Effects of two seminal fluid transcripts on post‐mating behaviour in the simultaneously hermaphroditic flatworm Macrostomum lignano

The seminal fluid proteins (SFPs) transferred to mating partners along with sperm often play crucial roles in mediating post‐mating sexual selection. One way in which sperm donors can maximize their own reproductive success is by modifying the partner's (sperm recipient's) post‐copulatory behaviour to prevent or delay re‐mating, thereby decreasing the likelihood or intensity of sperm competition. Here, we adopted a quantitative genetic approach combining gene expression and behavioural data to identify candidates that could mediate such a response in the simultaneously hermaphroditic flatworm Macrostomum lignano. We identified two putative SFPs—Mlig‐pro46 and Mlig‐pro63—linked to both mating frequency and ‘suck’ frequency, a distinctive behaviour, in which, upon ejaculate receipt, the worm places its pharynx over its female genital opening and apparently attempts to remove the received ejaculate. We, therefore, performed a manipulative experiment using RNA interference‐induced knockdown to ask how the loss of Mlig‐pro46 and Mlig‐pro63 expression, singly and in combination, affects mating frequency, partner suck propensity and sperm competitive ability. None of the knockdown treatments impacted strongly on the mating frequency or sperm competitive ability, but knockdown of Mlig‐pro63 resulted in a significantly decreased suck propensity of mating partners. This suggests that Mlig‐pro63 may normally act as a cue in the ejaculate to trigger recipient suck behaviour and—given that other proteins in the ejaculate have the opposite effect—could be one component of an ongoing arms race between donors and recipients over the control of ejaculate fate. However, the adaptive significance of Mlig‐pro46 and Mlig‐pro63 from a donor perspective remains enigmatic.

