, HALL and HALL 1980), was measured. In addition, the number of times that the male attempted copulation, making genital contact, was counted.

At the end of the observation period, males were discarded. Females and gynandromorphs were maintained individually in vials for 24 h, after which time they were retested with Canton-S males as described. Females and gynandromorphs that copulated during the second observation period were stored for 24 h, then tested a third time. After the second test or, if one was necessary, the

182 L. TOMPKINS AND J. C. HALL

third test, most of the females and gynandromorphs were maintained individually in vials for 3-7 days, after which time the presence of larvae in the culture medium was noted.

Analysis of gynnndromorphs: The distribution of male and female tissues was ascertained in those gynandromorphs that fulfilled certain behavioral criteria. The genotypes of the external structures were determined by observing the mosaics at approximately 50X magnification and scoring the presence of brown-colored or yellow cuticle and red or white eye tissue. Mosaics were then immediately frozen and cut into horizontal sections, 10 pm thick, which were stained for acid phosphatase according to the procedure of KANKEL and HALL (1976). The genotypes of clusters of cell bodies in the central nervous system, the locations of which are shown in KANKEL and HALL (1976), were determined by scoring cells as darkly staining (female) or unstained (male). On each side of a fly, the genotype of 48 external structures and 33 groups of cells in the central nervous system was scored.

To identify foci that are required to be of female genotype for a fly to be receptive to copulation with a male, each landmark in the selected mosaics was analyzed to determine (1) the fraction of copulating mosaics in which the landmark was female and (2) the fraction of those mosaics in which the landmark was female that copulated. A comparison of these values revealed the presence of foci that were necessary and sufficient to be of female genotype for receptivity to copulation.

RESULTS

The behavior of females: The standard procedure for determining receptivity is to test females with two males each for an hour (e.g., HOTTA and BENZER 1976; HALL 1977; NISSANI 1977); however, in these conditions, as few as 65% of the females copulate (HALL 1977). We observed in preliminary observations that females tested in the presence of one male were more likely to copulate than females with two males (data not shown). Moreover, many females that failed to copulate with one male after an hour of testing did mate when they were retested with another male 24 hr later. This is shown by experiments in which duplication-bearing females were tested twice with single males; of those females, 92% copulated by the end of the second hr, although only 76% had mated during the first test period. After copulation, 98% of the duplication- bearing females were fertile. None of the females that copulated remated 24 hr later, as expected, since females fertilized by normal males are unreceptive for several days after copulation (MANNING 1962). These results (Table 1) demonstrated that most females that are of the same genotype as female tissues in mosaics copulate and show normal postcopulatory behavior under conditions in which control females and gynandromorphs were tested.

In another series of experiments, in which yellow white females were tested twice with single males, 94% of the females copulated by the end of the second test period. As shown in Table 1,96% of the yellow white females that copulated were fertile, and none remated 24 hr later. These data indicate that the markers used to identify male tissue in gynandromorphs have no effect on receptivity or fertility.

A gynandromorph that fails to copulate may have insufficient sex appeal, since courtship is a prerequisite for copulation in both sexes. Males do not attempt copulation without having performed at least a few seconds of courtship (MANNING 1967a), and females must be courted before they will open their vaginal plates, a behavior that is required for intromission (BASTOCK and MANNING 1955). Thus, before analyzing the behavior of gynandromorphs, it was


TABLE 1

Sexual responses of females and gynandromorphs"

Percentage of copu- Percentage of flies lating flies that were ulating flies that

Percentage of cop-

Type of fly that copulated fertile remated

Duplication-bearing female 92 (99) 98 (88) 0 (9) Yellow white female 94 (33) 96 (27) 0 (28) Selected gynandromorph 26 (42) 100 (11) 9 (11)

a Genotypes of flies and criteria for selecting gynandromorphs are shown in the text. Number of flies tested is in parentheses.

TABLE 2

Receptivity of females that stimulated different amounts of courtship"

Duplication-bearing females Yellow white females

C.I. Copulation No copulation Copulation No copulation

1-10 11-20 21-30 31-40 41-50 51-60 61-70 71-80 81-60 91-100

1 0 2 8 8 8 16 22 23 2

6 3 2 4 6 6 4 1 0 0

0 0 0 1 0 3 5 10 11 1

0 0 0 2 0 2 0 2 1 0

For each range of C.1.s (the percentage of time during which males performed any courtship behavior), the number of tests that either did or did not terminate in copulation is shown. The results from the first and second testing periods are pooled (see text).

necessary to observe females to determine how much courtship normally precedes copulation. Without this information, it would be impossible to identify foci for receptivity, since mosaics that fail to copulate because they stimulate insufficient courtship could not be distinguished from those that fail to copulate because they are unreceptive.

