A targeted in situ hybridization screen identifies putative seminal fluid proteins in a simultaneously hermaphroditic flatworm

A targeted in situ hybridization screen identifies putative seminal fluid proteins in a... Background: Along with sperm, in many taxa ejaculates also contain large numbers of seminal fluid proteins (SFPs). SFPs and sperm are transferred to the mating partner, where they are thought to play key roles in mediating post-mating sexual selection. They modulate the partner’s behavior and physiology in ways that influence the reproductive success of both partners, thus potentially leading to sexual conflict. Despite the presumed general functional and evolutionary significance of SFPs, their identification and characterization has to date focused on just a few animal groups, predominantly insects and mammals. Moreover, until now seminal fluid profiling has mainly focused on species with separate sexes. Here we report a comprehensive screen for putative SFPs in the simultaneously hermaphroditic flatworm Macrostomum lignano. Results: Based on existing transcriptomic data, we selected 150 transcripts known to be (a) predominantly expressed in the tail region of the worms, where the seminal fluid-producing prostate gland cells are located, and (b) differentially expressed in social environments differing in sperm competition level, strongly implying that they represent a phenotypically plastic aspect of male reproductive allocation in this species. For these SFP candidates, we then performed whole-mount in situ hybridization (ISH) experiments to characterize tissue-specific expression. In total, we identified 98 transcripts that exhibited prostate-specific expression, 76 of which we found to be expressed exclusively in the prostate gland cells; additional sites of expression for the remaining 22 included the testis or other gland cells. Bioinformatics analyses of the prostate-limited candidates revealed that at least 64 are predicted to be secretory proteins, making these especially strong candidates to be SFPs that are transferred during copulation. Conclusions: Our study represents a first comprehensive analysis using a combination of transcriptomic and ISH screen data to identify SFPs based on transcript expression in seminal fluid-producing tissues. We thereby extend the range of taxa for which seminal fluid has been characterized to a flatworm species with a sequenced genome and for which several methods such as antibody staining, transgenesis and RNA interference have been established. Our data provide a basis for testing the functional and evolutionary significance of SFPs. Keywords: Seminal fluid, Flatworm, In situ hybridization, Prostate, Sex allocation, Sexual selection, Sperm competition, Sexual conflict, Allohormone * Correspondence: michael.weber1@uni-bielefeld.de Evolutionary Biology, Bielefeld University, Konsequenz 45, 33615 Bielefeld, Germany Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Weber et al. BMC Evolutionary Biology (2018) 18:81 Page 2 of 13 Background about putative functions of seminal fluid have been made, During ejaculate transfer, sperm cells are often accom- sincetheymight notonly affect thefemalesex function of panied by a complex mixture of additional substances – the mating partner (as in gonochorists), but also its male known collectively as seminal fluid – that is produced in function (with possible knock-on effects for the female the male accessory reproductive glands. Some of these function, if this induces changes in resource allocation to- components play important roles in nourishing and acti- wards the female function [48–50]). vating the sperm themselves [1], whereas others act – ei- Seminal fluid effects on the partner’smalefunctionin ther independently of, or in association with, sperm [2] simultaneous hermaphrodites are not just a theoretical – to influence subsequent female physiology and behav- possibility: in the only simultaneous hermaphrodite in iour in various ways, all of which can affect the outcome which seminal fluid has been studied in detail to date, the of sperm competition [3]. For example, specific seminal great pond snail Lymnaea stagnalis, effects on both the fluid proteins (SFPs) are known to modulate the recep- male and female functions have been observed. Several tivity of the female, reduce female attractiveness to fu- seminal fluid proteins were identified by HPLC and their ture potential mates, induce oviposition, change egg functions examined by injecting specific proteins intravag- laying rate, increase egg production, stimulate egg mat- inally [46, 51], revealing that seminal fluid receipt affects uration, change feeding behavior, and also increase fe- reproductive output in this species [46, 52–54]. More spe- male mortality rate [3–5]. Because all of these responses cifically, the receipt of one SFP (LyAcp10) had an effect to SFPs likely affect male reproductive success, SFPs are on egg laying [51]. The intravaginal injection of two other important targets of sexual selection, likely explaining SFPs (LyAcp8b and LyAcp5) resulted in a reduction of their rapid adaptive evolution [6–12]. Moreover, because sperm transferred in a subsequent mating by the recipient seminal fluid can modulate female physiology and repro- in L. stagnalis, and as a result, in a decrease in the pater- ductive behavior in so many ways – possibly to the advan- nity success in subsequent matings as a male [46]. This tage of the seminal fluid-donating individual (i.e. the male, study highlights that steering your partner away from its or more generally, the sperm donor), but not necessarily male function is a potentially adaptive strategy in simul- that of the seminal fluid-receiving individual (i.e. the fe- taneous hermaphrodites [48]. male, or more generally, the sperm recipient) – SFP de- Moreover, mating motivation in hermaphroditic indi- position is often expected to lead to sexual conflict [13], viduals is likely driven more by the opportunity it pro- an additional factor likely driving rapid SFP evolution [14]. vides to donate sperm to fertilize partners’ eggs rather In species with separate sexes, there are numerous than the opportunity to gain sperm to fertilize own eggs studies on seminal fluid composition and its functional [49, 55]. This means two simultaneous hermaphrodites effects. These mainly concern well-investigated species, will often agree on mating, even with the possible disad- mostly insects, including several Diptera [15–24], vari- vantage of receiving sperm (or of being unable to avoid ous Coleoptera [25–27], Orthoptera [8, 28], Lepidoptera receiving it because of reciprocal copulation). This fact, [29, 30], Hymenoptera [31, 32] and Hemiptera [33, 34]. and the expected scarcity of pre-mating sexual selection Besides insects, there are also studies on the [42, 56], leads to the assumption that there may be an characterization of seminal fluid in a few other taxa, most enhanced role for postcopulatory sexual selection in her- notably mammals, such as rodents [35, 36], livestock spe- maphrodites compared to gonochorists [55, 57]. To test cies [37–39] and humans [40, 41]. the generality of these insights, and those gained to date In simultaneous hermaphrodites (in which individuals in gonochorists, further hermaphroditic model systems possess both male and female sex functions at the same for studying seminal fluid-mediated postcopulatory ef- time), there are to date very few studies of seminal fluid. fects are clearly needed. One example in hermaphrodites for the transfer of In this study, we aimed to characterize seminal fluid in accessory gland substances is the shooting of so called a previously unstudied hermaphroditic group. Our study love darts in land snails [42, 43], where the accessory organism is the free-living flatworm Macrostomum lig- gland substances are transferred via hypodermic injection nano (Lophotrochozoa: Platyhelminthes: Rhabditophora) [44, 45]. But for seminal fluid proteins that are transferred , which has recently emerged as a model organism in together with the sperm in the ejaculate, the proteins have various other fields of biology [58, 59]. M. lignano is a partially been characterized only in a freshwater snail spe- simultaneous hermaphrodite with reciprocal copulation cies [46]. This scarcity is unfortunate, because simultan- (i.e., during mating, each partner both donates and re- eous hermaphroditism is a common reproductive mode ceives sperm and seminal fluid). This species has been throughout the animal kingdom [47], meaning that if we intensively studied in relation to sex allocation theory want to understand the effects of seminal fluid more [60–66]. Specifically, individuals can plastically allocate generally, we also need to understand them in hermaphro- their resources towards the male or the female sex func- dites [48]. Moreover, in hermaphrodites unique predictions tion [61, 65, 66] and we already know that they can Weber et al. BMC Evolutionary Biology (2018) 18:81 Page 3 of 13 plastically modify gene expression in several body regions, worms). In a second study, Marie-Orleach et al. [74] including their tail (the location of the SFP-producing sough to partition variance in reproductive success prostate glands cells, Fig. 1) in response to changes in so- via the male sex function into its component parts, find- cial group size (Ramm et al: Sex Allocation Plasticity on a ing that variance in male reproductive success mainly arises Transcriptome Scale: Socially-Sensitive Gene Expression from sperm transfer success and not from mating success. in the Hermaphroditic Flatworm Macrostomum lig- Besides the ability of individuals to have many sperm to nano, submitted), which reflects mating group size (i.e. transfer to their partner, sperm transfer success is expected the number of mating partners plus one) [61, 65, 67]. This to depend on the interactions between the ejaculates of result is expected according to the mating group size different donors and on the interactions between the ejacu- model [68, 69], which predicts that the optimal investment lates and the female reproductive tract [9, 75–77], both of in male function is low in the absence of sperm competi- which are potentially influenced by SFPs present in the tion but, as mating group size increases, sperm competi- ejaculate. Fertilization success could thus strongly depend tion drives the optimal male allocation up. upon the amount and composition of seminal fluid Until now, seminal fluid is not well studied in the genus transferred. Macrostomum, but recent results in M. lignanoboth hint Although the specific proteins found in the seminal at the potential for seminal fluid-mediated effects and pro- fluid of Macrostomum have not yet been investigated vide a basis on which to begin to identify individual sem- directly, two recent RNA-Seq datasets provide a basis inal fluid components. Regarding potential seminal fluid for investigating the seminal fluid composition of M. functions, Marie-Orleach et al. [70] found that individuals lignano. Firstly, Arbore et al. [78] examined differential mated to a virgin partner exhibit fewer instances of a so- gene expression in the head-, testis-, ovary- and tail-region called ‘suck behavior’, compared to individuals mated to a of the worms. Secondly, Ramm et al. (Ramm et al: Sex Allo- sexually experienced partner. This suck behavior is a post- cation Plasticity on a Transcriptome Scale: Socially- copulatory behavior that is thought to be involved in re- Sensitive Gene Expression in the Hermaphroditic Flatworm moving ejaculate components received during copulation Macrostomum lignano, submitted) characterized pheno- [71–73]. Marie-Orleach et al. [70] therefore concluded typic plasticity in M. lignano gene expression. Combining that seminal fluid could potentially inhibit the suck behav- information from these two studies, we here aimed to per- ior, based on the assumption that virgin individuals are form a comprehensive whole mount ISH screen (and asso- likely to transfer greater amounts of seminal fluid to their ciated bioinformatics analyses) of 150 transcripts that are mating partners during copulation (because they have both putatively tail-specific and exhibit plastic expression in more already-produced seminal fluid in storage prior to different social environments, making these strong candi- copulation, as measured by how visually prominent the dates for prostate-specific expression (for detailed descrip- prostate gland cells appear in vivo in these transparent tion of candidate selection see methods section). This Gut Testis Ovary Developing eggs bc Prostate gland cells Stylet Seminal vesicle Antrum Fig. 1 Schematic drawings and monoclonal antibody staining of specimens of Macrostomum lignano. a Habitus of M. lignano (ventral view); (b) Immunocytochemical staining of