Such functions do not inevitably lead to sexual conflict, but a good example of an SFP that acts in a sexually antagonistic manner is sex peptide in Drosophila melanogaster, which decreases female receptivity and stimulates egg production but lowers females' fitness and lifetime reproductive success (Wigby & Chapman, 2005).
Another example of a potential sexual conflict mediated through the seminal fluid is a mating-induced change of the partners' sexual receptivity. Decreased sexual receptivity of mated females occurs in a wide range of insects, and it is suggested that males benefit from this change because it decreases the likelihood or intensity of sperm competition. In D. melanogaster, mated females actively reject courting males and the SFP 'sex peptide' received with the ejaculate plays a central role in inducing this change (Chapman et al., 2003;Häsemeyer, Yapici, Heberlein, & Dickson, 2009;Liu & Kubli, 2003;Ram & Wolfner, 2009;Yang et al., 2009;Yapici, Kim, Ribeiro, & Dickson, 2008): females mated to sex peptide null males remain highly receptive to re-mating (Chapman et al., 2003;Liu & Kubli, 2003).
Although Drosophila has been the main model species to date for studying such seminal fluid-mediated effects, there are several other reported cases in a broad range of insects where females show a similarly reduced sexual receptivity after receiving full ejaculates or specific SFPs (reviewed in Avila et al., 2011;Simmons, 2001).
For example, females of the Queensland fruit fly Bactrocera tryoni show no difference in sexual receptivity when mated to irradiated males (deficient in sperm) compared with mates of nonirradiated males, suggesting that components of the seminal fluid, not sperm, are responsible for the decreased post-mating receptivity observed (Harmer, Radhakrishnan, & Taylor, 2006). Moreover, the injection of extracts obtained from male reproductive tracts to B. tryoni females leads to a decrease in sexual receptivity and shorter copulation times when subsequently mated, similar to behaviours seen in previously mated females (Radhakrishnan & Taylor, 2007). In Anopheles gambiae, injection of male accessory gland homogenates into virgin females results in a decreased likelihood of re-mating (Shutt, Stables, Aboagye-Antwi, Moran, & Tripet, 2010). Other examples of SFPs affecting post-mating behaviour include lady beetles (Perry & Rowe, 2008a, 2008b, seed beetles (Moya-Laraño & Fox, 2006;Rönn, Katvala, & Arnqvist, 2006;Yamane, Miyatake, & Kimura, 2008) and ground beetles (Takami, Sasabe, Nagata, & Sota, 2008). Furthermore, there is some evidence for similar seminal fluid-mediated effect in vertebrates; for example, the beta-endorphin found in rat seminal fluid has the capacity to suppress female receptivity (Forsberg, Bednar, Eneroth, & Södersten, 1990). Depending on any direct or indirect benefits females might accrue from additional matings, such effects may often be counter to female interests (Chapman et al.,1995;Hollis et al., 2019;Wigby & Chapman, 2005). Charnov (1979) first recognized that sexual conflict also occurs in hermaphrodites, since sperm donors can also potentially benefit from transferring ejaculate components manipulating sperm recipients and affecting the outcome of sperm competition (Charnov, 1979;Koene, 2006;Michiels, 1998;Schärer, Janicke, & Ramm, 2015). Moreover, as we explain in more detail below, simultaneous hermaphroditism might also create unique targets for seminal fluid action (Charnov, 1979;Schärer et al., 2015;Schärer & Ramm, 2016).
Such seminal fluid-mediated effects, with a potential beneficial effect for one but a potential harmful effect for the other partner, have already been detected in the pond snail Lymnaea stagnalis. The intravaginal injection of one L. stagnalis SFP (LyAcp10) led to a decrease in egg laying (Koene et al., 2010), and the injection of two other SFPs (LyAcp8b and LyAcp5) reduced the number of sperm transferred by the recipient in a subsequent mating as a donor and, as a result, decreased their paternity success (Nakadera et al., 2014; see also Schärer, 2014). This latter effect emphasizes that it is a potentially adaptive strategy in simultaneous hermaphrodites to steer your partner away from its male function and that the action of seminal fluid may be one means of doing so (Schärer & Ramm, 2016). There is also evidence for the manipulation of the re-mating frequency in a simultaneously hermaphroditic species, namely the love-dart shooting snail Euhadra quaesita. Before exchanging sperm, both mating partners attempt to drive their mucous-coated love-dart into their respective partner. In stabbed snails, the intermating interval is longer than in not-stabbed individuals, and so snail pairs injected with mucous subsequently mate less often than control pairs (Kimura, Shibuya, & Chiba, 2013).
In our model species Macrostomum lignano, the complement of SFPs has only just been characterized (Weber et al., 2018).
Nevertheless, there are already some indications for potential effects of SFPs. An initial screen of 18 putative SFPs selected without any prior functional information revealed that the RNAi-induced knockdown of at least some SFPs may modulate aspects of fertility and sperm competitiveness (Weber, Giannakara, & Ramm, 2019).
However, given that this screen identified only a minority of candidate SFPs modulating fertility or sperm competitiveness, and that all of these effects have not yet been verified after controlling for multiple testing, this suggests that more targeted methods may be beneficial to first prioritize candidates mediating specific effects. Patlar, Weber, Temizyürek, and Ramm (2020) adopted precisely such an approach, using quantitative genetics to identify six candidate SFPs likely to affect the post-mating 'suck' behaviour of sperm recipients, based on a negative genetic correlation between SFP transcript expression (Patlar, Weber, & Ramm, 2019) and suck propensity.
The suck behaviour in M. lignano is a striking and frequent response to ejaculate receipt (occurring after c. 50% of copulations), in which the worm places its pharynx over its female genital opening and appears to attempt to suck out its contents, suggesting this trait has evolved in the context of sexual conflict over control of the received ejaculate (Marie-Orleach, Schärer, Joss, & Sandner, 2004;Schärer, Littlewood, Waeschenbach, Yoshida, & Vizoso, 2011;Vizoso, Rieger, & Schärer, 2010). Whether worms are attempting to remove sperm or seminal fluid, or both, by this sucking behaviour, as well as the resulting fitness consequences, is so far unclear. RNAi-induced knockdown of two of the six SFP candidate genes, Mlig-pro31 and Mlig-pro32 (designated as 'suckless' genes), caused a substantially increased suck propensity of mating partners, suggesting that these transcripts indeed manipulate mating partners and may thereby mediate sexual conflict over ejaculate fate, but no clear effect on paternity outcomes in a standardized defensive sperm competition assay , but see Patlar & Ramm, 2020.
The quantitative genetics approach adopted by Patlar et al. (2020), linking transcript expression to downstream phenotypes, provides a powerful framework to identify candidates for subsequent screening of SFP-mediated effects in a targeted manner. We therefore adopted this same method, estimating genetic correlations between seminal fluid expression and partner behaviour to identify two candidate SFPs, Mlig-pro46 and Mlig-pro63, potentially affecting two behavioural phenotypes of interest: partner re-mating behaviour and the propensity for partners to exhibit the suck behaviour (see Materials and Methods). In order to test whether these transcripts indeed influence these behaviours and have downstream impacts on fitness, we then used RNAi to test these two putative SFPs for their effects on mating frequency in pairings with untreated partners, on the suck propensity of mating partners, as well as sperm competitive ability. Both putative SFPs were already included in the above-mentioned naïve RNAi screen , in which Mlig-pro46 showed some evidence for a reduced offensive sperm competitive ability (P 2 , see below), making it an even more promising candidate for more detailed characterization.

