The role of cuticular pheromones in courtship conditioning of Drosophila males

Abstract

Courtship conditioning is an associative learning paradigm in Drosophila melanogaster, wherein male courtship behavior is modified by experience with unreceptive, previously mated females. While the training experience with mated females involves multiple sensory and behavioral interactions, we hypothesized that female cuticular hydrocarbons function as a specific chemosensory conditioned stimulus in this learning paradigm. The effects of training with mated females were determined in courtship tests with either wild-type virgin females as courtship targets, or with target flies of different genotypes that express distinct cuticular hydrocarbon (CH) profiles. Results of tests with female targets that lacked the normal CH profile, and with male targets that expressed typically female CH profiles, indicated that components of this CH profile are both necessary and sufficient cues to elicit the effects of conditioning. Results with additional targets indicated that the female-specific 7,11-dienes, which induce naive males to court, are not essential components of the conditioned stimulus. Rather, the learned response was significantly correlated with the levels of 9-pentacosene (9-P), a compound found in both males and females of many Drosophila strains and species. Adding 9-P to target flies showed that it stimulates courting males to attempt to copulate, and confirmed its role as a component of the conditioned stimulus by demonstrating dose-dependent increases in the expression of the learned response. Thus, 9-P can contribute significantly to the conditioned suppression of male courtship toward targets that express this pheromone.

Figure 1.

Levels of the 24 most abundant hydrocarbons measured by gas chromatography in cuticular extracts of (A) Canton-S virgin females, (B) Canton-S males, and (C–H) six different genotypes used as courtship targets to assay the effects of courtship conditioning. The specific compounds represented by each bar number (#) are identified in Table 1. Data from 6–12 individual flies were averaged for each panel; there was very little variation within each of these genotypes (Savarit and Ferveur 2002a). Unsaturated compounds are graphed as black bars; saturated compounds as gray bars.

Figure 2.

The effects of training with mated females on subsequent courtship of Canton-S males toward different targets. The mean Courtship Indexes (CIs) are plotted (±SEM) for groups of Naive (black bars, N = 38–54 per group) and Trained (gray bars, N = 19–37 per group) male subjects tested with decapitated targets of the indicated genotypes, either 5 min (lighter gray bars) or 60 min (dark gray bars) after training. The test targets were (A) Canton-S virgin females, (B) hs-tra virgin females, (C) desat1¹⁵⁷³ virgin females, (D) 376-GAL4/+; UAS-tra/+ males, (E) Tp2602-GAL4/+; UAS-tra/+ males, (F) Tai2 males, and (G) desat1¹⁵⁷³ × Tai2 males. Differences between the CIs of Trained groups and Naive controls with each target were evaluated by ANOVA; (^**) P < 0.01; (^***) P < 0.001.

Figure 3.

The effects of training with mated females on males' attempts to copulate with different targets during 10-min courtship assays performed 5 min after training. The percentage of males that attempted to copulate with decapitated targets of the indicated genotypes is plotted for groups of Naive (black bars, N = 19–26 per group) and Trained (gray bars, N = 20–26 per group) male subjects. Fewer Trained males than Naives attempted to copulate with (A) Canton-S virgin females (χ² = 6.059, df = 1, [^*] P < 0.02) and (D) Tp2602-tra males (χ² = 6.584, df = 1, [^*] P < 0.02), but the frequencies of copulation attempts with other targets were independent of subjects' training status (by χ² tests of association). These targets were (B) hs-tra virgin females, (C) desat1 virgin females, (E) Tai2 males, and (F) desat1 × Tai2 males.

Figure 4.

Plots of Learning Index (LI) versus the levels of specific CHs in different target flies used to assay the effects of courtship conditioning. LI values were calculated as the percent reduction in the mean CI of trained males, tested 5 min after training, relative to naive controls, in a total of 16 experiments involving nine different target genotypes. These LI values are plotted versus the levels (nanograms/fly) of six individual unsaturated hydrocarbons in cuticular extracts of the target genotypes. CH profiles for seven of these targets are shown in Figure 1A, C–H; each of these was used in two experiments. Two additional pGAL4/+; UAS-tra/+ genotypes were used as targets in one experiment; these were Tp3502(GAL4)-tra males and Tp4778(GAL4)-tra females, which both had CH profiles similar to Canton-S females (Savarit and Ferveur 2002a). (A) LI versus 9-P, (B) LI versus 7-T, (C) LI versus 5-H, (D) LI versus 7-P, (E) LI versus 7,11-HD, and (F) LI versus 7,11-ND. Linear fits and correlation coefficients (R) are indicated only for the three CHs for which the simple regressions yielded P < 0.05. Also see Table 1.

Figure 5.

The effects of exogenous 9-pentacosene (9-P) on the Courtship Index (±SEM) of Naive (black bars) and Trained (gray bars, tested 5 min after training) males with (A) Canton-S virgin females (N = 20 per group), and three additional courtship targets that express no detectable 9-P among their cuticular hydrocarbons: (B) hs-tra virgin females (N = 11–28 per group), (C) desat1¹⁵⁷³ virgin females (N = 20–21 per group), and (D) immature Canton-S males (N = 20 per group). Two-way ANOVA indicated a significant effect of 9-P only in tests with desat1¹⁵⁷³ females (F_2,117 = 7.778, P < 0.01), where CIs with the high dose of 9-P were significantly greater than in the control or low dose conditions ([^**] P < 0.01, [^***] P < 0.001 by Fisher's PLSD). The two-way ANOVA also revealed significant effects of training on CIs with Canton-S females (F_1,89 = 143.2, P < 0.0001), with desat1¹⁵⁷³ females (F_1,117 = 16.22, P = 0.0001), and with immature Canton-S males (F_1,114 = 6.391, P < 0.05). No significant interactions were found between training and 9-P dose.

Figure 6.

The effects of exogenous 9-P on the frequency of copulation attempts of Naive (black bars) and Trained (gray bars, tested 5 min after training) males with different courtship targets, from the same experiment for which CIs are plotted in Figure 5. Possible effects of training and of 9-P were evaluated independently by χ² tests of association. The effect of 9-P (with Naive and Trained groups combined; thus brackets are positioned above each pair of bars) was to significantly increase the percent of males attempting to copulate with (A) Canton-S females (χ² = 6.988, df = 1, [^**] P < 0.01), (B) hs-tra females (χ² = 11.01, df = 2, [^**] P < 0.01), and (C) desat1¹⁵⁷³ virgin females (χ² = 8.200, df = 2, [^*] P < 0.02), but not with (D) immature Canton-S males (χ² = 2.800, df = 2, P = 0.2). Similarly, effects of training were assessed across all 9-P doses; these tests revealed a significant association between training status and attempts to copulate with (A) Canton-S females (χ² = 18.26, df = 1, P < 0.0001), (C) desat1¹⁵⁷³ females (χ² = 5.927, df = 1, P < 0.02), and (D) immature Canton-S males (χ² = 7.420, df = 1, P < 0.01), but not with (B) hs-tra females (χ² = 0.001, df = 1, P = 0.97).