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).