| Study organism and experimental subjects
Macrostomum lignano is a free-living, outcrossing simultaneous hermaphrodite found in the Northern Adriatic Sea and Eastern Mediterranean Zadesenets et al., 2016). The worms reach c. 1.5 mm in body length, and the testes and ovaries are located in the central part of the body on either side of a medial gut. The male copulatory organ, the female reproductive organ and the prostate gland cells (where seminal fluid is produced) are all located in the posterior part of the worms (Hyman,1951;Ladurner, Pfister, et al., 2005;Weber et al., 2018). M. lignano is obligately outcrossing; that is, self-fertilization does not occur, and copulations are reciprocal: both partners receive and donate sperm at the same time (Schärer & Ladurner, 2003). M. lignano shows an average mating rate of about 6-15 copulations per hour (Janicke & Schärer, 2009b;Marie-Orleach et al., 2013;Schärer, Joss, et al., 2004), and the high mating rate may lead to suck behaviour because individuals likely prefer donating sperm more than receiving (Schärer, Joss, et al., 2004;Vizoso et al., 2010). The worms are kept in cultures in glass petri dishes filled with artificial sea water (ASW, 32‰) or nutrient-enriched artificial sea water (Guillard's f/2 medium) (Guillard & Ryther, 1962) and fed with diatoms (Nitzschia curvilineata). They are kept under standard conditions on a 14:10 light:dark cycle at 60% relative humidity and a constant temperature of 20°C. All the animals used in this experiment as knockdown/control donors and as recipients (see below) belonged to the highly inbred DV1 line  that was previously used to identify and functionally characterize putative seminal fluid candidates Weber et al., 2018Weber et al., , 2019.
For the identification of transcripts that are genetically correlated with mating frequency, we analysed mating behaviour recorded in an earlier experiment by Patlar et al. (2019). Briefly, the worms used in this experiment originated from 12 highly inbred lines (hereafter genotypes) that belong to a larger set of inbred lines which was originally generated at the University of Innsbruck and is now maintained at the University of Basel (for details, see Vellnow, Vizoso, Viktorin, & Schärer, 2017).

