Behavioral The official journal of the Ecology ISBE International Society for Behavioral Ecology

Behavioral Ecology (2019), 30(4), 904–913. doi:10.1093/beheco/arz027

Original Article Plastic expression of seminal fluid

genes in a simultaneously Downloaded from https://academic.oup.com/beheco/article/30/4/904/5376642 by guest on 02 October 2021 hermaphroditic snail

Yumi Nakadera , Athina Giannakara, and Steven A. Ramm Evolutionary Biology Department, Bielefeld University, Germany Morgenbreede 45, 33615 Bielefeld, Germany Received 5 September 2018; revised 30 January 2019; editorial decision 31 January 2019; accepted 8 February 2019; Advance Access publication 18 March 2019.

Seminal fluid (SFPs) are components of the ejaculate that often induce drastic changes in female physiology, such as reduc- ing remating rate or shortening longevity. There is compelling evidence that these functions make SFPs a determinant of male repro- ductive success, and some evidence that males can strategically invest in their differential production. However, SFP-mediated effects have received relatively little attention in simultaneous hermaphrodites, that is, organisms that are male and female at the same time. Since this reproductive mode is widespread in animals and their SFPs have unique functions compared to separate-sexed species, examining SFPs of hermaphrodites would help generalize our understanding of the impact of SFPs. We therefore examined if individu- als strategically alter seminal fluid production in response to mate availability and competition in the freshwater snailLymnaea stagnalis. We exposed snails to different social group sizes, and measured the expression of 6 SFP genes. We found that the snails plastically elevated SFP expression in the presence of at least 1 mating partner. Specifically, paired snails showed higher SFP expres- sion than isolated snails, whereas SFP expression of snails exposed to , that is, in a larger group size, was equivalent to that of paired snails. Furthermore, 5 out of 6 SFP genes we examined responded to mate availability in a very similar way, implying that overall seminal fluid production increases when the snails have mating opportunities. The plastic expression of seminal fluid depending on mate availability supports that SFPs play important roles in post-copulatory processes in this hermaphroditic species. Key words: ejaculate investment, gene expression, mate availability, seminal fluid, sexual selection, sperm competition.

INTRODUCTION plastically adjust their investment in SFPs, for example, depending Variation in reproductive success arises not just from variation in on the presence of rivals or on the females’ mating history (Wigby mating success but also from the differential fertilizing ability of et al. 2009; Fedorka et al. 2011; Sirot et al. 2011; Ramm et al. transferred ejaculates (Parker 1970). Recent studies have revealed 2015; Simmons and Lovegrove 2017; Sloan et al. 2018). Such plas- that non-sperm components in the ejaculate, that is, seminal fluid tic investment in SFPs suggests that these proteins play important proteins (SFPs), play significant roles in determining male repro- roles in determining male reproductive success in post-copulatory ductive success. For example, SFPs induce essential responses sexual selection. in females for reproduction, such as ovulation or sperm trans- To date, most studies of seminal fluid have been conducted in port (Chapman 2001; Poiani 2006; Avila et al. 2011). Thus, their species with separate sexes, and the impact of SFPs has received positive effects on reproduction have also been emphasized in far less attention in simultaneous hermaphrodites, that is, organ- assisted reproductive technology (McGraw et al. 2015; Samanta isms that possess male and female functions within their body at et al. 2018). Moreover, SFPs have sometimes been shown to trig- the same time (hereafter called hermaphrodites). Hermaphroditism ger sexually antagonistic effects on females, seemingly exploiting is a widespread reproductive mode across animals and is found, for them for the male’s own benefits (e.g., shortening females’ life span: example, in snails, slugs, barnacles, flatworms, and fish (Jarne and Chapman et al. 1995, reviewed in Sirot et al. 2015). To further Auld 2006). Recent studies have demonstrated that hermaphrodites underline the significance of seminal fluid, males in several taxa are indeed under sexual selection (e.g., Anthes et al. 2010; Hoffer et al 2017). In particular, several studies in hermaphroditic snails have revealed the presence of multiple paternity in field popula- tions, suggesting that sperm competition occurs commonly (e.g., Address correspondence to Y. Nakadera. E-mail: yumi.nakadera@uni- bielefeld.de. Janssen and Baur 2015; Bürkli and Jokela 2017; Nakadera et al. 2017).

© The Author(s) 2019. Published by Oxford University Press on behalf of the International Society for Behavioral Ecology. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals. [email protected] Nakadera et al. • Plastic seminal fluid expression in hermaphrodites 905