cytoplasmic content within the prostate glands with the mAB MPr-1 (the red dot is likely an auto fluorescence artifact); (c) posterior part of the animal with female genitalia (antrum) & male genitalia (comprising stylet, seminal vesicle and prostate gland cells, the latter two which can overlap) Weber et al. BMC Evolutionary Biology (2018) 18:81 Page 4 of 13 strategy enabled us to identify a set of 76 putative seminal 366 transcripts were identified as increasing substantially fluid transcripts. (defined as a log expression fold-change > 2) in expres- sion in the only fragment containing the tail of the Methods worm compared to the fragment containing the head-, Study organism testis- and ovary-regions. This expression pattern makes The free-living flatworm Macrostomum lignano is an these transcripts promising candidates to be specific to outcrossing simultaneous hermaphrodite found in the the posteriorly-located prostate gland cells (as well as Northern Adriatic Sea and the Eastern Mediterranean other tail-specific structures). Moreover, for three of [58, 79]. As adults, the worms reach 1.5 mm in body those transcripts, follow-up assays with whole mount in length and the paired male and female gonads lay along situ hybridization (ISH) and RNA interference (RNAi) the body axis on both sides of a central gut (Fig. 1). The confirmed that these were indeed specifically expressed male and female genital organs are located in the poster- in the prostate gland cells, but they did not show evident ior part of the worms, and the former comprises the RNAi phenotypes when observed in vivo [78]. However, seminal vesicle (where sperm are stored prior to ejacula- for one transcript, namely RNA815_80.4, a phenotypic tion), the prostate gland cells (where seminal fluid is effect of the RNAi treatment was evident in that the produced) and the copulatory stylet (penis) [80]. The knock-down worms no longer showed labeling with the transparency of the worms permits the observation of prostate-specific antibody MPr-1 [83]. This could indi- internal organs and processes in vivo [58]. The worms cate that RNA815_80.4 codes for protein containing the are kept in cultures in glass petri dishes filled with artifi- epitope of MPr-1 but, as Arbore et al. [78] already cial sea water (32‰) or nutrient-enriched artificial sea- pointed out, it is also possible that the knock-down dis- water (Guillard’s f/2 medium) [81] and fed with diatoms rupted transcripts that are expressed earlier in the same (Nitzschia curvilineata). They are kept under a 14:10 pathway ultimately resulting in the synthesis of the pro- light:dark cycle at 60% relative humidity and a constant tein containing the antibody’s epitope. temperature of 20 °C. All the animals used in this ex- The second RNA-Seq study by Ramm et al. (Ramm et periment were adult worms coming from stock cultures al: Sex Allocation Plasticity on a Transcriptome Scale: kept either at the University of Innsbruck or the Biele- Socially-Sensitive Gene Expression in the Hermaphro- feld University and originated from the highly inbred ditic Flatworm Macrostomum lignano, submitted) fur- DV1 line [65] that was also used to generate the pos- ther refined the available expression information by itional RNA-Seq data [78], the socially-sensitive RNA- characterizing phenotypic plasticity in M. lignano gene Seq data (Ramm et al: Sex Allocation Plasticity on a expression. They allocated worms to four different treat- Transcriptome Scale: Socially-Sensitive Gene Expression ment groups, each representing different social environ- in the Hermaphroditic Flatworm Macrostomum lignano, ments favouring different optimal sex allocation submitted), the ML2 genome assembly [59], and the re- patterns. They were then able to identify which tran- cently published Mlig_3_7 genome and Mlig_RNA_3_7_ scripts are differentially expressed between these social DV1 transcriptome assembly [82]. The sequencing reads environments (a total of ca. 10–20% of all transcripts). from both the positional and the socially-sensitive RNA- Of particular interest for the current study are those Seq data described above were mapped to the M. lig- transcripts that are both tail-specific based on the pos- nano de novo transcriptome assembly MLRNA110815 itional classification of Arbore et al. [78] (i.e. 366 tran- [78] (available online at http://www.macgenome.org/ scripts) and differentially expressed between the two download/MLRNA110815). most extreme social environments (worms kept isolated vs. in groups of eight) studied by Ramm et al. (Ramm et Selection of candidates al: Sex Allocation Plasticity on a Transcriptome Scale: The selection of candidates for the screening for putative Socially-Sensitive Gene Expression in the Hermaphro- seminal fluid proteins is based on two recent transcrip- ditic Flatworm Macrostomum lignano, submitted). These tomic datasets. Firstly, Arbore et al. [78] examined dif- selection criteria yielded 150 differentially expressed ferential gene expression in the head-, testis-, ovary- and transcripts (i.e. 41% of the 366 transcripts), 140 of which tail-region of the worms using a ‘positional’ RNA-Seq exhibited significantly higher expression in octets than approach. To do so, they cut worms into fragments con- isolated individuals, with the remaining 10 showing sig- taining either (a) only the head-region, (b) the head- and nificantly lower expression. The former is exactly the testis-regions, (c) the head-, testis- and ovary-regions, or kind of phenotypically plastic expression pattern ex- (d) the whole worm (containing all regions).. The tran- pected if these transcripts represent an aspect of male scripts were then assigned, according to differences in allocation in this species, making them the most obvious their expression pattern between fragments, to putative candidates for prostate-specific components of seminal tissue-specific classes. Of most relevance here, a total of fluid. Indeed, all three prostate-specific transcripts found Weber et al. BMC Evolutionary Biology (2018) 18:81 Page 5 of 13 by Arbore et al. [78] were also found to be differentially according to Lengerer et al. [86], using ca. fifteen adult expressed in worms from different social group sizes in animals in each reaction. The signal was developed at 37 ° the study of Ramm et al. (Ramm et al: Sex Allocation C using the NBT/BCIP system (Roche). Processed speci- Plasticity on a Transcriptome Scale: Socially-Sensitive mens were mounted in Gelvatol medium, for bright field or Gene Expression in the Hermaphroditic Flatworm differential interference contrast visualization. Specimens Macrostomum lignano, submitted). However, besides the were examined with a Leica DM5000 or an Olympus prostate gland cells, the tail region is also the location of BX50. Images were taken with a Leica DFC495 digital cam- the developing eggs, the adhesive organs and the female era and Leica LAS software or a Canon EOS 600D digital and other male reproductive organs (seminal vesicle, camera and Zoom Browser EX version 6.9.0a software. copulatory stylet). In order to identify which of the dif- ferentially expressed tail-specific candidates are truly pu- Bioinformatics analysis tative seminal fluid components, we therefore needed to For downstream bioinformatics analyses, we focused on refine our picture of their expression by ascertaining only those transcripts that we found to be exclusively which are specifically and uniquely expressed in the expressed in the prostate gland cells – i.e. prostate- prostate gland cells. limited transcripts. This is a subset (n = 76, see Results) of all prostate-specific transcripts, the latter also includ- Experimental rationale ing transcripts that besides expression in the prostate The identification of the putative seminal fluid proteins is gland cells also exhibited expression in additional tissues based on performing an ISH screen of selected transcripts. located elsewhere. Gene Ontology (GO) categorization The transcripts were selected by combining information of the functional annotations of the top BLASTx hits (E- from both the ‘positional’ [78]and ‘social’ (Ramm et al: value cutoff = 1e-3) was performed using the program Sex Allocation Plasticity on a Transcriptome Scale: Blast2GO [87, 88]. Gene ontology enrichment analysis Socially-Sensitive Gene Expression in the Hermaphroditic was performed with Blast2GO mapping to determine Flatworm Macrostomum lignano, submitted) RNA-Seq protein functions in biological processes. data described in the previous section. Specifically, our For all 76 transcripts, all six putative open reading screening effort was targeted at the 140 putative tail- frames beginning with a start codon were generated in specific transcripts that show a significantly higher expres- ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf. sion in octets compared to isolated individuals, as well as html) and all resulting amino acid sequences were the 10 that show lower expression. blasted using the Blastp Nucleotide selection (nr/nt) database at the National Center for Biotechnology Infor- Whole-mount in situ hybridization screening mation (NCBI). Additionally, the reading frame of the nu- Of the 150 differentially expressed and tail-specific tran- cleotide sequences was identified via the Blastx Non- scripts mentioned in the previous section, 146 were se- redundant protein sequences (nr) database. The right ORF lected for the ISH screen. Four transcripts were was identified, in some cases by blasting against the M. excluded because of their low read number (< 30) in the lignano genome [59], and the translated amino acid se- differential expression analysis (Ramm et al: Sex Alloca- quences were then used to test for the presence of a pre- tion Plasticity on a Transcriptome Scale: Socially- dicted secretory signal peptide with SignalP v4.1 [89]orfor Sensitive Gene Expression in the Hermaphroditic Flat- indicators of non-classical secretion inferred via Secreto- worm Macrostomum lignano, submitted). Forward and meP v2.0 [90]. Additionally, the localization of the protein T7-reverse primer pairs were designed for each candi- was predicted with the program ProtComp v9.0 [91, 92] date transcript with Primer3 [84, 85] to obtain an opti- and we checked for a predicted transmembrane helix with mal probe length of about 700 bp. cDNA was the program TMHMM v2.0 [93, 94]. synthesized from total RNA extracted from 50 to 250 adult worms from a mass culture, using the peqGOLD Results cDNA Synthesis Kit H Plus (Peqlab). For probe synthe- Identification of transcripts with prostate-specific sis, the cDNA was amplified with the specific primer expression pairs for the transcript (PCR conditions: 95 °C 5 min, Our goal was to screen the candidate transcripts for the (95 °C 30 s, between 55 °C and 62 °C 30 s, 72 °C 1 min prostate-limited expression expected of seminal fluid 30s) × 32, 72 °C 7 min). After quality and size check by proteins. We performed ISH for 146 transcripts, to as- gel electrophoresis, the PCR products were purified with certain the site(s) of their expression. Overall, 76 of the the Wizard SV Gel and PCR Clean-Up System Kit (Pro- 146 tested transcripts show a prostate-limited expression mega) and the purified products were used to synthesize (Fig. 2a, and Additional file 1: Figure S1), indicating that single stranded anti-sense DIG-labeled RNA probes with more than half of our transcripts encode potential sem- the DIG RNA Labelling Kit (Roche). ISH was performed inal fluid proteins. A further 22 transcripts exhibit Weber et al. BMC Evolutionary Biology (2018) 18:81 Page 6 of 13 cd Fig. 2 Representative whole-mount in situ hybridization expression patterns found for transcripts in Macrostomum lignano that are both phenotypically plastic (sensu ‘social’ RNA-Seq) and tail-limited (sensu ‘positional’ RNA-Seq). Of the 146 transcripts investigated, we obtained tissue-specific expression patterns (visualized by ISH) that can be divided into three categories, namely (a) prostate-limited expression; (b) prostate-specific expression coupled with tissue-specific expression elsewhere in the worm; and (c) tissue-specific expression that did not include the prostate (note that the expression pattern of 10 transcripts could not be established). Note that the classification of the observed expression pattern is based on numerous images of multiple specimens per transcript and not only on the single image shown in this figure (single images can show a somewhat misleading expression pattern because of an overstaining of the specimens or an incomplete discolourisation after the staining process). The ISH patterns depicted here in the main text are for the most highly expressed transcripts in each category; a complete catalogue of ISH images for all 136 transcripts for which we obtained a tissue-specific expression pattern is