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.

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.

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.

Acoel Single-Cell Transcriptomics: Cell Type Analysis of a Deep Branching Bilaterian.

Bilaterian animals display a wide variety of cell types, organized into defined anatomical structures and organ systems, which are mostly absent in prebilaterian animals. Xenacoelomorpha are an early-branching bilaterian phylum displaying an apparently relatively simple anatomical organization that have greatly diverged from other bilaterian clades. In this study, we use whole-body single-cell transcriptomics on the acoel Isodiametra pulchra to identify and characterize different cell types. Our analysis identifies the existence of ten major cell type categories in acoels all contributing to main biological functions of the organism: metabolism, locomotion and movements, behavior, defense, and development. Interestingly, although most cell clusters express core fate markers shared with other animal clades, we also describe a surprisingly large number of clade-specific marker genes, suggesting the emergence of clade-specific common molecular machineries functioning in distinct cell types. Together, these results provide novel insight into the evolution of bilaterian cell types and open the door to a better understanding of the origins of the bilaterian body plan and their constitutive cell types.

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.

The free-living flatworm Macrostomum lignano.

Macrostomum lignano is a free-living flatworm that is emerging as an attractive experimental animal for research on a broad range of biological questions. One feature setting it apart from other flatworms is the successful establishment of transgenesis methods, facilitated by a steady supply of eggs in the form of single-cell zygotes that can be readily manipulated. This, in combination with the transparency of the animal and its small size, creates practical advantages for imaging and fluorescence-activated cell sorting in studies related to stem cell biology and regeneration. M. lignano can regenerate most of its body parts, including the germline, thanks to the neoblasts, which represent the flatworm stem cell system. Interestingly, neoblasts seem to have a high capacity of cellular maintenance, as M. lignano can survive up to 210 Gy of γ-irradiation, and partially offset the negative consequence of ageing. As a non-self-fertilizing simultaneous hermaphrodite that reproduces in a sexual manner, M. lignano is also used to study sexual selection and other evolutionary aspects of sexual reproduction. Work over the past several years has led to the development of molecular resources and tools, including high-quality genome and transcriptome assemblies, transcriptional profiling of the germline and somatic neoblasts, gene knockdown, and in situ hybridization. The increasingly detailed characterization of this animal has also resulted in novel research questions, such as bio-adhesion based on its adhesion-release glands and genome evolution due to its recent whole-genome duplication.

Metallomics reveals a persisting impact of cadmium on the evolution of metal-selective snail metallothioneins.

The tiny contribution of cadmium (Cd) to the composition of the earth's crust contrasts with its high biological significance, owing mainly to the competition of Cd with the essential zinc (Zn) for suitable metal binding sites in proteins. In this context it was speculated that in several animal lineages, the protein family of metallothioneins (MTs) has evolved to specifically detoxify Cd. Although the multi-functionality and heterometallic composition of MTs in most animal species does not support such an assumption, there are some exceptions to this role, particularly in animal lineages at the roots of animal evolution. In order to substantiate this hypothesis and to further understand MT evolution, we have studied MTs of different snails that exhibit clear Cd-binding preferences in a lineage-specific manner. By applying a metallomics approach including 74 MT sequences from 47 gastropod species, and by combining phylogenomic methods with molecular, biochemical, and spectroscopic techniques, we show that Cd selectivity of snail MTs has resulted from convergent evolution of metal-binding domains that significantly differ in their primary structure. We also demonstrate how their Cd selectivity and specificity has been optimized by the persistent impact of Cd through 430 million years of MT evolution, modifying them upon lineage-specific adaptation of snails to different habitats. Overall, our results support the role of Cd for MT evolution in snails, and provide an interesting example of a vestigial abiotic factor directly driving gene evolution. Finally, we discuss the potential implications of our findings for studies devoted to the understanding of mechanisms leading to metal specificity in proteins, which is important when designing metal-selective peptides.

Integrative Transcriptome and Proteome Analysis of the Tube Foot and Adhesive Secretions of the Sea Urchin Paracentrotus lividus.