These observations imply that SFPs in hermaphrodites could play Here we examined whether L. stagnalis alters SFP production important roles in sperm competition, just as they do in separate- in different social group sizes. We aimed to investigate the devel- sexed species (e.g., Clark et al. 1995; Chapman et al. 2003; Zhang opmental plasticity of SFP expression, so we exposed the snails to et al. 2013), since many of the mechanisms through which male different social group sizes continually prior to and after sexual mat- reproductive success can be maximized will be common to both uration. At the end of the experiment, we measured the abundance reproductive modes. Moreover, a recent study demonstrated that of mRNA from 6 SFP genes in the prostate glands of adult snails SFPs of hermaphrodites can have unique functions compared to using quantitative polymerase chain reaction (qPCR). This measure separate-sexed species. That is, the receipt of specific SFPs reduces serves as our estimate of SFP production. Since transcription is a the investment of male function in mating partners (Nakadera et al. key machinery of protein synthesis, we would expect that the abun- 2014), which may be one instance of an adaptive strategy in her- dance of mRNA broadly correlates with the abundance of protein maphrodites of targeting the sex allocation of a mating partner produced (e.g., Lovrić 2011; Bonilla et al. 2015) and so we assume (Charnov 1979; Schärer and Ramm 2016). Therefore, examining here that our measure of gene expression results in downstream SFPs in hermaphrodites could contribute to generalizing the cur- differences in SFP production. In our first experiment (Exp. 1), we Downloaded from https://academic.oup.com/beheco/article/30/4/904/5376642 by guest on 02 October 2021 rent insights about the functional and evolutionary significance of tested whether the snails exhibit higher SFP gene expression and seminal fluid to a wider range of animals and reproductive modes. thus presumably produce more seminal fluid when a mate is avail- As one aspect of investigating the role of SFPs in hermaphro- able (isolated vs. paired), as well as if isolated snails adjust SFP dites, we here focus on plasticity in SFP production under sperm gene expression within 24 h of being exposed to a potential mate competition. Based on the general framework of sperm competi- (isolated vs. iso-paired, see Material and Methods). The latter com- tion theory, males are expected to increase ejaculate production parison would thus inform us about the time window to adjust SFP when they are exposed to high sperm competition risk or inten- production in this species. In our second experiment (Exp. 2), we sity (e.g., Parker and Ball 2005). Our predictions in this study are tested if SFP production differs across varying social group sizes based on general sperm competition theory, rather than predictions (isolated vs. paired vs. grouped), to examine developmental plastic- concerning SFP allocation (e.g., Cameron et al. 2007; Alonzo and ity in seminal fluid production at different sperm competition levels. Pizzari 2010; Dhole and Servedio 2014), since the link between By comparing the isolated to the other 2 treatments (i.e., paired and such allocation decisions and measures of production is uncertain. grouped), we tested if the presence or absence of a mate (hereafter Several empirical studies on SFP production provide evidence for “mate availability”) affects SFP gene expression. The comparison plastic investment according to different sperm competition lev- between paired and grouped snails then allowed us to further test els (Fedorka et al. 2011; Mohorianu et al. 2017; Simmons and if the presence or absence of sperm competition alters SFP gene Lovegrove 2017; Patlar et al. forthcoming; Sloan et al. 2018; Ramm expression. et al. ­forthcoming). For example, a study on house mouse explicitly Note that, although the paired treatment enforced monogamy showed that males increase SFP production when they perceive a and so sperm competition is strictly absent, it is possible that this risk of sperm competition, and the composition of SFPs produced treatment results in a low perceived risk of sperm competition is altered under differential risk of sperm competition (Ramm (Parker et al. 1990, 1997), due to the following 2 reasons. First, et al. 2015, see also Simmons and Lovegrove 2017). Recent work since we used hermaphroditic snails, the male function of the part- in the hermaphroditic flatworm Macrostomum lignano also revealed ner in the paired treatment might represent a potential future rival evidence for both elevated expression of putative SFP transcripts should further snails be encountered. Second, in hermaphrodites, under the risk of sperm competition (Ramm et al. forthcoming, there is also the potential for competition between own sperm and but see Patlar et al. ­forthcoming), as well as differential expression sperm from mates (allosperm). However, we do not expect this of different transcripts likely affecting seminal fluid composition potential competition alone to be perceived as sperm competition (Patlar et al. forthcoming). By contrast, another study in Drosophila risk in L. stagnalis, since it is well established that this species prefers melanogaster found that males suppress the production of several allosperm to fertilize own eggs (i.e., outcrossing, Cain 1956; Koene SFPs when they perceive the risk of sperm competition (Fedorka et al. 2009b; Nakadera et al. 2017), though they can self-fertilize in et al. 2011, but see Mohorianu et al. 2017). In sum, empirical stud- the extended absence of mating partners without showing apparent ies demonstrated that males adjust SFP production according to inbreeding depression (e.g., Coutellec and Lagadic 2006; Puurtinen different sperm competition levels, but in order to see how males et al. 2007). Despite these additional considerations, we nevertheless generally alter the investment in SFPs, further experiments with expect that the perceived sperm competition level, even if assessed diverse model species are needed. at low rather than no risk, would still be far lower in paired snails We used the great pond snail Lymnaea stagnalis, to test if her- than in grouped snails, which in our case meant 5 snails. Thus, we maphrodites adjust SFP production in response to different sperm would consider it meaningful to compare SFP production under no competition levels. This species is one of the few hermaphrodites or low sperm competition risk and high sperm competition inten- to date in which SFPs have been identified and examined. Koene sity. In brief, we found that L. stagnalis increases SFP gene expres- et al. (2010) investigated the secretions of the prostate gland and sion in the presence of at least 1 mate. determined the partial amino acid sequence of several SFPs. Moreover, it has been shown that, in L. stagnalis, the receipt of one specific SFP reduces egg production (LyAcp10, Koene et al. 2009a, MATERIAL AND METHODS 2010), whereas receipt of 2 others reduces sperm transfer and Like many freshwater snail species, L. stagnalis matures as male paternity success of the recipient in a subsequent mating (LyAcp5, first, then becomes hermaphroditic (i.e., slight protandry,Jordaens LyAcp8b, Nakadera et al. 2014). However, it is unknown whether et al. 2007). This species copulates unilaterally, that is, at each mat- snails are able to adjust SFP production depending on the mating ing, 1 individual acts as a male to transfer its ejaculate to the part- conditions they face, as observed in other species (e.g., Ramm et al. ner acting as a female. L. stagnalis is highly promiscuous in both 2015; Patlar et al. forthcoming). sex roles throughout their life (Hermann et al. 2009; Hoffer et al. 906 Behavioral Ecology

