Molecular insights into the powerful mucus-based adhesion of limpets (Patella vulgata L.).

Limpets (Patella vulgata L.) are renowned for their powerful attachments to rocks on wave-swept seashores. Unlike adult barnacles and mussels, limpets do not adhere permanently; instead, they repeatedly transition between long-term adhesion and locomotive adhesion depending on the tide. Recent studies on the adhesive secretions (bio-adhesives) of marine invertebrates have expanded our knowledge on the composition and function of temporary and permanent bio-adhesives. In comparison, our understanding of the limpets' transitory adhesion remains limited. In this study, we demonstrate that suction is not the primary attachment mechanism in P. vulgata; rather, they secrete specialized pedal mucus for glue-like adhesion. Through combined transcriptomics and proteomics, we identified 171 protein sequences from the pedal mucus. Several of these proteins contain conserved domains found in temporary bio-adhesives from sea stars, sea urchins, marine flatworms and sea anemones. Many of these proteins share homology with fibrous gel-forming glycoproteins, including fibrillin, hemolectin and SCO-spondin. Moreover, proteins with potential protein- and glycan-degrading domains could have an immune defence role or assist degrading adhesive mucus to facilitate the transition from stationary to locomotive states. We also discovered glycosylation patterns unique to the pedal mucus, indicating that specific sugars may be involved in transitory adhesion. Our findings elucidate the mechanisms underlying P. vulgata adhesion and provide opportunities for future studies on bio-adhesives that form strong attachments and resist degradation until necessary for locomotion

Organ specific gene expression in the regenerating tail of Macrostomum lignano

Temporal and spatial characterization of gene expression is a prerequisite for the understanding of cell-, tissue-, and organ-differentiation. In a multifaceted approach to investigate gene expression in the tail plate of the free-living marine flatworm Macrostomum lignano, we performed a posterior-region-specific in situ hybridization screen, RNA sequencing (RNA-seq) of regenerating animals, and functional analyses of selected tail-specific genes. The in situ screen revealed transcripts expressed in the antrum, cement glands, adhesive organs, prostate glands, rhabdite glands, and other tissues. Next we used RNA-seq to characterize temporal expression in the regenerating tail plate revealing a time restricted onset of both adhesive organs and copulatory apparatus regeneration. In addition, we identified three novel previously unannotated genes solely expressed in the regenerating stylet. RNA interference showed that these genes are required for the formation of not only the stylet but the whole male copulatory apparatus. RNAi treated animals lacked the stylet, vesicula granulorum, seminal vesicle, false seminal vesicle, and prostate glands, while the other tissues of the tail plate, such as adhesive organs regenerated normally. In summary, our findings provide a large resource of expression data during homeostasis and regeneration of the morphologically complex tail regeneration and pave the way for a better understanding of organogenesis in M. lignano

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

Weber M, Wunderer J, Lengerer B, et al. A targeted in situ hybridization screen identifies putative seminal fluid proteins in a simultaneously hermaphroditic flatworm. BMC Evolutionary Biology. 2018;18(1): 81.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

Omics‐based molecular analyses of adhesion by aquatic invertebrates

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

Molecular biology approaches in bioadhesion research

The use of molecular biology tools in the field of bioadhesion is still in its infancy. For new research groups who are considering taking a molecular approach, the techniques presented here are essential to unravelling the sequence of a gene, its expression and its biological function. Here we provide an outline for addressing adhesion-related genes in diverse organisms. We show how to gradually narrow down the number of candidate transcripts that are involved in adhesion by (1) generating a transcriptome and a differentially expressed cDNA list enriched for adhesion-related transcripts, (2) setting up a BLAST search facility, (3) perform an in situ hybridization screen, and (4) functional analyses of selected genes by using RNA interference knock-down. Furthermore, latest developments in genome-editing are presented as new tools to study gene function. By using this iterative multi-technologies approach, the identification, isolation, expression and function of adhesion-related genes can be studied in most organisms. These tools will improve our understanding of the diversity of molecules used for adhesion in different organisms and these findings will help to develop innovative bio-inspired adhesives

The Involvement of Cell-Type-Specific Glycans in Hydra Temporary Adhesion Revealed by a Lectin Screen

