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

Abstract

BackgroundSexual 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.ResultsGFP-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.ConclusionThe 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.

A phylogenetically informed search for an alternative Macrostomum model species, with notes on taxonomy, mating behavior, karyology, and genome size

“…The free‐living flatworm Macrostomum lignano Ladurner, Schärer, Salvenmoser, Rieger 2005 (Rhabditophora, Macrostomorpha) has emerged as a model for a broad range of research topics (Ladurner, Schärer, Salvenmoser, & Rieger, ), including the biology of aging (Mouton, Grudniewska, Glazenburg, Guryev, & Berezikov, ; Mouton et al, ), bioadhesion (Lengerer, Hennebert, Flammang, Salvenmoser, & Ladurner, ; Lengerer et al, ; Wunderer et al, ), regeneration (Egger, Ladurner, Nimeth, Gschwentner, & Rieger, ; Lengerer et al, ), stem cell biology (Grudniewska et al, ; Ladurner et al, ), and sexual selection (Marie‐Orleach, Janicke, Vizoso, David, & Schärer, ; Schärer, Littlewood, Waeschenbach, Yoshida, & Vizoso, ; Sekii et al, ). This research has led to the establishment of many resources and tools that are crucial for a modern genetic and genomic model, including in situ hybridization (Pfister et al, ), RNA interference (Kuales et al, ; Sekii et al, ), gene expression (Arbore et al, ; Grudniewska et al, ; Lengerer et al, ), genome and transcriptome assemblies (Wasik et al, ; Wudarski et al, ), transgenesis (Marie‐Orleach, Janicke, Vizoso, Eichmann, & Schärer, ; Wudarski et al, ), and a clarification of the phylogenetic context (Janssen et al, ; Schärer et al, ). Jointly, these achievements make M. lignano an excellent model to complement the, in several aspects, more established planarian flatworm models (Newmark & Sánchez Alvarado, ; Pellettieri & Sánchez Alvarado, ; Rink, ; Rouhana et al, ).…”

Transcriptional signatures of somatic neoblasts and germline cells in Macrostomum lignano

“…Several techniques are developed for Macrostomum , including antibody labeling, in situ hybridization (ISH), RNA interference (RNAi), and gene expression analysis (Ladurner et al, 2000, 2005; Pfister et al, 2007; De Mulder et al, 2009; Arbore et al, 2015; Plusquin et al, 2016). Recently, the first genome and transcriptome assemblies were published (Wasik et al, 2015), and transgenesis utility was demonstrated (Marie-Orleach et al, 2014, 2016). Despite this available toolbox, the described molecular markers for proliferating cells in M. lignano are still limited to piwi and vasa , which are expressed in both somatic neoblasts and proliferating germline cells (Pfister et al, 2007, 2008; De Mulder et al, 2009; Zhou et al, 2015).…”

Genome and transcriptome of the regeneration-competent flatworm, Macrostomum lignano

“…Given its promise as a model for studying mechanisms governing pluripotency, a number of groups have worked to establish M. lignano as a model to study stem cell biology and regeneration (16,19,20), sexual selection and reproductive biology (21,22), bioadhesion (23), and neurobiology (24). Efforts of the M. lignano community have resulted in the development of a number of tools that can be used to study M. lignano biology (15,21,(25)(26)(27).…”

“…Efforts of the M. lignano community have resulted in the development of a number of tools that can be used to study M. lignano biology (15,21,(25)(26)(27).…”