2017). These snails become strongly motivated to copulate as a snails from the surface, gently stirred the water and blindly picked male after isolation of 8 days or longer (e.g., Van Duivenboden and 1 snail. After dissection, we placed the prostate gland into 500 µl Ter Maat 1985) and when 2 such snails meet, they often re-mate of RNAlater at 4 °C overnight and then at −20 °C until further immediately after their first mating and swap their sex roles (Koene processing. Due to a low level of background mortality during the and Ter Maat 2005). We used the laboratory strain of L. stagna- 10-week treatment (3 isolated snails and 2 snails in the paired treat- lis established at Bielefeld University, which is derived from ca. 20 ment died), our sample size consisted of 19 isolated, 10 iso-paired adult snails transferred from the laboratory strain maintained at and 18 pairs of snails at this stage. VU University Amsterdam, the Netherlands in 2014. The snails In Exp. 2, we assigned snails to 1 of 3 treatments: isolated, paired are kept under standardized culturing conditions in filtered water or grouped (together with 5 individuals). We kept snails accord- under L:D = 12:12 h light-dark conditions and are regularly fed ing to their assigned treatments in plastic containers (size: 15 × with broad leaf lettuce, sepia shell and fish food (Tetraphyll). 9 × 8 cm, 200 ml of water per capita) for 9 weeks, again starting before their maturation (shell length = ca. 1.5 cm, Supplementary Experimental design Figure S1). Although we did not monitor their mating activities, Downloaded from https://academic.oup.com/beheco/article/30/4/904/5376642 by guest on 02 October 2021 We conducted 2 experiments to examine SFP gene expression based on previous research we expect that the grouped snails under different social group size conditions differing in mate avail- copulated slightly more often than the paired (paired snails copu- ability and/or sperm competition. In Exp. 1, we tested whether the lated ca. 1.6 times and groups of 4 snails ca. 1.9 times on average snails adjust SFP production, as reflected in SFP gene expression, in a 24-h session, Koene and Ter Maat 2007). Due to logistical when they do or do not have a mate, and if so, how quickly they constraints, we split our experiment into 2, overlapping batches adjust SFP production upon commencing mating. In Exp. 2, we beginning 1 day apart, each consisting of 15 replicates of all 3 further examined if the snails adjust SFP production under differ- treatments. We applied the same procedure to both batches always ent sperm competition levels. Specifically, we kept snails isolated, 1 day apart, and we did not detect any significant effect of batch paired or together with 5 individuals, meaning we could again test on our measurements (e.g., body size, gene expression, data not if there was a difference in SFP gene expression due to mate avail- shown). During the experiment, we measured the shell length of ability (isolated vs. paired and grouped) but also whether expression all snails every 2 weeks using a caliper and recorded the onset of differed in the absence or presence of sperm competition, that is, egg laying and the number of egg masses every 4 days. We fol- paired versus groups (see Introduction for further explanation of lowed the same protocol for feeding, changing water and collecting the rationale behind these different group sizes). the prostate glands as in Exp. 1. Due to background mortality and In Exp. 1, we assigned snails to 1 of 3 treatments: isolated, iso- handling errors, our sample size after 9 weeks was 29 isolated, 28 paired and paired. The isolated snails did not have a mate, and the paired, and 27 grouped replicates. paired snails had one same mate throughout the experiment. The iso-paired snails had a mating opportunity (i.e., paired) only for the qPCR last 24 h at the end of the experiment. The rationale of the iso- In order to measure SFP gene expression, we performed reverse- paired treatment was to ascertain how rapidly the snails can adjust transcription quantitative PCR (hereafter qPCR) for 6 SFP genes SFP production once they are exposed to a mate. At the begin- (LyAcp5, LyAcp7a, LyAcp7b, LyAcp8a, LyAcp8b, LyAcp10). At the ning of the experiment, we isolated 42 snails for the isolated and start of our analyses, we had only 1 set of primers for LyAcp10 in iso-paired treatments and set up 20 pairs for the paired treatment. L. stagnalis, kindly shared by Elferra Swart and Joris Koene (Swart We started this setup far before their maturation (shell length = ca. et al. forthcoming, Supplementary Table S1). Thus, our first step 1.5 cm) and kept them in these treatments for 10 weeks, until was to design primers for the other SFP genes, based on published the snails were fully mature as indicated by the onset of egg lay- protein sequences and the genome database of L. stagnalis (Koene ing. Commencing the treatment prior to maturation ensured the et al. 2010). Note that these protein sequences have been obtained subjects had known mating histories corresponding to their desig- from the secretion of the prostate gland, not the prostate gland nated treatment (i.e., virgin or mated with 1 individual) and social tissue itself or the SFPs produced in other organs (e.g., seminal cues prior to maturation which may be relevant to SFP production vesicle). We obtained 4 candidate sequences with high matching strategies. We used plastic cups (diameter: 10 cm) with 150 ml of scores, using the built-in search program BLAT (LyAcp7a, LyAcp7b, filtered water per capita, to standardize the volume available for LyAcp8a, LyAcp8b). Also, the tentative sequence of LyAcp5 was each snail. We changed the water twice per week and provided a kindly shared by Dr. Joris Koene. Based on these sequences, we standardized amount of lettuce per capita as food (by the end of designed primers for qPCR using Primer3 (http://bioinfo.ut.ee/ the experiment, ca. 19.6 cm2 of lettuce per day: Zonneveld and primer3/, Supplementary Table S1). In order to confirm whether Kooijman 1989). As the snails started laying eggs from the fourth these genes are expressed predominantly in the prostate gland, we week on, we counted the number of egg masses laid in each con- dissected 2 snails to collect prostate glands, albumin glands, semi- tainer every week. In the tenth week, we measured the shell length nal vesicles, and foot muscles. In order to facilitate RNA stabiliza- of all the snails as a measure of body size using a caliper. Then, we tion, we cut the glandular organs using a razor blade and analyzed put 2 iso-paired snails together for 24 h, allowing them to copulate ca. 50% of prostate gland and ca. 30% of albumin gland. Given freely. This time window is expected to be sufficient for both snails that the prostate gland consists of different cell types distributed to mate at least once in both roles (e.g., Koene and Ter Maat 2007). along its anteroposterior axis (Koene et al. 2010), we cut it from Also, based on an SFP study in D. melanogaster, we expected that the top to bottom. The samples were either processed immediately or duration of 24 h could be sufficient to see a change in SFP gene stored in 500 µl of RNAlater® (Ambion™). We conducted RNA expression (Fedorka et al. 2011). The next day, we collected the extraction, cDNA synthesis, and PCR (see the details below) and prostate glands from all 3 treatments. To randomly select 1 indi- confirmed that the target genes are specifically expressed in the vidual from each container with more than 1 snail, we detached the prostate gland, but not the other organs. Thus, we established a Nakadera et al. • Plastic seminal