given in Additional file 1: Figure S1-S3 and the total number of transcripts in each category are given in (d) specific expression in the prostate gland cells, but are With this information on tissue-specificity at hand, we also expressed in other tissues (Fig. 2b, and Additional can now revisit the gene expression data that was used to file 1: Figure S2), with 50% also exhibiting expression in identify candidates. Doing so reveals a striking pattern: as the gonads (mostly testis, but also ovary, or both) and expected, phenotypically plastic expression represents a 15% in the pharyngeal glands. Additionally, 38 tran- signature of prostate-specificity, confirming our original scripts are not expressed in the prostate (Fig. 2c, and rationale. It is indeed those genes which showed the most Additional file 1: Figure S3); the expression pattern of plastic expression in the social RNA-Seq study (Ramm et the remaining 10 transcripts remains unknown, since no al: Sex Allocation Plasticity on a Transcriptome Scale: pattern could be found with ISH or it was not possible Socially-Sensitive Gene Expression in the Hermaphroditic to amplify these transcripts by PCR and therefore a hin- Flatworm Macrostomum lignano, submitted) that were drance for probe synthesis and subsequent ISH assays. most likely to be identified as prostate-limited or prostate- Overall, 67% of the tested transcripts therefore show ei- specific in our ISH screen (Fig. 3a). Based on this strong ther expression in the prostate exclusively or in the pros- agreement between the independently derived RNA-Seq tate and other tissue combined (Fig. 2d). We note here and ISH gene expression data, we therefore consider this that three of the ‘non-prostate’ transcripts (815_10124. subset of 76 transcripts with plastic, prostate-limited ex- 3, 815_35136 and 815_97.1) were found to have spe- pression as putative prostate-specific transcripts, naming cific expression in the adhesive glands, as do several them Mlig-pro1 through Mlig-pro76 (i.e. Macrostomum lig- of the tail-specific transcripts that are not plastically nano prostate transcripts 1–76), and numbering them in expressed in different social environments, as reported descending order of their overall expression level in octets elsewhere [95]. in the social RNA-Seq study (Ramm et al: Sex Allocation Weber et al. BMC Evolutionary Biology (2018) 18:81 Page 7 of 13 secretion SignalP SecretomeP SignalP ab expression (letters) c SecretomeP no signal yes no signal ProtComp fold-change (symbols) peptide peptide TMHMM 010 20 30 40 60 transcript candidate 25% 1 pr 9549.4 Mlig-pro39 pr,he 58.5% 41.5% 3730 59.4% 29683.1 pr Mlig-pro38 15.6% pr,go 1884.2 pr,go non- pr 9549.3 Mlig-pro7 classically 19868.1 pr Mlig-pro34 secreted pr 324.1 Mlig-pro14 pr 16008.2 Mlig-pro46 pr 9549.1 Mlig-pro8 de 38600.1 pr Mlig-pro49 ProtComp TMHMM te,ov other pr 14562 Mlig-pro42 outside inside plasma pr 15432.3 Mlig-pro40 7.7% membrane 18269 pr n/a Mlig-pro10 14% 10.8% pr 29684.1 Mlig-pro54 trans- pr 39625 Mlig-pro60 membrane 17% pr 80.4 Mlig-pro5 69% 16.9% pr 42719 Mlig-pro58 64.6% membrane- pr 16008.1 Mlig-pro37 extra- bound pr 21027 Mlig-pro22 cellular extracellular pr 9549.2 Mlig-pro12 (secreted) (secreted) pr 19361 Mlig-pro28 pr 15432.1 Mlig-pro19 pr 64228 n/a Mlig-pro69 2520.1 pr Mlig-pro20 7102.2 pr Mlig-pro1 go pr 14437.1 Mlig-pro24 SignalP expression (letters) 8281.1 go SecretomeP pr 14437.2 Mlig-pro23 ProtComp fold-change (symbols) pr 55723 Mlig-pro47 TMHMM pr 0 10203040 60 15432.2 Mlig-pro11 transcript mi pr 91 pr 32421 Mlig-pro53 7654.2 Mlig-pro50 pr 14220 Mlig-pro25 5875.2 pr,pg 19192 pr Mlig-pro6 30011 pr,ov pr pr 17071.2 Mlig-pro26 43041 Mlig-pro55 pr 41274 Mlig-pro35 34875 pr 15018.1 Mlig-pro4 59989 go 37400.2 pr Mlig-pro56 26811 go pr pr,he 18589.2 Mlig-pro65 324.3 pr pr pr 22046 Mlig-pro27 19312 n/a Mlig-pro33 18334 pr,go 7654.3 pr pr Mlig-pro62 5875.1 pr,te,ph 34551 pr pr,te 7102.3 Mlig-pro29 7654.1 dg go 10124.2 14954 23192.1 pr,pg 44529 pr,te pr pr n/a 7102.1 Mlig-pro36 57778 Mlig-pro75 pr go 11128.1 Mlig-pro13 20725 pr pr 39357 1884.2 Mlig-pro63 7258.5 Mlig-pro71 18395 1884.2 pr Mlig-pro44 43684 pr Mlig-pro76 pr 50706 te 30276 1884.2 Mlig-pro30 pr 22209 Mlig-pro2 24223 pr,te pr,te 51598 16433 18589.1 pr Mlig-pro52 10703.1 pr n/a Mlig-pro57 gu go 16678 7293.1 pr pr 17183 Mlig-pro9 57177 n/a Mlig-pro74 17219 pr,go 35586 rh 37400.1 pr,te 27832 an,go pr go,cg,gu 29684.2 Mlig-pro48 35153 pr rh 20163.1 Mlig-pro3 39096 41882 pr n/a Mlig-pro66 26191 pr cg,ov 37649 Mlig-pro67 8185 pr pr,an 2520.2 n/a Mlig-pro72 4584.2 pr go 11100.2 Mlig-pro32 19007 34081 pr Mlig-pro68 4584.1 pr,an pr,go,gu an,cg 35806 32122 pr 11100.1 Mlig-pro21 17241 pr 12565 n/a Mlig-pro18 16384 st 17071.1 pr Mlig-pro16 21876 cg pr un 38600.2 Mlig-pro59 35260 pr,pg st 11961 10687 pr an 8447 Mlig-pro31 24264 pr Mlig-pro61 35136 ad,gu pr an 35075 n/a Mlig-pro45 11627 pr an,ph,go 9549.5 Mlig-pro41 967.1 pr,go cg 29683.2 21837 pr Mlig-pro73 an 9549.6 5110.2 pr,te,ph an 5404.3 3346.1 32626.1 39099 pr ad 50917 n/a Mlig-pro64 97.1 pr ov,eg 45459 Mlig-pro15 2813.4 pr 26553 Mlig-pro51 2550 pr,ov,eg,te an 23759 345.1 pr Mlig-pro43 317.1 ad 5404.1 pr 16008.3 Mlig-pro70 56215 un 63864 716.3 go,pg ad 52031 16605 90 pr 146 62474 cg 2976 Mlig-pro17 Fig. 3 (See legend on next page.) Weber et al. BMC Evolutionary Biology (2018) 18:81 Page 8 of 13 (See figure on previous page.) Fig. 3 Summary of the expression patterns (visually identified by ISH) for all 146 tail-specific transcripts investigated and secretion predictions for those 76 with prostate-limited expression. In (a) the transcripts are listed in descending order of their fold-change (= solid vertical bar) observed in an earlier study comparing transcript expression in octets (groups of eight worms) compared to isolated worms (Ramm et al: Sex Allocation Plasticity on a Transcriptome Scale: Socially-Sensitive Gene Expression in the Hermaphroditic Flatworm Macrostomum lignano, submitted). Note that the 10 transcripts at the end of the list have fold-change values less than one (i.e. they had lower expression in octets than in isolated worms). For each transcript, we document its site of expression, indicated by the colour of the bar (green: prostate-limited; blue: prostate + elsewhere; orange: not prostate; grey: unknown) and by the abbreviated tissue assignments (pr: prostate; he: head; te: testis; ov: ovary; go: gonad; mi: unspecific in middle section; ph: pharynx; pg: pharyngeal glands; gu: gut; eg: egg; rh: rhabdites; an: antrum; cg: cement glands; st: stylet; un: unspecific; ad: adhesive glands). In addition, for each of the 76 prostate-limited transcripts we assign a unique number (Mlig-pro1–76, see main text for explanation) and sought bioinformatic evidence for secretion. For the 50 transcripts for which we could perform bioinformatic analyses, we indicate positive evidence for secretion based on predictions from SignalP (black circle: signal peptide present); SecretomeP (black circle: non-classically secreted); ProtComp (black circle: extra-cellularly secreted or membrane-bound extracellularly secreted; grey circle: plasma membrane); and TMHMM (black circle: outside the cell; grey circle: trans-membrane helix). Note that for 11 transcripts, it was not possible to identify the open reading frame and bioinformatics analyses could therefore not be performed (denoted ‘n/a’). The results from each of these analyses are summarized in panels (b-e) Plasticity on a Transcriptome Scale: Socially-Sensitive Gene SecretomeP analysis. So in total, 48 of the analyzed 65 Expression in the Hermaphroditic Flatworm Macrostomum prostate-limited proteins (74%) show evidence for secre- lignano, submitted) (Fig. 3a). tion, making these especially strong candidates as puta- tive seminal fluid proteins transferred during mating. Prediction of protein secretion and localization However, we note that this could be an underestimate, be- To predict whether or not the 76 identified transcripts cause for some proteins it is possible that a signal peptide that exhibited prostate-limited expression in our ISH was not detected, not because it is truly absent, but be- screen are likely to code for seminal fluid proteins that cause we do not know the full protein sequence. We note, are transferred to the mating partner – or alternatively however, that it is also possible that we currently overesti- for prostate-limited proteins that are not secretory, pre- mate the total number of SFPs, because some of the tran- sumably meaning they are some part of the seminal fluid script fragments in the current transcriptome assembly production machinery within the prostate gland cells – could ultimately prove to belong to the same protein. we sought evidence for secretion and final location using The predicted protein location was analyzed by Prot- bioinformatics tools. Specifically, the translated amino Comp to find candidates that are localized as ‘extracellu- acid sequences of each SFP candidate transcript were an- lar’ and/or have a ‘plasma membrane’ destination. Of the alyzed using SignalP to detect predicted signal peptides candidates, 42 are predicted to be extracellularly se- associated with classical secretion [89]. Additionally, creted, 11 are membrane-bound extracellular and 7 are candidate transcripts were analyzed with SecretomeP to a part of the plasma membrane (Fig. 3a, d). detect other motifs for secretion that are associated with Since the signal peptide can be recognized as a mem- non-classical secretory pathways [96]. Because we expect brane helix [97], TMHMM searches were also performed that seminal fluid proteins tend to be extracellularly se- to predict the cellular location of the protein as being ei- creted proteins, they can be expected to have a signal ther outside the cell, inside the cell or trans-membrane. peptide [6, 28]. Eleven of the candidates had a membrane helix, nine In total, we could predict the putative ORF for 65 of were predicted to be located inside of the membrane the 76 candidate transcripts and translated it into the and 45 to be outside (Fig. 3e). amino acid sequence. All of these 65 translated tran- Summing up these different bioinformatics analyses, scripts start with a start codon and, except for 6 candi- we posit that candidates are likely to be seminal fluid dates, stop at a stop codon. For the remaining 11 proteins if they show at least one of the following char- transcripts, it was not possible to identify the ORF. acteristics: (a) a predicted signal peptide inferred via Sig- According to analysis using SignalP, 38 of the 65 iden- nalP or a motif associated with a non-classical secretory tified proteins with prostate-limited expression were pre- pathway inferred from SecretomeP; (b) localization as dicted to be secreted by the signal peptide associated extracellular and/or with plasma membrane destination with the classical secretory pathway (Fig. 3a, b). Of the inferred via ProtComp; or (c) the recognition of a mem- 27 remaining proteins without such a signal peptide, 10 brane helix inferred via TMHMM. Almost all candidates were predicted to be secreted via a non-classical pathway analysed fulfil at least one of these criteria: a total of 64 according to analysis using SecretomeP (Fig. 3a, c). Note candidates (of 65 analysed) therefore currently are our that one of the transcripts had to be excluded from that top candidates to be seminal fluid proteins, though we analysis because its translated sequence was shorter than note that the transcripts that were not analyzed, be it be- the minimally required 40 amino acids needed for a cause they were shorter than 40AA or because we could Weber et al. BMC Evolutionary Biology (2018) 18:81 Page 9 of 13 not identify the ORF, should also not be excluded as pu- important roles in seminal fluid production. By far the most tative candidates. prevalent ‘mixed’ expression pattern was for expression in theprostateand thetestis, suggesting afunctionspecificto Gene ontology classification themalesex function,thoughthe precise significance of this To characterize the likely functions of our seminal fluid co-expression remains to be determined. In fact, we cannot candidates, we obtained Gene Ontology (GO) classifica- definitely exclude that transcripts with such a mixed expres- tions for the 76 transcripts with prostate-limited expression sion in the prostate gland cells and the testis are also trans- in three ontology domains: cellular component, molecular ferred SFPs, because there is the possibility that these function and biological process. It is important to note that proteins pass from the testis through the vas deferens to- a transcript could be included in several different categories gether with the sperm. and be associated with multiple GO annotations within a Several of the transcripts had no identifiable homo- single category. This therefore