Discussion
Our data reveal a much more dynamic and complex pattern of replenishment of SFPs than we predicted for this snail species. We found that L. stagnalis increases the transcription of a SFP gene 48 h after mating, supporting that transferring ejaculate indeed initiates SFP replenishment. However, three out of six SFP genes did not change their expression level after mating, implying that SFP replenishment occurs in a protein-specific manner. Lastly, even though seminal fluid reserves in the prostate gland are fully replenished after one week (Van Duivenboden and Ter Maat 1985; De Boer et al. 1997), the transcription of SFP genes seem high, contrasting with the low SFP expression of virgin snails previously reported (Nakadera et al. 2019). Below, we discuss the implications of these findings.
We found that the expression of the genes coding LyAcp5, LyAcp8a and LyAcp8b increased 48 h after mating in the male role, supporting the importance of the functions of these proteins that are known to reduce sperm transfer of recipients in their subsequent mating as sperm donor (Nakadera et al. 2014). Thus, increased production of LyAcp5 and LyAcp8b may hint at the intention of donors to reduce sperm transfer of their mating partners and, overall, supports the flexible and complex nature of SFP replenishment. Also, we did not detect signs of increased production after mating in the other three SFP genes studied here (Fig. 1). This may imply that SFP replenishment occurs in a protein-specific manner. In this species it has been established that, mating history indeed affects sperm transfer and SFP transcription (Loose and Koene 2008; Nakadera et al. 2019). Collectively, these studies suggest thatL. stagnalis allocates specific SFPs differently to an ejaculate, depending on the mating history of donors and recipients, which leads to protein specific SFP replenishment.
The timing of elevated SFP gene expression was rather unexpected, and currently we do not have a fully-satisfactory explanation for why this is the case. Single insemination should be sufficient to see the signal of SFP replenishment, because this species uses approximately one third of the amount of seminal fluid stored in the prostate gland for one insemination (Koene et al. 2010). Thus, we expected that this promiscuous species would refill its seminal fluid immediately after using up (part of) its supply, as shown in D. melanogaster (e.g., Monsma et al. 1990). Although, statistically speaking, we did detect the elevated expression of two SFP genes (LyAcp5 , LyAcp8a ), the up-regulation of LyAcp8b was observed 48 h after mating, which is much later than expected. Based on our knowledge about the biology of this species, we consider it unlikely that the up-regulation of SFP genes happened earlier than 3 h after mating in L. stagnalis , although in Drosophila , it occurs within 1 h after mating. The reproductive nature of L. stagnalis is slightly more promiscuous and much slower than D. melanogaster . For example, the courtship and insemination of L. stagnalis usually take several hours, and they can inseminate twice per day (Koene and Ter Maat 2007). Moreover, even if they elevated SFP gene expression immediately after mating, it would not cease within 3 h after mating. However, for the time being we do not have a suitable explanation nor reference to argue why they up-regulate SFP genes so late.
The discrepancy between the results from a previous study and ours suggests that SFP replenishment in L. stagnalis is affected by the mating history of female-mating snails (hereafter, recipient). Swart et al. (2019) examined the expression of one SFP gene, LyAcp10 , after mating. To do so, they let eight-day isolated donors inseminate non-isolated recipients. Then, they found that the expression ofLyAcp10 significantly increased 24 h after mating. In our experiment, however, we used both isolated donors and recipients, and we did not detect any change of LyAcp10 expression throughout our monitoring (Fig. 1, also see Nakadera et al. 2019). The comparison of the experimental setups and outcomes between these two studies implies that the mating history of recipients has strong impacts on SFP replenishment of donors. Although this hypothesis may be surprising, it is also supported from the perspective of their mating behaviour. When two isolated, male-mating motivated, snails meet, the recipient snails in the first mating tends to twist their body and grab the shells of their donors, so that the recipient can act as male immediately after the first mating (see photos in Koene and Ter Maat 2005). It is conceivable that this position of recipient snails squeezes the preputium of donors and might thereby reduce efficient seminal fluid transfer. The effect of squeezing is likely more relevant to SFP transfer than sperm transfer, since this species spends most of insemination duration for transferring non-sperm components (Weggelaar et al. 2019). Given this reasoning, we examined whether the gene expression of SFPs 48 h after mating correlated with insemination duration from our behavioural observation, but did not observe any association (data not shown). Nonetheless, these insights from other studies could explain why we did not see the expected increase ofLyAcp10 expression 24 h after mating as Swart et al. (2019), suggesting that this species alters SFP transfer and replenishment depending on the mating history of recipients.