| Putative seminal fluid transcripts in M. lignano
Transcripts encoding putative SFPs in M. lignano were previously identified in two stages. First, Ramm et al. (2019) used an RNA-Seq approach in combination with data on tail-limited expression from an earlier study (Arbore et al., 2015) to define a set of putatively tail-specific transcripts that are differentially expressed between an environment with a high sperm competition level (group of eight worms) compared to a no sperm competition level (isolated worms) (Social group size is a good predictor of mating group size and thus sperm competition intensity in M. lignano [Janicke & Schärer, 2009a;Schärer & Ladurner, 2003;Schärer, Ladurner, & Rieger, 2004]). The vast majority of these transcripts (140/150) exhibited higher expression in octets, meaning they likely represent an aspect of male allocation in this species. Based on M. lignano's anatomy, their most likely site of expression would be the prostate gland cells located around the male genital opening (stylet) that are responsible for seminal fluid production.
In a second step, Weber et al. (2018) then performed an in situ hybridization (ISH) screen to define which of these plastic tail-specific transcripts really are prostate-limited in their expression, identifying a subset of 76 transcripts expressed exclusively in the prostate gland cells making these putative SFP transcripts. Fifty-eight of these transcripts were included in a subsequent gene expression study characterizing genotypic, environmental and GEI sources of variation in transcriptional expression of seminal fluid (Patlar et al., 2019), and we selected candidates for this study from among these 58 transcripts.

| Selection of candidate transcripts
Based on this information, we could then identify candidate transcripts that affect mating frequency for subsequent RNAi. To do so, we re-used data on SFP transcript expression reported in Patlar et al. To examine the mating frequency, mating pairs were formed as one partner originated from a single inbred line (DV1) to be used as the virgin, standardized recipient grown under strict isolated conditions, and the other partner, the donor, originated from one of the different genotype/group size combinations. In total, 18 independent pair replicates and 18 independent octet replicates of each genotype were coupled with standardized recipients to observe mating behaviours. To be able to distinguish partners in mating pairs under normal light during observations, the recipient worms were exposed to the food colouring dye Grand Bleu [E131, E151] (Les Artistes-Paris), diluted to a concentration of 0.25 mg/ml in ASW, for 24 hr beforehand. Such a 24-hr exposure enables us to easily distinguish coloured from noncoloured worms and has previously been shown not to affect the mating frequency . Observations were done by video recording, and each donor worm and its coloured recipient partner was transferred into two-dimensional, mating observation chambers described in detail elsewhere (Schärer, Joss, et al., 2004). Briefly, the recipient worm was transferred with a 1.7 μl drop of ASW to the centre of a coated (Sigmacote ® ) microscope slide which has four HERMA photo stickers as spacers adhered to the sides (two per side). Then, the donor worm was transferred with 1.7 μl ASW to the recipient, merging the two drops. Each observation chamber consisted of 16 such pairs, each made by 3.4 μl drops of ASW, on a single glass slide. Once all mating pairs had been placed on the slide, another coated microscope slide was placed upon the first, to form each drop into a shallow three-dimensional pool that the worms can swim inside ( Figure S1).
To protect the drops from evaporation, the area was sealed with a thin line of Vaseline around the perimeter prior to adding the second slide. Immediately after the observation chamber was ready, it was placed under a camera for a video recording of 2.5 hr. Video recordings were obtained with a DFK 41AF02 camera (Imaging source GmbH) connected to a computer running the software Debut Video Capture Professional, version 2.02. The videos were captured at one frame per second, with a frame rate of 10 (a video of 2 hr was zipped into 12 min), in .mov format with 1920 × 1080 HD resolution. Videos were analysed using the Kinovea video player, version 0.8.15. For analysis of mating frequency and suck propensity, the number of copulations for each mating pair and the number of suck events of the recipient worm were counted throughout the full 2.5-hr period based on the videos recorded.
We then identified candidates in two steps. First, we used the first four principal components (PCs) describing variation in seminal fluid transcript expression from Patlar et al. (2019) to estimate which (if any) of these were genetically correlated with mating frequency, in both pairs and octets. Together, these four PCs explained c. 76% of the total variation in seminal fluid transcript expression. Because PC1 accounted for c. 42% of the total variance, including positive loadings for the majority of transcripts, with no corresponding negative loadings, it was interpreted as capturing overall investment in seminal fluid transcript expression. By contrast, PC2-PC4 each exhibited a mixture of positive and negative loadings for a smaller subset of SFP transcripts and therefore describe instead variation in seminal fluid composition (Patlar et al., 2019). Based on identifying one such PC, namely PC4, which was consistently negatively correlated with mating frequency at both social group sizes, we then investigated further those transcripts that were significantly loaded on PC4. We directly assessed how SFP expression level correlated with mating frequency, predicting that candidate mediators of a reduced mating frequency in partners should exhibit a negative genetic correlation between expression level and mating frequency. Two such candidates (Mlig-pro46 and Mlig-pro63, see Results) were then investigated further in an RNAi knockdown experiment.
Because the same two candidate transcripts, Mlig-pro46 and Mlig-pro63, were also positively loaded on an additional axis of seminal fluid variation, PC3, and this was positively correlated with suck propensity in the pairs environment , we further investigated whether RNAi knockdown also impacted on partner suck behaviour.