Accordingly, the receptivity of females as a function of the amount of courtship that they elicited was ascertained. As shown in Table 2, virtually all duplication-bearing and yellow white females that elicited courtship indices higher than 70 copulated during the test period; in contrast, many of the females that stimulated less courtship failed to mate. However, a re-analysis of these data suggested that the sexual responses of females during the first and second test periods were somewhat different. Specifically, 13 of 14 duplication-bearing females that stimulated courtship indices between 60 and 70 during the first test period copulated; in contrast, only one of five females that failed to mate during the first test period, then stimulated C.1.s between 60 and 70 during the second test period, eventually copulated. It was not possible to make this comparison for yellow white females, since only one fly of that genotype that

184 L. TOMPKINS A N D J. C. HALL

failed to copulate during the first test period subsequently elicited a C.I. between 60 and 70 during the second test period. Nevertheless, the results with the duplication-bearing flies suggested that females that failed to mate during the first test period might require more courtship stimulation during the second test period than females that copulated during the first test period did before mating.

Almost every male that courted females attempted copulation. Ninety-three percent (114 of 122) of the males tested with duplication-bearing females made genital contact at least once during the observation period, as did 100% (38 of 38) of the males tested with yellow white females. Although the probability that a male would attempt copulation was related to the C.I., less courtship stimulation was required for a male to make genital contact than for a female to respond by opening her vaginal plates. Most males with C.1.s between 20 and 60, and all males with C.1.s higher than 60, attempted copulation: in contrast, almost half of the males with C.1.s lower than 20 failed to attempt copulation (Table 3). Of the males that did make genital contact, very few achieved intromission the first time that they attempted to do so; 2 and 0% of the males tested with duplication-bearing and yellow white females, respectively, mated without having previously attempted copulation. As shown in Table 4, most males that mated attempted copulation two to three times before succeeding: in contrast, many of the males that failed to mate attempted copulation repeatedly during the test period.

The behavior of gynandromorphs: As described in the previous section, almost all virgin females that copulated stimulated C.1.s higher than 60 in the first test or C.1.s higher than 70 in the second test, if one was necessary. In addition, most females elicited attempted copulation two to three times before they mated. Accordingly, we required the gynandromorphs that were analyzed for receptivity to have female genitalia and, in addition, to fulfill one of two

TABLE 3

Attempted copulation by males that performed different amounts of courtship"

C.I.

1-10 11-20 21-30 31-40 41-50 51-60 61-70 71-80 81-90 91-100

Males tested with duplication-bearing females Males tested with yellow white females

Attempted copulation

No attempted copulation

Attempted copulation

No attempted copulation

4 2 4

11 13 12 20 23 23 2

3 1 0 1 1 2 0 0 0 0

0 0 0 3 0 5 5

12 12 1

0 0 0 0 0 0 0 0 0 0

"For each range of C.1.s the number of tests in which males either did or did not attempt copulation at least once is shown.


TABLE 4

Attempted copulations performed by males"

Males tested with duplication-bearing females Males tested with yellow white females

No. of attempted copulations Copulation No copulation Copulation No copulation

~~

0 8 0 1 2 5 0 3 2 46 3 17 1 3 39 2 13 0 4 3 1 1 0

5 or more 0 13 0 3

a The number of males that performed a given number of attempted copulations and either did or did not copulate is shown. For males that copulated, the successful attempt is included in the number of attempted copulations.

behavioral criteria: (1) stimulation of courtship, followed by copulation, or (2) stimulation of C.1.s higher than 60 and 70 m the first and second tests, respectively, and elicitation of at least three copulation attempts during each observation period. This selection was stringent, in the sense that a majority of gynandromorphs that failed to fulfill one of the behavioral criteria did stimulate as much courtship and as many copulation attempts as some normal females did before mating. However, the application of these criteria made it likely that most of the noncopulating gynandromorphs which were analyzed failed to mate because they lacked female tissue in foci for receptivity.

