Supplementary Figure 1. Thrombin-induced fluorescence (y axis) measured every minute for 150 minutes (x axis) for two representative samples:
DGA_4315, plug 1_1 from plate RP1 (red) and DJO_2668, plug 1_4 from plate
RP1 (blue). The duplicated standards are shown in green. Our method is specifically designed to ignore the asymptotic parts of the fluorescence and only estimate the slope for the respective lines indicated. Thrombin activity (i.e., the y-axis of Fig. 3) is measured as the estimated slope for a plug minus the average of the two slopes estimated from the standard curves of that particular plate. As shown, the red slope is steeper than the standards, suggesting that DGA plugs have endogenous protease activity.
Supplementary Figure 2. Plug mass by male genotype. Sample sizes shown above boxplots.
Supplementary Figure 3. A) An example of a coomassie-stained polyacrylamide gel. Strain names (i.e., DIK, DCA, DOT) plus individual identification number are shown as lane headers. There are four abundant proteins (numbered 1-4), which based on previous studies (Dean et al , 2011; Lin et al , 2002; Lundwall et al , 1997; Tseng et al , 2011) and the match to molecular mass according to Ensembl version 78 (www.ensembl.org), are likely SVS1 (93.5 kDa), SVS2 (40.8 kDa), SVS5 (13.0 kDa), and SVS4 (12.5 kDa).

Supplementary Figure 4.
“No thrombin” controls (see Discussion). Any fluorescence above zero indicates endogenous protease activity, which could be either male-derived or female-derived. DGA plugs, which were smaller than DJO plugs, did not elicit less fluorescence, arguing against the hypothesis that small plugs induce a less severe female proteolytic response.
Supplementary Table 1. Experimental matings. Column headers as follows:
Female= Female genotype; Male= Male genotype; Male No= Individual male's
ID; Time After Split (hrs)= Time (hrs) between mating and dissection; Plug (Y/N)=
Indicating if the plug was still present at time of dissection; Mating (Y/N)=
Indicates if a plug was present after experimental cross; Weight of Plug (g)= The absolute mass of the plug (NA indicates non successful mating); Weight of
Female (g): Weight of female at dissection; Female DOB: Female's date of birth;
Dissection Date= Date female was euthanized
Supplementary Table 2. Male morphological data. Column headers are as follows: Male= Male genotype; Male No= Individual male's ID; Date of Birth=
Male's date of birth; Body Weight (g)= Body weight at dissection; Seminal Vesicle
Weight (g)= Absolute weight of the left seminal vesicle; Right Testis Weight (g)=
Absolute weight of the right testis; Left Testis Weight (g)= Absolute weight of the left testis

Supplementary Table 3. Protein quantification across genotypes. Column headers are as follows: Male=Male genotype; SVS1:SVS5=abundance of each protein band/sum of the abundances of the four protein bands; Total=Absolute value of abundance of 4 protein bands