| Raising conditions for the RNAi knockdown experiment
Six to eight days post-hatching, a batch of same-age hatchlings (to be used as donors and recipients) was collected and distributed in glass petri dishes filled with ASW and fed ad libitum with algae at a density of c. 150 individuals per dish. Individuals were transferred once per week to new glass petri dishes filled with ASW and ad libitum algae until they underwent tail amputation (days 40 and 36 for the doors and recipients, respectively-see below).
The efficacy of RNAi knockdown was verified by performing wholemount ISH in a preliminary test ( Figure S1).
Before beginning the RNAi (or control) treatment, donor animals were tail-amputated between the antrum and ovaries. This takes advantage of the regenerative capacity of M. lignano (Egger, Ladurner, Nimeth, Gschwentner, & Rieger, 2006), allowing us to remove the antrum, with all potential previously received ejaculate in it, the seminal vesicle, with potential (own) stored sperm as well as the SFP-producing prostate gland cells. Through amputation and subsequent regeneration, we ensured that seminal fluid production was 'reset' prior to the RNAi/control treatment and the individuals have an equal amount and age of stored sperm and seminal fluid reserves and further that individuals also contained no received sperm or seminal fluid at the beginning of the mating trials (see below).
After amputation, individuals were randomly allocated to one of four treatment groups: either the negative (no dsRNA) control or one of three RNAi treatments containing the relevant dsRNA solution The GFP + donor worms used in the experiment as sperm competitors to the experimental subjects were also tail-amputated on the same day as the knockdown/control worms at the age of 40 (±1) days. Thereafter, they were also each kept individually in one well of a 60-well microtest plate in 10 µl ASW with ad libitum algae. GFP + worms were transferred to a new well containing 10 µl ASW with ad libitum algae once on day 45.
The recipient worms used in the experiment were 50 (±1)-dayold adult worms and were also tail-amputated, 14 days prior the mating assay at age 36 (±1) days. After amputation, they were also each kept individually in one well of a 60-well microtest plate in 10 µl ASW with ad libitum algae. Recipient worms were transferred to a new well containing 10 µl ASW with ad libitum algae once on day 43.

| Observation of mating and suck frequency of knockdown
For each RNAi/control treatment, we then conducted two separate assays with separate batches of donor, competitor and recipient worms, to measure (a) mating frequency (in both batches), (b) suck propensity (in both batches) and (c) either defensive (P 1 ) or offensive (P 2 ) sperm competitive ability (depending on the batch: in batch 1 we measured only P 1 and in batch 2 only P 2 ). Initially, worms in both assays (P 1 and P 2 ) were treated identically. The mating frequency and suck propensity were calculated by counting the number of copulations and the number of suck events per mating by the recipient during the 2.5-hr mating period which was filmed during the sperm competitive ability assays described in the next subsection. Video recording was performed using mating chambers using the same procedure as described above, with the only exception being that the mating period lasted for 2.5 hr here instead of the 2 hr used for the earlier trials identifying candidates.

