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). Moreover, SFPs play crucial roles in sperm
competition, e.g., by reducing remating rate of females or changing
sperm velocity (e.g., Fiumera et al. 2007; Bartlett et al. 2017).
Underlining the significance of SFP functions in sperm competition, a
few studies in insects reported that consecutive mating make males
deplete SFPs more quickly than they do sperm (Drosophila
melanogaster : Lefevre and Johnson 1962, bedbug Cimex
lectularius : Reinhardt et al. 2011, south American fruit flyAnastrepha fraterculus : Abraham et al. 2020). Furthermore,
previous studies observed that males adjust SFP production as well as
SFP transfer depending on the presence of rivals (e.g., D.
melanogaster : Fedroka et al. 2011; Mohorianu et al. 2017; Hopkins et
al. 2019, field cricket Teleogryllus oceanicus : Simmons and
Lovegrove 2017; Sloan et al. 2018, chinook salmon Oncorhynchus
tshawytscha : Bartlett et al. 2017, house mouse Mus musculus
domesticus : Ramm et al. 2015, flatworm Macrostomum lignano : Ramm
et al. 2019, pond snail Lymnaea stagnalis : Nakadera et al. 2019)
or mating status of partners (D. melanogaster : Sirot et al. 2011,
red junglefowl Gallus gallus : Alvarez-Fernandez et al. 2019).
This observed plasticity is often explained as males ‘tailoring’ SFP
composition of their ejaculate for each mating to optimize their
reproductive success under varying levels of expected sperm competition.
However, although SFP production and its transfer are well known to be
plastic in some taxa, 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 D.
melanogaster adjusts the amount of specific SFPs to transfer, depending
on whether the female is virgin or not (Sirot et al. 2011). Such
protein-specific adjustment of SFP transfer would affect the subsequent
SFP replenishment in the male’s accessory gland organ(s). That is, the
most recent usage of SFPs would affect which SFPs would be more
replenished than other SFPs. Also, males often alter SFP production
depending on prevailing sperm competition risk (e.g., Ramm et al. 2015;
Hopkins et al. 2019) as well as depending on on-going sperm competition
(e.g., Sloan et al. 2018; 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 in only a few Diptera species and our model
species, the great pond snail Lymnaea stagnalis (see below). InDrosophila , it has been well established that mating triggers
up-regulation of transcription and translation in male accessory glands,
likely to replenish SFPs (Bauman 1974; Schmidt et al. 1985; DiBenedetto
et al. 1990; Monsma et al. 1990; Bertram et al. 1992; Yamamoto et al.
1998; Herndon et al. 1997; Redhai et al. 2016; Leiblich et al. 2012,
2019). Several studies monitored the size of male accessory glands after
mating to see the time window of SFP replenishment (D.
melanogaster : Hopkins et al. 2019, Queensland fruit flyBactrocera tryoni : Radharkrishnan and Taylor 2008, stalk-eyed flyCyrtodiopsis dalmanni : Rogers et al. 2005, but seeBactrocera dorsalis : Wei et al. 2015), based on the correlation
between the size of male accessory glands and amount of secretion inDrosophila (Ravi Ram and Ramesh 2002). To date, only two studies
in D. melanogaster measured how long it takes to refill SFPs at
protein level (Coleman et al. 1995; Sirot et al. 2009). Sirot et al.
(2009) showed that full replenishment of two SFPs, sex peptide and
ovulin, was complete within three days (Sirot et al. 2009, see also
Hopkins et al. 2019). 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).
Given above, we consider that the knowledge of protein-specific
replenishment of SFPs would expand the understanding 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 L. stagnalis . To do so, we let the
snails copulate, then examined SFP gene expression at 3, 24, 48 and 192
h after mating. The rationale of finishing our monitoring after one week
is that previous studies show that these snails get highly motivated to
copulate as male after eight days of social isolation (Van Duivenboden
and Ter Maat 1985), and this male mating motivation is driven by the
fullness of the prostate gland (De Boer et al. 1997). Moreover, it has
been shown that this species increases the production of LyAcp10 one day
after mating (Swart et al. 2019). However, such an increase at 24 h
after mating was not 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 how
these SFPs get replenished 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.