results in more GO- logues in Blast searches and had also no conserved protein annotations than sequences annotated. Blast2GO revealed domains, indicating that these proteins are likely highly di- that 31 transcripts had no Blast hits against the non- verged compared to previously characterized proteins. redundant protein NCBI database. Of the remaining 45 This is perhaps due both to the relatively distant phylo- (59%) transcripts that had a Blast hit, we overall could genetic position of our study organism with respect to assign GO annotations to 44 transcripts. An overview of previously studied taxa, and/or because the proteins the distribution of the sequences in the three ontology themselves are rapidly evolving. Again, our data are con- domains can be seen in Fig. 4. In the cellular component sistent with the fact that many SFP sequences are thought category (Fig. 4a), there is a clear predominance of extracel- to evolve rapidly due to the fact that they are a target of lular region parts and cell parts compared to intracellular (sexual) selection [6–11, 14] and sexual conflict [13, 42]. regions. Regarding the molecular function classification Many previous studies seeking to identify seminal (Fig. 4b), the main functions are involved in binding, cata- fluid proteins have used proteomics approaches (e.g. lytic activity, transferase activity, hydrolase activity, receptor [17, 35, 36, 98–102]), but because of the small size of M. binding and protein kinase activity. In the biological process lignano, collecting proteins from ejaculates is currently category (Fig. 4c), the prevailing groups are associated with challenging. To overcome this limitation, we employed an metabolic processes, single-organism cellular processes and alternative approach to identify putative SFPs. Specifically, multicellular organism development. With a total number we prioritized transcripts as seminal fluid protein candi- of 6 each, the most often identified proteins were fungistatic dates based on three assumptions, namely (i) that they are metabolites or transmembrane receptors (For a more de- more highly expressed in larger groups, reflecting a tran- tailed description of the sequences and the BLAST hits see scriptional upregulation in response to the high level of Additional file 2:Table S1). sperm competition experienced at larger group size com- pared to the non-mating environment when isolated (a Discussion phenotypically plastic response that has been demon- Using a combination of transcriptomic data (Ramm et al: strated also in other taxa [103–105]; (ii) that they show an Sex Allocation Plasticity on a Transcriptome Scale: Socially- expression limited to the prostate glands; and (iii) that Sensitive Gene Expression in the Hermaphroditic Flatworm they exhibit positive evidence of being secreted. Of the 65 Macrostomum lignano,submitted)[78]and ISHexperi- prostate-limited transcripts investigated for signs of secre- ments (present study), we are able to identify 76 transcripts tion and their location in the cell, 74% were predicted to in M. lignano which show specific expression that is limited be secreted either via a signal pathway or by a non- to the prostate, as expected for SFPs. The identification of a classical secretory pathway. This is a much higher percent- large number of SFP candidates is in line with similarly large age than would be predicted for the whole proteome – in numbers of identified proteins in other species studied to humans, for example, only between 10 and 20% of all pro- date (e.g. 198 SFPs in Aedes albopictus [24], more than 200 teins are secreted [106] – but because we were looking for in Drosophila melanogaster [13], 69 in Mus domesticus [36] proteins that are part of the ejaculate and therefore are and several hundred in humans [40, 41]), supporting the no- transferred to the mating partner, this is in line with our tion that seminal fluid is a complex and diverse secretion [3, expectations. This result is also in concordance with the 35]. We consider it less likely that the 22 transcripts that are fact that 69% of the proteins we found by TMHMM ana- expressed in the prostate and additionally in other tissues lysis were located outside of the cell and that 82% of the are potential seminal fluid candidates, because we expect proteins were predicted to be either extracellularly seminal fluid proteins to be exclusively expressed in the secreted or to be extracellularly membrane-bound. In prostate (or in other accessory male reproductive organs, de- total, 64 of the 65 candidates fulfill our criteria for secre- pending on the taxon). Nevertheless, these are also of inter- tion (secretion, transmembrane, part of plasma membrane est in the current context, because they could play or extracellularly located), providing strong indirect Weber et al. BMC Evolutionary Biology (2018) 18:81 Page 10 of 13 number of transcripts 01234 56789 10 cellular component extracellular region part cell part membrane cytoplasm membrane-bounded organelle cell surface intracellular membrane-bounded organelle vesicle extracellular space integral components of membrane nuclear part number of transcripts 01234 56789 10 molecular function binding catalytic activity transferase activity hydrolase activity receptor binding protein kinase activity ion binding protein binding kinase activity metal ion binding DNA binding carbohydrate derivative binding oxidoreductase activity UDP-glycosyltransferase activity number of transcripts 01234 56789 10 biological process metabolic process single-organism cellular process multicellular organism development phosphate-containing compound metabolic process signal transduction protein phosphorylation regulation of cellular process response to stimulus localization cellular process negative regulation of cellular process cell differentiation protein metabolic process cellular component organization single-organism process single-multicellular organism process chondroitin sulfate proteoglycan biosynthetic process animal organ development heparan sulfate proteoglycan metabolic process system development activation of immune response immune system process transposition, DNA-mediated developmental process chondroitin sulfate metabolic process DNA integration transport lateral line development anatomical structure morphogenesis Fig. 4 Summary of Gene Ontology (GO) analysis using Blast2GO of 76 prostate-limited transcripts. a GO term distribution for the cellular component domain; (b) GO term distribution for the molecular function domain; and (c) GO term distribution for the biological processes domain support for their assignment as putative SFPs and a clear section, this conclusion comes with one important pro- vindication of our identification strategy. viso, namely that there is also potentially some redun- We note also that this list of putative SFPs is likely to be dancy in our candidate list, in that some of the different an underestimate, since 11 transcripts with confirmed transcript fragments tested could in fact belong to the prostate-limited expression could not be analyzed, due to same protein or represent different protein isoforms pro- the absence of conserved protein domains and homology duced by alternative splicing. Especially the transcripts by Blast search. As already mentioned in the Results with the same main number that end with different sub- Weber et al. BMC Evolutionary Biology (2018) 18:81 Page 11 of 13 numbers (e.g. 29684.1 and 29684.2) are likely to belong to three categories, namely (Figure S1.) prostate-limited expression; the same protein or different isoforms of that protein. (Figure S2.) prostate-specific expression coupled with tissue-specific expression elsewhere in the worm; and (Figure S3.) tissue-specific They show a high sequence similarity with each other and expression that did not include the prostate. The expression patterns when we blasted them against the M. lignano genome of 10 transcripts could not be established. Within each category, (ML2 assembly) [59] they always align to the same region pictures are arranged (from top-left to bottom-right) in descending order of fold-change in expression in octets versus isolated worms. within the same contig. One representative picture per transcript is included. (ZIP 5699 kb) To begin to characterize potential functions of the puta- Additional file 2: Table S1. Summary of top blast hits identified by tive seminal fluid proteins, we used Blast2Go to classify Blast2Go. (PDF 55 kb) them according to their predicted molecular functions, involvement in biological processes, and cellular compo- Abbreviations nents. However, the fact that many of the functional ISH: Whole-mount in situ hybridization; SFP: Seminal fluid protein categories are extremely broad (such as ‘binding’ or ‘cata- Acknowledgements lytic activity’), and that the same putative proteins can be We thank Athina Giannakara and Bahar Patlar for helpful comments on the assigned to several categories, makes interpreting these manuscript. The computational results presented have been achieved (in part) using the HPC infrastructure LEO and MACH of the University of Innsbruck. classifications far from straightforward. Nevertheless, the fact that in the cellular components classification the Funding biggest fraction belongs to the category “extracellular This work was supported by the German Research Foundation (DFG) grant RA 2468/1–1 to SAR; Austrian Science Fund (FWF) grant P25404-B25 to PL; Swiss region part” is in concordance with the findings just dis- National Science Foundation (SNSF) grants 31003A-127503 and 31003A-143732 cussed before that the majority is extracellularly secreted. to LS. The funding bodies had no role in the design of the study or collection, To move beyond these broad functional classifications, analysis, and interpretation of data or in writing the manuscript. we need to directly assess the roles of specific SFPs. The Availability of data and materials next steps are therefore to evaluate which of these SFP A collection of 3–5 ISH pictures for each transcript is available in the Dryad candidates are actually transferred to the mating partner repository doi:https://doi.org/10.5061/dryad.316c7h6. during insemination, and especially to elucidate what Authors’ contributions effect they have on the mating partner and the repro- MW performed in situ hybridization experiments, analysed results and ductive success of the sperm donor. Due to the availabil- drafted the manuscript together with SAR; BL, RP, JW, MR and PL performed ity of applying RNAi in Macrostomum it is possible to in situ hybridization experiments; LS and SAR conceived the study and analysed RNA-Seq data to generate candidates. All authors contributed to knock-down the expression of specific transcripts and to manuscript revisions, and approved the final manuscript. test for their specific functions in mating experiments (cf. [86, 107–110]). Moreover, due to the availability of a GFP- Ethics approval and consent to participate Not applicable. expressing line there is also the possibility to readily assign paternity following double-mating experiments. In this Competing interests way, we can examine the fitness consequences of knock- The authors declare that they have no competing interests. ing down SFP expression, to test directly for the ability of a donor worm, missing a specific SFP, to compete against Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in arival [65, 74, 111]. published maps and institutional affiliations. Author details Conclusions Evolutionary Biology, Bielefeld University, Konsequenz 45, 33615 Bielefeld, In summary, our study represents the first large-scale Germany. Institute of Zoology and Center of Molecular Biosciences screen to identify putative SFPs in a flatworm, identify- Innsbruck, University of Innsbruck, Technikerstr. 25, A-6020 Innsbruck, Austria. Evolutionary Biology, Zoological Institute, University of Basel, Vesalgasse 1, ing 76 transcripts in M. lignano with prostate-limited 4051 Basel, Switzerland. Current address: School of Natural and expression. Of these, at least 64 also exhibit evidence of Environmental Sciences, Ridley Building, Newcastle University, Newcastle being secreted and therefore of being transferred SFPs. upon Tyne, England NE1 7RU, UK. These putative SFPs are now exciting candidates for Received: 18 January 2018 Accepted: 30 April 2018 future genetic and behavioral studies to examine the function of this important class of proteins. References 1. Mann T, Lutwak-Mann C. Male reproductive function and semen. Additional files Andrologia. 1982;14:76. 2. Peng J, Chen S, Büsser S, Liu H, Honegger T, Kubli E. 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A targeted in situ hybridization screen identifies putative seminal fluid proteins in a simultaneously hermaphroditic flatworm

