Comparative development of the serotonin- and FMRFamide-immunoreactive components of the nervous system in two distantly related ribbon worm species (Nemertea, Spiralia).

Introduction: Neurodevelopment in larval stages of non-model organisms, with a focus on the serotonin- and FMRFamide-immunoreactive components, has been in the focus of research in the recent past. However, some taxonomic groups remain understudied. Nemertea (ribbon worms) represent such an understudied clade with only few reports on nervous system development mostly from phylogenetically or developmentally derived species. It would be insightful to explore neurodevelopment in additional species to be able to document the diversity and deduce common patterns to trace the evolution of nervous system development. Methods: Fluorescent immunohistochemical labeling with polyclonal primary antibodies against serotonin and FMRF-amide and a monoclonal antibody against synapsin performed on series of fixed larval stages of two nemertean species Cephalothrix rufifrons (Archinemertea, Palaeonemertea) and Emplectonema gracile (Monostilifera, Hoplonemertea) were analyzed with confocal laser scanning microscopy. Results: This contribution gives detailed accounts on the development of the serotonin- and FMRFamide-immunoreactive subsets of the nervous system in two nemertean species from the first appearance of the respective signals. Additionally, data on synapsin-like immunoreactivity illustrates the general structure of neuropil components. Events common to both investigated species are the appearance of serotonin-like immunoreactive signals before the appearance of FMRF-like immunoreactive signals and the strict progression of the development of the lateral nerve cords from the anteriorly located, ring-shaped brain toward the posterior pole of the larva. Notable differences are (1) the proboscis nervous system that is developing much earlier in investigated larval stages of E. gracile and (2) distinct early, but apparently transient, serotonergic neurons on the frontal and caudal pole of the larva in E. gracile that seem to be absent in C. rufifrons. Discussion: According to the results from this investigation and in line with previously published accounts on nervous system development, the hypothetical last common ancestor of Nemertea had a ring-shaped brain arranged around the proboscis opening, from which a pair of ventro-lateral nerve cords develops in anterior to posterior progression. Early frontal and caudal serotonergic neurons that later degenerate or cease to express serotonin are an ancestral character of Nemertea that they share with several other spiralian clades.

Genome Size Dynamics in Marine Ribbon Worms (Nemertea, Spiralia).

Nemertea is a phylum consisting of 1300 mostly marine species. Nemertea is distinguished by an eversible muscular proboscis, and most of the species are venomous. Genomic resources for this phylum are scarce despite their value in understanding biodiversity. Here, we present genome size estimates of Nemertea based on flow cytometry and their relationship to different morphological and developmental traits. Ancestral genome size estimations were done across the nemertean phylogeny. The results increase the available genome size estimates for Nemertea three-fold. Our analyses show that Nemertea has a narrow genome size range (0.43-3.89 pg) compared to other phyla in Lophotrochozoa. A relationship between genome size and evolutionary rate, developmental modes, and habitat was found. Trait analyses show that the highest evolutionary rate of genome size is found in upper intertidal, viviparous species with direct development. Despite previous findings, body size in nemerteans was not correlated with genome size. A relatively small genome (1.18 pg) is assumed for the most recent common ancestor of all extant nemerteans. The results provide an important basis for future studies in nemertean genomics, which will be instrumental to understanding the evolution of this enigmatic and often neglected phylum.

Proteo-Transcriptomic Analysis Identifies Potential Novel Toxins Secreted by the Predatory, Prey-Piercing Ribbon Worm Amphiporus lactifloreus.

Nemerteans (ribbon worms) employ toxins to subdue their prey, but research thus far has focused on the small-molecule components of mucus secretions and few protein toxins have been characterized. We carried out a preliminary proteotranscriptomic analysis of putative toxins produced by the hoplonemertean Amphiporus lactifloreus (Hoplonemertea, Amphiporidae). No variants were found of known nemertean-specific toxin proteins (neurotoxins, cytotoxins, parbolysins or nemertides) but several toxin-like transcripts were discovered, expressed strongly in the proboscis, including putative metalloproteinases and sequences resembling sea anemone actitoxins, crown-of-thorn sea star plancitoxins, and multiple classes of inhibitor cystine knot/knottin family proteins. Some of these products were also directly identified in the mucus proteome, supporting their preliminary identification as secreted toxin components. Two new nemertean-typical toxin candidates could be described and were named U-nemertotoxin-1 and U-nemertotoxin-2. Our findings provide insight into the largely overlooked venom system of nemerteans and support a hypothesis in which the nemertean proboscis evolved in several steps from a flesh-melting organ in scavenging nemerteans to a flesh-melting and toxin-secreting venom apparatus in hunting hoplonemerteans.

