The ultrastructure of the apical organ of Curini-Galletti's larva, a new polyclad larval type.

Polycladida are the only free-living flatworms with a planktonic larval stage in some species. Currently, it is not clear if a larval stage is ancestral in polyclads, and which type of larva that would be. Known polyclad larvae are Müller's larva, Kato's larva and Goette's larva, differing by body shape and the number of lobes and eyes. A valuable character for the comparison and characterisation of polyclad larval types is the ultrastructural composition of the apical organ. This organ is situated at the anterior pole of the larva and is associated with at least one ciliary tuft. The larval apical organ of Theama mediterranea features two multiciliated apical tuft sensory cells. Six unfurcated apical tuft gland cell necks are sandwiched between the apical tuft sensory cells and two anchor cells and have their cell bodies located lateral to the brain. Another type of apical gland cell necks is embedded in the anchor cells. Ventral to the apical tuft, ciliated sensory neurons are present, which are neighbouring the cell necks of two furcated apical tuft gland cells. Based on the ultrastructural organisation of the apical organ and other morphological features, like a laterally flattened wedge-shaped body and three very small lobes, we recognise the larva of T. mediterranea as a new larval type, which we name Curini-Galletti's larva after its first discoverer. The ultrastructural similarities of the apical organ in different polyclad larvae support their possible homology, that is, all polyclad larvae have likely evolved from a common larva.

A new species of Cycloporus from the Adriatic Sea, with an updated phylogeny of the families Euryleptidae and Stylostomidae (Polycladida, Platyhelminthes).

We describe Cycloporus pinkipus sp. n., a new polyclad flatworm species from the Adriatic coast of Croatia using live images, histological sections, and a molecular marker. It is the fifteenth described species of Cycloporus Lang, 1884 and the second described congener in the Mediterranean. The genus Cycloporus is characterised by a small oval body, tentacular bumps and the name-giving marginal pores. Cycloporus pinkipus sp. n. has a smooth dorsal surface, which is transparent creamy white with light brown to yellow spots, covered with prominent serial pink spots on the inner rim of the body margin. There is little variation of the genital organs between different species of the genus, therefore we recognise C. pinkipus sp. n. as a new species in particular due to its unique coloration, and a unique partial large nuclear ribosomal subunit (28S) sequence. In recent years the family Euryleptidae Stimpson, 1857, which also contains the genus Cycloporus, was discussed and revised in several molecular studies. In an updated molecular phylogeny of the Polycladida based on partial 18S and 28S rDNA marker genes, C. pinkipus sp. n. was recovered in a clade of many other Cycloporus species within Euryleptidae.

A chromosome-scale epigenetic map of the Hydra genome reveals conserved regulators of cell state.

The epithelial and interstitial stem cells of the freshwater polyp Hydra are the best-characterized stem cell systems in any cnidarian, providing valuable insight into cell type evolution and the origin of stemness in animals. However, little is known about the transcriptional regulatory mechanisms that determine how these stem cells are maintained and how they give rise to their diverse differentiated progeny. To address such questions, a thorough understanding of transcriptional regulation in Hydra is needed. To this end, we generated extensive new resources for characterizing transcriptional regulation in Hydra, including new genome assemblies for Hydra oligactis and the AEP strain of Hydra vulgaris, an updated whole-animal single-cell RNA-seq atlas, and genome-wide maps of chromatin interactions, chromatin accessibility, sequence conservation, and histone modifications. These data revealed the existence of large kilobase-scale chromatin interaction domains in the Hydra genome that contain transcriptionally coregulated genes. We also uncovered the transcriptomic profiles of two previously molecularly uncharacterized cell types: isorhiza-type nematocytes and somatic gonad ectoderm. Finally, we identified novel candidate regulators of cell type-specific transcription, several of which have likely been conserved at least since the divergence of Hydra and the jellyfish Clytia hemisphaerica more than 400 million years ago.

Do Not Lose Your Head Over the Unequal Regeneration Capacity in Prolecithophoran Flatworms.

One of the central questions in studying the evolution of regeneration in flatworms remains whether the ancestral flatworm was able to regenerate all body parts, including the head. If so, this ability was subsequently lost in most existent flatworms. The alternative hypothesis is that head regeneration has evolved within flatworms, possibly several times independently. In the well-studied flatworm taxon Tricladida (planarians), most species are able to regenerate a head. Little is known about the regeneration capacity of the closest relatives of Tricladida: Fecampiida and Prolecithophora. Here, we analysed the regeneration capacity of three prolecithophoran families: Pseudostomidae, Plagiostomidae, and Protomonotresidae. The regeneration capacity of prolecithophorans varies considerably between families, which is likely related to the remaining body size of the regenerates. While all studied prolecithophoran species were able to regenerate a tail-shaped posterior end, only some Pseudostomidae could regenerate a part of the pharynx and pharynx pouch. Some Plagiostomidae could regenerate a head including the brain and eyes, provided the roots of the brain were present. The broad spectrum of regeneration capacity in Prolecithophora suggests that head regeneration capacity is not an apomorphy of Adiaphanida.