Echinoderms, such as the rock-boring sea urchin Paracentrotus lividus, attach temporarily to surfaces during locomotion using their tube feet. They can attach firmly to any substrate and release from it within seconds through the secretion of unknown molecules. The composition of the adhesive, as well as the releasing secretion, remains largely unknown. This study re-analyzed a differential proteome dataset from Lebesgue et al. by mapping mass spectrometry-derived peptides to a P. lividus de novo transcriptome generated in this study. This resulted in a drastic increase in mapped proteins in comparison to the previous publication. The data were subsequently combined with a differential RNAseq approach to identify potential adhesion candidate genes. A gene expression analysis of 59 transcripts using whole mount in situ hybridization led to the identification of 16 transcripts potentially involved in bioadhesion. In the future these data could be useful for the production of synthetic reversible adhesives for industrial and medical purposes.

Cadmium Pathways in Snails Follow a Complementary Strategy between Metallothionein Detoxification and Auxiliary Inactivation by Phytochelatins.

Metal detoxification is crucial for animals to cope with environmental exposure. In snails, a pivotal role in protection against cadmium (Cd) is attributed to metallothioneins (MTs). Some gastropod species express, in a lineage-specific manner, Cd-selective MTs devoted exclusively to the binding and detoxification of this single metal, whereas other species of snails possess non-selective MTs, but still show a high tolerance against Cd. An explanation for this may be that invertebrates and in particular snails may also synthetize phytochelatins (PCs), originally known to be produced by plants, to provide protection against metal or metalloid toxicity. Here we demonstrate that despite the fact that similar mechanisms for Cd inactivation exist in snail species through binding of the metal to MTs, the actual detoxification pathways for this metal may follow different traits in a species-specific manner. In particular, this depends on the detoxification capacity of MTs due to their Cd-selective or non-specific binding features. In the terrestrial slug Arion vulgaris, for example, Cd is solely detoxified by a Cd-selective MT isoform (AvMT1). In contrast, the freshwater snail Biomphalaria glabrata activates an additional pathway for metal inactivation by synthesizing phytochelatins, which compensate for the insufficient capacity of its non-selective MT system to detoxify Cd. We hypothesize that in other snails and invertebrate species, too, an alternative inactivation of the metal by PCs may occur, if their MT system is not Cd-selective enough, or its Cd loading capacity is exhausted.

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'.

Sex allocation plasticity on a transcriptome scale: Socially sensitive gene expression in a simultaneous hermaphrodite.

Phenotypic plasticity can enable organisms to produce optimal phenotypes in multiple environments. A crucial life history trait that is often highly plastic is sex allocation, which in simultaneous hermaphrodites describes the relative investment into the male versus female sex functions. Theory predicts-and morphological evidence supports-that greater investment into the male function is favoured with increasing group size, due to the increasing importance of sperm competition for male reproductive success. Here, we performed a genome-wide gene expression assay to test for such sex allocation plasticity in a model simultaneous hermaphrodite, the free-living flatworm Macrostomum lignano. Based on RNA-Seq data from 16 biological replicates spanning four different group size treatments, we demonstrate that at least 10% of the >75,000 investigated transcripts in M. lignano are differentially expressed according to the social environment, rising to >30% of putative gonad-specific transcripts (spermatogenesis and oogenesis candidates) and tail-specific transcripts (seminal fluid candidates). This transcriptional response closely corresponds to the expected shift away from female and towards male reproductive investment with increasing sperm competition level. Using whole-mount in situ hybridization, we then confirm that many plastic transcripts exhibit the expected organ-specific expression, and RNA interference of selected testis- and ovary-specific candidates establishes that these indeed function in gametogenesis pathways. We conclude that a large proportion of sex-specific transcripts in M. lignano are differentially expressed according to the prevailing ecological conditions and that these are functionally relevant to key reproductive phenotypes. Our study thus begins to bridge organismal and molecular perspectives on sex allocation plasticity.

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.

Differential response to Cadmium exposure by expression of a two and a three-domain metallothionein isoform in the land winkle Pomatias elegans: Valuating the marine heritage of a land snail.

