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). Although
this species is a simultaneous hermaphrodite, 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
relatively 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), and 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, and the sample size is not fully balanced due to a few handling
errors (total N : 3 h = 4, 24 h = 4, 48 h = 6, 192 h = 5).
To estimate the expression level of SFP genes, we sacrificed the donor
snails to collect their prostate glands 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 a generalized linear model (GLM) with gamma
distribution, due to the expression data being skewed. We used
expression levels as the dependent variable, and Hours after mating and
experimental block (Exp, N = 2) as fixed, categorical factors.
Subsequently, we corrected the p values using false discovery
rate (FDR) correction. When there was a significant difference between
Hours after mating, we used Tukey’s Honest Significant Differences
(Tukey HSD) test. To visualize the overall change in SFP gene expression
over time, we reduced the dimensions of expression data using principal
component analysis (PCA). In addition, we tested the created PC scores
using GLM with gaussian distribution with the same model above and FDR
correction. We performed all the analyses with R (ver. 4.0.3, R Core
Team).