Fluorescent sperm in a transparent worm: validation of a GFP marker to study sexual selection

Background: Sexual selection has initially been thought to occur exclusively at the precopulatory stage in terms of contests among males and female mate choice, but research over the last four decades revealed that it often continues after copulation through sperm competition and cryptic female choice. However, studying these postcopulatory processes remains challenging because they occur internally and therefore are often difficult to observe. In the transparent free-living flatworm Macrostomum lignano, a recently established transgenic line that expresses green fluorescent protein (GFP) in all cell types, including sperm, offers a unique opportunity to non-invasively visualise and quantify the sperm of a GFP-expressing donor inside the reproductive tract of wild-type recipients in vivo. We here test several aspects of the reproductive performance of the transgenic individuals and the accuracy of the techniques involved in assessing the GFP-expressing worms and their sperm. We then show the usefulness of these methods in a study on sperm displacement. Results: GFP-expressing worms do not differ from wild-type worms in terms of morphology, mating rate and reproductive success. In addition, we show that the GFP signal is reliably and unequivocally expressed by all GFP-expressing individuals observed under epifluorescence illumination. However, the intensity of the GFP signal emitted by sperm of GFP expressing donors can vary (which we show to be at least in part due to sperm ageing) and the GFP marker is inherited according to Mendel’s laws in most, but not all, of the individuals. Nevertheless, we argue these two issues can be addressed with an appropriate experimental design. Finally, we demonstrate the value of the GFP-techniques by comparing the number of GFP-expressing sperm in a wild-type recipient before and after mating with a competing sperm donor, providing clear experimental evidence for sperm displacement in M. lignano. This result suggests that sperm donors can displace previously stored sperm and replace it with their own. Conclusion: The availability of the GFP-techniques in a transparent organism provide unique opportunities to visualise and quantify internal processes in the female reproductive tract after mating, which opens new avenues in the study of sexual selection.

背景：长期以来，性选择被认为仅发生于交配前阶段，即雄性间的竞争与雌性的配偶选择。但近四十年的研究表明，性选择往往在交配后仍会通过精子竞争与隐蔽雌性选择（cryptic female choice）持续进行。然而，由于这些交配后过程发生于生物体内，往往难以直接观测，因此对其开展研究仍颇具挑战。在营自由生活的透明扁形动物林氏大口涡虫（Macrostomum lignano）中，近期构建出了一种在包括精子在内的所有细胞类型中均表达绿色荧光蛋白（green fluorescent protein，以下简称GFP）的转基因品系，这为我们在活体状态下无创可视化观测并定量野生型受体生殖道内表达GFP的供体精子提供了独特契机。本研究首先检测了转基因个体繁殖性能的多个维度，以及评估表达GFP的涡虫及其精子的相关技术的准确性；随后验证了这些方法在精子置换（sperm displacement）研究中的实用性。
结果：表达GFP的涡虫在形态、交配率与繁殖成功率上均与野生型涡虫无显著差异。此外，我们证实，所有经落射荧光照明观测的表达GFP的个体，均能稳定且清晰地发出GFP信号。不过，GFP供体精子所发出的GFP信号强度存在差异（我们证实该差异至少部分源于精子衰老）；且尽管多数个体的GFP标记符合孟德尔定律（Mendel’s laws）遗传，但仍有部分个体并非如此。不过我们认为，通过合理的实验设计可解决上述两个问题。最后，我们通过对比野生型受体在与竞争性精子供体交配前后体内GFP阳性精子的数量，验证了GFP相关技术的应用价值，为林氏大口涡虫中的精子置换现象提供了明确的实验证据。该结果表明，精子供体可置换受体此前储存的精子，并用自身精子取而代之。
结论：在透明模式生物中应用GFP相关技术，为交配后雌性生殖道内的体内过程可视化与定量分析提供了独特契机，也为性选择研究开辟了全新的研究方向。