Of 389 gynandromorphs with female genitalia, 42 mosaics fulfilled the behavioral criteria and were thus selected for analysis. Although all of the selected gynandromorphs were like normal females in terms of their ability to stimulate high levels of courtship and to elicit repeated copulation attempts, the sexual behavior performed or elicited by the mosaics differed from that of females in two respects. First, almost half of the selected gynandromorphs (19 of 42) performed male courtship in response to one or both of the males with which they were tested, a behavior not observed in any of the duplication-bearing or yellow white females. This behavior usually resulted in the abrupt termination of the male's courtship bout, since a courting gynandromorph assumed a position immediately behind the male and thus became inaccessible to courtship, The other aberrant behavior observed was that several of the gynandromorphs (9 of 42) repeatedly stimulated males to court inappropriate parts of their bodies. Specifically, instead of tapping these mosaics' abdomens, then licking and attempting to copulate with their genitalia, males courted and in some cases attempted copulation with the mosaics' wings or eyes. Surprisingly, all of the gynandromorphs that elicited this behavior had boundaries between male and female tissue such that the mosaics had one yellow wing, one white eye or one of each; these male wings and eyes were the targets of the misdirected courtship. Since it was not known whether tapping, licking or attempting copulation with wings or eyes provided normal courtship stimulation to either


the courting male or the gynandromorph, the occurrence of these behaviors was not included in the compilation of the C.I.

In spite of the fact that many gynandromorphs performed or stimulated abnormal courtship behaviors, neither courtship of males or elicitation of misdirected courtship necessarily precluded copulation. As shown in Table 1, 26% (11 of 42) of the selected gynandromorphs copulated, one of which courted males and four of which elicited courtship of their wings or eyes. All but one of the mosaics that mated were subsequently fertile and were unreceptive to copulation when tested 24 hr later (Table 1). The exceptional gynandromorph copulated during the first and second observation periods. When tested a third time, this mosaic stimulated little courtship (C.I. = 6), extruded its ovipositor when courted, and was unreceptive to copulation, responses that are character- istic of fertilized females (BASTOCK and MANNING 1955; MANNING 1962; CON- NOLLY and COOK 1973). Examination of the vial in which the mosaic had been stored between the first and second tests revealed no fertilized eggs or larvae, although the gynandromorph did produce progeny after the second copulation. On the basis of these observations, it is likely that the mosaic remated because it initially copulated with a sterile male, an event that causes normal females to be transiently unreceptive for 12-48 hr (MANNING 1967a).

Analysis of gynandromorphs: The foregoing experiments demonstrated that all of the mosaics that copulated subsequently performed normal postcopulatory behavior. However, most of the selected mosaics did not copulate, in spite of the fact that they were tested under conditions in which female flies virtually always do so. All of the 42 selected gynandromorphs had external abdomens that were at least partially female. Furthermore, all of the gynanaromorphs elicited vigorous courtship, suggesting that their internal abdomens were also female (e.g., JALLON and HOTTA 1979). Since most of the selected gynandromorphs did not copulate, these observations implied that there was a requirement for female tissue in parts of the fly other than the abdomen for receptivity to copulation. Accordingly, the selected mosaics were analyzed to determine where the putative foci for receptivity were and whether they were required to be female unilaterally or bilaterally. First, the degree of femaleness was determined for each of the 81 landmarks by classifying the genotype of each structure as entirely female, entirely male, or mixed, then dividing the number of instances in which a structure was female by the number of instances in which it was either completely male or completely female. As expected, since the selected gynandromorphs had female genitalia and female tissue within the abdomen but were otherwise dissimilar to each other, the degrees of femaleness for different structures varied widely, ranging from 1.00 for the genitalia to 0.38 for the median ocellus (Table 5). The average degree of femaleness for all landmarks was 0.62 k 0.16 (& s.D.).