A simple method for long-term vital-staining of ciliated epidermal cells in aquatic larvae.

Observing the process of growth and differentiation of tissues and organs is of crucial importance for the understanding of the evolution of organs in animals. Unfortunately, it is notoriously difficult to continuously monitor developmental processes due to the extended time they take. Long-term labeling of the tissues of interest represents a promising alternative to raise these pivotal data. In the case of the prototroch, a band of ciliated cells typical of marine, planktotrophic trochophora larvae, we were able to apply a long-term fluorescent vital-staining to the prototroch cells that remains detectable throughout further larval life. We were able to stain ciliated cells of planktonic larvae from different spiralian clades by using long-chain dialkylcarbocyanine dyes that are detectable in different fluorescent emission spectra in combination with a non-ionic surfactant. The larvae survived and developed normally, their ciliated cells retaining the originally applied fluorescent labels. Combined with additional fluorescent staining of the larvae after fixation, we provide an easy, versatile, and broadly applicable method to investigate the processes of the differentiation of epidermal organs in various aquatic larvae.

Development of the Nervous System of Carinina ochracea (Palaeonemer-tea, Nemertea).

The various clades of Lophotrochozoa possess highly disparate adult morphologies. Most of them, including Nemertea (ribbon worms), are postulated to develop via a pelagic larva of the trochophora type, which is regarded as plesiomorphic in Lophotrochozoa. With respect to the nervous system, the trochophora larva displays a set of stereotypic features, including an apical organ and trochal neurites, both of which are lost at the onset of metamorphosis. In the investigated larvae of Nemertea, the nervous system is somewhat divergent from the postulated hypothetical trochophore-like pattern. Moreover, no detailed data is available for the "hidden" trochophore larva, the hypothetical ancestral larval type of palaeonemertean species. Therefore, the development of the nervous system in the larva of Carinina ochracea, a basally branching palaeonemertean species, was studied by means of immunofluorescence and confocal laserscanning microscopy. Like in the other investigated nemertean larvae, the prospective adult central nervous system in C. ochracea develops in an anterior to posterior direction, as an anterior brain with paired longitudinal nerve cords. Thus, development of the adult nervous system in Nemertea is largely congruent with currently accepted hypotheses of nervous system development in Spiralia. In early development, transitory apical, serotonin-like immunoreactive flask-shaped cells are initially present, but the trochal neurites that have been considered as pivotal to lophotrochozoan development, are absent. In the light of the above stated hypothesis, trochal neurites have to be interpreted as reduced in Nemertea. On the other hand, due to the unsettled systematic status of Palaeonemertea, more comparative data are desirable to answer the remaining questions regarding the evolution of nervous system development in Nemertea.

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.

Arenogigas armoricus, a New Genus and Species of a Monostiliferous Hoplonemertean (Nemertea) from the North-West Coast of France.

A new genus and species of an endobenthic, unusually large eumonostiliferous hoplonemertean, Arenogigas armoricus gen. et sp. nov., is described from an intertidal sandflat in Pouldohan Bay near Concarneau, France. Morphological characters of the species and genus include a prominent connective tissue that divides the anterior longitudinal musculature, an extremely branched vascular system, the absence of a pre-cerebral septum, a pair of eyes situated at the anterior tip of the head, small cerebral organs positioned far anterior to the brain, 10 proboscidial nerves, and nine accessory stylet pouches.

Disentangling ribbon worm relationships: multi-locus analysis supports traditional classification of the phylum Nemertea.

The phylogenetic relationships of selected members of the phylum Nemertea are explored by means of six markers amplified from the genomic DNA of freshly collected specimens (the nuclear 18S rRNA and 28S rRNA genes, histones H3 and H4, and the mitochondrial genes 16S rRNA and cytochrome c oxidase subunit I). These include all previous markers and regions used in earlier phylogenetic analyses of nemerteans, therefore acting as a scaffold to which one could pinpoint any previously published study. Our results, based on analyses of static and dynamic homology concepts under probabilistic and parsimony frameworks, agree in the non-monophyly of Palaeonemertea and in the monophyly of Heteronemerta and Hoplonemertea. The position of Hubrechtella and the Pilidiophora hypothesis are, however, sensitive to analytical method, as is the monophyly of the non-hubrechtiid palaeonemerteans. Our results are, however, consistent with the main division of Hoplonemertea into Polystilifera and Monostilifera, the last named being divided into Cratenemertea and Distromatonemertea, as well as into the main division of Heteronemertea into Baseodiscus and the remaining species. The study also continues to highlight the deficient taxonomy at the family and generic level within Nemertea and sheds light on the areas of the tree that require further refinement. © The Willi Hennig Society 2011.