fluid expression in hermaphrodites 907 protocol to examine the expression of all 6 SFP genes that are cur- To compare the expression of SFP genes, we excluded several rently available. individuals either because none of the snails in a container laid For extracting RNA, we followed the manufacturer’s protocol for eggs (Exp. 1: 3 isolated, Exp. 2: 9 isolated, 1 paired), or because the TRI reagent (Sigma). In order to remove DNA from RNA extracts, paired snails showed more than 0.7 cm difference in shell length we further processed samples with RQ1 DNase (Promega) follow- within a pair (Exp. 1: 2 paired, Exp. 2: 3 paired). The absence of ing the manufacturer’s protocol. After confirming the quality and egg laying might have been either because the isolated snails were quantity of nucleic acid using NanoDrop™ (ND 1000, NanoDrop), still waiting for a potential mate, or because they were incapable we synthesized cDNA from the RNA samples using the GoScript™ of laying eggs. The pairs with such a large body size difference Reverse Transcription system (Promega) according to the manufac- would have been physically impeded or prevented from copulat- turer’s protocol. The absence of DNA in RNA samples was further ing with each other, especially if the larger one tried to inseminate confirmed by running a PCR with the samples before and after the smaller one. In both cases, the status of seminal fluid produc- cDNA synthesis. The cDNA samples were stored at −20 °C until tion was expected to differ from the other replicates, and so these further processing. individuals were excluded from further analyses. Also, a few sam- Downloaded from https://academic.oup.com/beheco/article/30/4/904/5376642 by guest on 02 October 2021 For running the qPCR, we prepared a master mix (4.5 µl ples were omitted due to the low quality of the stored sample or of H2O, 1.25 µl of 10 µM forward and reverse primer, 2 µl of extracted RNA, as indicated by low and inconsistent amplifica- EvaGreen® [SsoFast™ Supermix with Low ROX, Bio-Rad]) to tion of SFP and reference genes (Exp. 1: 1 isolated, 1 iso-paired, which we added 1 µl of each diluted cDNA sample. The ther- Exp. 2: 1 isolated). Lastly, we excluded 1 outlier, due to extremely mal cycling program (following initial denaturation at 95 °C for low measured expression of SFP genes (Exp. 1, 1 paired, Grubb’s 3 min, 39 cycles of [95 °C for 10 s, 60 °C for 30 s]) was run on test, LyAcp7a: G = 3.69, P < 0.001, LyAcp7b: G = 3.85, P < 0.001, a CFX Connect Real-Time System (Bio-Rad). In a 96 well plate, LyAcp8a: G = 3.54, P = 0.002, LyAcp8b: G = 3.61, P = 0.001, we measured the gene expression of 5 SFP genes (LyAcp7a-10), LyAcp10: G = 3.23, P = 0.010). The final sample size for the gene plus 3 housekeeping genes as reference (Histone 2a [Lhis], ubiq- expression analysis was therefore 39 in Exp. 1 (15 isolated, 9 iso- uitin-conjugating enzyme E2 [Lubi], 14-3-3 protein zeta [Lyh]: paired, 15 paired), and 70 in Exp. 2 (19 isolated, 24 paired, 27 Davison et al. 2016, Supplementary Table S1). The rationale of grouped). selecting these housekeeping genes is that they are expressed in We compared the relative expression of SFP genes between every cell, and not primarily involved in cell structure (e.g., actin), treatments, by normalizing the cycle threshold (Ct) values of the since the size of prostate gland in this species changes depending target SFP genes using reference gene expression. We obtained the on mating opportunities (De Boer et al. 1997). We had 3 techni- raw Ct values from the software CFX Connect (ver 3, Bio-rad). We cal replicates for each gene on the same plate, so we obtained standardized the differences between plates using the amplifica- data of 8 genes from 3 individuals per plate. In addition to nega- tion efficiency of plate controls. Next, we tested the difference of tive controls, we prepared the plate controls to standardize the reference gene expression across treatment using Anovas, in order difference between plates using amplification efficiency (cDNA to establish if we could use the expression of these genes for nor- from single individual throughout an experiment, 3 dilution malization of SFP gene expression (Livak and Schmittgen 2001). factors, 2 technical replicates, LyAcp10). Since we obtained the For the analysis of Exp. 1, we used only one of the housekeeping sequence of LyAcp5 at a later stage of processing, we ran qPCR genes (Lhis) as reference, since we could not detect a significant for this gene separately. For Exp. 1, we ran the samples for mea- difference across treatments using a post hoc test, though Anova suring LyAcp5 expression together with 1 reference gene (Lhis), test indicated so (Anovas, Lhis: F2, 36 = 3.60, P = 0.038, Lubi: F2, whereas for Exp. 2, we used all 3 reference genes (see below, 36 = 4.91, P = 0.013, Lyh: F2, 36 = 6.92, P = 0.003; Lhis for LyAcp5: Supplementary Figure S3). Since the patterns of reference gene F2, 34 = 2.05, P = 0.144, N = 39: Supplementary Figure S3a and b). expression were similar to the first runs, technical deviation due The expression of housekeeping genes was low in isolated snails, to the different timing appears negligible, and we were in any which hints that the relative expression of SFP genes could be case primarily interested here in between-treatment, within-gene rather underestimated in isolated snails of Exp. 1. Despite the dif- effects, not comparing between genes. ference of reference gene expression, the expressions of SFP genes are comparable in Exp. 1 and 2 (see Results). Therefore, we con- sider that, although the reference gene pattern clearly urges caution Statistical analysis in interpretation of Exp. 1, the outcome is still likely to be repre- In these experiments, we measured body size, the onset of egg sentative of SFP expression. In Exp. 2, as there was no significant laying, the number of egg masses laid and the relative expression difference in the expression of housekeeping genes (Supplementary of 6 SFP genes. To compare the body size between treatments at Figure S3c and d), we used the average expression of all 3 genes as the end of Exp. 1 (Week 10) and the beginning and end of Exp. 2 reference. For each SFP gene from each sample, we calculated the (Week 0 and 8), we used nested Anovas with containers nested average Ct values from 3 technical replicates. In cases where one within treatment, since we inputted the data of all the individuals of the technical replicates had more than 1.0 difference in Ct value in a container. To test if the onset of egg laying differed between from the average, we excluded this replicate and re-calculated the treatments, we used Kruskal-Wallis rank sum tests, as our mea- average using the remaining 2 (Exp. 1: N = 26 out of 1056 [2.5%]: surements were not continuous (Exp. 1: every week, Exp. 2: every Exp. 2: N = 17 out of 1680 [1.0%]). In a few cases in Exp. 1, 4 days). Also, we calculated the number of egg masses per individ- where all 3 replicates differed substantially from one another, all ual per given time (Exp. 1: 1 week, Exp. 2: 1 day), then compared 3 replicates for the particular gene were excluded (N = 4). We the difference between treatments using Anovas. Because we han- note that body size positively correlates with the dry weight of the dled the isolated and iso-paired snails in exactly the same manner prostate gland in this species (Koene and Ter Maat 2004; Hoffer while collecting the data on body size and egg laying, we combined et al. 2010), and so the prostate gland of isolated snails was prob- these 2 groups for the analyses. ably larger than that of paired and grouped snails (see Results). 908 Behavioral Ecology