Next, the average degree of femaleness for all landmarks was compared with the fraction of mosaics that copulated to determine whether the foci were required to be of female genotype unilaterally or bilaterally. In general, since the average degree of femaleness represents the probability that landmarks in a gynandromorph are of female genotype on either side of the fly, this value


TABLE 5

Sexual behavior and genotype of landmarks in the selected gynandromorphs"

Landmark

Proboscis Antenna Vibrissa bristle Carina bristle Palp Anterior postorbital bristle Posterior postorbital bristle Anterior vertical bristle Posterior vertical bristle Anterior orbital bristle Median orbital bristle Posterior orbital bristle Ocellar bristle Median ocellus Lateral ocellus Postvertical bristle Eye SPl SP2 SP3 s p l l sp12

ogl

og3 sp21 sp22

sp13

sp23 sp24 og1l og12 sbl sb2 og21 og22 Og23 Dorsal humeral bristle Ventral humeral bristle Presutural bristle Anterior notopleural bristle Posterior notopleural bristle Anterior supra-alar bristle Posterior supra-alar bristle Anterior postalar bristle Posterior postalar bristle Anterior dorsocentral bristle

Fraction of copulating

Frequency mosaics that of unilateral are bilaterally

Location femaleness female

External head External head External head External head External head External head External head External head External head External head External head External head External head External head External head External head External head Brain Brain Brain Brain Brain Brain Optic lobe Optic lobe Optic lobe Brain Brain Brain Brain Optic lobe Optic lobe Brain Brain Optic lobe Optic lobe Optic lobe External thorax External thorax External thorax External thorax External thorax External thorax External thorax External thorax External thorax External thorax

0.55 0.49 0.51 0.52 0.56 0.55 0.54 0.40 0.42 0.41 0.47 0.45 0.39 0.38 0.48 0.45 0.51 0.44 0.44 0.43 0.40 0.45 0.46 0.54 0.50 0.50 0.44 0.48 0.54 0.51 0.54 0.48 0.51 0.56 0.51 0.51 0.48 0.64 0.63 0.69 0.69 0.70 0.69 0.64 0.65 0.61 0.62

0.91 0.64 0.55 0.73 0.91 0.91 0.73 0.73 0.82 0.82 1.00 0.82 0.82 0.82 0.91 0.73 0.82 0.91 0.91 0.91 1.00 0.91 0.91 0.91 0.91 0.91 0.82 0.82 0.91 0.91 0.91 0.91 0.91 0.91 0.82 0.82 0.82 0.73 0.73 0.73 0.64 0.64 0.55 0.27 0.36 0.27 0.36

Fraction of bilaterally female mosaics that copulate

0.55 0.58 0.50 0.57 0.59 0.59 0.53 0.80 0.75 0.75 0.79 0.79 0.90 0.69 0.67 0.73 0.75 0.71 0.77 0.77 0.92 0.77 0.71 0.63 0.63 0.63 0.64 0.75 0.59 0.63 0.63 0.63 0.77 0.59 0.56 0.56 0.64 0.50 0.47 0.40 0.37 0.35 0.33 0.20 0.25 0.23 0.27

188 L . TOMPKINS AND J. C. HALL

TABLE 5-Continued

Landmark

Posterior dorsocentral bristle Anterior scutellar bristle Posterior scutellar bristle Anterior sternopleural bristle Posterior sternopleural bristle Wing Prothoracic leg Mesothoracic leg Metathoracic leg tgl tgl l tg12

tg21 tg22

tg13

tg23 tg24 tg31 tg32 tg41 tg42 tg43 First tergite Second tergite Third tergite Fourth tergite Fifth tergite Sixth tergite First sternite Second sternite Third sternite Fourth sternite Fifth sternite Genitalia

Location

External thorax External thorax External thorax External thorax External thorax External thorax External thorax External thorax External thorax Thoracic ganglia Thoracic ganglia Thoracic ganglia Thoracic ganglia Thoracic ganglia Thoracic ganglia Thoracic ganglia Thoracic ganglia Thoracic ganglia Thoracic ganglia Thoracic ganglia Thoracic ganglia Thoracic ganglia Abdomen Abdomen Abdomen Abdomen Abdomen Abdomen Abdomen Abdomen Abdomen Abdomen Abdomen Abdomen

Fraction of Fraction of copulating bilaterally

Frequency mosaics that female mo- of unilateral are bilaterallv saics that

__ femaleness female copulate

0.61 0.62 0.63 0.67 0.70 0.72 0.69 0.74 0.71 0.71 0.63 0.60 0.74 0.66 0.67 0.69 0.87 0.73 0.85 0.75 0.73 0.83 0.82 0.82 0.80 0.83 0.84 0.90 0.88 0.87 0.88 0.96 0.99 1.00

0.27 0.36 0.45 0.55 0.64 0.64 0.73 0.82 0.73 1.00 0.64 0.36 0.91 0.73 0.64 0.64 1 .oo 0.73 0.91 0.82 0.64 0.82 0.73 0.73 0.73 0.64 0.64 0.82 0.91 0.91 0.91 1.00 1.00 1.00

