Post-mating change in excretion by mated Drosophila melanogaster females is a long-term response that depends on sex peptide and sperm

Abstract

Drosophila seminal fluid proteins elicit physiological and behavioral changes in the female after mating. For example, the seminal protein sex peptide (SP) causes females to lay more eggs, reduce receptivity to re-mating, consume more food and produce more concentrated excreta upon mating. It has been reported that SP indirectly increases food consumption as a result of its stimulation of egg production, but its role in producing more concentrated excreta in the mated female was reported to be independent of egg production. Additionally, it has been shown that SP's effect on food consumption persists for several days after mating, while it is unknown whether this is true for its effect on excretion. SP can have both transient and long-term effects on mated females; the latter occur because of the peptide's binding to, and slow release from, sperm in the female. Here we used timed measures of excretion by female flies that had mated to males mutant in SP or in its regulators, to test the duration of SP's effect on excretion. We found that SP's effect on excretion persists for at least ~1 week after mating, and that this persistence requires that SP bind to and be released from sperm. Although these binding/release requirements of SP are similar to those for increased egg production (and consequent increased food intake) following mating, we find that the long-term change in excretion phenotype is only partially dependent on the presence of eggs in the female. Our data indicate that a change in intestinal transit is part of the long-term post-mating response elicited by the gradual release of sperm-bound SP in the female after mating, even though it is not fully dependent on other long-term responses elicited by SP.

Keywords: Drosophila melanogaster; Excretion; Feeding; Post-mating response; Sex peptide; Sperm.

Figure 1

The effect of mating on excreta produced by Drosophila females at 24 hours, 48 hours and 72 hours post-mating. Bars with the same letter are statistically similar; MF = mated females, VF = virgin females. (A) The proportion of reproductive oblong deposits (RODs) was calculated at each time point. Females mated to CS males (CS MF) produced more RODs than CS virgin females (CS VF) at 24 hours (p=0.01), 48 hours (p<0.0001) and 72 hours (p<0.0001). (B) The integrated optical density (IOD) of excreta despoits per fly was calculated by totaling the IOD (pixel area x optical density) for the entire petri dish and dividing by the number of flies. The IOD of excreta per fly for CS MF was significantly higher than CS VF at 24 hours (p<0.0001), 48 hours (p<0.0001) and 72 hours (p=0.0424). Total numbers of flies used are as follows: for 24 hours, n_CSMF = 39 and n_CSVF = 40, for 48 hours, n_CSMF = 36 and n_CSVF = 36, for 72 hours, n_CSMF = 35 and n_CSVF = 29.

Figure 2

Sex peptide elicits long-term post-mating excretion change in females. (A) Females mated to control males (SP+ MF) produced significantly more RODs than virgin females (CS VF) at 24 hours (p=0.0016), 48 hours (p=0.0014) and 72 hours (p=0.0014) and than CS females that had mated to SP null males (SP0 MF) at 48 hours (p=0.001) and 72 hours (p=0.0014). SP null-mated females produced significantly more RODs than virgin females at 24 hours (p=0.0235). (B) The mean IOD of excreta per fly for SP+ mated females was significantly higher than SP0 mated females at 24 hours (p=0.017), 48 hours (p=0.0306) and 72 hours (p=0.001) and virgin females at each time point (p=0.0012, p=0.0299 and p=0.0042, respectively), while there was no difference between SP0 mated females and virgin females at any time point. In panels A and B, bars with the same letter are statistically similar. Total numbers of flies used are as follows: for 24 hours, n_SP0MF = 40, n_SP+MF = 39 and n_CSVF = 40, for 48 hours, n_SP0MF = 38, n_SP+MF = 39 and n_CSVF = 38, for 72 hours, n_SP0MF = 38, n_SP+MF = 39 and n_CSVF = 37. (C) Females mated to control males (SP+ MF) produced significantly more RODs than virgin females (CS VF) 1 day (p=0.0224), 2+3 days (p<0.0001), 4+5 days (p<0.0001) and 6+7 days (p<0.0001) ASM and more RODs than females mated to SP null males (SP0 MF) at each time point (p=0.0061, p<0.0001, p<0.0001, and p<0.0001, respectively). (D) The mean IOD of excreta per fly for SP+ mated females was significantly higher than SP0 mated females at 1 day (p=0.036), 2+3 days (p=0.0002), 4+5 days (p<0.0001) and 6+7 days (p<0.0001) ASM and higher than virgin females at each time point (p=0.0488, p=0.0002, p=0.0003 and p<0.0001, respectively). In panels C and D, measurements were taken at 1 day ASM, 3 days ASM, 5 days ASM and 7 days ASM. p<0.05, p**<0.01, p***<0.0001. Total numbers of flies used in C and D are as follows: for day 1, n_SP0MF = 36, n_SP+MF = 39 and n_CSVF = 44, for days 2+3, n_SP0MF = 35, n_SP+MF = 39 and n_CSVF = 39, for days 4+5, n_SP0MF = 31, n_SP+MF = 38 and n_CSVF = 38, for days 6+7, n_SP0MF = 31, n_SP+MF = 38 and n_CSVF = 38.

