Palps across the tree - the neuronal innervation and development of sensory head appendages in Annelida.

Polychaetes inhabit a wide variety of habitats and show a great morphological diversity. In this context, a key morphological structure for adapting to their individual lifestyles and ecological niches are the prominent head appendages. In the last years more and more studies focused on the mainly sensory annelid head appendages - namely the antennae, palps, buccal lips and cirri - to unravel the evolutionary origin and phylogeny of Annelida. Unfortunately, comparable data for most of the polychaete families are lacking so far, especially when it comes to features of the larval anterior nervous system and the related innervation and potential homology of these head appendages. In this study, we therefore use an integrative morphological approach including immunohistochemistry and confocal laser scanning microscopy in combination with histological serial sections and 3D-visualizations. With special focus on the palp-like appendages, our data provides a closer look into the development of the larval anterior nervous system and the related sensory structures of three polychaete families representing major groups of the annelid tree of life. Hence, we investigate members of the palaeoannelid Magelonidae as well as basally-branching Amphinomidae, and the pleistoannelid Spionidae forming a taxon deeply nested within Sedentaria. Our comparative data of larval and adult neuronal features support the homology of feeding-palps across the annelid tree. Furthermore, our observations show that larval palps gradually transform into the adult ones while keeping a very similar neuronal innervation pattern. Solely for Amphinomidae a loss of larval palps during ontogenesis has to be assumed. Therefore, our investigations uncover important and so far unknown details in terms of structural homology across Annelida and provide important results necessary for our understanding of annelid evolution.

Ultrastructure of cerebral eyes in Oweniidae and Chaetopteridae (Annelida) - implications for the evolution of eyes in Annelida.

BACKGROUND: Recent phylogenomic studies have revealed a robust, new hypothesis of annelid phylogeny. Most surprisingly, a few early branching lineages formed a basal grade, whereas the majority of taxa were categorized as monophyletic Pleistoannelida. Members of these basal groups show a comparatively simple organization lacking certain characters regarded to be annelid specific. Thus, the evolution of organ systems and the characteristics probably present in the last common annelid ancestor require reevaluation. With respect to light-sensitive organs, a pair of simple larval eyes is regarded as being present in their last common ancestor. However, the evolutionary origin and structure of adult eyes remain obscure. Typically, adult eyes are multicellular pigment cups or pinhole eyes with or without a lens comprising rhabdomeric photoreceptor cells (PRCs) and pigmented supportive cells (PSCs) in converse design. However, in the most basal lineages, eyes are only present in a few taxa, and thus far, their ultrastructure is unknown. RESULTS: Ultrastructural investigations of members of Oweniidae and Chaetopteridae reveal a corresponding design of adult cerebral eyes and PRCs. The eyes in species of these groups are simple pigment spot eyes, either forming a flat patch or embedded in a tube-like invagination. They are part of the epidermis and comprise two cell types, PSCs and rhabdomeric PRCs. Both cell types bear microvilli and one more or less reduced cilium. However, the PRCs showed only a moderate increase in the apical membrane surface in the form of irregularly arranged microvilli intermingling with those of the PSCs; a densely arranged brush border of rhabdomeric microvilli was absent. Additionally, both cell types show certain characteristics elsewhere observable in typical epidermal supportive cells. CONCLUSIONS: These findings shed new light on the evolutionary history of adult eyes in Annelida. Most likely, the adult eye of the annelid stem species was a pair of simple pigment spot eyes with only slightly specialized PSCs and PRCs being an integrative part of the epidermis. As is the case for the nuchal organs, typical pigment cup adult eyes presumably evolved later in the annelid phylogeny, namely, in the stem lineages of Amphinomida and Pleistoannelida.

Getting two birds with one stone: Combining immunohistochemistry and Azan staining in animal morphology.

Classical histological stained sections have the disadvantage that fine structures, like individual neurites, or specific macromolecules, like neurotransmitters cannot be visualized. Due to its highly specific staining of only one target molecule within the cell, the visualization of delicate structures, which would be superimposed by other tissue layers in classical Azan staining, is possible with immunohistochemistry. However, using immunohistological methods not all tissues of a specimen can be visualized at once. In contrast, density specific stains like Azan allow for a whole staining of the tissues. We provide a step by step protocol of how to combine immunohistochemistry and Azan staining in the same serial paraffin sections. The combination of both methods allows for a highly detailed investigation of structures of interest. The spatial detection of the previous, to Azan staining, gained antibody-labeled signal allows for a much better understanding of animal organ systems. By using serial sections, it is possible to create an aligned image stack that is both Azan stained and also antibody-labeled. Thus enabling a correlative approach that bridges traditional histology with immunohistochemistry in animal morphology.

