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.
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