| Sperm competitive ability assays (P 1 and P 2 )
All mating trials were conducted on day 11 post-amputation.
Recipients were kept after their mating trial in 60-well plates in ASW with ad libitum algae and were transferred to a new well every second day until day 11 (6 wells in total), where they remained until day 21 (after which no further offspring were detected). The resulting offspring were counted and categorized as either GFP − (sired by first knockdown or control donor) or GFP + (sired by the competitor donor) until day 21, based on expression of GFP assessed at age 7-10 days using a Nikon SMZ-18 stereomicroscope with a C-HGFI Intensilight fluorescence light source and GFP filter cube (Nikon GmbH).
To estimate defensive sperm competitive ability (P 1 ), either knockdown or control worms were mated and filmed for 2.5 hr with a randomly selected recipient worm in the above-described mating observation chamber. After 2.5 hr the recipient worms were each put individually in a well of a 60-well microtest plate. 30 min after separating the mating partners, a GFP + sperm competitor worm was added to the well containing the already-mated recipient worm, and the pair was allowed to mate for a further 2.5 hr. After the 2.5-hr mating period, the recipient and the GFP + sperm competitor were separated into an individual well as described above.
To estimate offensive sperm competitive ability (P 2 ), the sperm competition assay was carried out exactly like the P 1 assay, except that the GFP + worm was paired with the recipient first and the knockdown/control worm second. Again, the mating period with the knockdown/control individual was conducted in the mating observation chamber and filmed.
Each treatment group started with 36 donor worms at the beginning of the RNAi treatment. With some replicates excluded due to death of the donor before the mating trial, the absence of resulting offspring or the observation of five or fewer successful matings, the final realized sample sizes for each treatment group ranged from 22-34 (see Table 1).

| Statistical analysis
The effect of genotype on mating frequency was assessed by performing two-way ANOVA using genotype as a fixed effect and, in addition, group size and genotype × group size interaction effects.
Genetic correlation estimates were based on the Pearson correlation coefficients calculated between mean trait values of genotypes.
We calculated the corresponding genetic correlations as r G = Cov(x1, x2)/[Var(x1) × Var(x2)] and tested for statistical significance by comparing the z-scores to two-tailed significance levels derived from a standard normal distribution.
For analysis of the P 1 and P 2 assays, the paternity share of knockdown and control individuals (GFP − ) were compared against the GFP + competitor using a generalized linear model with a quasibinomial distribution and a logit link function (Engqvist, 2013). For analysis of the mating frequency, we compared the number of copulations of knockdown and control mating pairs, using a separate linear model for each of the three treatment-control comparisons. For analysis of suck propensity, the frequency of sucking events (i.e. the proportion of matings followed by a suck) of the mating partners of knockdown and control individuals were compared using a generalized linear model with a binomial distribution and a logit link function. Analyses were conducted using the lme4 package (Bates, Maechler, Bolker, & Walker, 2015) in R (R version 3.1.3,2015).