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.

Sticking Together an Updated Model for Temporary Adhesion.

Non-parasitic flatworms are known to temporarily attach to the substrate by secreting a multicomponent bioadhesive to counteract water movements. However, to date, only species of two higher-level flatworm taxa (Macrostomorpha and Proseriata) have been investigated for their adhesive proteins. Remarkably, the surface-binding protein is not conserved between flatworm taxa. In this study, we sequenced and assembled a draft genome, as well as a transcriptome, and generated a tail-specific positional RNA sequencing dataset of the polyclad Theama mediterranea. This led to the identification of 15 candidate genes potentially involved in temporary adhesion. Using in situ hybridisation and RNA interference, we determined their expression and function. Of these 15 genes, 4 are homologues of adhesion-related genes found in other flatworms. With this work, we provide two novel key components on the flatworm temporary adhesion system. First, we identified a Kringle-domain-containing protein (Tmed-krg1), which was expressed exclusively in the anchor cell. This in silico predicted membrane-bound Tmed-krg1 could potentially bind to the cohesive protein, and a knockdown led to a non-adhesive phenotype. Secondly, a secreted tyrosinase (Tmed-tyr1) was identified, which might crosslink the adhesive proteins. Overall, our findings will contribute to the future development of reversible synthetic glues with desirable properties for medical and industrial applications.

(Un)expected Similarity of the Temporary Adhesive Systems of Marine, Brackish, and Freshwater Flatworms.

Many free-living flatworms have evolved a temporary adhesion system, which allows them to quickly attach to and release from diverse substrates. In the marine Macrostomum lignano, the morphology of the adhesive system and the adhesion-related proteins have been characterised. However, little is known about how temporary adhesion is performed in other aquatic environments. Here, we performed a 3D reconstruction of the M. lignano adhesive organ and compared it to the morphology of five selected Macrostomum, representing two marine, one brackish, and two freshwater species. We compared the protein domains of the two adhesive proteins, as well as an anchor cell-specific intermediate filament. We analysed the gene expression of these proteins by in situ hybridisation and performed functional knockdowns with RNA interference. Remarkably, there are almost no differences in terms of morphology, protein regions, and gene expression based on marine, brackish, and freshwater habitats. This implies that glue components produced by macrostomids are conserved among species, and this set of two-component glue functions from low to high salinity. These findings could contribute to the development of novel reversible biomimetic glues that work in all wet environments and could have applications in drug delivery systems, tissue adhesives, or wound dressings.

The serotonergic nervous system of prolecithophorans shows a closer similarity to fecampiids than to triclads (Platyhelminthes).

Prolecithophora is a poorly studied flatworm order belonging to the adiaphanidan clade, together with Tricladida and Fecampiida. The phylogenetic position of the three orders within this clade is not yet resolved. Additionally, no obvious synapomorphy other than an opaque epidermis could be found so far. In this study, the serotonergic nervous system of six different prolecithophoran species has been studied for the first time with a fluorescent immunocytochemical technique. We found that all six species show a similar pattern of the serotonergic nervous system. The typical prolecithophoran serotonergic nervous system consists of a cephalic ganglion in the anterior body part from which a pair of dorsal, ventral, and lateral longitudinal nerve cords originate. Furthermore, the three longitudinal nerve cords of one body side are connected to each other at the posterior body part by a conspicuous commissure. The ventral cords, which we consider the main cords, are most prominent and show double brain roots. A comparison of the nervous system within Adiaphanida shows clearly that prolecithophorans and fecampiids are much more similar in this regard than prolecithophorans and triclads.

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.

Regeneration of the flatworm Prosthiostomum siphunculus (Polycladida, Platyhelminthes).

Fueled by the discovery of head regeneration in triclads (planarians) two and a half centuries ago, flatworms have been the focus of regeneration research. But not all flatworms can regenerate equally well and to obtain a better picture of the characteristics and evolution of regeneration in flatworms other than planarians, the regeneration capacity and stem cell dynamics during regeneration in the flatworm order Polycladida are studied. Here, we show that as long as the brain remained at least partially intact, the polyclad Prosthiostomum siphunculus was able to regenerate submarginal eyes, cerebral eyes, pharynx, intestine and sucker. In the complete absence of the brain only wound closure was observed but no regeneration of missing organs. Amputated parts of the brain could not be regenerated. The overall regeneration capacity of P. siphunculus is a good fit for category III after a recently established system, in which most polyclads are currently classified. Intact animals showed proliferating cells in front of the brain which is an exception compared with most of the other free-living flatworms that have been observed so far. Proliferating cells could be found within the regeneration blastema, similar to all other flatworm taxa except triclads. No proliferation was observed in epidermis and pharynx. In pulse-chase experiments, the chased cells were found in all regenerated tissues and thereby shown to differentiate and migrate to replace the structures lost upon amputation.