Hydra is a freshwater solitary polyp, capable of temporary adhesion to underwater surfaces. The reversible attachment is based on an adhesive material that is secreted from its basal disc cells and left behind on the substrate as a footprint. Despite Hydra constituting a standard model system in stem cell biology and tissue regeneration, few studies have addressed its bioadhesion. This project aimed to characterize the glycan composition of the Hydra adhesive, using a set of 23 commercially available lectins to label Hydra cells and footprints. The results indicated the presence of N-acetylglucosamine, N-acetylgalactosamine, fucose, and mannose in the adhesive material. The labeling revealed a meshwork-like substructure in the footprints, implying that the adhesive is mainly formed by fibers. Furthermore, lectins might serve as a marker for Hydra cells and structures, e.g., many labeled as glycan-rich nematocytes. Additionally, some unexpected patterns were uncovered, such as structures associated with radial muscle fibers and endodermal gland cells in the hypostome of developing buds

In the footsteps of sea stars: deciphering the catalogue of proteins involved in underwater temporary adhesion

Sea stars adhere strongly but temporarily to underwater substrata via the secretion of a blend of proteins, forming an adhesive footprint that they leave on the surface after detachment. Their tube feet enclose a duo-gland adhesive system comprising two types of adhesive cells, contributing different layers of the footprint and de-adhesive cells. In this study, we characterized the catalogue of sea star footprint proteins (Sfps) in the species Asterias rubens to gain insights in their potential function. We identified 16 Sfps and mapped their expression to type 1 and/or type 2 adhesive cells or to de-adhesive cells by double fluorescent in situ hybridization. Based on their cellular expression pattern and their conserved functional domains, we propose that the identified Sfps serve different functions during attachment, with two Sfps coupling to the surface, six providing cohesive strength and the rest forming a binding matrix. Immunolabelling of footprints with antibodies directed against one protein of each category confirmed these roles. A de-adhesive gland cell-specific astacin-like proteinase presumably weakens the bond between the adhesive material and the tube foot surface during detachment. Overall, we provide a model for temporary adhesion in sea stars, including a comprehensive list of the proteins involved

Additional file 4: Figure S3. of Adhesive organ regeneration in Macrostomum lignano

GSL I labelling of Macrostomum lignano. (A) Overview of a GSL I stained adult animal with (A1) a confocal projection, (A2) DIC image, and (A3) overlay. (B1-3) Detail the most anterior part of the rostrum. The openings of frontal glands 1 emerge from the epidermis on the ventral side of the rostrum, whereas the frontal glands 2 emerge at the margin. (C1-3) Higher magnification of a head, with stained frontal glands 1, 2 and 4. An antrum, rb rhabdites, fg frontal glands, mo mouth, ph pharyngeal glands. Scale bars: (A) 100 μm, (B-C) 20 μm. (TIF 5127 kb

Additional file 3: Figure S2. of Adhesive organ regeneration in Macrostomum lignano

SBA labelling of Macrostomum lignano. (A) Overview of a SBA stained adult animal with (A1) a confocal projection, (A2) DIC image, and (A3) overlay. (B1-3) Detail of the posterior end showing the intensive labelled antrum, single stained cement glands, and the weakly stained prostate glands. (C1-3) Higher magnification of a head, revealing a dotted staining in frontal glands 1 and a ubiquitous staining in the pharyngeal gland cell bodies and frontal glands 2 and 4. An antrum, cg cement glands, fg frontal glands, pg prostate glands, ph pharyngeal glands, st stylet. Scale bars: (A) 100 μm, (B-C) 20 μm. (TIF 5721 kb

Additional file 9: Figure S8. of Adhesive organ regeneration in Macrostomum lignano

PNA labelling of Macrostomum lignano. (A) Overview of a PNA stained adult animal with (A1) confocal projection, (A2) DIC image, and (A3) overlay. Arrow indicates weakly stained sperm at the centre of testes. (B1-3) Detail of the stained antrum and surrounding cement glands. (C1-3) Detail of a head with stained pharyngeal glands and frontal glands 1, 2, and 4. (D) Control PNA staining and (E) staining with PNA pre-incubated with its inhibitory monosaccharide D-Galactose. Dotted lines indicate the outline of the animals. Acg adhesive gland cells, an antrum, cg cement glands, fg frontal glands, ph pharyngeal glands. Scale bars: (A, E) 100 μm, (B, C) 20 μm. (TIF 7252 kb