w¹¹¹⁸ males did not display any discernible ethanol-induced intermale courtship upon recurring ethanol treatment, which was reversed by transgenic w expression in mw⁺/+, mw⁺/mw⁺, and hs-mw⁺/+ without (hs-mw⁺/+) and with heat shock (hs-mw⁺/+ Hs). Diamonds denote no courtship observed. General linear model revealed the significant effects of exposure, genotype, and interaction (exposure effect, F_3,167 = 266.85, p<0.0001; genotype effect, F_5,167 = 181.04, p<0.0001; double asterisks, significant difference by Tukey-Kramer tests in each exposure; interaction, F_15,167 = 28.23, p<0.0001; n = 3–11).

(A) Courtship. The wings of CS males or females were clipped to distinguish the sex. The ethanol-naïve or ethanol-treated males were housed with an equal number of virgin females and subjected to ethanol exposure in 95% Flypub. Two-factor ANOVA revealed the significant effects of partner, exposure, and interaction in both sets of experiments (intact male with wing-clipped female: partner effect, F_1,27 = 75.8, p<0.0001; exposure effect, F_1,27 = 93.9, p<0.0001; interaction, F_1,27 = 53.2, p<0.0001; n = 7. Wing-clipped male with intact female: partner effect, F_1,27 = 120.8, p<0.0001; exposure effect, F_1,27 = 239.5, p<0.0001; interaction, F_1,27 = 43.2, p<0.0001; n = 7). Double asterisks, significant difference by planned Student t-tests. (B) Copulation. CS males under the influence of ethanol on the 1^st and 6^th exposures displayed significantly reduced copulation with virgin females (ANOVA, F_2,20 = 47.7, p<0.0001; double asterisk, significant difference by Tukey-Kramer; n = 7).

(A) TH-GAL4/UAS-Shi^ts and DDC-GAL4/UAS-Shi^ts males, when subjected to daily ethanol exposure at 32°C to block synaptic output of dopamine neurons, displayed drastically reduced intermale courtship compared to the control mw⁺/UAS-Shi^ts males on all exposures (1^st exposure, F_2,21 = 18.24, p<0.0001; 2^nd, F_2,21 = 35.26, p<0.0001; 3^rd, F_2,21 = 27.02, p<0.0001; n = 7–8). Double asterisks, significant difference by Tukey-Kramer tests. (B) TH-GAL4/UAS-Shi^ts males, when subjected to daily ethanol exposure at room temperature, showed intermale courtship activities comparable to those of the control TH-GAL4/UAS-GFP males, indicating that Shi^ts expressed in dopamine neurons without the dominant negative activity has no effect on the ethanol-induced intermale courtship (Student t-test on all exposures, p>0.5, n = 7).

fru¹ and fru³ males showed vigorous intermale courtship in Flypub without ethanol (No EtOH). Upon daily ethanol treatments in 95% Flypub, their courtship activities did not change with additional ethanol treatment while the courtship levels under ethanol were lower than those without ethanol (ANOVA: fru¹, F_3,27 = 3.24, p = 0.0396, Tukey-Kramer showed a significant difference between No EtOH and 3^rd EtOH; fru³, F_3,27 = 16.94, p<0.0001, No EtOH was significantly different from the others by Tukey-Kramer; n = 7). NS, not significant. CS males tested together with fru males as a control showed negligible intermale courtship in the absence of ethanol and on the 1^st ethanol exposure (marked by diamonds); however, their courtship activities were enhanced under the influence of ethanol on the 2^nd and 3^rd ethanol treatments (F_3,27 = 42.86, p<0.0001; double asterisks, significant difference by Tukey-Kramer).

(A) Sedation profile. Flies were exposed to ethanol vapor in 70% Flypub (diamond) or 95% Flypub (triangle). MST in 70% Flypub was higher than that in 95% Flypub on all exposures and recurring treatment in both ethanol concentrations increased MST. Two-way ANOVA revealed the significant effects of ethanol concentration and exposure, and a marginal interaction of two factors (concentration effect, F_1,72 = 383.4, p<0.0001; exposure effect, F_5,72 = 40.5, p<0.0001; interaction, F_5,72 = 2.7, p = 0.03; n = 7). Post hoc Tukey-Kramer tests revealed the significant difference of the 1^st from the other exposures in both ethanol concentrations. All data are reported as mean±standard error of the mean. (B) Ethanol concentrations. CS males were subjected to ethanol treatment for 1, 2 or 6 days (1^st, 2^nd, 6^th) in 95% Flypub and ethanol contents were measured at 16 or 30 min after the onset of ethanol exposure. CS males without ethanol treatment (No EtOH) were used to measure the basal level. There was no significant difference in the ethanol contents of the males on the 1^st, 2^nd and 6^th exposure at 16 min (ANOVA, F_2,11 = 1.75, p = 0.23, n = 4) or 30 min (F_2,11 = 3.98, p = 0.06, n = 4).

(A) The percentage of males engaged in intermale courtship progressively increased upon additional ethanol treatments in 95% Flypub. Least squares regression showed the significant effect of exposure (r² = 0.68, p<0.0001, n = 10). (B) CS males subjected to daily ethanol exposure in 70% Flypub exhibited the exposure-dependent increase in intermale courtship but at the significantly reduced levels compared to those challenged with 95% ethanol. Two-way ANOVA revealed the significant effects of ethanol concentration, exposure, and interaction (concentration effect, F_1,24 = 54.02, p<0.0001; exposure effect, F_3,24 = 138.2, p<0.0001; interaction, F_3,24 = 6.7, p = 0.0019; n = 4). Post hoc two-tailed Student t-test showed the significant difference of the courtship scores on the 2^nd (p<0.005) or 3^rd exposure (p<0.0005) (marked by double asterisks). (C) The chronic-ethanol-treated males displayed active courtship toward the decapitated previously-ethanol-naïve males under the influence of ethanol (chronic to naïve), whereas the ethanol-naïve males, on the 1^st ethanol exposure, displayed negligible courtship toward the decapitated chronic-ethanol-exposed males (naïve to chronic). Two-tailed Student t-test showed a significant difference (p<0.0001, n = 6, marked by double asterisks). (D) Two or 4 wk-old CS males exhibited the increased levels of intermale courtship compared to 4 d-old males when tested in 95% Flypub. Two-way ANOVA revealed the significant effects of age and exposure, and a marginal interaction (age effect, F_2,36 = 16.6, p<0.0001; exposure effect, F_3,36 = 61.7, p<0.0001; interaction, F_6,36 = 2.58, p = 0.035; n = 4). Tukey-Kramer tests showed that the intermale courtship activities of 2 and 4 wk-old males were significantly different from that of 4 d-old males on the 2^nd exposure (marked by double asterisks).