| Genetic correlations between SFP expression, mating frequency and suck propensity
We estimated genetic correlations between mating frequency and the overall seminal fluid investment (PC1) and relative composition (PC2-4) axes reported in Patlar et al. (2019) at two different group sizes (pairs and octets). We found that mating frequency was not genetically correlated with overall seminal fluid investment, but there was a highly significant negative genetic correlation between mating frequency and PC4 in both pairs and octets (Figure 1a).
Having established that mating frequency is highly negatively correlated with PC4, we therefore next estimated the genetic correlations between mating frequency and the five seminal fluid transcripts which were significantly loaded on PC4 (Patlar et al., 2019).
Among these, we found three transcripts which exhibit a significant negative correlation between mating frequency and SFP transcript expression: Mlig-pro63, Mlig-pro46 and Mlig-pro37 (Figure 1b; here we illustrated these correlations only for octets, which is anyway the more relevant environment for sperm competition, because both group sizes show a very similar pattern). In fact, according to their highly similar sequence with overlapping regions, and the fact that when blasted against the M. lignano genome assembly ML2 (Wasik et al., 2015) they align to the same regions within the same protein-coding gene in the genome, Mlig-pro37 and Mlig-pro46 appear to belong to the same gene. For that reason, for our RNAi screen we selected just Mlig-pro46, which was already investigated in the previous screen , plus the independent candidate

Mlig-pro63.
Notably, these same candidates were also significantly loaded on PC3, as reported previously in Patlar et al. (2019), which was found to be positively correlated with suck behaviour (Figure 1a), at least in the pair group size without rivals .

| RNAi knockdown effects on mating frequency
When we compared the mating frequency of mating pairs including an SFP knockdown donor to those including a control donor, none of the knockdowns impacted strongly on mating frequency, as measured by the total number of copulation events in the 2.5-hr mating period (all p ≥ .1, Figure 2; for full statistical details for each knockdown, see Table 1).

| RNAi knockdown effects on suck propensity
When we compared the mean suck propensity of mating pairs including an SFP knockdown donor to those including a control donor, one of the individual knockdowns exhibited a strongly reduced suck propensity by the mating partner (Mlig-pro63, t = −3.032, p = .0035; Figure 2; Table 1), as measured by the TA B L E 1 Descriptive statistics and tests for treatment effects on mating rate, suck propensity and sperm competitive ability following RNAi knockdown of two seminal fluid transcripts   sucking events per mating in the 2.5-hr mating period, although only in the P 1 assay (i.e. when they mated as the first partner with a recipient worm who subsequently mated with a rival sperm donor). None of the other knockdowns had a significant effect on suck propensity, although in both the P 1 and P 2 assays the combined RNAi showed a nonsignificant trend towards reduced suck propensity.

| RNAi knockdown effects on P 1 and P 2
When we compared recipient worms mated to SFP knockdown donors to those mated to the control donors, none of the knockdowns impacted strongly on defensive sperm competitive ability (P 1 ) or offensive sperm competitive ability (P 2 ), as measured by the paternity share between knockdown/control individuals and the competitor (all p ≥ .3, Figure 2; Table 1).