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Life Sciences; Evolutionary Biology; Animal Systematics/Taxonomy/Biogeography; Entomology; Genetics and Population Dynamics; Life Sciences, general
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Abstract

Background: Along with sperm, in many taxa ejaculates also contain large numbers of seminal fluid proteins (SFPs). SFPs and sperm are transferred to the mating partner, where they are thought to play key roles in mediating post-mating sexual selection. They modulate the partner’s behavior and physiology in ways that influence the reproductive success of both partners, thus potentially leading to sexual conflict. Despite the presumed general functional and evolutionary significance of SFPs, their identification and characterization has to date focused on just a few animal groups, predominantly insects and mammals. Moreover, until now seminal fluid profiling has mainly focused on species with separate sexes. Here we report a comprehensive screen for putative SFPs in the simultaneously hermaphroditic flatworm Macrostomum lignano. Results: Based on existing transcriptomic data, we selected 150 transcripts known to be (a) predominantly expressed in the tail region of the worms, where the seminal fluid-producing prostate gland cells are located, and (b) differentially expressed in social environments differing in sperm competition level, strongly implying that they represent a phenotypically plastic aspect of male reproductive allocation in this species. For these SFP candidates, we then performed whole-mount in situ hybridization (ISH) experiments to characterize tissue-specific expression. In total, we identified 98 transcripts that exhibited prostate-specific expression, 76 of which we found to be expressed exclusively in the prostate gland cells; additional sites of expression for the remaining 22 included the testis or other gland cells. Bioinformatics analyses of the prostate-limited candidates revealed that at least 64 are predicted to be secretory proteins, making these especially strong candidates to be SFPs that are transferred during copulation. Conclusions: Our study represents a first comprehensive analysis using a combination of transcriptomic and ISH screen data to identify SFPs based on transcript expression in seminal fluid-producing tissues. We thereby extend the range of taxa for which seminal fluid has been characterized to a flatworm species with a sequenced genome and for which several methods such as antibody staining, transgenesis and RNA interference have been established. Our data provide a basis for testing the functional and evolutionary significance of SFPs. Keywords: Seminal fluid, Flatworm, In situ hybridization, Prostate, Sex allocation, Sexual selection, Sperm competition, Sexual conflict, Allohormone * Correspondence: michael.weber1@uni-bielefeld.de Evolutionary Biology, Bielefeld University, Konsequenz 45, 33615 Bielefeld, Germany Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Weber et al. BMC Evolutionary Biology (2018) 18:81 Page 2 of 13 Background about putative functions of seminal fluid have been made, During ejaculate transfer, sperm cells are often accom- sincetheymight notonly affect thefemalesex function of panied by a complex mixture of additional substances – the mating partner (as in gonochorists), but also its male known collectively as seminal fluid – that is produced in function (with possible knock-on effects for the female the male accessory reproductive glands. Some of these function, if this induces changes in resource allocation to- components play important roles in nourishing and acti- wards the female function [48–50]). vating the sperm themselves [1], whereas others act – ei- Seminal fluid effects on the partner’smalefunctionin ther independently of, or in association with, sperm [2] simultaneous hermaphrodites are not just a theoretical – to influence subsequent female physiology and behav- possibility: in the only simultaneous hermaphrodite in iour in various ways, all of which can affect the outcome which seminal fluid has been studied in detail to date, the of sperm competition [3]. For example, specific seminal great pond snail Lymnaea stagnalis, effects on both the fluid proteins (SFPs) are known to modulate the recep- male and female functions have been observed. Several tivity of the female, reduce female attractiveness to fu- seminal fluid proteins were identified by HPLC and their ture potential mates, induce oviposition, change egg functions examined by injecting specific proteins intravag- laying rate, increase egg production, stimulate egg mat- inally [46, 51], revealing that seminal fluid receipt affects uration, change feeding behavior, and also increase fe- reproductive output in this species [46, 52–54]. More spe- male mortality rate [3–5]. Because all of these responses cifically, the receipt of one SFP (LyAcp10) had an effect to SFPs likely affect male reproductive success, SFPs are on egg laying [51]. The intravaginal injection of two other important targets of sexual selection, likely explaining SFPs (LyAcp8b and LyAcp5) resulted in a reduction of their rapid adaptive evolution [6–12]. Moreover, because sperm transferred in a subsequent mating by the recipient seminal fluid can modulate female physiology and repro- in L. stagnalis, and as a result, in a decrease in the pater- ductive behavior in so many ways – possibly to the advan- nity success in subsequent matings as a male [46]. This tage of the seminal fluid-donating individual (i.e. the male, study highlights that steering your partner away from its or more generally, the sperm donor), but not necessarily male function is a potentially adaptive strategy in simul- that of the seminal fluid-receiving individual (i.e. the fe- taneous hermaphrodites [48]. male, or more generally, the sperm recipient) – SFP de- Moreover, mating motivation in hermaphroditic indi- position is often expected to lead to sexual conflict [13], viduals is likely driven more by the opportunity it pro- an additional factor likely driving rapid SFP evolution [14]. vides to donate sperm to fertilize partners’ eggs rather In species with separate sexes, there are numerous than the opportunity to gain sperm to fertilize own eggs studies on seminal fluid composition and its functional [49, 55]. This means two simultaneous hermaphrodites effects. These mainly concern well-investigated species, will often agree on mating, even with the possible disad- mostly insects, including several Diptera [15–24], vari- vantage of receiving sperm (or of being unable to avoid ous Coleoptera [25–27], Orthoptera [8, 28], Lepidoptera receiving it because of reciprocal copulation). This fact, [29, 30], Hymenoptera [31, 32] and Hemiptera [33, 34]. and the expected scarcity of pre-mating sexual selection Besides insects, there are also studies on the [42, 56], leads to the assumption that there may be an characterization of seminal fluid in a few other taxa, most enhanced role for postcopulatory sexual selection in her- notably mammals, such as rodents [35, 36], livestock spe- maphrodites compared to gonochorists [55, 57]. To test cies [37–39] and humans [40, 41]. the generality of these insights, and those gained to date In simultaneous hermaphrodites (in which individuals in gonochorists, further hermaphroditic model systems possess both male and female sex functions at the same for studying seminal fluid-mediated postcopulatory ef- time), there are to date very few studies of seminal fluid. fects are clearly needed. One example in hermaphrodites for the transfer of In this study, we aimed to characterize seminal fluid in accessory gland substances is the shooting of so called a previously unstudied hermaphroditic group. Our study love darts in land snails [42, 43], where the accessory organism is the free-living flatworm Macrostomum lig- gland substances are transferred via hypodermic injection nano (Lophotrochozoa: Platyhelminthes: Rhabditophora) [44, 45]. But for seminal fluid proteins that are transferred , which has recently emerged as a model organism in together with the sperm in the ejaculate, the proteins have various other fields of biology [58, 59]. M. lignano is a partially been characterized only in a freshwater snail spe- simultaneous hermaphrodite with reciprocal copulation cies [46]. This scarcity is unfortunate, because simultan- (i.e., during mating, each partner both donates and re- eous hermaphroditism is a common reproductive mode ceives sperm and seminal fluid). This species has been throughout the animal kingdom [47], meaning that if we intensively studied in relation to sex allocation theory want to understand the effects of seminal fluid more [60–66]. Specifically, individuals can plastically allocate generally, we also need to understand them in hermaphro- their resources towards the male or the female sex func- dites [48]. Moreover, in hermaphrodites unique predictions tion [61, 65, 66] and we already know that they can Weber et al. BMC Evolutionary Biology (2018) 18:81 Page 3 of 13 plastically modify gene expression in several body regions, worms). In a second study, Marie-Orleach et al. [74] including their tail (the location of the SFP-producing sough to partition variance in reproductive success prostate glands cells, Fig. 1) in response to changes in so- via the male sex function into its component parts, find- cial group size (Ramm et al: Sex Allocation Plasticity on a ing that variance in male reproductive success mainly arises Transcriptome Scale: Socially-Sensitive Gene Expression from sperm transfer success and not from mating success. in the Hermaphroditic Flatworm Macrostomum lig- Besides the ability of individuals to have many sperm to nano, submitted), which reflects mating group size (i.e. transfer to their partner, sperm transfer success is expected the number of mating partners plus one) [61, 65, 67]. This to depend on the interactions between the ejaculates of result is expected according to the mating group size different donors and on the interactions between the ejacu- model [68, 69], which predicts that the optimal investment lates and the female reproductive tract [9, 75–77], both of in male function is low in the absence of sperm competi- which are potentially influenced by SFPs present in the tion but, as mating group size increases, sperm competi- ejaculate. Fertilization success could thus strongly depend tion drives the optimal male allocation up. upon the amount and composition of seminal fluid Until now, seminal fluid is not well studied in the genus transferred. Macrostomum, but recent results in M. lignanoboth hint Although the specific proteins found in the seminal at the potential for seminal fluid-mediated effects and pro- fluid of Macrostomum have not yet been investigated vide a basis on which to begin to identify individual sem- directly, two recent RNA-Seq datasets provide a basis inal fluid components. Regarding potential seminal fluid for investigating the seminal fluid composition of M. functions, Marie-Orleach et al. [70] found that individuals lignano. Firstly, Arbore et al. [78] examined differential mated to a virgin partner exhibit fewer instances of a so- gene expression in the head-, testis-, ovary- and tail-region called ‘suck behavior’, compared to individuals mated to a of the worms. Secondly, Ramm et al. (Ramm et al: Sex Allo- sexually experienced partner. This suck behavior is a post- cation Plasticity on a Transcriptome Scale: Socially- copulatory behavior that is thought to be involved in re- Sensitive Gene Expression in the Hermaphroditic Flatworm moving ejaculate components received during copulation Macrostomum lignano, submitted) characterized pheno- [71–73]. Marie-Orleach et al. [70] therefore concluded typic plasticity in M. lignano gene expression. Combining that seminal fluid could potentially inhibit the suck behav- information from these two studies, we here aimed to per- ior, based on the assumption that virgin individuals are form a comprehensive whole mount ISH screen (and asso- likely to transfer greater amounts of seminal fluid to their ciated bioinformatics analyses) of 150 transcripts that are mating partners during copulation (because they have both putatively tail-specific and exhibit plastic expression in more already-produced seminal fluid in storage prior to different social environments, making these strong candi- copulation, as measured by how visually prominent the dates for prostate-specific expression (for detailed descrip- prostate gland cells appear in vivo in these transparent tion of candidate selection see methods section). This Gut Testis Ovary Developing eggs bc Prostate gland cells Stylet Seminal vesicle Antrum Fig. 1 Schematic drawings and monoclonal antibody staining of specimens of Macrostomum lignano. a Habitus of M. lignano (ventral view); (b) Immunocytochemical