The sex peptide of Drosophila melanogaster: female post-mating responses analyzed by using RNA interference

Abstract

Mating induces profound changes in female insect behavior and physiology. In Drosophila melanogaster, mating causes a reduction in sexual receptivity and an elevation in egg production for at least 5 days. Injection of the seminal fluid sex peptide (SP) induces both responses in virgin females, but only for 1-2 days. The role of SP in eliciting the responses to mating remains to be elucidated. Functional redundancy between seminal fluid components may occur. In addition, mating with spermless males results in brief (1- to 2-day) post-mating responses, indicating either that there is a "sperm effect" or that sperm act as carriers for SP or other seminal fluid components. Here we used RNA interference to suppress SP expression, to determine whether SP is required to elicit full post-mating responses, the magnitude of responses due to other seminal fluid components, and whether SP accounts for the "sperm effect." Receptivity was higher and egg production lower in females mated to SP knock-down males than in controls. Comparison with virgins showed that the responses were brief. SP is therefore required for normal magnitude and persistence of postmating responses. Sperm transfer and use were normal in mates of SP knock-down males, yet their post-mating responses were briefer than after normal matings, and similar to those reported in mates of spermless son-of-tudor males. The prolonged "sperm effect" on female receptivity and egg production is therefore entirely attributable to SP, but sperm are necessary for its occurrence.

Fig. 1.

Specificity of expression driven by the Acp26Aa-P-Gal4 transgene, demonstrated by LacZ expression in Acp26Aa-P-Gal4;UAS-lacZ males. (a) Acp26Aa-P-Gal4 drives expression of UAS-lacZ in male accessory glands (AG) but not the ejaculatory duct (ED) or ejaculatory bulb (EB). (b) No LacZ expression in is detected in control males, homozygous for UAS-lacZ but lacking the driver.

Fig. 2.

Western blot showing levels of SP in SP knock-down and control males in 3-day-old virgin males (a) and in 3-day-old males mated when 2 days old (b). From left, in lanes 1–4, homozygous UAS-SP-IR1, UAS-SP-IR2, UAS-SP-IR3, and Acp26Aa-P-Gal4 control males produced SP [molecular mass 4,428 Da (22)]. In lanes 5, 7, and 9, control males carrying the inverted repeat insert without the Gal4 driver (+;UAS-SP-IR1, +;UAS-SP-IR2, and +;UAS-SP-IR3) produced SP. In lanes 6, 8, and 10, males with the inverted repeat and the Gal4 driver (Acp26Aa-P-Gal4;UAS-SP-IR1, Acp26Aa-P-Gal4;UAS-SP-IR2, and Acp26Aa-P-Gal4;UAS-SP-IR3) produced no detectable SP.

Fig. 3.

Effect of SP on female receptivity. Shown is percentage of females remating within1hina receptivity test with wild-type males, 24 and 48 h after initial matings to Acp26Aa-P-Gal4;UAS-SP-IR1 SP knock-down males, +;UAS-SP-IR1 control males, Acp26Aa-P-Gal4;UAS-SP-IR2 SP knock-down males, or +;UAS-SP-IR2 control males. The receptivity of virgin females is also shown. The numbers of females that did and did not mate after each type of initial mating were analyzed in 2 × 2 contingency tables by using Fisher exact tests. Females mated to SP knock-down males (Acp26Aa-P-Gal4;UAS-SP-IR1 and Acp26Aa-P-Gal4;UAS-SP-IR2) were significantly more receptive than their respective controls (i.e., mates of +;UAS-SP-IR1 and +;UAS-SR-IR2 males), P < 0.0001, all tests. Females mated to both lines of SP knock-down males were significantly less receptive than virgin females at 24 h (P < 0.0001, both comparisons). At 48 h, females initially mated to Acp26Aa-P-Gal4;UAS-SP-IR1 males were only marginally less receptive than virgins (P = 0.03), and females mated to Acp26Aa-P-Gal4;UAS-SP-IR2 males did not differ significantly in receptivity compared with virgin females.

