Female receptivity to second mating.

<p>Females were mated first to control or RNAi knockdown males from the line shown (either seminase line 1 or 2, or CG4815). CG4815 is included as a control for the VDRC strain background. The time given is the number of days after the first mating that the second mating was attempted. Females who remated to a male from a wildtype strain (Canton-S) during a one hour observation period are shown as a percentage with the total number of potential mating pairs assayed in brackets. P values in bold are significant.</p

Females mated to seminase knockdown males lay fewer eggs.

<p>(A) The average number of eggs laid per female in a given treatment over 10 days. Seminase Line 1: Control N = 55, RNAi N = 59; CG4815: Control N = 20, RNAi N = 18. (B) Hatchability data for the corresponding experiments in (A). Hatchability is defined as the proportion of eggs that yielded adult progeny. CG4815 (a serine protease SFP) is included as a control for strain background. (A),(B) Asterisks indicate p<0.0001. (C) The data from (A) plotted as average number of eggs laid by females in each group on individual days of the experiment. (D) CG4815 egg laying data plotted as in (C). Asterisks indicate level of significance after Bonferroni correction (*p<0.05, **p<0.01, ***p<0.0001). Error bars indicate standard error of the raw data pooled over experiments.</p

Females mated to seminase knockdown males retain more sperm 4 and 10 days after mating.

<p>Sperm counts for seminase line 1 are shown for sperm stored at three timepoints after mating, as given on the x-axis. Line 2 yielded similar results. Asterisks indicate level of significance (*p<0.05; **p<0.01). (A) Total average number of sperm stored in both storage organs (2 h: t = −0.09, p = 0.93; 4 d: t = −1.6, p = 0.13; 10 d: t = −3.2, p<0.01). (B) Average number of sperm stored in the seminal receptacle only (2 h: t = −0.06, p = 0.96; 4 d: t = −2.8, p<0.01; 10 d: t = −3.2, p<0.01). (C) Average number of sperm stored in the paired spermathecae only; numbers are the sum of sperm stored in each spermatheca (2 h: t = −0.11, p = 0.91; 4 d: t = 2.2, p<0.05; 10 d: t = −1.1, p = 0.26). Samples sizes are, for bars from left to right: (A) 12, 14, 16, 10, 7, 15; (B) 17, 21, 18, 19, 7, 15; (C) 13, 14, 19, 10, 7, 15. Abbreviations: 2 h, 2 hours; 4 d, four days; 10 d, 10 days. Error bars indicate standard error.</p

Seminase is produced in the accessory glands and is processed during and after mating.

<p>(A) Western blot probed with seminase antibody. Seminase in the male accessory glands (AG) has an apparent molecular weight of ∼29-kDa. No seminase was detected in testes (T), the ejaculatory duct and bulb (ED/EB), or in virgin female reproductive tracts. AG and T: tissue from 10 virgin males. ED/EB: tissue from 20 virgin males. Female: tissue from 4 virgin females. Tubulin is shown as a loading control. Total protein loaded is shown in micrograms as measured by BCA assay. (B) Western blot probed with seminase antibody. Lane 1: full-length seminase in AG. Lane 2: ED/EB dissected from 20 males at 8–10 minutes ASM. Lane 3: Reproductive tracts (RT) from 20 females dissected 8–10 minutes ASM. Lanes 4–8: Female RTs dissected at the times ASM indicated above the lanes. (C) Western blots probed for seminase (top) and ovulin (bottom). Lane 1: full length protein in AG. Lanes 2 and 3: RT from females mated to CG11864 control (+) or knockdown (−) males at 1 hour ASM.</p

Processing of SFPs is defective in the absence of seminase.

<p>(A) Western blot probed with ovulin antibody. Lane 1: full-length ovulin in male accessory glands (AG). Lanes 2–9: female reproductive tracts (RT) dissected after mating to control (+) or RNAi (−) males for the gene given above the lanes. Females were dissected at 45 minutes after the start of mating (ASM) (lanes 2–5) or 2 hours ASM (lanes 6–9). All lanes are from the same gel with extraneous lanes removed for clarity. (B) Western blot probed with ovulin antibody. All lanes are female RT dissected at 30 minutes ASM. Lanes 1 and 2 are from females mated to seminase control (+) or RNAi (−) males. Lanes 3 and 4 are from females mated to CG10587 control (+) or RNAi (−) males. (C) Western blot probed with Acp36DE antibody. All lanes are from the same gel, with extraneous lanes cut out for clarity. Lanes 1–8 contain RT from females mated to males of the given genotype as in (A). Females were dissected at 1 hour ASM (lanes 2–5) or 3 hours ASM (lanes 6–9). Un-processed (full-length) Acp36DE runs at ∼122 kDa. (D) Western blot probed with CG11864 antibody. Female RTs dissected at 45 minutes ASM to seminase control (lane 1) or knockdown (lane 2) males. Numbers to the left of blots indicate approximate band size in kDa.</p

Seminase is required for two post-mating pathways.

<p>Seminase is cleaved during mating within the male reproductive tract where it is required for CG11864 cleavage. CG11864 then regulates processing of two SFPs, ovulin and Acp36DE (left branch). Seminase is also necessary for the long term response (LTR) pathway (right branch). Proteins/pathways downstream of seminase are involved in the processes shown in boxes, based on earlier knockout or knockdown studies. The consequences of proteolytic processing of ovulin and Acp36DE remain unknown.</p