0.21 0.31 0.33 0.35 0.37 0.39 0.40 0.43 0.36 0.44 0.37 0.36 0.40 0.40 0.44 0.35 0.33 0.36 0.32 0.36 0.44 0.30 0.31 0.30 0.32 0.25 0.23 0.26 0.31 0.31 0.31 0.27 0.27 0.26

__ For locations of the landmarks in the brain, optic lobes and thoracic ganglia, see KANKEL and

HALL (1976).

would be expected to be similar to the fraction of mosaics that copulated if the receptivity foci only needed to be female unilaterally (domineering foci). If, on the other hand, female tissue were required bilaterally (submissive foci), the average degree of femaleness for the selected mosaics would be expected to be higher than the fraction of mosaics that copulated. In the selected gynandromorphs, the average degree of femaleness for all landmarks (0.62) was markedly higher than the fraction of mosaics that copulated (0.26), suggesting that the foci were submissive. However, one assumption in this comparison is that the average degree of femaleness for all landmarks is similar to the degree of


femaleness of landmarks encompassing the foci. This assumption is valid for unselected gynandromorphs, in which all of the landmarks have been shown to have similar degrees of femaleness (e.g., KANKEL and HALL 1976); therefore, the average degree of femaleness is a reasonably accurate reflection of the degree of femaleness for any specific landmark in such mosaics. In the selected gynandromorphs, however, the degrees of femaleness for different structures varied widely, raising the possibility that the degree of femaleness in the vicinity of the receptivity foci could be much lower than average. Since the locations of the receptivity foci were not known a priori, it was not possible to ascertain the degree of femaleness for landmarks encompassing the foci. However, the lowest degree of femaleness for any landmark (0.38) was still higher than the fraction of mosaics that copulated (0.26), which implies that the receptivity foci are in fact submissive.

To map the foci roughly, the behavior of gynandromorphs with abdomens, thoraces or heads that were unilaterally or bilaterally of one genotype was ascertained (see HOTTA and BENZER 1972; HALL 1978). The population of selected gynandromorphs included mosaics with entirely male heads or thoraces. Since all of these mosaics stimulated vigorous courtship, this observation implies that there is no focus for sex appeal in the brain or thoracic ganglion. With regard to receptivity, gynandromorphs that were bilaterally split into male and female halves except for the genitalia (which were female) did not copulate; this provides additional evidence for the submissive nature of the receptivity foci. Most of the 23 gynandromorphs with female abdomens failed to copulate, as did all of the five mosaics whose thoraces were virtually or totally female, implying that the presence of female tissue in the abdomen or thorax was insufficient for receptivity. In contrast, all of the eight gynandromorphs with entirely female brains and optic lobes, including seven mosaics with completely female heads and one with a single male head bristle, were receptive to copulation. Conversely, no copulation was observed in any of the 11 gynandromorphs with male brains and optic lobes, all of which had entirely male heads with the exception of one mosaic that had a single female head bristle. These results, summarized in Table 6, suggested that the foci for receptivity to copulation are on or in the head.

Having determined that there is apparently a focus on the head or in the brain or optic lobes for receptivity, we analyzed the selected gynandromorphs to determine whether the presence of bilateral female tissue in one landmark in the head was necessary and sufficient for copulation (Table 5). First, we calculated the frequency at which each landmark was bilaterally female in the copulating mosaics, a value that would be 1.00 if female tissue in a landmark were necessary for receptivity. Seven structures were bilaterally female in all of the copulating mosaics: the genitalia, the fifth and sixth sternites, two groups of cells in the thoracic ganglion, the median orbital bristle on the head and one group of cells in the dorsal anterior brain. Next, we identified the selected gynandromorphs that were bilaterally female at each landmark and then calculated the fraction of those mosaics that copulated; this value would be approximately equal to the fraction of female flies that copulated if bilateral

190 L. TOMPKINS A N D J. C. HALL

female tissue in a landmark were sufficient for copulation in the selected gynandromorphs. Of the seven landmarks that were bilaterally female in all of the copulating mosaics, all but one were also bilaterally female in many of those mosaics that failed to copulate. This observation indicates that female tissue in those landmarks was insufficient for receptivity. With regard to the remaining landmark, designated s p l l (site 11 in the supraesophageal ganglion; see KANKEL and HALL 1976), none of the 30 mosaics that had male tissue in sp l l copulated. Figure 1 shows an example of such a mosaic, in which most of the brain is female, whereas s p l l as well as more dorsal and more ventral sites on one side of the brain are male.

