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.