Conditioning Mutations in Drosophila Behavioral Modification in Courting Males Melanogaster Affect an Experience-dependent

One aspect of courtship in1 male Drosophila melanogaster has been reported to be experience dependent. Males that have courted fertilized females are virtually unresponsive to virgin females for 2-3 hr. Here, this response was utilized as an assay for the effects of conditioning mutations on experiencedependent courtship. Seven strains expressing conditioning mutations (previously isolated and characterized for learning or memory defects in an electrical shock-odor association paradigm, independent of courtship) were all found to be mutant in expression of this experience-dependent change in courtship behavior. By comparison, three control strains that were unselected for conditioning defects all expressed normal experience-dependent courtship. Other results indicate that males of the conditioning-defective strains are able to elicit necessary cues from fertilized females, yet do not then modify their courtship with virgin females. Thus, it is suggested that experience-dependent modification of courtship and the previously reported associative olfactory conditioning with electric shock share common elements of processing. The possibility that experience-dependent courtship represents adaptive behavior is discussed. ERTILIZED Drosophila melunoguster females stimulate males to court them F (BASTOCK and MANNING 1955), but this courtship is less vigorous than that elicited by virgin females (COOK and COOK 1975; SIEGEL and HALL 1979) and rarely results in copulation (CONNOLLY and COOK 1973). Males that have courted fertilized females express a temporary modification of their courtship behavior with subsequent females. For 2-3 hr they remain virtually unresponsive to virgins (SIEGEL and HALL 1979), and for nearly 1 day they avoid courtship with other fertilized females (D. A. 'GAILEY, unpublished results). These and other examples of experience-dependent courtship behavior in Drosophila (O'HARA, PRUZAN and EHRMAN 1976; VON SCHILCHER 1976; GAILEY, JACKSON and SIEGEL 1982) imply that certain components of courtship are modifiable, in contrast to the usual view that the courtship response in this genus is made up entirely of sequences of fixed action patterns (e.g., SPIETH 1968). This report represents a further analysis of the modified response that ' Current address: Department of Biology, Brandeis University, Waltham, Massachusetts 02254. 'Current address: Department of Genetics, Albert Einstein College of Medicine, Bronx, New York 10461. Genetics 106: 613-623 April, 1984. 614 D. A. GAILEY, F. R. JACKSON AND R. W. SIEGEL follows courtship experience with fertilized females; we refer to this phenomenon as "experience-dependent courtship." After courting virgin females, males continue to court other virgins with no change in their "ardor." By comparison, after courting fertilized females, males do not court virgins. We show here that this response serves as a reliable assay for the effects of learning and memory mutations on experience-dependent courtship, which can be revealed in individual male flies. In this respect, results will be presented that indicate that this phenomenon is amenable to mutational analysis. In a different courtship situation, males can be induced to avoid otherwise courtship-stimulating, newly emerged males (GAILEY, JACKSON and SIEGEL 1982). Aspects that distinguish the effects of courting fertilized females us. immature males will be discussed, along with the possibility that the capacity to express experience-dependent courtship represents adaptive behavior. MATERIALS AND METHODS Normal D. melunoguster males were obtained either from a stock of the Canton-S strain (hereafter designated "wild type"), or from "gel-16," a stock established in late 1979 from a single inceminated female caught near Gelendzik, USSR. Mutant males were obtained from the following stocks, which were isolated on the basis of their inability to associate a chemical odor with electric shock. cabbage (cub, X-linked; ACEVES-PIKJA and QUINN 1979). dunce (dnr ' , dnr', dn~~ '~) -mutant dnc alleles, mapping to the 3D region of the X chromosome (BYERS, DAVIS and KICER 1981). The dnc' mutation is maintained in a homozygous mutant stock; dnc2 is a female-sterile mutation (e.g., SALZ, DAVIS and KICER 1982), so males of this strain were maintained with C(l)DX, y f females. The dnd"14 allele was isolated as a female-sterile mutation (MOHLER 1973) and was later found to affect shock-odor-associative conditioning (BYERS, DAVIS and KICER 1981). The d n F 4 stock carries the recessive markers y (yellow cuticle), N (crossveinless wings), v (vermilion eye color) and f (forked bristles). Mutant dunce flies have increased levels of cyclic-AMP compared with wild type, attributable to defects in one form of cyclic-AMP phosphodiesterase (PDE) (reviewed by DAVIS and KAUVAR 1984). rutabaga (rut, 1:46; M. S. LIVINGSTONE and R. E. FORRESTAL, unpublished results): Flies from this strain have subnormal adenylate cyclase activity and decreased levels of cyclic-AMP compared with wild type (LIVINGSTONE, SZIBER and QUINN 1982). turnip (tur, near cur on the X chromosome; BOOKER and QUI" 1981). Flies expressing the amnesiac mutation (amn, near f on the X chromosome) condition as wild type in shock-odor association tests but retain the effects of training for a relatively brief time compared with wild type (QUINN, SZIBER and BOOKER 1979). Males from the y n, vfstrain were tested for conditioned courtship, since they carry the same genetic markers as the d~1L'"'~ stock but express the dnc+ allele. Females