(A) To measure male courtship behavior, one male fly is placed with 2 females, one mated (M), and one virgin (V). (B) D. arizonae (Dari) and D. mojavensis (Dmoj) prefer to court virgin females over recently mated females (n = 20, Fisher’s exact test, p=0.00123; n = 31, p=0.0105). **: p<0.01; ns: not significant. A preference score of 1 indicates all males initiate courtship first with the virgin female; −1 indicates all males initiated courtship first with the mated female. (C) Levels of male-transferred TAGs and long OAcs on the female cuticle after first mating decreases by 2 hr post-mating. (D) Females are significantly less attractive for up to 2 hr after mating. By 4 hr, males do not exhibit significant preference for courting mated vs virgin females. **: Fisher’s exact test, p=0.00123; *: p=0.0256. (E) D. arizonae males are more reluctant to initiate courtship with females perfumed with the contents of [1.25] ejaculatory bulb (eb) (n = 27, Fisher’s exact test, p=0.000624) or [0.5] eb (n = 28, p<0.0001) but not [0.25] eb (n = 28, p=0.176). Extracts from immature male ebs were ineffective at inhibiting male courtship (n = 28, p=0.000389). D. mojavensis and D. navojoa (Dnav) males also avoided virgin females perfumed with eb contents (Dmoj: n = 21, p=0.00160; Dnav: n = 21, p<0.0002). **: p<0.002; ***: p<0.0001. C: solvent control; P: perfumed. (F) Suppression of D. arizonae courtship initiation is elicited only when TAGs and long OAcs are combined (n = 28, Fisher’s exact test, p<0.0001). TAGs alone are ineffective (n = 28, p=0.593). Long OAcs on their own could be attractive to males (n = 28, p=0.006). *: p<0.05; ***: p<0.0001. (G) D. arizonae copulation is suppressed in the presence of TAGs alone (n = 28; Fisher’s exact probability test, p=0.0287) or TAGs combined with long OAcs (n = 28; p<0.0001), but not long OAcs alone (n = 28; p=0.0543). *: p<0.05; ***: p<0.0001. A copulation choice score of 1 indicates all males copulated with solvent-perfumed females; −1 indicates all males copulated with TAG-perfumed females. (H) Perfuming with synthetic TAGs recapitulates copulation suppression. Oleic acid (C18:1)-containing TAGs produced significant effects at high and low doses (750 ng: n = 21, Fisher’s exact test, p<0.0001; 75 ng: n = 21, p=0.00167). Only the (R)-18:1 stereoisomer was bioactive (75 ng: n = 21, p=0.00480); the (S)-18:1 stereoisomer did not elicit a significant behavioral response (75 ng: n = 21, p=1). *: p<0.01; **: p<0.002; ***: p<0.0001. (I) Two combinations of TAGs produced synergistic effects on copulation suppression: oleic acid-TAG paired with stearic acid-TAG (n = 21, p=0.000139) and stearic acid-TAG paired with linoleic acid-TAG (n = 19, p=0.022). The oleic acid-containing TAG is not bioactive at a dose of 37.5 ng/fly (n = 20, Fisher’s exact test, p=0.751). *: p<0.05; ***: p<0.0005. (J) UV-LDI MS spectra of females perfumed with TLC fractions or synthesized TAGs. Signals for TAGs and long OAcs are indicated in red and blue, respectively. *: C35:2 Pentatriacontadiene reference peak, m/z 527.5 [M + K]+.