The flatwormMacrostomum lignanofeatures a duo-gland adhesive system that allows it to repeatedly attach to and release from substrates in seawater within a minute. However, little is known about the molecules involved in this temporary adhesion. In this study, we show that the attachment ofM. lignanorelies on the secretion of two large adhesive proteins,M. lignanoadhesion protein 1 (Mlig-ap1) and Mlig-ap2. We revealed that both proteins are expressed in the adhesive gland cells and that their distribution within the adhesive footprints was spatially restricted. RNA interference knockdown experiments demonstrated the essential function of these two proteins in flatworm adhesion. Negatively charged modified sugars in the surrounding water inhibited flatworm attachment, while positively charged molecules impeded detachment. In addition, we found thatM. lignanocould not adhere to strongly hydrated surfaces. We propose an attachment–release model where Mlig-ap2 attaches to the substrate and Mlig-ap1 exhibits a cohesive function. A small negatively charged molecule is secreted that interferes with Mlig-ap1, inducing detachment. These findings are of relevance for fundamental adhesion science and efforts to mitigate biofouling. Further, this model of flatworm temporary adhesion may serve as the starting point for the development of synthetic reversible adhesion systems for medicinal and industrial applications.

BackgroundFree-living flatworms, in both marine and freshwater environments, are able to adhere to and release from a substrate several times within a second. This reversible adhesion relies on adhesive organs comprised of three cell types: an adhesive gland cell, a releasing gland cell, and an anchor cell, which is a modified epidermal cell responsible for structural support. However, nothing is currently known about the molecules that are involved in this adhesion process.ResultsIn this study we present the detailed morphology of the adhesive organs of the free-living marine flatworm Macrostomum lignano. About 130 adhesive organs are located in a horse-shoe-shaped arc along the ventral side of the tail plate. Each organ consists of exactly three cells, an adhesive gland cell, a releasing gland cell, and an anchor cell. The necks of the two gland cells penetrate the anchor cell through a common pore. Modified microvilli of the anchor cell form a collar surrounding the necks of the adhesive- and releasing glands, jointly forming the papilla, the outer visible part of the adhesive organs. Next, we identified an intermediate filament (IF) gene, macif1, which is expressed in the anchor cells. RNA interference mediated knock-down resulted in the first experimentally induced non-adhesion phenotype in any marine animal. Specifically, the absence of intermediate filaments in the anchor cells led to papillae with open tips, a reduction of the cytoskeleton network, a decline in hemidesmosomal connections, and to shortened microvilli containing less actin.ConclusionOur findings reveal an elaborate biological adhesion system in a free-living flatworm, which permits impressively rapid temporary adhesion-release performance in the marine environment. We demonstrate that the structural integrity of the supportive cell, the anchor cell, is essential for this adhesion process: the knock-down of the anchor cell-specific intermediate filament gene resulted in the inability of the animals to adhere. The RNAi mediated changes of the anchor cell morphology are comparable to situations observed in human gut epithelia. Therefore, our current findings and future investigations using this powerful flatworm model system might contribute to a better understanding of the function of intermediate filaments and their associated human diseases.

Background The genus Macrostomum consists of small free-living flatworms and contains Macrostomum lignano, which has been used in investigations of ageing, stem cell biology, bioadhesion, karyology, and sexual selection in hermaphrodites. Two types of mating behaviour occur within this genus. Some species, including M. lignano, mate via reciprocal copulation, where, in a single mating, both partners insert their male copulatory organ into the female storage organ and simultaneously donate and receive sperm. Other species mate via hypodermic insemination, where worms use a needle-like copulatory organ to inject sperm into the tissue of the partner. These contrasting mating behaviours are associated with striking differences in sperm and copulatory organ morphology. Here we expand the genomic resources within the genus to representatives of both behaviour types and investigate whether genes vary in their rate of evolution depending on their putative function. Results We present de novo assembled transcriptomes of three Macrostomum species, namely M. hystrix, a close relative of M. lignano that mates via hypodermic insemination, M. spirale, a more distantly related species that mates via reciprocal copulation, and finally M. pusillum, which represents a clade that is only distantly related to the other three species and also mates via hypodermic insemination. We infer 23,764 sets of homologous genes and annotate them using experimental evidence from M. lignano. Across the genus, we identify 521 gene families with conserved patterns of differential expression between juvenile vs. adult worms and 185 gene families with a putative expression in the testes that are restricted to the two reciprocally mating species. Further, we show that homologs of putative reproduction-related genes have a higher protein divergence across the four species than genes lacking such annotations and that they are more difficult to identify across the four species, indicating that these genes evolve more rapidly, while genes involved in neoblast function are more conserved. Conclusions This study improves the genus Macrostomum as a model system, by providing resources for the targeted investigation of gene function in a broad range of species. And we, for the first time, show that reproduction-related genes evolve at an accelerated rate in flatworms.

Many aquatic invertebrates are associated with surfaces, using adhesives to attach to the substratum for locomotion, prey capture, reproduction, building or defence. Their intriguing and sophisticated biological glues have been the focus of study for decades. In all but a couple of specific taxa, however, the precise mechanisms by which the bioadhesives stick to surfaces underwater and (in many cases) harden have proved to be elusive. Since the bulk components are known to be based on proteins in most organisms, the opportunities provided by advancing 'omics technologies have revolutionised bioadhesion research. Time-consuming isolation and analysis of single molecules has been either replaced or augmented by the generation of massive data sets that describe the organism's translated genes and proteins. While these new approaches have provided resources and opportunities that have enabled physiological insights and taxonomic comparisons that were not previously possible, they do not provide the complete picture and continued multi-disciplinarity is essential. This review covers the various ways in which 'omics have contributed to our understanding of adhesion by aquatic invertebrates, with new data to illustrate key points. The associated challenges are highlighted and priorities are suggested for future research.

BackgroundAlong 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.ResultsBased 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (10.1186/s12862-018-1187-0) contains supplementary material, which is available to authorized users.