Integrative anatomical study of the branched annelid Ramisyllis multicaudata (Annelida, Syllidae).

The sponge-dwelling Syllidae Ramisyllis multicaudata and Syllis ramosa are the only annelid species for which a branched body with one head and multiple posterior ends is known. In these species, the head is located deep within the sponge, and the branches extend through the canal system of their host. The morphology of these creatures has captivated annelid biologists since they were first discovered in the late XIXth century, and their external characteristics have been well documented. However, how their branched bodies fit within their symbiotic host sponges and how branches translate into internal anatomy has not been documented before. These features are crucially relevant for understanding the body of these animals, and therefore, the aim of this study was to investigate these aspects. In order to assess these questions, live observation, as wells as histology, immunohistochemistry, micro-computed tomography, and transmission electron microscopy techniques were used on specimens of R. multicaudata. By using these techniques, we show that the complex body of R. multicaudata specimens extends greatly through the canal system of their host sponges. We demonstrate that iterative external bifurcation of the body is accompanied by the bifurcation of the longitudinal organ systems that are characteristic of annelids. Additionally, we also highlight that the bifurcation process leaves an unmistakable fingerprint in the form of newly-described "muscle bridges." These structures theoretically allow one to distinguish original and derived branches at each bifurcation. Last, we characterize some of the internal anatomical features of the stolons (reproductive units) of R. multicaudata, particularly their nervous system. Here, we provide the first study of the internal anatomy of a branched annelid. This information is not only crucial to deepen our understanding of these animals and their biology, but it will also be key to inform future studies that try to explain how this morphology evolved.

May the palps be with you - new insights into the evolutionary origin of anterior appendages in Terebelliformia (Annelida).

BACKGROUND: Head appendages in Annelida contribute significantly to the immense morphological diversity in this spiralian taxon. Nevertheless, the evolutionary origin of annelid antennae, palps, cirri and tentacles are part of vast theories and debates that took place over decades. One of these heavily discussed groups are the Terebelliformia, which bear numerous anterior tentacles originating from different regions of the head. The question, whether these tentacles are homologous to feeding palps in other annelids or if these structures evolved convergently in terebellids and the remaining taxa, has been highly debated in the past. RESULTS: By using morphological methods including immunohistochemistry, confocal microscopy, Azan-stained serial sections and 3D-visualisation, we are able to shed new light and a fresh look on the old question of the evolutionary origin of the buccal tentacles and their associated head structures in Terebelliformia. Our investigations show that the brains of the ampharetid Hypania invalida and the aulophora larvae of Lanice conchilega (Terebellidae) consist of a dorsal, more prominent and a more slender, ventral brain region. Neurite bundles innervating the buccal tentacles split off from the ventral and dorsal root within the ventral brain region and thus originate from the dorsal and ventral root of the circumoesophageal connectives. Hence, the observed neurite bundles fulfil the morphological criteria for the innervating neurite bundles of feeding palps known from Paleoannelida. CONCLUSIONS: We disagree with former conclusions that buccal tentacles are part of the alimentary canal. Based on the presented data, the buccal tentacles of terebelliform taxa are innervated by neurite bundles and can be homologized with peristomial feeding palps of other Annelida. Our comparative investigations reveal important insights into morphological changes during the evolution of anterior head appendages in Terebelliformia and Annelida in general. Nevertheless, our analyses also illustrate the gaps in knowledge and that more investigations throughout the annelid tree are necessary to explain and understand the huge diversity of annelid anterior appendages.

The anatomy and development of the nervous system in Magelonidae (Annelida) - insights into the evolution of the annelid brain.