Fig. 4.

Effect of SP on oviposition and ovulation. (a) The median (± interquartile range) numbers of eggs laid per 24 h by females over 5 days, after mating to SP knock-down or control males. Data for unmated virgin females are also shown. The data were analyzed by using Wilcoxon tests. Females mated to Acp26Aa-P-Gal4;UAS-SP-IR1 SP knock-down males did not differ at 24 h in egg production from mates of their respective controls (mates of +;UAS-SP-IR1 males), but laid significantly fewer eggs than their mated controls on days 2–5 (P < 0.0001, all comparisons). Females mated to Acp26Aa-P-Gal4;UAS-SP-IR2 males laid significantly fewer eggs than their controls (mates of +;UAS-SP-IR2 males) on all days (P < 0.0001, all comparisons). Females mated to both SP knock-down line males produced significantly more eggs than virgin females on the first day after mating (P < 0.001, both tests). Females mated to Acp26Aa-P-Gal4;UAS-SP-IR1 males also produced significantly more eggs than virgin females on day 2 after mating (P < 0.0001), but the egg production of these females then became similar to that of virgin females on days 3 (P = 0.3), 4 (P = 0.02), and 5 (P = 0.7). Egg production of females mated to Acp26Aa-P-Gal4;UAS-SP-IR2 males was not significantly different from that of virgins on days 2–5(P > 0.1, all tests). (b) The percentage of females with a egg in the uterus 6, 24, and 48 h after mating to SP knock-down or control males. Data for virgin females are also shown. The numbers of females that did and did not have an egg in the uterus were analyzed in 2 × 2 contingency tables by using Fisher exact tests. Females mated to SP knock-down males were significantly less likely to have an egg in the uterus than were their respective control females (females mated to Acp26Aa-P-Gal4;UAS-SP-IR1 males versus their controls, at 6 h P = 0.1, at 24 h P = 0.03, at 48 h P = 0.0003; females mated to Acp26Aa-P-Gal4;UAS-SP-IR2 males versus their controls, at 6 h P = 0.02, at 24 h P = 0.003, at 48 h P < 0.0001). Females mated to SP knock-down males were also significantly more likely (in all comparisons except one) to have an egg in the uterus than were virgin females (females mated to Acp26Aa-P-Gal4;UAS-SP-IR1 males versus virgins, at 6 h P = 0.03, at 24 h P < 0.0001, at 48 h P = 0.002; females mated to Acp26Aa-P-Gal4;UAS-SP-IR2 males versus virgins, at 6h P = 0.002, at 24 h P < 0.0001, at 48 h P = 0.1).

Fig. 5.

Effect of SP on egg fertility. Egg fertility [(number of pupae/number of eggs laid) × 100, ± interquartile range] of females 1, 3, and 5 days after mating to SP knock-down or control males. The data were analyzed by using Wilcoxon tests. On days 1 and 3, the fertility of eggs produced by females mated to SP knock-down males was not significantly different from the fertility of eggs produced by females mated to control males (females mated to Acp26Aa-P-Gal4;UAS-SP-IR1 males versus their controls, on day 1 P = 0.2, on day 3 P = 0.05; females mated to Acp26Aa-P-Gal4;UAS-SP-IR2 males versus their controls, on day 1 P = 0.5, on day 3 P = 0.2). On day 5, the fertility of eggs laid by control females was significantly lower than that of females mated to SP knock-down males (females mated to Acp26Aa-P-Gal4;UAS-SP-IR1 males versus their controls, P < 0.0001, females mated to Acp26Aa-P-Gal4;UAS-SP-IR2 males versus their controls, P = 0.0007). On day 5, females of all lines produced nonsignificantly different numbers of fertile eggs (females mated to Acp26Aa-P-Gal4;UAS-SP-IR1 males versus their controls, P = 0.8, females mated to Acp26Aa-P-Gal4;UAS-SP-IR2 males versus their controls, P = 0.6).