staining of cytoplasmic content within the prostate glands with the mAB MPr-1 (the red dot is likely an auto fluorescence artifact); (c) posterior part of the animal with female genitalia (antrum) & male genitalia (comprising stylet, seminal vesicle and prostate gland cells, the latter two which can overlap) Weber et al. BMC Evolutionary Biology (2018) 18:81 Page 4 of 13 strategy enabled us to identify a set of 76 putative seminal 366 transcripts were identified as increasing substantially fluid transcripts. (defined as a log expression fold-change > 2) in expres- sion in the only fragment containing the tail of the Methods worm compared to the fragment containing the head-, Study organism testis- and ovary-regions. This expression pattern makes The free-living flatworm Macrostomum lignano is an these transcripts promising candidates to be specific to outcrossing simultaneous hermaphrodite found in the the posteriorly-located prostate gland cells (as well as Northern Adriatic Sea and the Eastern Mediterranean other tail-specific structures). Moreover, for three of [58, 79]. As adults, the worms reach 1.5 mm in body those transcripts, follow-up assays with whole mount in length and the paired male and female gonads lay along situ hybridization (ISH) and RNA interference (RNAi) the body axis on both sides of a central gut (Fig. 1). The confirmed that these were indeed specifically expressed male and female genital organs are located in the poster- in the prostate gland cells, but they did not show evident ior part of the worms, and the former comprises the RNAi phenotypes when observed in vivo [78]. However, seminal vesicle (where sperm are stored prior to ejacula- for one transcript, namely RNA815_80.4, a phenotypic tion), the prostate gland cells (where seminal fluid is effect of the RNAi treatment was evident in that the produced) and the copulatory stylet (penis) [80]. The knock-down worms no longer showed labeling with the transparency of the worms permits the observation of prostate-specific antibody MPr-1 [83]. This could indi- internal organs and processes in vivo [58]. The worms cate that RNA815_80.4 codes for protein containing the are kept in cultures in glass petri dishes filled with artifi- epitope of MPr-1 but, as Arbore et al. [78] already cial sea water (32‰) or nutrient-enriched artificial sea- pointed out, it is also possible that the knock-down dis- water (Guillard’s f/2 medium) [81] and fed with diatoms rupted transcripts that are expressed earlier in the same (Nitzschia curvilineata). They are kept under a 14:10 pathway ultimately resulting in the synthesis of the pro- light:dark cycle at 60% relative humidity and a constant tein containing the antibody’s epitope. temperature of 20 °C. All the animals used in this ex- The second RNA-Seq study by Ramm et al. (Ramm et periment were adult worms coming from stock cultures al: Sex Allocation Plasticity on a Transcriptome Scale: kept either at the University of Innsbruck or the Biele- Socially-Sensitive Gene Expression in the Hermaphro- feld University and originated from the highly inbred ditic Flatworm Macrostomum lignano, submitted) fur- DV1 line [65] that was also used to generate the pos- ther refined the available expression information by itional RNA-Seq data [78], the socially-sensitive RNA- characterizing phenotypic plasticity in M. lignano gene Seq data (Ramm et al: Sex Allocation Plasticity on a expression. They allocated worms to four different treat- Transcriptome Scale: Socially-Sensitive Gene Expression ment groups, each representing different social environ- in the Hermaphroditic Flatworm Macrostomum lignano, ments favouring different optimal sex allocation submitted), the ML2 genome assembly [59], and the re- patterns. They were then able to identify which tran- cently published Mlig_3_7 genome and Mlig_RNA_3_7_ scripts are differentially expressed between these social DV1 transcriptome assembly [82]. The sequencing reads environments (a total of ca. 10–20% of all transcripts). from both the positional and the socially-sensitive RNA- Of particular interest for the current study are those Seq data described above were mapped to the M. lig- transcripts that are both tail-specific based on the pos- nano de novo transcriptome assembly MLRNA110815 itional classification of Arbore et al. [78] (i.e. 366 tran- [78] (available online at http://www.macgenome.org/ scripts) and differentially expressed between the two download/MLRNA110815). most extreme social environments (worms kept isolated vs. in groups of eight) studied by Ramm et al. (Ramm et Selection of candidates al: Sex Allocation Plasticity on a Transcriptome Scale: The selection of candidates for the screening for putative Socially-Sensitive Gene Expression in the Hermaphro- seminal fluid proteins is based on two recent transcrip- ditic Flatworm Macrostomum lignano, submitted). These tomic datasets. Firstly, Arbore et al. [78] examined dif- selection criteria yielded 150 differentially expressed ferential gene expression in the head-, testis-, ovary- and transcripts (i.e. 41% of the 366 transcripts), 140 of which tail-region of the worms using a ‘positional’ RNA-Seq exhibited significantly higher expression in octets than approach. To do so, they cut worms into fragments con- isolated individuals, with the remaining 10 showing sig- taining either (a) only the head-region, (b) the head- and nificantly lower expression. The former is exactly the testis-regions, (c) the head-, testis- and ovary-regions, or kind of phenotypically plastic expression pattern ex- (d) the whole worm (containing all regions).. The tran- pected if these transcripts represent an aspect of male scripts were then assigned, according to differences in allocation in this species, making them the most obvious their expression pattern between fragments, to putative candidates for prostate-specific components of seminal tissue-specific classes. Of most relevance here, a total of fluid. Indeed, all three prostate-specific transcripts found Weber et al. BMC Evolutionary Biology (2018) 18:81 Page 5 of 13 by Arbore et al. [78] were also found to be differentially according to Lengerer et al. [86], using ca. fifteen adult expressed in worms from different social group sizes in animals in each reaction. The signal was developed at 37 ° the study of Ramm et al. (Ramm et al: Sex Allocation C using the NBT/BCIP system (Roche). Processed speci- Plasticity on a Transcriptome Scale: Socially-Sensitive mens were mounted in Gelvatol medium, for bright field or Gene Expression in the Hermaphroditic Flatworm differential interference contrast visualization. Specimens Macrostomum lignano, submitted). However, besides the were examined with a Leica DM5000 or an Olympus prostate gland cells, the tail region is also the location of BX50. Images were taken with a Leica DFC495 digital cam- the developing eggs, the adhesive organs and the female era and Leica LAS software or a Canon EOS 600D digital and other male reproductive organs (seminal vesicle, camera and Zoom Browser EX version 6.9.0a software. copulatory stylet). In order to identify which of the dif- ferentially expressed tail-specific candidates are truly pu- Bioinformatics analysis tative seminal fluid components, we therefore needed to For downstream bioinformatics analyses, we focused on refine our picture of their expression by ascertaining only those transcripts that we found to be exclusively which are specifically and uniquely expressed in the expressed in the prostate gland cells – i.e. prostate- prostate gland cells. limited transcripts. This is a subset (n = 76, see Results) of all prostate-specific transcripts, the latter also includ- Experimental rationale ing transcripts that besides expression in the prostate The identification of the putative seminal fluid proteins is gland cells also exhibited expression in additional tissues based on performing an ISH screen of selected transcripts. located elsewhere. Gene Ontology (GO) categorization The transcripts were selected by combining information of the functional annotations of the top BLASTx hits (E- from both the ‘positional’ [78]and ‘social’ (Ramm et al: value cutoff = 1e-3) was performed using the program Sex Allocation Plasticity on a Transcriptome Scale: Blast2GO [87, 88]. Gene ontology enrichment analysis Socially-Sensitive Gene Expression in the Hermaphroditic was performed with Blast2GO mapping to determine Flatworm Macrostomum lignano, submitted) RNA-Seq protein functions in biological processes. data described in the previous section. Specifically, our For all 76 transcripts, all six putative open reading screening effort was targeted at the 140 putative tail- frames beginning with a start codon were generated in specific transcripts that show a significantly higher expres- ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf. sion in octets compared to isolated individuals, as well as html) and all resulting amino acid sequences were the 10 that show lower expression. blasted using the Blastp Nucleotide selection (nr/nt) database at the National Center for Biotechnology Infor- Whole-mount in situ hybridization screening mation (NCBI). Additionally, the reading frame of the nu- Of the 150 differentially expressed and tail-specific tran- cleotide sequences was identified via the Blastx Non- scripts mentioned in the previous section, 146 were se- redundant protein sequences (nr) database. The right ORF lected for the ISH screen. Four transcripts were was identified, in some cases by blasting against the M. excluded because of their low read number (< 30) in the lignano genome [59], and the translated amino acid se- differential expression analysis (Ramm et al: Sex Alloca- quences were then used to test for the presence of a pre- tion Plasticity on a Transcriptome Scale: Socially- dicted secretory signal peptide with SignalP v4.1 [89]orfor Sensitive Gene Expression in the Hermaphroditic Flat- indicators of non-classical secretion inferred via Secreto- worm Macrostomum lignano, submitted). Forward and meP v2.0 [90]. Additionally, the localization of the protein T7-reverse primer pairs were designed for each candi- was predicted with the program ProtComp v9.0 [91, 92] date transcript with Primer3 [84, 85] to obtain an opti- and we checked for a predicted transmembrane helix with mal probe length of about 700 bp. cDNA was the program TMHMM v2.0 [93, 94]. synthesized from total RNA extracted from 50 to 250 adult worms from a mass culture, using the peqGOLD Results cDNA Synthesis Kit H Plus (Peqlab). For probe synthe- Identification of transcripts with prostate-specific sis, the cDNA was amplified with the specific primer expression pairs for the transcript (PCR conditions: 95 °C 5 min, Our goal was to screen the candidate transcripts for the (95 °C 30 s, between 55 °C and 62 °C 30 s, 72 °C 1 min prostate-limited expression expected of seminal fluid 30s) × 32, 72 °C 7 min). After quality and size check by proteins. We performed ISH for 146 transcripts, to as- gel electrophoresis, the PCR products were purified with certain the site(s) of their expression. Overall, 76 of the the Wizard SV Gel and PCR Clean-Up System Kit (Pro- 146 tested transcripts show a prostate-limited expression mega) and the purified products were used to synthesize (Fig. 2a, and Additional file 1: Figure S1), indicating that single stranded anti-sense DIG-labeled RNA probes with more than half of our transcripts encode potential sem- the DIG RNA Labelling Kit (Roche). ISH was performed inal fluid proteins. A further 22 transcripts exhibit Weber et al. BMC Evolutionary Biology (2018) 18:81 Page 6 of 13 cd Fig. 2 Representative whole-mount in situ hybridization expression patterns found for transcripts in Macrostomum lignano that are both phenotypically plastic (sensu ‘social’ RNA-Seq) and tail-limited (sensu ‘positional’ RNA-Seq). Of the 146 transcripts investigated, we obtained tissue-specific expression patterns (visualized by ISH) that can be divided into three categories, namely (a) prostate-limited expression; (b) prostate-specific expression coupled with tissue-specific expression elsewhere in the worm; and (c) tissue-specific expression that did not include the prostate (note that the expression pattern of 10 transcripts could not be established). Note that the classification of the observed expression pattern is based on numerous images of multiple specimens per transcript and not only on the single image shown in this figure (single images can show a somewhat misleading expression pattern because of an overstaining of the specimens or an incomplete discolourisation after the staining process). The ISH patterns depicted here in the main text are for the most highly expressed transcripts in each category; a complete catalogue of ISH images for all 136 transcripts for which we obtained a tissue-specific