However, the normalization based on the expression of housekeep- to iso-paired and isolated snails (MANOVA, Pillai’s trace = 0.61, ing gene expression should standardize this variation in gland size P = 0.038, Figure 1, Table 1). We further examined how SFP gene (i.e., larger prostate glands would show lower Ct values [= higher expression differed between treatments, by creating PCs using the expression] for both SFP and housekeeping genes). 5 SFP genes responding to different mating opportunities (Table 2, To calculate the relative expression of SFP genes, we inputted LyAcp7a-10). PC1 explained 82.9% of the total variance, indicating the average Ct values of target SFP genes and reference genes into that the expression responses of these genes are highly correlated. the formula 2Ct(Reference) – Ct(Target) (Livak and Schmittgen 2001). We Based on the positive loadings of all SFP genes, this variable mainly then log-transformed the relative expression ratio of each gene and captures variation in overall transcript abundance and therefore pre- compared between treatments using Anovas and, if applicable, sumably overall investment in the production of SFPs (Table 2). In Tukey multiple comparison tests. In order to visualize and compare turn, the remaining PCs showed a mixture of positive and nega- the overall SFP gene expression pattern across treatments in fewer tive loadings for the different transcripts, likely representing varia- dimensions, we applied principal component analysis (PCA). For tion in relative SFP gene expression, that is, resulting in differential PCA, we did not include 1 SFP gene, LyAcp5, since we could not Downloaded from https://academic.oup.com/beheco/article/30/4/904/5376642 by guest on 02 October 2021 properly evaluate the influence of social group sizes (see Results). LyAcp5 LyAcp7a LyAcp7b LyAcp8a LyAcp8b LyAcp10 Since the first 3 PCs explained almost all variance in the data aabaaaabb aabaab aab (Exp. 1: 97.9%, Exp. 2: 98.6%), we examined only these variables 4 using Anovas. All the statistical analyses were performed in R (ver 3.4.4, R Core Team 2018).

