Cost-efficient PCR based DNA barcoding of marine invertebrate specimens with NovaSeq amplicon sequencing.

BACKGROUND: The marine environment harbors high biodiversity; however, it is poorly understood. Nucleotide sequence data of all marine organisms should be accumulated before natural and/or anthropogenic environmental changes jeopardize the marine environment. In this study, we report a cost-effective and easy DNA barcoding method. This method can be readily adopted without using library preparation kits. It includes multiplex PCR of short targets, indexing PCR, and outsourcing to a sequencing service using the NovaSeq system. METHODS AND RESULTS: We targeted four mitochondrial genes [cytochrome c oxidase subunit I (COI), COIII, 16S rRNA (16S), and 12S rRNA (12S)] and three nuclear genes [18S rRNA (18S), 28S rRNA (28S), internal transcribed spacer 2 (ITS2)] in 95 marine invertebrate specimens, which were primarily annelids. The primers, including adapters and indices for NovaSeq sequencing, were newly designed. Two PCR runs were conducted. The 1st PCR amplified specific loci with universal primers and the 2nd added sequencing adapters and indices to the 1st PCR products. The gene sequences obtained from the FASTQ files were subjected to BLAST search and phylogenetic analyses. One run using 95 specimens yielded sequences averaging 2816 bp per specimen for a total length of six loci. Nuclear genes were more successfully assembled compared with mitochondrial genes. A weak but significantly negative correlation was observed between the average length of each locus and success rate of the assembly. Some of the sequences were almost identical to the sequences obtained from specimens collected far from Japan, indicating the presence of potentially invasive species identified for the first time. CONCLUSIONS: We obtained gene sequences efficiently using next-generation sequencing rather than Sanger sequencing. Although this method requires further optimization to increase the success rate for some loci, it is used as a first step to select specimens for further analyses by determining the specific loci of the targets.

Transcriptomic profiling of immune-associated molecules in the coelomocytes of lugworm Arenicola marina (Linnaeus, 1758).

Organization and functioning of immune system remain unevenly studied in different taxa of lophotrochozoan animals. We analyzed transcriptomic data on coelomocytes of the lugworm Arenicola marina (Linnaeus, 1758; Annelida, Polychaeta) to gain insights into the molecular mechanisms involved in polychaete immunity. Coelomocytes are specialized motile cells populating coelomic fluid of annelids, responsible for cellular defense reactions and providing humoral immune factors. The transcriptome was enriched with immune-related transcripts by challenging the cells in vitro with lipopolysaccharides of Escherichia coli and Zymosan from Saccharomyces cerevisiae. Our analysis revealed a multifaceted and complex internal defense system of the lugworm. A. marina possesses orthologs of proto-complement-like factors: six thioester-containing proteins, a complement-like receptor, and a MASP-related serine protease (MReM2). A. marina coelomocytes employ pattern-recognition receptors to detect pathogens and regulate immune responses. Among them, there are 18 Toll-like receptors and various putative lectin-like proteins with evolutionary conserved and taxa-specific domains. C-type lectins and a novel family of Gal-binding and CUB domains containing receptors were the most abundant in the transcriptome. The array of pore-forming proteins in the coelomocytes was surprisingly reduced compared to that of other invertebrate species. We characterized a set of conserved proteins metabolizing reactive oxygen species and nitric oxide and expanded the arsenal of potential antimicrobial peptides. Phenoloxidase activity in immune cells of lugworm is mediated only by laccase enzyme. The described repertoire of immune-associated molecules provides valuable candidates for further functional and comparative research on the immunity of annelids.

A New Species of Branchellion (Hirudinea: Piscicolidae) Parasitizing the Gills of Short-Tail Stingrays (Batoidea: Dasyatidae) from the West Pacific.

A new fish leech, Branchellion brevicaudatae sp. n., is described based on specimens parasitizing the gills of the short-tail stingray, Bathytoshia brevicaudata (Hutton, 1875), collected from Japanese waters. The new species can be distinguished from other congeners by having: i) pulsating vesicles emerging from posterior base of branchiae, one pair per somite; ii) dorsal white spots, not arranged in longitudinal row; and iii) blackish body. A phylogenetic tree based on partial sequences of the mitochondrial cytochrome c oxidase subunit I gene from the new species and other piscicolid worms showed that the new species is sister to Branchellion torpedinis Savigny, 1822. This is the first record of Branchellion Savigny, 1822 from Japanese waters.

Sources of Polycyclic Aromatic Hydrocarbons in Fecal Pellets of a Marphysa Species (Annelida: Eunicidae) in the Yoro Tidal Flat, Japan.

