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

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 whole-mount 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.研究背景：在诸多动物类群中，精液除精子外，还含有大量精液蛋白（seminal fluid proteins, SFPs）。SFPs与精子一同被转移至交配伴侣体内，此前被认为在介导交配后性选择过程中发挥关键作用。它们可调控伴侣的行为与生理状态，进而影响双方的生殖成功率，因此可能引发性冲突。尽管SFPs被认为具有广泛的功能与进化意义，但迄今为止，对其的鉴定与表征仅聚焦于少数动物类群，主要为昆虫与哺乳动物。此外，此前的精液蛋白分析主要集中于雌雄异体物种。本研究首次针对同步雌雄同体的林氏大环口涡虫*Macrostomum lignano*的潜在SFPs开展全面筛选。 研究结果：基于已有的转录组数据（transcriptomic data），本研究筛选出150个符合以下特征的转录本：其一，主要在涡虫尾部区域表达——该区域正是产生精液的前列腺腺细胞的定位处；其二，在精子竞争水平不同的社会环境中存在差异表达，这强烈提示这些转录本代表了该物种雄性生殖资源分配的表型可塑性特征。针对这些候选SFPs，我们开展了整体原位杂交（whole mount in situ hybridization, ISH）实验以表征其组织特异性表达模式。最终共鉴定出98个呈现前列腺特异性表达的转录本，其中76个仅在前列腺腺细胞中表达；剩余22个转录本的额外表达位点包括精巢或其他腺细胞。对前列腺限制性表达的候选转录本进行生物信息学分析后发现，至少有64个被预测为分泌蛋白，这使其成为交配过程中转移的SFPs的强有力候选者。 研究结论：本研究首次结合转录组数据与整体原位杂交筛选数据，基于精液产生组织的转录本表达特征鉴定SFPs。我们将已完成精液表征的动物类群范围拓展至一种拥有已测序基因组、且可开展抗体染色、转基因（transgenesis）及RNA干扰（RNA interference, RNAi）等多种实验操作的涡虫物种。本研究数据为验证SFPs的功能与进化意义提供了坚实的研究基础。