Almost every mosaic (11 of 12) that was bilaterally female at sp l l was receptive. Since the fraction of mosaics with bilateral female tissue in s p l l that copulated (0.92) was virtually identical with the fraction of duplication-bearing and yellow white females that copulated (0.91 and 0.94, respectively), this result suggests that there are bilateral foci for receptivity in or very close to sp11 in the dorsal anterior brain.

Although we were unable to determine exactly where the receptivity foci were in relation to sp l l , one gynandromorph did provide some information about the location of the foci. In this mosaic, which was unreceptive, sp l l on the right side of the brain was completely female; on the left side, the medial cells of sp l l were female and the lateral cells within this site were male. Although it is possible that the mosaic was bilaterally female in the foci for

TABLE 6

Behavior of bilaterally split gynandromorphs and those with abdomens, thoraces and heads of one genotype

Type of gynandromorph No. observed No. that copulated

Bilaterally split Female abdomen Male abdomen Female thorax Male thorax Female head Male head

4 23 0 5" 3 ab

1lC

0 6

a Two of these had two male bristles each on the thorax. * Two of these had two male bristles each on the head.

One of these had one female bristle on the head.

FIGURE 1.-Mosaicism in the brain of a gynandromorph. Each photograph is from a 10-pm frozen section, cut horizontally through the head of a diplo-X/haplo-X mosaic that was not receptive to copulation. This gynandromorph did elicit vigorous courtship. A represents a dorsal section, B is ventral with respect to A, and C is ventral with respect to B. In each photograph, the anterior part of the head is at the top. spl l is a specific group of cells in the anterior cortex of the brain. In this mosaic, spl l on the right side did not stain for acid phosphatase and is thus identified as male: s p l l on the left side did stain for the enzyme and is thus identified as female. A group of cells ventral to spl1, which is also male on the right side and female on the left side, has been labeled accordingly. es-esophagus. Scale bar (in A) = 100 pm for all three photographs.

-. . -<---- .- I .-

191


receptivity, the fact that nearly all female flies and mosaics in which both foci were entirely female copulated suggested that the gynandromorph had unilateral male tissue in a critical part of the brain. Accordingly, if the foci are within s p l l , they either encompass the entire landmark or are restricted to the lateral cells. In any event, the possibility that the foci represent only the medial cells of s p l l is probably excluded.

DISCUSSION

The sexual behavior of Drosophila females is complex. Although female flies appear to be passive, they attract males by moving, which provides a visual stimulus (TOMPKINS et al. 1982), and by releasing a sex pheromone (TOMPKINS, HALL and HALL 1980; VERNARD and JALLON 1980). Males react to these visual and olfactory cues by performing the courtship behaviors, which stimulate females to respond to attempted copulation by opening their vaginal plates. From this description, it is obvious that a fly must have female genitalia and, in addition, must stimulate vigorous courtship to copulate with a male. For sex appeal, it is essential that there be female tissue within the abdomen (HALL 1977; NISSANI 1977; JALLON and HOTTA 1979), although we have shown that there is no requirement for female tissue within the head or thorax for this behavior.

To identify foci for receptivity, it was thus necessary to analyze gynandromorphs in which the genitalia and most of the abdomen were female. Such mosaics are systematically biased: the genitalia are always female, other abdominal structures have a high probability of being female, and the likelihood that head and thoracic landmarks are female is inversely related to the distance of those structures from abdominal landmarks on a fate map (see HALL 1978). The fact that the frequency at which structures were of female genotype was dependent on their anatomical location put severe constraints on the procedures that could be used to localize the receptivity foci. HOTTA and BENZER’S (1972) original method for mapping foci determines the distance between a focus and a landmark by calculating the number of instances in which the landmark is male and the behavior is female or vice versa. One assumption of this method is that mosaics are, on the average, half male. Since the average frequency of maleness in the selected mosaics was 0.38 (1-0.62, the average frequency of femaleness), this procedure was clearly not applicable. A modification of HOTTA and BENZER’S method, which makes no assumption about the proportion of male tissue in mosaics, is the contour mapping procedure (HOTTA and BENZER 1976; J. FEITELSON and L. HALL, cited in HALL 1978), in which the distance between a focus and a landmark is determined by calculating the number of instances in which mosaics with female behavior are male with respect to the landmark. Application of this procedure to the selected gynandromorphs leads to the erroneous conclusion that there are seven pairs of foci for receptivity, since there were seven pairs of landmarks that were bilaterally female in all of the copulating mosaics. Although most of those landmarks would undoubtedly be eliminated from consideration if more mosaics were analyzed, use ‘of the contour mapping procedure would yield a spurious focus regardless of sample size, since the gynandromorphs were selected on the basis of their having