Through evolution, marine snails have adapted several times independently to terrestrial life. A prime example for such transitions is the adaptation to terrestrial conditions in members of the gastropod clade of Littorinoidea (Caenogastropoda). Some species of this lineage like the periwinkle (Littorina littorea), live in intertidal habitats, where they are intermittently exposed to semi-terrestrial conditions. Pomatias elegans is a close relative of Littorina littorea that has successfully colonized terrestrial habitats. Evolutionary transitions from marine to terrestrial conditions have often been fostered in marine ancestors by acquisition of physiological pre-adaptations to terrestrial life. Such pre-adaptations are based, among others, on the optimization of a wide repertoire of stress resistance mechanisms, such as the expression of metal inactivating metallothioneins (MTs). The objective of our study was to explore the Cd handling strategy in the terrestrial snail Pomatias elegans in comparison to that observed previously in Littorina littorea. After Cd exposure, the metal is accumulated mainly in the midgut gland of Pomatias elegans, in a similar way as in its marine relative. Upon Cd exposure, Pomatias elegans expresses Cd-specific MTs, as also described from Littorina littorea. In contrast to the latter species, however, the detoxification of Cd in Pomatias elegans is mediated by two different MT isoforms, one two-domain and one three-domain MT. Although the MT proteins of both species are homologous and clearly originate from one common ancestor, the three-domain MT isoform of Pomatias elegans has evolved independently from the three-domain MT of its marine counterpart, probably by addition of a third domain to the pre-existing two-domain MT. Obviously, the occurrence of homologous MT structures in both species is a hereditary character, whereas the differentiation into two distinct MT isoforms with different upregulation capacities in Pomatias elegans is an adaptive feature that probably emerged upon transition to life on land.

Papillae revisited and the nature of the adhesive secreting collocytes.

Ascidian papillae (palps) constitute a transient sensory adhesive organ that assures larval settlement and the onset of metamorphosis to the filterfeeding adult. Despite the importance of papillae for the ascidian development, their cellular composition is only roughly described. For Ciona intestinalis/robusta, a clear definition of cell numbers and discriminative molecular markers for the different cell types is missing. While some attention was given to neural cell types and their connectivity little is known about the adhesive producing collocytes. We converge serial-section electron microscopy and confocal imaging with various marker combinations to document the 3D organization of the Ciona papillae. We show the papillar development with 4 axial columnar cells (ACCs), 4 lateral primary sensory neurons (PSNs) and 12 central collocytes (CCs). We propose molecular markers for each cell type including novel ones for collocytes. The subcellular characteristics are suggestive of their role in papillar function: the ACCs featuring apical protrusions and microvilli, also contain neuroactive and endocytic vesicles indicative of a chemosensory role. They are clearly distinct from the ciliated glutamatergic PSNs. CCs encircle the ACCs and contain microvilli, small endocytic vesicles and notably a large numbers of adhesive granules that, according to element analysis and histochemistry, contain glycoproteins. Interestingly, we detect two different types of collocyte granules, one of them containing fibrous material and larger quantities of polysaccharides. Consistently, carbohydrate specific lectins label the papillar apex, the granules within CCs and the adhesive plaques upon larval attachment. We further propose CCs to derive from an evolutionary ancient neurosecretory cell type. Our findings contribute to understanding the development of the anterior ('new head') region of the Ciona larva and notably the adhesive secreting cells which has implications for developmental biology, cell differentiation and evolution, but also bioadhesion.

Metal binding functions of metallothioneins in the slug Arion vulgaris differ from metal-specific isoforms of terrestrial snails.

Arion vulgaris is a land-living European slug belonging to the gastropod clade of Stylommatophora. The species is known as an efficient pest organism in vegetable gardening and horticulture, which may in part be the consequence of its genetically based innate immunity, along with its high ability to withstand toxic metal stress by intracellular detoxification. Like many species of terrestrial snails, slugs possess a distinct capacity for Cd accumulation in their midgut gland, where the metal is stored and inactivated, conferring to these animals an increased metal tolerance. Although midgut gland Cd fractions in slugs have been shown to be variably allocated between different metal-binding protein pools, depending on the level of environmental metal contamination, a true metallothionein (MT) was so far never characterized from slugs. Instead, the Cd binding proteins identified so far were described as Metallothionein-like proteins (MTLPs). In the present study, the slug A. vulgaris was used as a model organism, in order to verify the presence of true MTs in experimentally metal-exposed slugs. We wanted to find out if these suggested slug MTs have similar metal binding properties and metal-selective features like those previously reported from helicid snails. To this aim, two MT isoform genes (AvMT1 and AvMT2) were characterized from midgut gland extracts and localized in the cells of this tissue. The AvMT1 and AvMT2 proteins were purified and partially sequenced, and their metal-binding features analysed after recombinant expression. Eventually, we wanted to understand if and by how much the metal binding features of the two MT isoforms of A. vulgaris may be related, owing to their reciprocal amino acid sequence similarities, to the binding properties of metal-specific MTs from terrestrial snails.