Phylogeny and mitochondrial gene order variation in Lophotrochozoa in the light of new mitogenomic data from Nemertea.

BACKGROUND: The new animal phylogeny established several taxa which were not identified by morphological analyses, most prominently the Ecdysozoa (arthropods, roundworms, priapulids and others) and Lophotrochozoa (molluscs, annelids, brachiopods and others). Lophotrochozoan interrelationships are under discussion, e.g. regarding the position of Nemertea (ribbon worms), which were discussed to be sister group to e.g. Mollusca, Brachiozoa or Platyhelminthes. Mitochondrial genomes contributed well with sequence data and gene order characters to the deep metazoan phylogeny debate. RESULTS: In this study we present the first complete mitochondrial genome record for a member of the Nemertea, Lineus viridis. Except two trnP and trnT, all genes are located on the same strand. While gene order is most similar to that of the brachiopod Terebratulina retusa, sequence based analyses of mitochondrial genes place nemerteans close to molluscs, phoronids and entoprocts without clear preference for one of these taxa as sister group. CONCLUSION: Almost all recent analyses with large datasets show good support for a taxon comprising Annelida, Mollusca, Brachiopoda, Phoronida and Nemertea. But the relationships among these taxa vary between different studies. The analysis of gene order differences gives evidence for a multiple independent occurrence of a large inversion in the mitochondrial genome of Lophotrochozoa and a re-inversion of the same part in gastropods. We hypothesize that some regions of the genome have a higher chance for intramolecular recombination than others and gene order data have to be analysed carefully to detect convergent rearrangement events.

Larval development with transitory epidermis in Paranemertes peregrina and other hoplonemerteans.

We describe development of the hoplonemertean Paranemertes peregrina from fertilization to juvenile, using light, confocal, and electron microscopy. We discovered that the uniformly ciliated lecithotrophic larva of this species has a transitory epidermis, which is gradually replaced by the definitive epidermis during the course of planktonic development. The approximately 90 large multiciliated cleavage-arrested cells of the transitory larval epidermis become separated from each other by intercalating cells of the definitive epidermis, then gradually diminish in size and disappear more or less simultaneously. Rudiments of all major adult structures-the gut, proboscis, cerebral ganglia, lateral nerve cords, and cerebral organs-are already present in 4-day-old larvae. Replacement of the epidermis is the only overt metamorphic transformation of larval tissue; larval structures otherwise prefigure the juvenile body, which is complete in about 10 days at 7-10 degrees C. Our findings on development of digestive system, nervous system, and proboscis differ in several ways from previous descriptions of hoplonemertean development. We report development with transitory epidermis in two other species, review evidence from the literature, and suggest that this developmental type is the rule for hoplonemerteans. The hoplonemertean planuliform larva is fundamentally different both from the pilidium larva of the sister group to the Hoplonemertea, the Pilidiophora, and from the hidden trochophore of palaeonemerteans. We discuss the possible function and homology of the larval epidermis in development of other nemerteans and spiralians in general.

Ultrastructure and development of the rhabdomeric eyes in Lineus viridis (Heteronemertea, Nemertea).

Nemerteans are undoubtedly members of the Spiralia, although their phylogenetic relationships are still a matter of debate. The apparently acoelomate organization suggests a relationship with the platyhelminths, whereas the blood-vascular system has been interpreted as an equivalent to coelomic cavities of annelids, indicating a close relation between annelids and nemerteans. Like other spiralians, most nemertean species are known to have one or several pairs of rhabdomeric and subepidermally situated eyes when adult. The development of these eyes as well as the mode in which the eyes are multiplied is as yet unknown. This is the first attempt to investigate eye formation in a nemertean. In the heteronemertean Lineus viridis (Müller, 1774) the everse rhabdomeric eyes are located deeply underneath the epidermis and consist of a few pigment cells that form a cup-like structure with interdigitating processes that contain numerous pigment granules. In hatchlings, the optical cavity contains processes of 12 sensory cells, each bearing a single cilium and various microvilli. The perikarya of these cells are located distally from the pigment cup. During further development the number of cells increases. Eye development starts with a small anlage situated underneath the epidermis, irrespective of whether this is the first eye or any additional one. The anlage consists of five unpigmented cells and three dendritic processes, each bearing apical microvilli and a single cilium. There is no evidence for an epidermal origin of the eyes. In L. viridis eye formation resembles that described in platyhelminths in which eyes also develop as cerebral derivatives. Although this result has the potential to influence the discussion on the position of Nemertea, the data have to be interpreted with care, since development of L. viridis is derived within the Nemertea.