Introduction
Seminal fluid proteins (SFPs, also referred to as accessory gland
proteins or ACPs) are part of the non-sperm component of an ejaculate,
and consist of up to several hundreds of proteins (Sirot et al. 2015).
Although SFPs were initially considered as merely assisting the
functioning of sperm, it has since become clear that they also mediate
other important and diverse processes in reproduction (McGraw et al.
2016). For example, SFPs facilitate the initiation of healthy pregnancy
in humans (Bromfield 2014) and induce oviposition after mating in many
insects (Avila et al. 2011). Underlining these significant functions,
males have been shown to adjust SFP production as well as SFP transfer
depending on the presence of rivals (e.g., Ramm et al. 2015; Nakadera et
al. 2019) or mating status of partners (Sirot et al. 2011). Thus, this
is often explained as males ‘tailoring’ SFP composition of their
ejaculate for each mating to optimize their reproductive success.
However, although SFP production is well known to be plastic, their
replenishment has received surprisingly little attention. This is a
non-trivial knowledge-gap in multiple mating species, as refilling
seminal fluid is expected to be dynamic depending on their past and
future copulations. For instance, male Drosophila melanogasteradjust the amount of specific SFPs to transfer, depending on whether the
female is virgin or not (Sirot et al. 2011). Such protein-specific
adjustment would affect the subsequent SFP replenishment in the male’s
accessory gland organ(s). Also, males often alter SFP production
depending on prevailing sperm competition risk (e.g., Fedorka et al.
2011; Ramm et al. 2015; Bartlett et al. 2017; Mohorianu et al. 2017;
Simmons and Lovegrove 2017; Hopkins et al. 2019) as well as depending on
on-going sperm competition (e.g., Sloan et al. 2018; Ramm et al. 2019;
Nakadera et al. 2019). This plastic SFP production and transfer implies
that males predict and prepare for future mating opportunities. Thus, it
is likely that refilling seminal fluid after mating is highly plastic,
although empirical data for such patterns over time are largely missing
up to now.
To the best of our knowledge, SFP replenishment within the accessory
gland has been investigated only in D. melanogaster and our model
species, the great pond snail Lymnaea stagnalis (see below). InD. melanogaster , transferring ejaculate indeed triggers the
replenishment of Acp95EF (Herndon et al. 1997). Furthermore, it takes at
least three days to fully replenish two SFPs, sex peptide and ovulin
(Sirot et al. 2009). Also, when enlarging our scope to general protein
replenishment, this yields very few studies. One example comes from
snake venom, also a complex mixture of proteins, for which it was
reported that the production of the different classes of protein occur
in parallel when the venom gland is refilled (Currier et al. 2012).
Therefore, we consider that filling this knowledge gap of SFP
replenishment would not only expand the knowledge of SFP expression and
male reproductive strategies, but also stimulate studying the
replenishment of other proteins in various biological contexts.
In this study, we examined the dynamics of SFP replenishment after
mating in the great pond snail Lymnaea stagnalis . To do so, we
let the snails copulate, then examined SFP gene expression at 3, 24, 48
and 192 h after mating. A previous study showed that this species
increases the production of LyAcp10 one day after mating (Swart et al.
2019). However, such an increase was not always observed in another
study (Nakadera et al. 2019). In this experiment, we included all SFP
genes identified in this species (N = 6, Koene et al. 2010;
Nakadera et al. 2019), to monitor if all SFPs replenish in parallel
after mating. It has also been shown that virgin snails express SFP
genes lower than snails with mating opportunities (Nakadera et al. 2019;
2020). This expression pattern led us to predict that SFP production
would be low after a long absence of mating. In sum, we predicted that,
in this species, (1) insemination triggers SFP production, and (2) the
expression of all SFP genes decreases when they are fully replenished in
the seminal fluid producing prostate gland. Furthermore, we examined
whether SFP replenishment occurs in parallel across all SFP genes.