The structural and chemical basis of temporary adhesion in the sea star Asterina gibbosa.

Background: Marine biological adhesives are a promising source of inspiration for biomedical and industrial applications. Nevertheless, natural adhesives and especially temporary adhesion systems are mostly unexplored. Sea stars are able to repeatedly attach and detach their hydraulic tube feet. This ability is based on a duo-gland system and, upon detachment, the adhesive material stays behind on the substrate as a 'footprint'. In recent years, characterization of sea star temporary adhesion has been focussed on the forcipulatid species Asterias rubens. Results: We investigated the temporary adhesion system in the distantly related valvatid species Asterina gibbosa. The morphology of tube feet was described using histological sections, transmission-, and scanning electron microscopy. Ultrastructural investigations revealed two adhesive gland cell types that both form electron-dense secretory granules with a more lucid outer rim and one de-adhesive gland cell type with homogenous granules. The footprints comprised a meshwork on top of a thin layer. This topography was consistently observed using various methods like scanning electron microscopy, 3D confocal interference microscopy, atomic force microscopy, and light microscopy with crystal violet staining. Additionally, we tested 24 commercially available lectins and two antibodies for their ability to label the adhesive epidermis and footprints. Out of 15 lectins labelling structures in the area of the duo-gland adhesive system, only one also labelled footprints indicating the presence of glycoconjugates with α-linked mannose in the secreted material. Conclusion: Despite the distant relationship between the two sea star species, the morphology of tube feet and topography of footprints in A. gibbosa shared many features with the previously described findings in A. rubens. These similarities might be due to the adaptation to a benthic life on rocky intertidal areas. Lectin- and immuno-labelling indicated similarities but also some differences in adhesive composition between the two species. Further research on the temporary adhesive of A. gibbosa will allow the identification of conserved motifs in sea star adhesion and might facilitate the development of biomimetic, reversible glues.

Properties of temporary adhesion systems of marine and freshwater organisms.

Underwater adhesive secretions are a promising source of inspiration for biomedical and industrial applications. Although marine permanent adhesives have been extensively investigated, reversible adhesion, e.g. as used for locomotion and feeding, is still poorly understood. Here, we summarise the current knowledge on secretion-based, temporary adhesive systems in aquatic environments, with a special emphasis on the morphology and structure of adhesive organs and adhesive material. Many animals employing temporary adhesion to the substratum rely on so-called duo-gland adhesive organs, consisting of two secretory gland cells and one supportive cell. We give a detailed depiction of a basic duo-gland adhesive organ and variations thereof. Additionally, we discuss temporary adhesive systems with an alternative building plan. Next, the topography of secreted adhesive footprints is described based on examples. The limited data on the composition of temporary adhesives are summarised, separating known protein components and carbohydrate residues. There are still large gaps in our understanding of temporary adhesion. We discuss three proposed models for detachment, although the actual mechanism of voluntary detachment is still a matter for debate.

Xenacoelomorpha Survey Reveals That All 11 Animal Homeobox Gene Classes Were Present in the First Bilaterians.

Homeodomain transcription factors are involved in many developmental processes across animals and have been linked to body plan evolution. Detailed classifications of these proteins identified 11 distinct classes of homeodomain proteins in animal genomes, each harboring specific sequence composition and protein domains. Although humans contain the full set of classes, Drosophila melanogaster and Caenorhabditis elegans each lack one specific class. Furthermore, representative previous analyses in sponges, ctenophores, and cnidarians could not identify several classes in those nonbilaterian metazoan taxa. Consequently, it is currently unknown when certain homeodomain protein classes first evolved during animal evolution. Here, we investigate representatives of the sister group to all remaining bilaterians, the Xenacoelomorpha. We analyzed three acoel, one nemertodermatid, and one Xenoturbella transcriptomes and identified their expressed homeodomain protein content. We report the identification of representatives of all 11 classes of animal homeodomain transcription factors in Xenacoelomorpha and we describe and classify their homeobox genes relative to the established animal homeodomain protein families. Our findings suggest that the genome of the last common ancestor of bilateria contained the full set of these gene classes, supporting the subsequent diversification of bilaterians.

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

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.