Material and Methods
We used the lab culture of L. stagnalis maintained at Vrije
Universiteit Amsterdam. All the snails are kept in a flow-through tank
with low copper water maintained at 20 ± 1 °C under dark:light cycle of
12:12h. In this experiment, we used adult snails (4-month-old). As
mentioned, this species is a simultaneous hermaphrodite, but individuals
copulate unilaterally. That is, one individual acts in the male role,
and the other in the female role. Afterwards, they can swap their sex
roles and copulate again (Koene and Ter Maat, 2005). In addition, this
species is promiscuous as exemplified by the fact that they can
inseminate more than once within 24 hours (Koene and Ter Maat, 2007).
To estimate the expression level of SFP genes at several time points
after mating, we let the snails copulate under observation. First, to
increase their male mating motivation, we isolated the snails for eight
days, by keeping one individual per 460-ml perforated container placed
in a flow-through tank (Van Duivenboden and Ter Maat 1985; De Boer et
al. 1997). During isolation, we fed ca. 19.6 cm2 of
broad leaf lettuce per day per capita, which is slightly less than their
maximum food intake (Zonneveld and Kooijman 1989). Next, we placed two
individuals together in a container to let them mate. We size-matched
pairs of snails to reduce the effect of body size on sex role decision
(Nakadera et al. 2015), marked snails on their shell with waterproof
marker for identification during observations. During the mating
observation, we recorded their mating behavior every 15 min (No contact,
mounting, probing, intromission: see Jarne et al. 2010). After
insemination finished, we immediately separated the pair to prevent a
second copulation, and isolated the male-acting snails (hereafter called
donor) until their designated sampling time. We ran this experiment
twice (total N : 3 h = 4, 24 h = 4, 48 h = 6, 192 h = 5).
To estimate the expression level of SFP genes, we collected prostate
glands of donors in four different time intervals, which were 3, 24, 48,
192 h after mating in the male role. First, we injected ca. 2 ml of 50
mM MgCl2 into foot for anesthetization. Then, we quickly
dissected out a prostate gland, placed the tissue into an 1.5 ml
Eppendorf tube, and immediately after the collection, we snap froze the
collected samples using liquid nitrogen. The samples were stored at -80
°C until further processing.
Next, we isolated total RNA using trizol-chloroform, following the
classic protocol. In brief, we homogenized the tissue with trizol, added
chloroform for phase separation, and precipitated RNA pellet using
2-propanol. After washing the pellet using 75 % ethanol, we applied
DNAse treatment. After the quality control of extracted total RNA using
Nanodrop and electrophoresis, we synthesized cDNA using the MML-V
Reverse transcriptase kit (Promega). Then, we conducted quantitative PCR
(qPCR) to estimate the relative expression levels of SFP genes, using
NO-ROX SYBR® Green mix (BioLine) and thermal cycler (CFX-96, Bio-Rad).
We examined all the known SFP genes (N = 6) with two technical
replicates, and used two house-keeping genes as reference (Beta-tubulin,
Ubiquitin, Davison et al. 2016; Young et al. 2019; Table S1). For primer
designing, we applied the following thresholds: annealing temperature
59-60 °C, GC contents = 40-45 %, amplicon melting temperature = 80-85
°C. To calculate the relative, normalized gene expression
(2-∆∆Ct, Livak and Schmittgen 2001), we used the
software CFX Manager v3.1. We confirmed that the expression of reference
genes was not significantly different across treatments (Fig. S1).
To examine the temporal expression changes of each SFP gene after
mating, we used two-way ANOVAs. We used Hours after mating and
experimental block (Exp) as fixed factors. When there was a significant
difference between time after mating, we used Tukey’s Honest Significant
Differences test (HSD). To visualize the overall change in SFP gene
expression over time, we reduced the dimensions of expression data using
principal component analysis (PCA), and tested the expression pattern
over time using two-way ANOVAs again. We performed all the analyses with
R (ver. 4.0.3, R Core Team).