| D ISCUSS I ON
By selecting two putative seminal fluid transcripts with prostate-limited expression (Weber et al., 2018) that exhibit genetic correlations with both mating frequency and partner suck propensity (Patlar et al., 2019, and subjecting these to RNAi knockdown (individually and combined) followed by behavioural and competitive paternity assays, we aimed to test whether these transcripts directly influence partner behaviour. In fact, we did not detect any significant seminal fluid-mediated effect on mating frequency. However, we did find that one of the seminal fluid transcripts, Mlig-pro63, appears to impact positively on the frequency of the post-copulatory suck behaviour often exhibited by ejaculate recipients in M. lignano.
Finally, we also did not detect any difference in paternity share between knockdown and control individuals, neither when the knockdown individuals were the first mating partners (P 1 ) nor when the knockdown individuals were the second mating partners (P 2 ) in controlled sperm competition assays. In the following, we discuss each of these three main results in turn.
Firstly, one reason why RNAi knockdown of Mlig-pro46 and/or  the longer term. In a screen of 25 D. melanogaster SFP knockdowns, none appeared to modulate the receptivity of the mated female at 24 hr post-mating (Ram & Wolfner, 2007b), with an equally low receptivity to re-mating regardless of whether females mated to control or knockdown males. But three of these SFP knockdowns showed a significant long-term effect on female receptivity, with females mated to males from these three knockdown treatments being significantly more receptive to re-mating at 4 days post-mating than were mates of control males. Nevertheless, the way that we identified candidates in M. lignano involved testing for correlations between transcript expression and mating frequency over a similarly short timescale, so the lack of effect in our assay is still surprising.
Perhaps another explanation could be that while Patlar et al. (2019) used 12 different inbred lines, differing in their genotypes, we used in our experiment only one genotype. The 12 inbred lines could differ in other, correlated ways, which in combination with Mlig-pro46 and/or Mlig-pro63 affect mating frequency but are not incorporated in our RNAi experimental design.
Our results indicate that Mlig-pro63 appears to play a role in promoting the suck behaviour of the mating partner. Although it can occur by various taxon-specific means, ejection of previously received ejaculates by recipients (females) could be quite a common phenomenon (see, e.g., Pizzari & Birkhead, 2000;Rodriguez, 1995;Snook & Hosken, 2004) and is usually interpreted in the context of cryptic female choice and/or sexual conflict over ejaculate fate (Arnqvist & Rowe, 2005;Simmons, 2001 himself (Chapman et al., 2000). This highlights one methodological difficulty with performing such double mating assays, in that SFPmediated effects presumably intended to influence the utilization of own sperm can, under certain study designs that may not well reflect the situation in nature, actually influence that of rivals, and vice versa. In Drosophila, the ejaculates of the first and the second male to mate can both contribute to the second male advantage. Second male SFPs contribute to the ejection or incapacitation of residing first male's sperm, whereas SFPs from the first male still increase the longevity of also the second male's sperm (Nguyen & Moehring, 2018; see also Hodgson & Hosken, 2006).
The absence of detectable impacts of SFP knockdown on sperm competitiveness could be a direct result of the fact the time window we investigated does not well reflect the action of these transcripts. Ram and Wolfner (2007b) tested 25 SFPs knockdowns in Drosophila for their impact on re-mating, none of them appeared to modulate the receptivity of the mated female at 24 hr post-mating. Females showed equally low receptivity to re-mating independently of being mated to either control or knockdown males. But they could identify three SFPs which showed an effect on long-term receptivity of females: at 4 days post-mating, mates of these three knockdown males were significantly more receptive to re-mating than mates of control males.
Also, because in our experiment the competitor was introduced either very soon following the knockdown/control individuals (P 1 assay), or else there was no competitor following the knockdown/ control individuals (P 2 assay), there was perhaps no opportunity to influence sperm competition outcomes via (eventually) reduced receptivity. The surprising result that we did not, contrary to the previous RNAi screen , find an effect of the Mlig-pro46 knockdown on paternity share, certainly suggests we should remain cautious about interpreting the role of Mlig-pro46 in sperm competition until we have gained a greater understanding of its mechanism of action. However, the different outcomes of the two studies could perhaps be due to the slightly different experimental designs regarding mating duration and length of the break between the two competitors. While in the previous experiment there was 1 hr between removing the first donor and adding the second, as well as a longer mating period of 3 instead of 2.5 hr, in this experiment this gap between pairings was just half an hour.
The shorter mating period presumably reduced the opportunity for cumulative effects on sperm competitiveness, and the shorter gap is likely to have exacerbated any carry-over effects alluded to above, meaning effects of SFPs presumably intended for own ejaculates actually also impact on rival ejaculates, or vice versa, tending to equalize paternity success between competitors.
In conclusion, we found evidence for seminal fluid-mediated effects on suck propensity in the simultaneously hermaphrodite M. lignano, but no indication that the two candidate transcripts Mlig-pro46 and Mlig-pro63 affect mating frequency. Further research will be

ACK N OWLED G M ENTS
We thank L. Schärer from the University of Basel for providing the GFP-expressing Macrostomum lignano BAS1 strain used in this study.

SAR is a member of the editorial board of Journal of Evolutionary
Biology.