female genitalia. The fact that the genitalia were female in all of the selected mosaics also precluded use of FLANAGAN’S (1977) focusing method for localizing foci, since one of the variables in an equation used to determine the distance between a focus and a landmark assumes an infinite value for landmarks which are always of one genotype.

In contrast to the foregoing procedures, our “necessity and sufficiency” criteria for localizing behavioral foci can be used to analyze mosaics in which landmarks are always of one genotype, or mosaics in which the frequency at which a structure is male or female varies widely from landmark to landmark. For receptivity to copulation, the foci are localized to the landmark that is bilaterally female in all copulating mosaics and that, when bilaterally female, is usually sufficient for copulation. However, use of these criteria is not restricted to submissive, discrete foci. For domineering foci, unilateral male or female tissue at a landmark is necessary and sufficient for the performance of a behavior; for diffuse foci, the genotype of a group of landmarks in one part of the fly is crucial. Accordingly, this technique may be generally useful for identifying foci for behaviors that can only be analyzed in selected gynandromorphs, such as behaviors that are performed in an obligatory sequence or those for which more than one part of the fly is required to be of a specific genotype.

With regard to receptivity, we have used the necessity and sufficiency criteria to identify a group of cells in the dorsal anterior brain that must be bilaterally female for copulation with a male. These foci are anatomically distinct from the cells in the dorsal posterior brain which are required to be at least unilaterally male for tapping, following, and wing extension, three of the male courtship behaviors (HALL 1977, 1979). This finding is in agreement with our finding that one of the selected gynandromorphs performed courtship and was receptive to copulation and with the observations of COOK (1978) and SZABAD and FAJSZI (1982) that some mosaics are able to court females and, in separate tests, copulate with males.

In contrast, no mosaics have been observed to lick female genitalia and copulate with males (e.g., COOK 1978). These findings suggested the possibility that the behaviors were mutually exclusive, in the sense that the same foci in the dorsal posterior brain which are required to be bilaterally male for licking (HALL 1979) were required to be bilaterally female for receptivity. Localization of the receptivity foci to the anterior brain thus provides the first evidence that the foci for receptivity and licking are in separate parts of the central nervous system. The lack of mosaics that lick females and copulate with males can be explained by the fact that the landmarks associated with the foci for the two behaviors are close to each other on a fate map (see KANKEL and HALL 1976) and that both foci are submissive. In addition, there is the aforementioned requirement that abdominal tissues be female for the courtship that is a prerequisite for copulation. Hence, the probability that a mosaic dividing line would partition a gynandromorph in such a way that the anterior brain is bilaterally female, the posterior brain is bilaterally male, and the abdomen is female is vanishingly small.


The landmark associated with the receptivity foci, spl l , includes cell bodies that project their axons into the alpha lobe of the mushroom bodies (STRAUSFELD 1976). This part of the brain is of interest as a region in which input from chemoreceptors and mechanoreceptors converges; olfactory input, in particular, is processed primarily in the mushroom bodies (HEISENBERG 1980). Female flies that cannot perceive olfactory or auditory stimuli have a high probability of being unreceptive to copulation (MANNING 1967b; BURNETT, EASTWOOD and CONNOLLY 1977; TOMPKINS et al. 1982). In light of this anatomical and behavioral evidence, the receptivity foci may represent parts of the female brain that integrate input from receptors stimulated by the courting male’s sex pheromones and song.

We thank RICHARD ROTHMAN for doing some preliminary work on this project. We also thank THOMAS CLINE and RALPH HILLMAN for their comments on the manuscript. Supported by United States Public Health Service grant GM 21473 to J. C. H.

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identification of brain sites controlling …€¦laurie tompkins*.' and jeffrey c. hall**...

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