Sustained post-mating response in Drosophila melanogaster requires multiple seminal fluid proteins

Abstract

Successful reproduction is critical to pass genes to the next generation. Seminal proteins contribute to important reproductive processes that lead to fertilization in species ranging from insects to mammals. In Drosophila, the male's accessory gland is a source of seminal fluid proteins that affect the reproductive output of males and females by altering female post-mating behavior and physiology. Protein classes found in the seminal fluid of Drosophila are similar to those of other organisms, including mammals. By using RNA interference (RNAi) to knock down levels of individual accessory gland proteins (Acps), we investigated the role of 25 Acps in mediating three post-mating female responses: egg production, receptivity to remating and storage of sperm. We detected roles for five Acps in these post-mating responses. CG33943 is required for full stimulation of egg production on the first day after mating. Four other Acps (CG1652, CG1656, CG17575, and CG9997) appear to modulate the long-term response, which is the maintenance of post-mating behavior and physiological changes. The long-term post-mating response requires presence of sperm in storage and, until now, had been known to require only a single Acp. Here, we discovered several novel Acps together are required which together are required for sustained egg production, reduction in receptivity to remating of the mated female and for promotion of stored sperm release from the seminal receptacle. Our results also show that members of conserved protein classes found in seminal plasma from insects to mammals are essential for important reproductive processes.

Figure 1. Example Western Blots and RT-PCR Showing the Levels of Acps in Knockdown Males (RNAi) Compared to Control Males

In western blots, the extents of Acp knockdowns were quantified by running serial dilutions to the level of 2.5% (lane 1), 5% (lane 2), 10% (lane 3), 15% (lane 4) 20% (lane 5), 25% (lane 6) and 100% (lane 7) of accessory gland proteins from control males in parallel with accessory gland proteins equivalent to the amount in one knockdown male (RNAi). Two independent sets of samples were analyzed to confirm the level of knockdown. Additional multiple nonquantitative westerns were carried out on these lines to confirm their knockdown prior to each assay. Knockdowns were specific to the targeted Acp except for the related C-type lectins (CG1652 and CG1656). RNAi of CG1652 also knocks down CG1656, as shown. Similarly, knockdown of CG1656 knocks down CG1652; an example is shown using RT-PCR. For PCR amplification, cDNA prepared from RNA extracts of 20 control males or experimental males was used. RPL32 primers [30] were used as positive control for the quality/quantity of cDNA. The panel on the extreme right in the “RT-PCR” panel represents an example of knockdown specific to the targeted Acp (CG33943). We observed similar results for all other Acps tested either by western or RT-PCR (unpublished data).

Figure 2. The Effect of Knockdown of Different Acps on Egg Laying by Mated Females

Shown here is the number (mean ± SE) of eggs laid per day by the mates of knockdown males (RNAi) or control males over a period of 10 days. Egg laying response by the mates of most knockdown males was not significantly different from their control mates (p > 0.5). However, mates of CG1652/CG1656, CG17575, or CG9997 laid significantly fewer eggs compared to their controls (p < 0.0001). Number of females ranged from 15–45 depending on the Acp analyzed.

Figure 3. Mean Number (Mean ± SE) of Eggs Laid by the Mates of Knockdown Males (RNAi) or Control Males from Day 1 to Day 10

Egg laying by the mates of CG1652, CG1656, CG9997, or CG17575 knockdown males (RNAi) was not significantly different from their controls (p > 0.2) at 24 h ASM but from day 2 onwards, mates of CG1652, CG1656, CG17575, or CG9997 knockdown males laid significantly fewer eggs compared to their controls (p < 0.0001; n = 22–27). On the contrary, mates of CG33943 knockdown males laid significantly fewer eggs only at 24 h ASM (p < 0.0003; n = 30 for control and 27 for knockdown) compared to their controls; from day 2 onwards their levels were similar to the controls (overall p > 0.6). The other Acps did not differ from controls. One example is shown here (CG10852 panel; overall p > 0.8).

Figure 4. Number of Sperm Stored (Mean ± SE) in Seminal Receptacle or Spermathecae of Mates of CG1652, CG1656, CG9997, or CG17575 Knockdown Males (RNAi) Compared to their Controls at 2 h ASM, 4 d ASM, and 10 d ASM.

(A) We did not detect a significant difference in the number of sperm stored by mates of CG1652, CG1656, or CG17575 knockdown males compared to their controls in the seminal receptacles at 2 h ASM (p > 0.25) or 4 d ASM (p > 0.2) but we observed significant difference at 10 d ASM (p < 0.0001). Mates of CG9997 knockdown males had sperm storage levels similar to their controls at 2 h ASM (p > 0.2) but at 4 d and 10 d ASM, the number of sperm stored in the seminal receptacle of mates of CG9997 knockdown males was significantly higher than their controls (p < 0.0001). (B) We did not detect significant difference in the number of sperm stored by mates of CG1652, CG1656, CG9997, or CG17575 knockdown males compared to their controls in the spermathecae at 2 h ASM (p > 0.2) or 4 d ASM (p > 0.2) or 10 d ASM (p > 0.5). Number of females ranged from 15–35 per treatment depending on the Acp analyzed or the storage organ counted.