BACKGROUND: The annelid anterior central nervous system is often described to consist of a dorsal prostomial brain, consisting of several commissures and connected to the ventral ganglionic nerve cord via circumesophageal connectives. In the light of current molecular phylogenies, our assumptions on the primary design of the nervous system in Annelida has to be reconsidered. For that purpose we provide a detailed investigation of the adult nervous system of Magelonidae - a putatively basally branching annelid family - and studied early stages of the development of the latter. RESULTS: Our comparative investigation using an integrative morphological approach shows that the nervous system of Magelonidae is located inside the epidermis. The brain is composed of an anterior compact neuropil and posteriorly encircles the prostomial coelomic cavities. From the brain two lateral medullary cords branch off which fuse caudally. Prominent brain structures such as nuchal organs, ganglia or mushroom bodies are absent and the entire nervous system is medullary. Our investigations also contradict previous investigations and present an updated view on established assumptions and descriptions. CONCLUSION: The comprehensive dataset presented herein enables a detailed investigation of the magelonid anterior central nervous system for the first time. The data reveal that early in annelid evolution complexity of brains and anterior sensory structures rises. Polymorphic neurons in clusters and distinct brain parts, as well as lateral organs - all of which are not present in outgroup taxa and in the putative magelonid sister group Oweniidae - already evolved in Magelonidae. Commissures inside the brain, ganglia and nuchal organs, however, most likely evolved in the stem lineage of Amphinomidae + Sipuncula and Pleistoannelida (Errantia+ Sedentaria). The investigation demonstrates the necessity to continuously question established descriptions and interpretations of earlier publications and the need for transparent datasets. Our results also hint towards a stronger inclusion of larval morphology and developmental investigations in order to understand adult morphological features, not only in Annelida.

The central nervous system of Oweniidae (Annelida) and its implications for the structure of the ancestral annelid brain.

BACKGROUND: Recent phylogenomic analyses congruently reveal a basal clade which consists of Oweniidae and Mageloniidae as sister group to the remaining Annelida. These results indicate that the last common ancestor of Annelida was a tube-dwelling organism. They also challenge traditional evolutionary hypotheses of different organ systems, among them the nervous system. In textbooks the central nervous system is described as consisting of a ganglionic ventral nervous system and a dorsally located brain with different tracts that connect certain parts of the brain to each other. Only limited information on the fine structure, however, is available for Oweniidae, which constitute the sister group (possibly together with Magelonidae) to all remaining annelids. RESULTS: The brain of Oweniidae is ring- shaped and basiepidermal. Ganglia, higher brain centers or complex sensory organs do not exist; instead the central nervous system is medullary. Posterior to the brain the ventral medullary cord arises directly from the ventral region of the brain in Myriowenia sp. while in Owenia fusiformis two medullary cords arise perpendicular to the brain ring, extend caudally and fuse posterior. The central nervous system is composed of a central neuropil and surrounding somata of the neurons. According to ultrastructural and histological data only one type of neuron is present in the central nervous system. CONCLUSION: The central nervous system of Oweniidae is the simplest in terms of enlargement of the dorsal part of the brain and neuron distribution found among Annelida. Our investigation suggests that neither ganglia nor commissures inside the brain neuropil or clusters of polymorphic neurons were present in the annelid stem species. These structures evolved later within Annelida, most likely in the stem lineage of Amphinomidae, Sipuncula and Pleistoannelida. Palps were supposedly present in the last common ancestor of annelids and innervated by two nerves originating in the dorsal part of the brain. A broader comparison with species of each major spiralian clade shows the medullary nervous system to be a common feature and thus possibly representing the ancestral state of the spiralian nervous system. Moreover, ganglia and clusters of polymorphic neurons seemingly evolved independently in the compared taxa of Spiralia and Annelida.

Convergent evolution of the ladder-like ventral nerve cord in Annelida.

BACKGROUND: A median, segmented, annelid nerve cord has repeatedly been compared to the arthropod and vertebrate nerve cords and became the most used textbook representation of the annelid nervous system. Recent phylogenomic analyses, however, challenge the hypothesis that a subepidermal rope-ladder-like ventral nerve cord (VNC) composed of a paired serial chain of ganglia and somata-free connectives represents either a plesiomorphic or a typical condition in annelids. RESULTS: Using a comparative approach by combining phylogenomic analyses with morphological methods (immunohistochemistry and CLSM, histology and TEM), we compiled a comprehensive dataset to reconstruct the evolution of the annelid VNC. Our phylogenomic analyses generally support previous topologies. However, the so far hard-to-place Apistobranchidae and Psammodrilidae are now incorporated among the basally branching annelids with high support. Based on this topology we reconstruct an intraepidermal VNC as the ancestral state in Annelida. Thus, a subepidermal ladder-like nerve cord clearly represents a derived condition. CONCLUSIONS: Based on the presented data, a ladder-like appearance of the ventral nerve cord evolved repeatedly, and independently of the transition from an intraepidermal to a subepidermal cord during annelid evolution. Our investigations thereby propose an alternative set of neuroanatomical characteristics for the last common ancestor of Annelida or perhaps even Spiralia.