Figure 3

The gradual release of sperm-bound sex peptide is responsible for the long-term increase in ROD production after mating. (A) Females mated to control males (Control MF) produced significantly more RODs than females mated to spermless males (Spermless MF) and virgin females (CS VF) at 24 hours (p=0.0018 and p=0.0005), 48 hours (p=0.0019 and p=0.0002) and 72 hours after mating (p<0.0001 and p<0.0001). Spermless mated-females produced significantly more RODs than virgin females only at 48 hours (p=0.0243). (B) Mean IOD of excreta per fly for control-mated females was higher than virgin females at 24 hours (p=0.0008), 48 hours (p<0.0001) and 72 hours (p<0.0001) and higher than spermless-mated females at 48 hours (p=0.0023) and 72 hours (p<0.0001). (C) Females mated to control males produced more RODs than virgin females at all 3 time points ASM (p=0.0026, p<0.0001 and p<0.0001) and more RODs than females mated to males with non-cleavable SP (SP-TG^QQ MF) at 48 and 72 hours (p=0.0179 and p<0.0001). SP-TG^QQ -mated females also produced more RODs than virgin females at all 3 time points (p=0.0004, p<0.0001, p=0.0065). (D) Mean IOD of excreta per fly for control mated females was significantly higher than SP-TG^QQ -mated females at 48 and 72 hours ASM (p=0.0101 and p=0.0216). (E) Control-mated females produced more RODs than virgin females at each time point after mating (p=0.0197, p<0.0001 and p<0.0001) and more RODs than females mated to CG1656 knockdown males (1656 KD MF) at 48 hours (p=0.0002) and 72 hours (p=0.0003). (F) Females mated to control males and females mated to CG1656 knockdown males had a higher IOD of excreta per fly than virgin females at 24 hours (p=0.007 and p=0.0034), 48 hours (p=0.0154 and p=0.0185) and 72 hours (p=0.0007 and p=0.0005) while there was no significant difference between females mated to control or knockdown males. Bars with the same letter are statistically similar. Total numbers of flies used are as follows: for 24 hours, n_SPERMLESS = 64, n_CONTROL = 63, n_CSVF = 64, n_SP-TGQQ = 52, n_CONTROL = 67, n_CSVF = 59, n_1656KD = 35, n_CONTROL = 36, n_CSVF = 24, for 48 hours, n_SPERMLESS = 59, n_CONTROL = 61, n_CSVF = 63, n_SP-TGQQ = 52, n_CONTROL = 67, n_CSVF = 58, n_1656KD = 31, n_CONTROL = 35, n_CSVF = 24, for 72 hours, n_SPERMLESS = 56, n_CONTROL = 53, n_CSVF = 61, n_SP-TGQQ = 51, n_CONTROL = 64, n_CSVF = 58, n_1656KD = 27, n_CONTROL = 34, n_CSVF = 24.

Figure 4

SP elicits long-term ROD production partially independent of egg production. (A) Mated control females (M Control F) produced significantly more RODs than virgin control females (V Control F) and mated eggless females (M Eggless F) at 24 hours (p=0.0026 and p=0.0054), 48 hours (p<0.0001 and p=0.0003) and 72 hours ASM (p=0.0001 and p=0.0008), while mated eggless females produced more RODs than virgin eggless females (V Eggless F) only at 72 hours after mating (p=0.0156). (B) Mated control females had a significantly higher IOD of excreta per fly than virgin control females and mated eggless females at 24 hours (p=0.0004 and p=0.002), 48 hours (p=0.0005 and p=0.0003) and 72 hours (p<0.0001 and p<0.0001). There was no significant difference between eggless mated females and eggless virgin females. (C) Eggless females mated to control males (SP+ M) produced more RODs than eggless females mated to SP0 males (SP0 M) and eggless virgin females at 48 hours (p=0.014 and p=0.0167) and 72 hours (p=0.0045 and p=0.0095) ASM. Control females mated to SP+ males produced significantly more RODs than eggless females mated to SP+ males at all three time points ASM (p=0.01, p=0.0034, and p=0.0002, respectively). (D) There was no significant difference in IOD of excreta per fly between eggless females mated to SP0 males, eggless females mated to SP+ males and eggless virgin females at 24 hours, 48 hours and 72 hours ASM. Control females mated to SP+ males at a higher IOD of excreta per fly than control females mated to SP0 males at 48 hours (p=0.0006) and 72 hours (p=0.0007) and control virgin females at all three time points ASM (p=0.0052, p=0.0003 and p<0.0001, respectively). Bars with the same letter are statistically similar. Total numbers of flies used for A and B are as follows: for 24 hours, n_MEF = 51, n_VEF = 52, n_MCF = 52 and n_VCF = 52, for 48 hours, n_MEF = 49, n_VEF = 50, n_MCF = 49 and n_VCF = 50, for 72 hours, n_MEF = 44, n_VEF = 48, n_MCF = 47 and n_VCF = 45. Sample sizes for B and C are as follows: for eggless females at 24 hours, n_SP0M = 30, n_SP+M = 33, n_V = 28, for 48 hours, n_SP0M = 29, n_SP+M = 33, n_V = 27, for 72 hours, n_SP0M = 28, n_SP+M = 33, n_V = 22. For control females at 24 hours, n_SP0M = 27, n_SP+M = 41, n_V = 42, for 48 hours n_SP0M = 27, n_SP+M = 40, n_V = 41, for 72 hours, n_SP0M = 25, n_SP+M = 39, n_V = 38.