expression pattern is given in Additional file 1: Figure S1-S3 and the total number of transcripts in each category are given in (d) specific expression in the prostate gland cells, but are With this information on tissue-specificity at hand, we also expressed in other tissues (Fig. 2b, and Additional can now revisit the gene expression data that was used to file 1: Figure S2), with 50% also exhibiting expression in identify candidates. Doing so reveals a striking pattern: as the gonads (mostly testis, but also ovary, or both) and expected, phenotypically plastic expression represents a 15% in the pharyngeal glands. Additionally, 38 tran- signature of prostate-specificity, confirming our original scripts are not expressed in the prostate (Fig. 2c, and rationale. It is indeed those genes which showed the most Additional file 1: Figure S3); the expression pattern of plastic expression in the social RNA-Seq study (Ramm et the remaining 10 transcripts remains unknown, since no al: Sex Allocation Plasticity on a Transcriptome Scale: pattern could be found with ISH or it was not possible Socially-Sensitive Gene Expression in the Hermaphroditic to amplify these transcripts by PCR and therefore a hin- Flatworm Macrostomum lignano, submitted) that were drance for probe synthesis and subsequent ISH assays. most likely to be identified as prostate-limited or prostate- Overall, 67% of the tested transcripts therefore show ei- specific in our ISH screen (Fig. 3a). Based on this strong ther expression in the prostate exclusively or in the pros- agreement between the independently derived RNA-Seq tate and other tissue combined (Fig. 2d). We note here and ISH gene expression data, we therefore consider this that three of the ‘non-prostate’ transcripts (815_10124. subset of 76 transcripts with plastic, prostate-limited ex- 3, 815_35136 and 815_97.1) were found to have spe- pression as putative prostate-specific transcripts, naming cific expression in the adhesive glands, as do several them Mlig-pro1 through Mlig-pro76 (i.e. Macrostomum lig- of the tail-specific transcripts that are not plastically nano prostate transcripts 1–76), and numbering them in expressed in different social environments, as reported descending order of their overall expression level in octets elsewhere [95]. in the social RNA-Seq study (Ramm et al: Sex Allocation Weber et al. BMC Evolutionary Biology (2018) 18:81 Page 7 of 13 secretion SignalP SecretomeP SignalP ab expression (letters) c SecretomeP no signal yes no signal ProtComp fold-change (symbols) peptide peptide TMHMM 010 20 30 40 60 transcript candidate 25% 1 pr 9549.4 Mlig-pro39 pr,he 58.5% 41.5% 3730 59.4% 29683.1 pr Mlig-pro38 15.6% pr,go 1884.2 pr,go non- pr 9549.3 Mlig-pro7 classically 19868.1 pr Mlig-pro34 secreted pr 324.1 Mlig-pro14 pr 16008.2 Mlig-pro46 pr 9549.1 Mlig-pro8 de 38600.1 pr Mlig-pro49 ProtComp TMHMM te,ov other pr 14562 Mlig-pro42 outside inside plasma pr 15432.3 Mlig-pro40 7.7% membrane 18269 pr n/a Mlig-pro10 14% 10.8% pr 29684.1 Mlig-pro54 trans- pr 39625 Mlig-pro60 membrane 17% pr 80.4 Mlig-pro5 69% 16.9% pr 42719 Mlig-pro58 64.6% membrane- pr 16008.1 Mlig-pro37 extra- bound pr 21027 Mlig-pro22 cellular extracellular pr 9549.2 Mlig-pro12 (secreted) (secreted) pr 19361 Mlig-pro28 pr 15432.1 Mlig-pro19 pr 64228 n/a Mlig-pro69 2520.1 pr Mlig-pro20 7102.2 pr Mlig-pro1 go pr 14437.1 Mlig-pro24 SignalP expression (letters) 8281.1 go SecretomeP pr 14437.2 Mlig-pro23 ProtComp fold-change (symbols) pr 55723 Mlig-pro47 TMHMM pr 0 10203040 60 15432.2 Mlig-pro11 transcript mi pr 91 pr 32421 Mlig-pro53 7654.2 Mlig-pro50 pr 14220 Mlig-pro25 5875.2 pr,pg 19192 pr Mlig-pro6 30011 pr,ov pr pr 17071.2 Mlig-pro26 43041 Mlig-pro55 pr 41274 Mlig-pro35 34875 pr 15018.1 Mlig-pro4 59989 go 37400.2 pr Mlig-pro56 26811 go pr pr,he 18589.2 Mlig-pro65 324.3 pr pr pr 22046 Mlig-pro27 19312 n/a Mlig-pro33 18334 pr,go 7654.3 pr pr Mlig-pro62 5875.1 pr,te,ph 34551 pr pr,te 7102.3 Mlig-pro29 7654.1 dg go 10124.2 14954 23192.1 pr,pg 44529 pr,te pr pr n/a 7102.1 Mlig-pro36 57778 Mlig-pro75 pr go 11128.1 Mlig-pro13 20725 pr pr 39357 1884.2 Mlig-pro63 7258.5 Mlig-pro71 18395 1884.2 pr Mlig-pro44 43684 pr Mlig-pro76 pr 50706 te 30276 1884.2 Mlig-pro30 pr 22209 Mlig-pro2 24223 pr,te pr,te 51598 16433 18589.1 pr Mlig-pro52 10703.1 pr n/a Mlig-pro57 gu go 16678 7293.1 pr pr 17183 Mlig-pro9 57177 n/a Mlig-pro74 17219 pr,go 35586 rh 37400.1 pr,te 27832 an,go pr go,cg,gu 29684.2 Mlig-pro48 35153 pr rh 20163.1 Mlig-pro3 39096 41882 pr n/a Mlig-pro66 26191 pr cg,ov 37649 Mlig-pro67 8185 pr pr,an 2520.2 n/a Mlig-pro72 4584.2 pr go 11100.2 Mlig-pro32 19007 34081 pr Mlig-pro68 4584.1 pr,an pr,go,gu an,cg 35806 32122 pr 11100.1 Mlig-pro21 17241 pr 12565 n/a Mlig-pro18 16384 st 17071.1 pr Mlig-pro16 21876 cg pr un 38600.2 Mlig-pro59 35260 pr,pg st 11961 10687 pr an 8447 Mlig-pro31 24264 pr Mlig-pro61 35136 ad,gu pr an 35075 n/a Mlig-pro45 11627 pr an,ph,go 9549.5 Mlig-pro41 967.1 pr,go cg 29683.2 21837 pr Mlig-pro73 an 9549.6 5110.2 pr,te,ph an 5404.3 3346.1 32626.1 39099 pr ad 50917 n/a Mlig-pro64 97.1 pr ov,eg 45459 Mlig-pro15 2813.4 pr 26553 Mlig-pro51 2550 pr,ov,eg,te an 23759 345.1 pr Mlig-pro43 317.1 ad 5404.1 pr 16008.3 Mlig-pro70 56215 un 63864 716.3 go,pg ad 52031 16605 90 pr 146 62474 cg 2976 Mlig-pro17 Fig. 3 (See legend on next page.) Weber et al. BMC Evolutionary Biology (2018) 18:81 Page 8 of 13 (See figure on previous page.) Fig. 3 Summary of the expression patterns (visually identified by ISH) for all 146 tail-specific transcripts investigated and secretion predictions for those 76 with prostate-limited expression. In (a) the transcripts are listed in descending order of their fold-change (= solid vertical bar) observed in an earlier study comparing transcript expression in octets (groups of eight worms) compared to isolated worms (Ramm et al: Sex Allocation Plasticity on a Transcriptome Scale: Socially-Sensitive Gene Expression in the Hermaphroditic Flatworm Macrostomum lignano, submitted). Note that the 10 transcripts at the end of the list have fold-change values less than one (i.e. they had lower expression in octets than in isolated worms). For each transcript, we document its site of expression, indicated by the colour of the bar (green: prostate-limited; blue: prostate + elsewhere; orange: not prostate; grey: unknown) and by the abbreviated tissue assignments (pr: prostate; he: head; te: testis; ov: ovary; go: gonad; mi: unspecific in middle section; ph: pharynx; pg: pharyngeal glands; gu: gut; eg: egg; rh: rhabdites; an: antrum; cg: cement glands; st: stylet; un: unspecific; ad: adhesive glands). In addition, for each of the 76 prostate-limited transcripts we assign a unique number (Mlig-pro1–76, see main text for explanation) and sought bioinformatic evidence for secretion. For the 50 transcripts for which we could perform bioinformatic analyses, we indicate positive evidence for secretion based on predictions from SignalP (black circle: signal peptide present); SecretomeP (black circle: non-classically secreted); ProtComp (black circle: extra-cellularly secreted or membrane-bound extracellularly secreted; grey circle: plasma membrane); and TMHMM (black circle: outside the cell; grey circle: trans-membrane helix). Note that for 11 transcripts, it was not possible to identify the open reading frame and bioinformatics analyses could therefore not be performed (denoted ‘n/a’). The results from each of these analyses are summarized in panels (b-e) Plasticity on a Transcriptome Scale: Socially-Sensitive Gene SecretomeP analysis. So in total, 48 of the analyzed 65 Expression in the Hermaphroditic Flatworm Macrostomum prostate-limited proteins (74%) show evidence for secre- lignano, submitted) (Fig. 3a). tion, making these especially strong candidates as puta- tive seminal fluid proteins transferred during mating. Prediction of protein secretion and localization However, we note that this could be an underestimate, be- To predict whether or not the 76 identified transcripts cause for some proteins it is possible that a signal peptide that exhibited prostate-limited expression in our ISH was not detected, not because it is truly absent, but be- screen are likely to code for seminal fluid proteins that cause we do not know the full protein sequence. We note, are transferred to the mating partner – or alternatively however, that it is also possible that we currently overesti- for prostate-limited proteins that are not secretory, pre- mate the total number of SFPs, because some of the tran- sumably meaning they are some part of the seminal fluid script fragments in the current transcriptome assembly production machinery within the prostate gland cells – could ultimately prove to belong to the same protein. we sought evidence for secretion and final location using The predicted protein location was analyzed by Prot- bioinformatics tools. Specifically, the translated amino Comp to find candidates that are localized as ‘extracellu- acid sequences of each SFP candidate transcript were an- lar’ and/or have a ‘plasma membrane’ destination. Of the alyzed using SignalP to detect predicted signal peptides candidates, 42 are predicted to be extracellularly se- associated with classical secretion [89]. Additionally, creted, 11 are membrane-bound extracellular and 7 are candidate transcripts were analyzed with SecretomeP to a part of the plasma membrane (Fig. 3a, d). detect other motifs for secretion that are associated with Since the signal peptide can be recognized as a mem- non-classical secretory pathways [96]. Because we expect brane helix [97], TMHMM searches were also performed that seminal fluid proteins tend to be extracellularly se- to predict the cellular location of the protein as being ei- creted proteins, they can be expected to have a signal ther outside the cell, inside the cell or trans-membrane. peptide [6, 28]. Eleven of the candidates had a membrane helix, nine In total, we could predict the putative ORF for 65 of were predicted to be located inside of the membrane the 76 candidate transcripts and translated it into the and 45 to be outside (Fig. 3e). amino acid sequence. All of these 65 translated tran- Summing up these different bioinformatics analyses, scripts start with a start codon and, except for 6 candi- we posit that candidates are likely to be seminal fluid dates, stop at a stop codon. For the remaining 11 proteins if they show at least one of the following char- transcripts, it was not possible to identify the ORF. acteristics: (a) a predicted signal peptide inferred via Sig- According to analysis using SignalP, 38 of the 65 iden- nalP or a motif associated with a non-classical secretory tified proteins with prostate-limited expression were pre- pathway inferred from SecretomeP; (b) localization as dicted to be secreted by the signal peptide associated extracellular and/or with plasma membrane destination with the classical secretory pathway (Fig. 3a, b). Of the inferred via ProtComp; or (c) the recognition of a mem- 27 remaining proteins without such a signal peptide, 10 brane helix inferred via TMHMM. Almost all candidates were predicted to be secreted via a non-classical pathway analysed fulfil at least one of these criteria: a total of 64 according to analysis using SecretomeP (Fig. 3a, c). Note candidates (of 65 analysed) therefore currently are our that one of the transcripts had to be excluded from that top candidates to be seminal fluid proteins, though we analysis because its translated sequence was shorter than note that the transcripts that were not analyzed, be it be- the minimally required 40 amino acids needed for a cause they were shorter than 40AA or because we could Weber et al. BMC Evolutionary Biology (2018) 18:81 Page 9 of 13 not identify the ORF, should also not be excluded as pu- important roles in seminal fluid production. By far the most tative candidates. prevalent ‘mixed’ expression pattern was for expression in theprostateand thetestis, suggesting afunctionspecificto Gene ontology classification themalesex function,thoughthe precise significance of this To characterize the likely functions of our seminal fluid co-expression remains to be determined. In fact, we cannot candidates, we obtained Gene Ontology (GO) classifica- definitely exclude that transcripts with such a mixed expres- tions for the 76 transcripts with prostate-limited expression sion in the prostate gland cells and the testis are also trans- in three ontology domains: cellular component, molecular ferred SFPs, because there is the possibility that these function and biological process. It is important to note that proteins pass from the testis through the vas deferens to- a transcript could be included in several different categories gether with the sperm. and be associated with multiple GO annotations within a Several of the transcripts had no identifiable homo- single