used in courtship tests were C(I)DX, y J attached-X. Stocks were maintained at 25" on a medium of cornmeal, sucrose, dextrose, wheat germ, yeast and agar. Males and females to be tested as sexually mature flies were collected under light ether anesthesia within 10 hr after eclosion. Males were stored singly in food vials; females were stored ten per vial. These flies were tested 5 days later in the same room in which they had been maintained on a 12-hr light-12-hr dark cycle. A humidifier was in operation continually in this room, and the relative humidity ranged from 30-60%. Fertilized females were obtained by pairing 5-day-old wild-type males with 4-day-old virgin females, observing copulation and then using the females in experiments the following day. Certain tests required that mature males be paired with sexually immature, newly eclosed males (which stimulate the similar high level of courtship that mature virgin females do; e.g., JALLON and COURTSHIP IN DROSOPHILA 615 HOTTA 1979). Immature males were collected by aspiration and used in tests within 4 hr after their eclosion. In other tests, males were paired with sexually immature virgin females, which also stimulate as much courtship as mature virgin females but do not copulate (MANNING 1967). Immature females were collected under light ether anesthesia and then aged 14-20 hr before use in courtship tests. All conditioning and test pairings were carried out in the cylindrical chambers (0.4 cm3) of a ‘mating wheel” (HOTTA and BENZER 1976). A 6-mm diameter circlet of no. 42 Whatman filter paper moistened with distilled water was placed into each chamber to ensure a humid environment. This negates otherwise spurious effects on conditioned courtship apparently brought about by low humidity (R. W. SIEGEL, unpublished results). Quantijcation of experience-dependent courtship: The courtship response of each male being tested was measured by a courtship index (CI, e.g., TOMPKINS, HALL and HALL 1980), that is, the percentage of time within an observation period that a male displayed courtship behavior. A standard test for experience-dependent courtship was carried out by determining the response of each test male to an ether-immobilized, 5-day-old virgin female; immobilizing the female reduces variability in the assay (see discussion in SIEGEL and HALL 1979). The length of an observation period was determined by the time required for the etherized fly to regain mobility (usually about 8-10 min). Experience-dependent courtship responses of the mutant strains relative to wild type were determined by comparing indices of experience-dependent courtship (A,) calculated for each strain. X, is a ratio given by: x,= 1 3 X I 0 0 ( Cl,) where CI, = mean CI with mature virgin females of 20 males that have first courted immature virgin females; CI, = mean CI with mature virgin females of 20 males that have first courted fertilized females. CI, was determined by individually pairing courtship-naive test males with immature virgins for a 30-min period, then transferring each test male into a fresh chamber by gentle aspiration, allowing a 5-min accommodation and then measuring the CI with an added ether-immobilized virgin female. To test for a modified response, CI, was recorded for other males that had been individually paired with fertilized females for 30 min. The test for thP ability of males to elicit from firtilized females the cues that mediate experience-dependent courtship: We have isolated and begun characterization of a mutant strain we call don giovanni, dg (D. A. GAILEY and R. W. SIEGEL, unpublished results). One aspect of the dg phenotype is that fertilized females do not induce experience-dependent courtship in dg males. Males from this strain do not express experience-dependent courtship apparently because they are unable to elicit appropriate cues from fertilized females. This interpretation is based, in part, on the fact that fertilized females courted by dg males do not then induce experience-dependent courtship in wildtype males (D. A. GAILEY and R. W. SIEGEL, unpublished results). The following experiment was carried out to test whether males from conditioning-defective strains are, like dg, not conditioned simply because they are unable to elicit the appropriate cues from fertilized females. Twenty fertilized females were individually paired with wild-type males for a 30-niin period. The central nervous system (CNS) of each fertilized female was then cauterized by crushing the head with hot no. 5 Dumont forceps under COS anesthesia (such flies live for hours and can express conditioned behavior; BOOKER and QUINN 1981). Twenty additional wild-type males were next paired for 30 min with these fertilized females (now rendered overtly ”unresponsive” to male courtship) and were then tested for their CIS with virgin females in the usual way. A series of experiments of this type was carried out with dg, amn, cub, dnc’, rut or tur males being first paired with fertilized females. The remainder of each experiment was then carried out in the same way, testing whether these fertilized females, “primed” by prior courtship performed by the various types of males, had the capacity to bring about experience-dependent courtship in wild-type males. Statistics: Determination of significant difference among CI or A, values was accomplished by Student’s two-tailed t-test. All C1 and A, values are reported as means f SEM. The SEM for a A, value was computed from a formula for the variance of A, as a function of CI, and CI, with known variances (equation A.9.14 of CROW and KIMURA 1970). 616 D. A. GAILEY, F. R. JACKSON AND R. W. SIEGEL