Discussion
Our data reveal a much more dynamic and complex pattern of replenishment of SFP than we predicted for this snail species. We found that L. stagnalis increases the transcription of two SFP genes 48 h after mating, supporting that transferring ejaculate indeed initiates SFP replenishment. However, four 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, 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 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). The first study that identified these SFPs showed that this species uses approximately one third of the amount of seminal fluid stored in the prostate gland for one insemination (Koene et al. 2010). For promiscuous species, it is very intuitive that they refill their seminal fluid after using up (part of) their supply, as shown in D. melanogaster (Herndon et al. 1997). Nevertheless, we did not detect signs of increased production after mating in the other four SFP genes studied here (Fig. 1). This may imply that SFP replenishment occurs in a protein-specific manner. Furthermore, this species might replenish specific SFPs depending on its own mating history as well as that of its partner(s). In this species, mating history indeed affects sperm transfer and SFP transcription (Loose and Koene 2008; Nakadera et al. 2019). These studies suggest that L. stagnalis allocates specific SFPs differently to an ejaculate, depending on the mating history of donors and recipients, which leads to protein specific SFP replenishment. Also, it has been shown that receiving LyAcp5 and LyAcp8b reduces sperm transfer in a subsequent mating (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.
Comparing our results to a previous study also points out that SFP replenishment in L. stagnalis is affected by the status of female-mating snails (hereafter, recipient). Given the data of Swart et al. (2019), we predicted that the expression of LyAcp10 increases 24 h after mating, but we did not observe such a pattern (see also Nakadera et al. 2019, Fig. 1). The cause of this deviation probably lies in that we decided to standardize the mating history of donors and recipients by isolating them for eight days, while Swart et al. (2019) used eight-day isolated individuals as donors, and non-isolated individuals as recipients. Such a difference in mating history in recipients could cause differential allocation of SFPs due to the following details of mating behaviours in L. stagnalis . When two isolated, male-mating motivated, snails meet, the recipient snails in the first mating tends to twist their body and grab the shell of the donor, so that the recipient can act as male immediately after the first mating (Koene and Ter Maat 2005). It is conceivable that this position squeezes the preputium of the donor and might thereby reduce efficient seminal fluid transfer. Also, this species transfers sperm at the very end of the insemination phase (Weggelaar et al. 2019), but it is still possible that donors transfer non-sperm components, including seminal fluid, prior to sperm during the preceding insemination duration. 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 details could explain why we did not see the expected increase of LyAcp10 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. 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 do 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 (White et al. 2021).
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 investigated SFP replenishment of L. stagnalis to contribute to filling the knowledge gap in SFP research. 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 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 this might also be the case for other glandular systems involving protein replenishment.
Acknowledgements
We appreciate the support from Carool Popelier and Omar Bellaoui for maintaining the lab culture of snails. This work was supported by NWO Open Competition Grant OCENW.KLEIN.062 (JMK, YN).
Author contribution
JMK conceived and designed the study. YK and JM conducted experiments and processed the samples. YN and JMK analysed the data and wrote the manuscript with input from JM and YK.
Data accessibility
All data of this research will be deposited in an open-access and permanent data depository (e.g., Dryad), upon the acceptance of publication.
Competing interests.
None.
Reference
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