Early evolution of radial glial cells in Bilateria.

Bilaterians usually possess a central nervous system, composed of neurons and supportive cells called glial cells. Whereas neuronal cells are highly comparable in all these animals, glial cells apparently differ, and in deuterostomes, radial glial cells are found. These particular secretory glial cells may represent the archetype of all (macro) glial cells and have not been reported from protostomes so far. This has caused controversial discussions of whether glial cells represent a homologous bilaterian characteristic or whether they (and thus, centralized nervous systems) evolved convergently in the two main clades of bilaterians. By using histology, transmission electron microscopy, immunolabelling and whole-mount in situ hybridization, we show here that protostomes also possess radial glia-like cells, which are very likely to be homologous to those of deuterostomes. Moreover, our antibody staining indicates that the secretory character of radial glial cells is maintained throughout their various evolutionary adaptations. This implies an early evolution of radial glial cells in the last common ancestor of Protostomia and Deuterostomia. Furthermore, it suggests that an intraepidermal nervous system-composed of sensory cells, neurons and radial glial cells-was probably the plesiomorphic condition in the bilaterian ancestor.

On the role of the proventricle region in reproduction and regeneration in Typosyllis antoni (Annelida: Syllidae).

BACKGROUND: Syllids are a species rich annelid family possessing remarkable regenerative ability, which is not only the response after traumatic injury, but also a key step during the life cycle of several syllid taxa. In these animals the posterior part of the body becomes an epitoke and is later detached as a distinct unit named stolon. Such a sexual reproductive mode is named schizogamy or stolonization. The prostomium and the proventricle, a modified foregut structure, have been proposed to have a control function during this process, though the concrete mechanisms behind it have never been elucidated. RESULTS: By using different experimental set-ups, histology and immunohistochemistry combined with subsequent cLSM analyzes, we investigate and document the regeneration and stolonization in specimens of Typosyllis antoni that were amputated at different levels throughout the antero-posterior body axis. The removal of the anterior end including the proventricle implies an incomplete anterior regeneration as well as severe deviations from the usual reproductive pattern, i.e. accelerated stolonization, masculinization and the occurrence of aberrant stolons. The detailed anatomy of aberrant stolons is described. A histological study of the proventricle revealed no signs of glandular or secretory structures. The ventricle and the caeca are composed of glandular tissue but they are not involved in the reproductive and regenerative processes. CONCLUSIONS: As in other investigated syllids, the proventricle region has a significant role during stolonization and reproduction processes in Typosyllis antoni. When the proventricle region is absent, anterior and posterior regeneration are considerably deviated from the general patterns. However, proventricle ultrastructure does not show any glandular component, thereby questioning a direct involvement of this organ itself in the control of reproduction and regeneration. Our findings offer a comprehensive starting point for further studies of regeneration and reproductive control in syllids as well as annelids in general.

Myoanatomy and anterior muscle regeneration of the fireworm Eurythoe cf. complanata (Annelida: Amphinomidae).

Amphinomidae or so-called "fireworms" are known for their inflammatory substances and their regeneration ability. Recent transcriptome-based molecular analyses revealed that these remarkable annelids are a basal branching taxon outside the annelid main radiation (Pleistoannelida). Although several studies dealing with analyses of the morphology of these annelids have been published, detailed investigations of the anterior muscle regeneration and the musculature in general are largely lacking for amphinomids. Using histology, phalloidin labeling together with subsequent confocal laser scanning microscopy (cLSM), and further light microscopic image acquisition of different regeneration stages, we here present the first morphological study describing the myoanatomy and muscular regeneration. During anterior muscular regeneration, longitudinal muscle bundles develop prior to transverse muscle fibers and segment boundaries. Additionally, Eurythoe cf. complanata develops an independent muscular ring surrounding the mouth opening in an early stage of regeneration. Detailed investigation of adult body wall musculature and the parapodial muscle complex in amphinomids show that E. cf. complanata bears well-developed dorsal and ventral longitudinal muscle bundles as well as outer transverse muscles comparable to the pattern described for several Pleistoannelida. Furthermore, the biramous parapodia possess a complex meshwork of distinct muscle fibers allowing detailed comparisons with other annelid families.

Observations and Experiments on the Biology and Life History of Riseriellus occultus (Heteronemertea: Lineidae).