RESULTS 3

Exp. 1: SFP plasticity in response to mate ) availability ∆ct

At the end of the experiment, isolated and iso-paired snails were Log (2 significantly larger than paired ones (isolated + iso-paired vs. 2 paired, nested Anova, N = 75, F1, 71 = 14.96, P < 0.001, mean shell length in cm [SD]: isolated = 2.90 [0.24], iso-paired = 2.92 [0.12], paired = 2.70 [0.29]). Consistent with previous reports (e.g., Escobar et al. 2011), isolated snails delayed egg laying, compared to paired snails (isolated + iso-paired vs. paired, Kruskal-Wallis test, N = 54, 1 2 χ1 = 11.46, P < 0.001). The explanation usually invoked for this response is that isolated snails wait for a potential mate before start- ing self-ferlitization, to avoid inbreeding depression (Tsitrone et al. IIPIPPIIP IIPP IIPP IIPP I P P 2003, but see Coutellec and Lagadic 2006; Puurtinen et al. 2007). Treatment The average number of egg masses per week also differed between Figure 1 isolated and paired snails (isolated + iso-paired vs. paired, Anova, Relative expression of SFP genes in relation to mating opportunities in N = 54, F1, 52 = 52.70, P < 0.001, Supplementary Figure S2a). Lymnaea stagnalis (Exp. 1). Gene expression was compared between isolated We estimated the relative expression of SFP genes, using one (I), iso-paired (IP), and paired (P) snails. Letters above plots indicate of the housekeeping genes (Lhis) as reference (see Material and significant differences according to post hoc tests (P < 0.05, Table 1). Methods, Supplementary Figure S3a and b). We found that almost Abbreviation: ∆Ct, difference of Ct values between target and reference all SFP genes showed higher expression in the paired, compared genes.

Table 1 Summary statistics in the comparison of SFP expression between treatments

Exp. 1 (N = 39) Exp. 2 (N = 70)

F P Post hoc (P < 0.05) F P Post hoc (P < 0.05)

SFP gene LyAcp5 0.01 0.994 4.38 0.016 P > I LyAcp7a 5.42 0.009 P > (IP = I) 2.01 0.142 LyAcp7b 5.53 0.008 P > (IP = I) 2.14 0.125 LyAcp8a 6.62 0.004 P > (IP = I) 6.80 0.002 (G = P) > I LyAcp8b 14.85 <0.001 P > (IP = I) 8.77 < 0.001 (G = P) > I LyAcp10 5.37 0.009 P > (IP = I) 5.03 0.009 G > I PCA PC1 5.96 0.006 P > (IP = I) 4.81 0.011 (G = P) > I PC2 0.24 0.792 1.66 0.198 PC3 5.56 0.008 P > IP 27.99 < 0.001 G > P > I

We tested the difference of SFP gene expression between treatments using 1-way Anovas and if necessary, Tukey multiple comparison tests (P < 0.05). We applied the same tests for PCs. Abbreviation: I, isolated; IP, iso-paired; P, paired; G, grouped. Nakadera et al. • Plastic seminal fluid expression in hermaphrodites 909

Table 2 The outcome of principal component analysis

Exp. 1 Exp. 2

PC1 PC2 PC3 PC1 PC2 PC3

Eigenvalue 4.144 0.630 0.121 4.526 0.329 0.075 Standard deviation 2.036 0.794 0.347 2.127 0.574 0.273 Proportion of variance 0.829 0.126 0.024 0.905 0.066 0.015 Cumulative proportion 0.829 0.955 0.979 0.905 0.971 0.986 Loadings LyAcp10 0.433 0.547 0.088 0.441 −0.528 −0.476 LyAcp7a 0.467 −0.271 −0.503 0.452 0.302 0.640 Downloaded from https://academic.oup.com/beheco/article/30/4/904/5376642 by guest on 02 October 2021 LyAcp7b 0.431 0.559 −0.103 0.437 −0.577 0.405 LyAcp8a 0.449 −0.462 −0.279 0.452 0.411 −0.144 LyAcp8b 0.454 −0.317 0.807 0.454 0.358 −0.422

seminal fluid composition. We found that the PC1 scores of paired LyAcp5 LyAcp7a LyAcp7b LyAcp8a LyAcp8b LyAcp10 aba,babb abbaa,bb snails differed significantly from that of the iso-paired and isolated 4 4 ones, as for the general expression pattern of each of the SFP genes (Supplementary Figure S4a, Table 1). We also found a significant difference in PC3, though this variable explained only 2.4% of the total variance (Tables 1 and 2). In sum, we found that paired 3 snails had on average higher SFP gene expression, suggesting they produced more SFPs than isolated ones, but the SFP composition variation likely indicated by subsequent PCs was similar across treat- )