RNA-Seq of three free-living flatworm species suggests rapid evolution of reproduction-related genes.

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.

No head regeneration here: regeneration capacity and stem cell dynamics of Theama mediterranea (Polycladida, Platyhelminthes).

Research on the regeneration potential of flatworms (Platyhelminthes) has been mainly undertaken with planarians (Tricladida), where most species can regenerate a head and no proliferation takes place in the blastema, i.e. the early undifferentiated regenerative tissue. Only few studies are available for an early-branching group within the Platyhelminthes, the Polycladida. Head regeneration in polyclads is not possible, with a single exception from a study performed more than 100 years ago: Cestoplana was reported to be able to regenerate a head if cut a short distance behind the brain. Here, we show that 'Cestoplana' was misdetermined and most likely was the small interstitial polyclad Theama mediterranea. We revisited regeneration capacity and dynamics of T. mediterranea with live observations and stainings of musculature, nervous system, and proliferating and differentiating stem cells. In our experiments, after transversal amputation, only animals retaining more than half of the brain could fully restore the head including the brain. If completely removed, the brain was never found to regenerate to any extent. Different from planarians, but comparable to other free-living flatworms we detected cell proliferation within the posterior regeneration blastema in T. mediterranea. Similar to other free-living flatworms, proliferation did not occur within, but only outside, the differentiating organ primordia. Our results strongly imply that brain regeneration in the absence of the latter is not possible in any polyclad studied so far. Also, it appears that proliferation of stem cells within the regeneration blastema is a plesiomorphy in flatworms and that planarians are derived in this character.

Temporary adhesion of the proseriate flatworm Minona ileanae.

Flatworms can very rapidly attach to and detach from many substrates. In the presented work, we analysed the adhesive system of the marine proseriate flatworm Minona ileanae. We used light-, scanning- and transmission electron microscopy to analyse the morphology of the adhesive organs, which are located at the ventral side of the tail-plate. We performed transcriptome sequencing and differential RNA-seq for the identification of tail-specific transcripts. Using in situ hybridization expression screening, we identified nine transcripts that were expressed in the cells of the adhesive organs. Knock-down of five of these transcripts by RNA interference led to a reduction of the animal's attachment capacity. Adhesive proteins in footprints were confirmed using mass spectrometry and antibody staining. Additionally, lectin labelling of footprints revealed the presence of several sugar moieties. Furthermore, we determined a genome size of about 560 Mb for M. ileanae. We demonstrated the potential of Oxford Nanopore sequencing of genomic DNA as a cost-effective tool for identifying the number of repeats within an adhesive protein and for combining transcripts that were fragments of larger genes. A better understanding of the molecules involved in flatworm bioadhesion can pave the way towards developing innovative glues with reversible adhesive properties. This article is part of the theme issue 'Transdisciplinary approaches to the study of adhesion and adhesives in biological systems'.

The adult musculature of two pseudostomid species reveals unique patterns for flatworms (Platyhelminthes, Prolecithophora).

We analyzed the adult musculature of two prolecithophoran species, Cylindrostoma monotrochum (von Graff, 1882) and Monoophorum striatum (von Graff, 1878) using a phalloidin-rhodamine technique. As in all rhabdithophoran flatworms, the body-wall musculature consisted of three muscle layers: on the outer side was a layer of circular muscle fibers and on the inner side was a layer of longitudinal muscle fibers; between them were two different types of diagonally orientated fibers, which is unusual for flatworms. The musculature of the pharynx consisted of a basket-shaped grid of thin longitudinal and circular fibers. Thick anchoring muscle fibers forming a petal-like shape connected the proximal parts of the pharynx with the body-wall musculature. Male genital organs consisted of paired seminal vesicles, a granular vesicle, and an invaginated penis. Peculiar ring-shaped muscles were only found in M. striatum, predominantly in the anterior body part. In the same species, seminal vesicles and penis only had circular musculature, while in C. monotrochum also longitudinal musculature was found in these organs. Female genital organs were only present in M. striatum, where we characterized a vagina interna, and a bursa seminalis. Transverse, crossover, and dorsoventral muscle fibers were lacking in the middle of the body and greatly varied in number and position in both species.

A mechanism for temporary bioadhesion.

The flatworm Macrostomum lignano features 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 of M. lignano relies on the secretion of two large adhesive proteins, M. lignano adhesion 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 that M. lignano could 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.