Studies on the biology and life history of nemerteans are scarce, mostly because these animals are nocturnal. In order to broaden the knowledge base on the life history of nemerteans as a prerequisite for comparative analyses, we studied a population of Riseriellus occultus (Heteronemertea: Lineidae) inhabiting the rocky intertidal in southern Brittany near Concarneau (France) for more than 10 years. Our studies show that R. occultus is an iteroparous, perennial species exclusively inhabiting rocky shore crevices that result from onionskin weathering of the granite. From September through October R. occultus reproduces by external fertilization and develops via a planktonic pilidium larva, which, under laboratory conditions, metamorphoses after about six weeks. Adults of R. occultus are nocturnal macrophagous predators that preferentially feed on the gastropods Gibbula umbilicalis and Patella species, but also consume the bivalve Mytilus edulis. Since R. occultus devours the snail inside the shell, we fixed individuals while feeding, and serially sectioned them. Reconstruction of the sections shows that R. occultus swallows the entire soft body and finally detaches the columellar muscle from the shell. Estimates on the density of R. occultus inside the rock crevices provide evidence for clustered distribution and locally high abundance on the rocky shore. These data strongly suggest that R. occultus affects the structure of the rocky shore gastropod community. Although our data are still fragmentary with respect to the ecology of this species and its role in the local food web, our knowledge has grown to such extent that R. occultus can now be regarded as one of the few well characterized nemertean species.

The nervous systems of basally branching nemertea (palaeonemertea).

In recent years, a lot of studies have been published dealing with the anatomy of the nervous system in different spiralian species. The only nemertean species investigated in this context probably shows derived characters and thus the conditions found there are not useful in inferring the relationship between nemerteans and other spiralian taxa. Ingroup relationships within Nemertea are still unclear, but there is some agreement that the palaeonemerteans form a basal, paraphyletic grade. Thus, palaeonemertean species are likely the most informative when comparing with other invertebrate groups. We therefore analyzed the nervous system of several palaeonemertean species by combining histology and immunostaining. 3D reconstructions based on the aligned slices were performed to get an overall impression of the central nervous system, and immunohistochemistry was chosen to reveal fine structures and to be able to compare the data with recently published results. The insights presented here permit a first attempt to reconstruct the primary organization of the nemertean nervous system. This comparative analysis allows substantiating homology hypotheses for nerves of the peripheral nervous system. This study also provides evidence that the nemertean brain primarily consists of two lobes connected by a strong ventral commissure and one to several dorsal commissures. During nemertean evolution, the brain underwent continuous compartmentalization into a pair of dorsal and ventral lobes interconnected by commissures and lateral tracts. Given that this conclusion can be corroborated by cladistic analyses, nemerteans should share a common ancestor with spiralians that primarily have a simple brain consisting of paired medullary, frontally commissurized and reinforced cords. Such an organization resembles the situation found in presumably basally branching annelids or mollusks.

Lophotrochozoan neuroanatomy: An analysis of the brain and nervous system of Lineus viridis(Nemertea) using different staining techniques.

BACKGROUND: The now thriving field of neurophylogeny that links the morphology of the nervous system to early evolutionary events relies heavily on detailed descriptions of the neuronal architecture of taxa under scrutiny. While recent accounts on the nervous system of a number of animal clades such as arthropods, annelids, and molluscs are abundant, in depth studies of the neuroanatomy of nemerteans are still wanting. In this study, we used different staining techniques and confocal laser scanning microscopy to reveal the architecture of the nervous system of Lineus viridis with high anatomical resolution. RESULTS: In L. viridis, the peripheral nervous system comprises four distinct but interconnected nerve plexus. The central nervous system consists of a pair of medullary cords and a brain. The brain surrounds the proboscis and is subdivided into four voluminous lobes and a ring of commissural tracts. The brain is well developed and contains thousands of neurons. It does not reveal compartmentalized neuropils found in other animal groups with elaborate cerebral ganglia. CONCLUSIONS: The detailed analysis of the nemertean nervous system presented in this study does not support any hypothesis on the phylogenetic position of Nemertea within Lophotrochozoa. Neuroanatomical characters that are described here are either common in other lophotrochozoan taxa or are seemingly restricted to nemerteans. Since detailed descriptions of the nervous system of adults in other nemertean species have not been available so far, this study may serve as a basis for future studies that might add data to the unsettled question of the nemertean ground pattern and the position of this taxon within the phylogenetic tree.