∆ct 3 ments. Also, SFP gene expression of previously isolated snails after 24 h with access to a mate was still lower than that of paired snails. 2 Log (2 Exp. 2: SFP plasticity in response to sperm competition level At the start of Exp. 2, there was no difference in body size 1 between treatments (Anova, N = 220, F2, 214 = 2.02, P = 0.136, Supplementary Figure S1). At the end of the experiment, how- 2 ever, body sizes were significantly different (Anova, N = 220, 0 F2, 214 = 55.80, P < 0.001). The isolated snails were larger than the paired snails and the paired were larger than the grouped IPGGIPP IPG IPG IPG I G (Supplementary Figure S1). Again, the onset of egg laying was Treatment delayed for ca. 20 days in isolated snails, and paired and grouped snails started laying eggs at a similar time (Kruskal-Wallis test, Figure 2 Relative expression of SFP genes in relation to sperm competition levels in N = 74, χ2 = 31.23, P < 0.001, Supplementary Figure S5). Egg 2 Lymnaea stagnalis (Exp. 2). Gene expression was compared between isolated mass production differed significantly between treatments in that (I), paired (P), and grouped (G) snails. Note that the y axis for LyAcp5 has grouped snails produced the least number of egg masses per capita different scale than that for other 5 genes. Letters above plots indicate (Anova, N = 74, F2, 71 = 144.10, P < 0.001, Tukey multiple com- significant differences according to post hoc tests (P < 0.05, Table 1). parison: isolated > paired > grouped, Supplementary Figure S2b). Abbreviation: ∆Ct, difference of Ct values between target and reference We found that SFP gene expression was usually higher in paired genes. and grouped snails compared to isolated (MANOVA, Pillai’s trace = 0.79, P < 0.001, Table 1, Figure 2). Regarding the variation We found that isolated individuals deviated from the paired and in expression for LyAcp5, we observed a clear bimodal distribution grouped snails for PC1 (Table 1), suggesting a difference in over- (Figure 2). This pattern suggested there was an additional explana- all SFP production consistent with Exp. 1. However, there was no tory factor, but we did not find any significant association with any significant difference between the paired and grouped treatments other traits we measured in this experiment (e.g., body size, onset (Table 1, Supplementary Figure S4b). We also detected significant of egg laying, egg production, other SFP gene expression; results differences between treatments in PC3 (Table 1), but this variable not shown). We thus ran PCA using 5 SFP genes (LyAcp7a-10), to explained only 1.5% of the overall variance, and based on the PC examine any differences between paired and grouped snails. PC1 loadings from Exp. 1 and 2, PC3 was not equivalent in both experi- explained 90.5% of overall variance. As in Exp. 1, all SFP genes ments (Table 2). were positively loaded on PC1, suggesting that it represents over- all SFP gene expression (Table 2). PC2 again likely captured the relative composition of SFPs produced, and with exactly opposite DISCUSSION signs but similar absolute loadings for all transcripts, PC2 likely We found that SFP gene expression is plastic in L. stagnalis, depend- also captures the same axis of variation across the 2 experiments. ing on mate availability. Snails invested more in overall SFP 910 Behavioral Ecology production when they had mating opportunities (i.e., paired or been too short for L. stagnalis. Another possibility is that these virgin grouped), but SFP gene expression did not differ between the pres- snails do not actually shift investment into the male function once ence or absence of sperm competition. Further, it appears that the they have started selfing, even if they are afforded mating opportu- up-regulation of SFP genes upon commencing mating takes longer nities. In other words, the iso-paired snails would not have elevated than 24 h after exposure to a potential mate. In the following, we their SFP gene expression as high as the paired snails, even if they first discuss the effects of mate availability and sperm competition had more time to copulate. As in many freshwater snails (Escobar level on SFP gene expression, then discuss the timing and corre- et al. 2011), L. stagnalis waits for a potential mate before starting lated nature of these expression responses, and then focus on the self-fertilization, presumably in order to avoid inbreeding depres- effects of varying social group sizes on other traits (body size, egg sion (e.g., Tsitrone et al. 2003, but see Coutellec and Lagadic 2006; production) in this hermaphroditic species. Puurtinen et al. 2007). Because all iso-paired snails had started self- The availability of a mate, which in hermaphrodites could also fertilization, that is, finished waiting, they had likely invested more lead to a perception of increased future sperm competition risk, in their female function. This biased sex allocation might poten- induced a marked increase in SFP gene expression, but there was tially hinder them from allocating their resources to the male func- Downloaded from https://academic.oup.com/beheco/article/30/4/904/5376642 by guest on 02 October 2021 no significant difference in SFP expression between snails under no tion. Conceivably, this difference in early reproductive situation realized sperm competition and high sperm competition intensity. could thus have a carry-over effect on SFP expression in later life We found that L. stagnalis increased SFP production when they had stages of this species. at least 1 mating partner (Exp. 1 and 2). The increased SFP pro- In both experiments, we found that the snails increased over- duction fits with the intuitive prediction that the snails need SFPs all SFP production, except for LyAcp5, when they have at least 1 to transfer ejaculates, and also with the prediction from sperm mate, but they do not make apparent changes to the relative com- competition theory that males increase ejaculate production when position of SFPs produced. It has been shown that SFPs form a they are exposed to sperm competition (e.g., Parker and Ball 2005; complex network with each other to function (Ayroles et al. 2011; Lemaître et al. 2011; Ramm et al. 2015; Simmons and Lovegrove Findlay et al. 2014; Mohorianu et al. 2018; Patlar et al. forthcom- 2017). Moreover, although we might have expected that the snails ing). Since these 5 SFPs (LyAcp7a-10) showed similar responses to increase SFP production even more under intense sperm competi- the different mate availability regimes (Figures 1 and 2), and PC1 tion, there was no apparent difference in overall SFP expression or captures almost all the variation in gene expression (Table 2), this its composition between paired and grouped snails. These results pattern suggests that LyAcp7a-10 are part of a single, closely func- might imply that the snails optimally adjust the