We originally predicted that SFP expression would be reduced 192 h after mating, but this was not fully supported. Our prediction stemmed from previous study in D. melanogaster (Sirot et al. 2009), as well as following reproductive biology of this species. 192 h is sufficient for these snails to become fully motivated to copulate as male (Van Duivenboden and Ter Maat 1985), based on the completed filling state of their prostate glands (De Boer et al. 1997). Moreover, previous studies showed that virgin snails show reduced SFP production (Nakadera et al. 2019, 2020). Therefore, we predicted that SFP production would be very low one week after mating in this species. However, our data did not fully reflect that (Fig. 1). This pattern either suggests that one week was too short for this species to down-regulate SFP production, or past mating experience had changed their reproductive physiology to produce SFPs permanently. The latter is not such a far-fetched hypothesis, since mated females often experience drastic changes triggered by SFPs (e.g., White et al. 2021). Also, we like to emphasize that our study species is simultaneously hermaphroditic, and we cannot rule out long-lasting effects of receiving SFPs, next to the known short-term effects (Nakadera et al. 2014). Therefore, in future studies we will also need to consider that mating experience might mediate long-lasting effects on SFP expression. Moreover, we want to point out that there is a lack of study focusing on this feature of SFP expression, although relatively high expression of SFP genes long after mating was reported in a previous study in mice showing that that SFPs undergo considerable turnover even without copulation or presence of rivals (Claydon et al. 2012).
Our study also provides several cautionary pointers for predicting and interpreting gene regulation patterns of SFPs. First, we estimated the abundance of mRNA, which indicates the degree to which the protein production machinery is at work, but does not strictly reflect the amount of protein produced and/or present in the gland; a standard caveat when using qPCR (Futcher et al. 1999). For example, post-transcriptional regulation, translation efficiencies and turnover rate of each protein could disturb the direct relationship between the amount of mRNA and protein products (Futcher et al. 1999; Pratt et al. 2002). Second, SFP expression can be highly flexible and as we explained above, a slight change of experimental design can already have unexpectedly strong impact on the transcriptome. In our case, a slight deviation of protocol using snails directly from our mass culture as recipient did reveal the potential high plasticity on SFP expression depending on the mating history of recipients (Swart et al. 2019). Lastly, timing is essential to capture the expected up- and down-regulation of target genes. Based on our previous study (Swart et al. 2019), we expected that most expression changes would occur one day after mating. However, it turned out that this rather occurs between 24-48 h after mating, or not at all. Therefore, it is vital to carefully plan and conduct pilot experiments before investigating SFPs using extensive and expensive approaches, such as RNAseq.
In sum, we measured SFP gene expression after mating in L. stagnalis to expand the knowledge of protein-specific SFP replenishment. Our investigation indeed supported that insemination triggers up-regulation of SFP genes, but the result also suggested that it proceeds in a SFP-specific manner. Furthermore, our results showed that SFP replenishment is plastic depending on the mating history of recipient snails. Lastly, we found that not all SFP genes are down-regulated 192 h after mating, although the seminal fluid producing prostate gland is fully replenished by then. Given these outcomes, we believe our study expands the understanding of SFP dynamics and reproductive strategies in animals and suggests that protein-specific replenishment might also be the case in other glandular systems involving protein replenishment.