Effects of two seminal fluid proteins on post-mating behavior in the simultaneously hermaphroditic flatworm Macrostomum lignano

Along with sperm, in many taxa male ejaculates also contain a complex mixture of proteins, peptides and other substances found in seminal fluid. Once seminal fluid proteins (SFPs) are transferred to the mating partner, they play crucial roles in mediating post-mating sexual selection, since they can modulate the partner’s behavior and physiology in ways that influence the reproductive success of both partners. One way in which sperm donors can maximize their own reproductive success is by changing the partners’ (sperm recipient’s) postcopulatory behavior to prevent or delay re-mating, thereby decreasing the likelihood or intensity of sperm competition. We therefore adopted a quantitative genetic approach combining gene expression and behavioral 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 - that exhibit a negative genetic correlation between transcript expression and mating frequency. Importantly, however, in one of the two different group sizes, differing in their sperm competition level, in which we measured genetic correlations, these same two transcripts are also linked to a second post-mating behavior in M. lignano, namely the ‘suck’ behavior of recipients in which, upon ejaculate receipt, the worm places its pharynx over its female genital opening and appears to attempt to remove ejaculate components. To therefore investigate directly whether these two candidates manipulate partner behavior, and test whether this impacts on competitive fertilization success, we performed a manipulative experiment using RNA interference-induced knockdown to ask how loss of Mlig-pro46 and Mlig-pro63 expression, singly and in combination, affects mating frequency, partner suck propensity and both defensive and offensive sperm competitive ability (P1 and P2, respectively). None of the knock-down treatments impacted strongly on mating frequency or sperm competitive ability, but the knock-down 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 behavior, though the functional and adaptive significance of these two seminal proteins from a donor perspective remains enigmatic.


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In polyandrous species, when females store sperm from multiple males, post-mating 51 sexual selection can occur if sperm from different males compete with each other for 52 fertilization (i.e. sperm competition; (Parker 1970)) and/or females choose specific 53 sperm to fertilize eggs (i.e. cryptic female choice; (Thornhill 1983;Eberhard 1996)). As a sexual receptivity and shorter copulation times when subsequently mated, similar to 96 behaviors seen in previously mated females (Radhakrishnan and Taylor 2007). In 97 Anopheles gambiae, injection of male accessory gland homogenates into virgin females 98 results in a decreased likelihood of re-mating (Shutt et al. 2010). There is also growing   (Nakadera et al. 2014; see also Schärer 2014). This latter effect emphasizes that it is a 120 potentially adaptive strategy in simultaneous hermaphrodites to steer your partner away 121 from its male function, and that the action of seminal fluid may be one means of doing 122 so (Schärer and Ramm 2016). There is also evidence for the manipulation of the re-123 mating frequency in a simultaneously hermaphroditic species, namely the love-dart 124 shooting snail Euhadra quaesita. Before exchanging sperm, both mating partners 125 attempt to drive their mucous-coated love-dart into their respective partner. In stabbed 126 snails, the intermating interval is longer than in not-stabbed individuals, and so snail 127 pairs injected with mucous subsequently mate less often than control pairs ( M. lignano is a striking response to ejaculate recipient, in which the worm places its 143 pharynx over its female genital opening and appears to attempt to suck out its contents, 144 suggesting this trait has evolved in the context of sexual conflict over control of the

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To estimate defensive sperm competitive ability (P 1 ), either knockdown or control worms 348 were mated and filmed for 2.5 hours with a randomly selected recipient worm in the already-mated recipient worm, and the pair was allowed to mate for a further 2.5 hours.

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After the 2.5 hours mating period, the recipient and the GFP + sperm competitor were 354 separated into an individual well as described above.

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To estimate offensive sperm competitive ability (P 2 ), the sperm competition assay was 356 carried out exactly like the P 1 assay, except that the GFP + worm was paired with the  Table 1).

Statistical analysis 366
The effect of genotype on mating frequency was assessed by performing two-way 367 ANOVA with interaction after testing for homogeneity of variance for mating group size

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(2018) at two different group sizes (pairs and octets). We found that mating frequency 389 was not genetically correlated with overall seminal fluid investment, but there was a 390 highly significant negative genetic correlation between mating frequency and PC4 in 391 both pairs and octets (Fig. 1a).

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Having established that mating frequency is highly negatively correlated with PC4, we plus the independent candidate Mlig-pro63. 406 Notably, these same candidates were also significantly loaded on PC3, as reported 407 before in Patlar et al. 2018, which was found as to be positively correlated with suck 408 behavior (Fig. 1a), at least in the pair group size (Patlar et al submitted).  Also with respect to suck propensity, we note that the fact we observed an effect of 483 Mlig-pro63 knockdown in the P 1 but not the P 2 assay suggests that Mlig-pro63