investment in SFP tioning unit, although the examination of the full SFP network production for any situation, when they have at least 1 mate. The will require a more comprehensive omics-scale approach than we apparent absence of increased SFP production in a sperm com- attempted here. petitive situation differs from previous studies that have shown that LyAcp5 is reported as one of the SFPs responsible for reduc- some SFPs appear to be down-regulated at high perceived sperm ing sperm transfer of the mate in a subsequent mating (Nakadera competition intensity (Fedorka et al. 2011; Sloan et al. 2018), which et al. 2014), and in this study, it showed a somewhat different pat- could suggest that transcript expression reflects immediate alloca- tern compared to other SFP genes. The expression of LyAcp5 tion decisions (cf. Parker and Ball 2005). However, the slightly showed high variance and did not respond consistently to differ- increased mating rate in grouped treatment might have reduced ent social group sizes. For example, in Exp. 2, the expression of SFPs allocated in the ejaculate (Koene and Ter Maat 2007), though LyAcp5 clearly exhibited a bimodal distribution in all 3 treatments mating rates would not proportionally increase as the number of (Figure 2). Thus, this variation is not so much a consequence of available mates (e.g., Janicke et al. 2013). In addition, L. stagnalis mating, as isolated snails showed a bimodal distribution as well. We prostate gland secretion reserves are not exhausted at each mating explored but did not find a clear explanation for this pattern, with (Koene et al. 2009a). Thus, slightly increased mating rate would not no obvious association with body size, the date the snails started considerably affect SFP production in this species. Also, the pattern laying eggs, relative egg production or expression of other SFP of SFP expression we observed is quite similar to a recent study genes, or with other technical factors such as experimental batch or in another hermaphroditic species, the flatworm M. lignano (Patlar plate (results not shown). In short, at this moment, we do not have et al. forthcoming; but see Ramm et al. forthcoming). Thus, further sufficient knowledge to explain the variation in LyAcp5 expression, investigations of SFPs in diverse species and reproductive modes though this remains an intriguing pattern. would be required to draw a general conclusion about the relation- Although our results are compatible with that of another her- ship between SFP production, mate availability and sperm compe- maphroditic species examined to date for SFP expression (Patlar tition levels. et al. forthcoming; but see Ramm et al. forthcoming), the reasons In Exp. 1, the isolated snails did not change SFP gene expres- we did not see the expected differences in SFP expression between sion after being given the opportunity to mate for 24 h, for which paired and grouped snails could also be due to technical issues. For we suggest 2 possible explanations. First, 24 h might simply have instance, although we would expect that the amount of mRNA been too short for a change in SFP gene expression to occur in positively correlates with the amount of protein produced (e.g., this species, although it seems to be a sufficient time window for Bonilla et al. 2015), there are several other factors which could D. melanogaster (Fedorka et al. 2011). For their first matings, the affect the estimate for the amount of protein produced from the snails presumably use seminal fluid they accumulated during the abundance of mRNA (Lovrić 2011). Any weakening of the cor- isolation period and thus some lag period may not be too surpris- relation between the abundance of mRNA and protein might ing. In the case of the hermaphroditic flatworm M. lignano, it takes explain the discrepancy between gene expression and expected several days to fully adjust their plastic sex allocation according to biological functions of SFPs in some cases (e.g., Smith et al. 2009, their mating group size as captured by testis and ovary size (Brauer 2012), though manipulating the expression of specific SFP genes et al. 2007). Compared to morphological changes, gene expression has been reported to trigger the expected biological responses changes would be expected to occur faster, but 24 h might still have (Chapman et al. 2003; Simmons and Lovegrove 2017). Another Nakadera et al. • Plastic seminal fluid expression in hermaphrodites 911 caveat we would like to emphasize regards the normalization using SUPPLEMENTARY MATERIAL housekeeping genes, particularly in Exp. 1. We detected differential Supplementary data are available at Behavioral Ecology online. expression of housekeeping genes across treatments in Exp. 1, but not in Exp. 2. We are also more confident with the outcome of Exp. 2, as we optimized the rearing condition in this experiment FUNDING by providing larger space per capita. Since we found a comparable This work was supported by the Alexander von Humboldt Foundation pattern of SFP production in these 2 experiments, the effect of (Humboldt Research Fellowship for Postdoctoral Researchers to YN). differential reference gene expression might have been negligible. Nonetheless, for further investigation of SFP expression in L. stag- We appreciate the support from Marie-Agnès Coutellec for permission to nalis, complementary transcriptomic and proteomic approaches access Lymnaea stagnalis Genome database; Joris Koene for providing us are required, and the current study provides vital information for with the snail culture; Bahar Patlar, Michael Weber, Renate Feist and Klaus applying these more comprehensive methodologies. Reinhold for help conducting experiments; and P. Smiseth and 2 anony- Whilst our main focus was on seminal fluid, we also observed mous reviewers for feedback to improve the manuscript. We thank the par- Downloaded from https://academic.oup.com/beheco/article/30/4/904/5376642 by guest on 02 October 2021 effects of different social group sizes on body size, the onset of ticipants of the 2018 Simultaneously Hermaphroditic Organism Workshop (SHOW) in Paris for helpful comments and encouragement. egg laying and egg mass production in our experiments. Isolated snails were on average significantly larger than paired in Exp. 1 Data accessibility: Analyses reported in this article can be reproduced using and 2, and grouped snails were smaller than the paired. One the data provided by Nakadera et al. (2019). potential explanation for this pattern is the different timing to Handling Editor: Per Smiseth start laying egg masses in the different treatments (Supplementary Figure S5). When their female function became mature, it is likely that paired and grouped snails allocated their resource to REFERENCES reproduction, rather than to somatic growth (Koene et al. 2008). 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