category. This therefore results in more GO- logues in Blast searches and had also no conserved protein annotations than sequences annotated. Blast2GO revealed domains, indicating that these proteins are likely highly di- that 31 transcripts had no Blast hits against the non- verged compared to previously characterized proteins. redundant protein NCBI database. Of the remaining 45 This is perhaps due both to the relatively distant phylo- (59%) transcripts that had a Blast hit, we overall could genetic position of our study organism with respect to assign GO annotations to 44 transcripts. An overview of previously studied taxa, and/or because the proteins the distribution of the sequences in the three ontology themselves are rapidly evolving. Again, our data are con- domains can be seen in Fig. 4. In the cellular component sistent with the fact that many SFP sequences are thought category (Fig. 4a), there is a clear predominance of extracel- to evolve rapidly due to the fact that they are a target of lular region parts and cell parts compared to intracellular (sexual) selection [6–11, 14] and sexual conflict [13, 42]. regions. Regarding the molecular function classification Many previous studies seeking to identify seminal (Fig. 4b), the main functions are involved in binding, cata- fluid proteins have used proteomics approaches (e.g. lytic activity, transferase activity, hydrolase activity, receptor [17, 35, 36, 98–102]), but because of the small size of M. binding and protein kinase activity. In the biological process lignano, collecting proteins from ejaculates is currently category (Fig. 4c), the prevailing groups are associated with challenging. To overcome this limitation, we employed an metabolic processes, single-organism cellular processes and alternative approach to identify putative SFPs. Specifically, multicellular organism development. With a total number we prioritized transcripts as seminal fluid protein candi- of 6 each, the most often identified proteins were fungistatic dates based on three assumptions, namely (i) that they are metabolites or transmembrane receptors (For a more de- more highly expressed in larger groups, reflecting a tran- tailed description of the sequences and the BLAST hits see scriptional upregulation in response to the high level of Additional file 2:Table S1). sperm competition experienced at larger group size com- pared to the non-mating environment when isolated (a Discussion phenotypically plastic response that has been demon- Using a combination of transcriptomic data (Ramm et al: strated also in other taxa [103–105]; (ii) that they show an Sex Allocation Plasticity on a Transcriptome Scale: Socially- expression limited to the prostate glands; and (iii) that Sensitive Gene Expression in the Hermaphroditic Flatworm they exhibit positive evidence of being secreted. Of the 65 Macrostomum lignano,submitted)[78]and ISHexperi- prostate-limited transcripts investigated for signs of secre- ments (present study), we are able to identify 76 transcripts tion and their location in the cell, 74% were predicted to in M. lignano which show specific expression that is limited be secreted either via a signal pathway or by a non- to the prostate, as expected for SFPs. The identification of a classical secretory pathway. This is a much higher percent- large number of SFP candidates is in line with similarly large age than would be predicted for the whole proteome – in numbers of identified proteins in other species studied to humans, for example, only between 10 and 20% of all pro- date (e.g. 198 SFPs in Aedes albopictus [24], more than 200 teins are secreted [106] – but because we were looking for in Drosophila melanogaster [13], 69 in Mus domesticus [36] proteins that are part of the ejaculate and therefore are and several hundred in humans [40, 41]), supporting the no- transferred to the mating partner, this is in line with our tion that seminal fluid is a complex and diverse secretion [3, expectations. This result is also in concordance with the 35]. We consider it less likely that the 22 transcripts that are fact that 69% of the proteins we found by TMHMM ana- expressed in the prostate and additionally in other tissues lysis were located outside of the cell and that 82% of the are potential seminal fluid candidates, because we expect proteins were predicted to be either extracellularly seminal fluid proteins to be exclusively expressed in the secreted or to be extracellularly membrane-bound. In prostate (or in other accessory male reproductive organs, de- total, 64 of the 65 candidates fulfill our criteria for secre- pending on the taxon). Nevertheless, these are also of inter- tion (secretion, transmembrane, part of plasma membrane est in the current context, because they could play or extracellularly located), providing strong indirect Weber et al. BMC Evolutionary Biology (2018) 18:81 Page 10 of 13 number of transcripts 01234 56789 10 cellular component extracellular region part cell part membrane cytoplasm membrane-bounded organelle cell surface intracellular membrane-bounded organelle vesicle extracellular space integral components of membrane nuclear part number of transcripts 01234 56789 10 molecular function binding catalytic activity transferase activity hydrolase activity receptor binding protein kinase activity ion binding protein binding kinase activity metal ion binding DNA binding carbohydrate derivative binding oxidoreductase activity UDP-glycosyltransferase activity number of transcripts 01234 56789 10 biological process metabolic process single-organism cellular process multicellular organism development phosphate-containing compound metabolic process signal transduction protein phosphorylation regulation of cellular process response to stimulus localization cellular process negative regulation of cellular process cell differentiation protein metabolic process cellular component organization single-organism process single-multicellular organism process chondroitin sulfate proteoglycan biosynthetic process animal organ development heparan sulfate proteoglycan metabolic process system development activation of immune response immune system process transposition, DNA-mediated developmental process chondroitin sulfate metabolic process DNA integration transport lateral line development anatomical structure morphogenesis Fig. 4 Summary of Gene Ontology (GO) analysis using Blast2GO of 76 prostate-limited transcripts. a GO term distribution for the cellular component domain; (b) GO term distribution for the molecular function domain; and (c) GO term distribution for the biological processes domain support for their assignment as putative SFPs and a clear section, this conclusion comes with one important pro- vindication of our identification strategy. viso, namely that there is also potentially some redun- We note also that this list of putative SFPs is likely to be dancy in our candidate list, in that some of the different an underestimate, since 11 transcripts with confirmed transcript fragments tested could in fact belong to the prostate-limited expression could not be analyzed, due to same protein or represent different protein isoforms pro- the absence of conserved protein domains and homology duced by alternative splicing. Especially the transcripts by Blast search. As already mentioned in the Results with the same main number that end with different sub- Weber et al. BMC Evolutionary Biology (2018) 18:81 Page 11 of 13 numbers (e.g. 29684.1 and 29684.2) are likely to belong to three categories, namely (Figure S1.) prostate-limited expression; the same protein or different isoforms of that protein. (Figure S2.) prostate-specific expression coupled with tissue-specific expression elsewhere in the worm; and (Figure S3.) tissue-specific They show a high sequence similarity with each other and expression that did not include the prostate. The expression patterns when we blasted them against the M. lignano genome of 10 transcripts could not be established. Within each category, (ML2 assembly) [59] they always align to the same region pictures are arranged (from top-left to bottom-right) in descending order of fold-change in expression in octets versus isolated worms. within the same contig. One representative picture per transcript is included. (ZIP 5699 kb) To begin to characterize potential functions of the puta- Additional file 2: Table S1. Summary of top blast hits identified by tive seminal fluid proteins, we used Blast2Go to classify Blast2Go. (PDF 55 kb) them according to their predicted molecular functions, involvement in biological processes, and cellular compo- Abbreviations nents. However, the fact that many of the functional ISH: Whole-mount in situ hybridization; SFP: Seminal fluid protein categories are extremely broad (such as ‘binding’ or ‘cata- Acknowledgements lytic activity’), and that the same putative proteins can be We thank Athina Giannakara and Bahar Patlar for helpful comments on the assigned to several categories, makes interpreting these manuscript. The computational results presented have been achieved (in part) using the HPC infrastructure LEO and MACH of the University of Innsbruck. classifications far from straightforward. Nevertheless, the fact that in the cellular components classification the Funding biggest fraction belongs to the category “extracellular This work was supported by the German Research Foundation (DFG) grant RA 2468/1–1 to SAR; Austrian Science Fund (FWF) grant P25404-B25 to PL; Swiss region part” is in concordance with the findings just dis- National Science Foundation (SNSF) grants 31003A-127503 and 31003A-143732 cussed before that the majority is extracellularly secreted. to LS. The funding bodies had no role in the design of the study or collection, To move beyond these broad functional classifications, analysis, and interpretation of data or in writing the manuscript. we need to directly assess the roles of specific SFPs. The Availability of data and materials next steps are therefore to evaluate which of these SFP A collection of 3–5 ISH pictures for each transcript is available in the Dryad candidates are actually transferred to the mating partner repository doi:https://doi.org/10.5061/dryad.316c7h6. during insemination, and especially to elucidate what Authors’ contributions effect they have on the mating partner and the repro- MW performed in situ hybridization experiments, analysed results and ductive success of the sperm donor. Due to the availabil- drafted the manuscript together with SAR; BL, RP, JW, MR and PL performed ity of applying RNAi in Macrostomum it is possible to in situ hybridization experiments; LS and SAR conceived the study and analysed RNA-Seq data to generate candidates. All authors contributed to knock-down the expression of specific transcripts and to manuscript revisions, and approved the final manuscript. test for their specific functions in mating experiments (cf. [86, 107–110]). Moreover, due to the availability of a GFP- Ethics approval and consent to participate Not applicable. expressing line there is also the possibility to readily assign paternity following double-mating experiments. In this Competing interests way, we can examine the fitness consequences of knock- The authors declare that they have no competing interests. ing down SFP expression, to test directly for the ability of a donor worm, missing a specific SFP, to compete against Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in arival [65, 74, 111]. published maps and institutional affiliations. Author details Conclusions Evolutionary Biology, Bielefeld University, Konsequenz 45, 33615 Bielefeld, In summary, our study represents the first large-scale Germany. Institute of Zoology and Center of Molecular Biosciences screen to identify putative SFPs in a flatworm, identify- Innsbruck, University of Innsbruck, Technikerstr. 25, A-6020 Innsbruck, Austria. Evolutionary Biology, Zoological Institute, University of Basel, Vesalgasse 1, ing 76 transcripts in M. lignano with prostate-limited 4051 Basel, Switzerland. Current address: School of Natural and expression. Of these, at least 64 also exhibit evidence of Environmental Sciences, Ridley Building, Newcastle University, Newcastle being secreted and therefore of being transferred SFPs. upon Tyne, England NE1 7RU, UK. These putative SFPs are now exciting candidates for Received: 18 January 2018 Accepted: 30 April 2018 future genetic and behavioral studies to examine the function of this important class of proteins. References 1. Mann T, Lutwak-Mann C. Male reproductive function and semen. Additional files Andrologia. 1982;14:76. 2. Peng J, Chen S, Büsser S, Liu H, Honegger T, Kubli E. 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BMC Evolutionary BiologySpringer Journals

Published: May 30, 2018

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