The fecal pellets of Marphysa sp. E sensu Abe et al. (2019) (Annelida, Eunicidae) living in the Yoro tidal flat (Ichihara, Chiba, Japan) contain high levels of polycyclic aromatic hydrocarbons (PAHs), and the concentrations rapidly decrease over time. To investigate the origin of the high-concentration PAHs in the fecal pellets and food sources of the worms, the PAH concentrations, carbon and nitrogen stable isotope ratios (δ13C and δ15N), total organic carbon, and total nitrogen for two types of sediment (sands and reduced muds), fecal pellets, and the body of the worms were determined. The PAH concentrations and chemical properties of the fecal pellets were similar to those of the reduced muds (20-30 cm sediment depth). The δ13C, δ15N, and C/N values of reduced muds were the same as the typical values of terrestrial C3 plants, suggesting that reduced muds were derived from terrestrial plants. These data indicated that the worms selectively take up reduced muds containing high levels of PAHs. The δ13C and δ15N values of the worm bodies indicated that the worms did not use the organic carbon derived from terrestrial C3 plants as primary nutrition. Taking into consideration their selective uptake of reduced muds, excretion, and subsequent rapid decrease of PAHs in the fecal pellets, the worms could contribute to the remediation of chemical pollutants in the tidal flat sediments.

Novel BRICHOS-Related Antimicrobial Peptides from the Marine Worm Heteromastus filiformis: Transcriptome Mining, Synthesis, Biological Activities, and Therapeutic Potential.

Marine polychaetes represent an extremely rich and underexplored source of novel families of antimicrobial peptides (AMPs). The rapid development of next generation sequencing technologies and modern bioinformatics approaches allows us to apply them for characterization of AMP-derived genes and the identification of encoded immune-related peptides with the aid of genome and transcriptome mining. Here, we describe a universal bioinformatic approach based on the conserved BRICHOS domain as a search query for the identification of novel structurally unique AMP families in annelids. In this paper, we report the discovery of 13 novel BRICHOS-related peptides, ranging from 18 to 91 amino acid residues in length, in the cosmopolitan marine worm Heteromastus filiformis with the assistance of transcriptome mining. Two characteristic peptides with a low homology in relation to known AMPs-the α-helical amphiphilic linear peptide, consisting of 28 amino acid residues and designated as HfBRI-28, and the 25-mer ß-hairpin peptide, specified as HfBRI-25 and having a unique structure stabilized by two disulfide bonds-were obtained and analyzed as potential antimicrobials. Interestingly, both peptides showed the ability to kill bacteria via membrane damage, but mechanisms of their action and spectra of their activity differed significantly. Being non-cytotoxic towards mammalian cells and stable to proteolysis in the blood serum, HfBRI-25 was selected for further in vivo studies in a lethal murine model of the Escherichia coli infection, where the peptide contributed to the 100% survival rate in animals. A high activity against uropathogenic strains of E. coli (UPEC) as well as a strong ability to kill bacteria within biofilms allow us to consider the novel peptide HfBRI-25 as a promising candidate for the clinical therapy of urinary tract infections (UTI) associated with UPEC.

Modelling the effects of the pyrethroid insecticide cypermethrin on the life cycle of the soil dwelling annelid Enchytraeus crypticus, an original experimental design to calibrate a DEB-TKTD model.

The Dynamic Energy Budget theory (DEB) enables ecotoxicologists to model the effects of chemical stressors on organism life cycles through the coupling of toxicokinetic-toxicodynamic (TK-TD) models. While good progress has been made in the application of DEB-TKTD models for aquatic organisms, applications for soil fauna are scarce, due to the lack of dedicated experimental designs suitable for collecting the required time series effect data. Enchytraeids (Annelida: Clitellata) are model organisms in soil ecology and ecotoxicology. They are recognised as indicators of biological activity in soil, and chemical stress in terrestrial ecosystems. Despite this, the application of DEB-TKTD models to investigate the impact of chemicals has not yet been tested on this family. Here we assessed the impact of the pyrethroid insecticide cypermethrin on the life cycle of Enchytraeus crypticus. We developed an original experimental design to collect the data required for the calibration of a DEB-TKTD model for this species. E. crypticus presented a slow initial growth phase that has been successfully simulated with the addition of a size-dependent food limitation for juveniles in the DEB model. The DEB-TKTD model simulations successfully agreed with the data for all endpoints and treatments over time. The highlighted physiological mode of action (pMoA) for cypermethrin was an increase of the growth energy cost. The threshold for effects on survival was estimated at 73.14 mg kg- 1, and the threshold for effects on energy budget (i.e., sublethal effects) at 19.21 mg kg- 1. This study demonstrates that DEB-TKTD models can be successfully applied to E. crypticus as a representative soil species, and may improve the ecological risk assessment for terrestrial ecosystems, and our mechanistic understanding of chemical effects on non-target species.

Distribution of Siboglinids (Annelida, Siboglinidae) in the Laptev Sea and Adjacent Areas of the Arctic Basin.

Biodiversity in the Laptev Sea was assessed for gutless marine worms of the family Siboglinidae (Annelida), whose metabolism is provided by symbiotic bacteria that oxidize hydrogen sulfide and methane. Seven siboglinid species were found within the geographical boundaries of the Laptev Sea, and another species was found in an adjacent sector of the Arctic Basin. The largest number of finds and the greatest biological diversity of siboglinids were observed in the eastern part of the Laptev Sea in a field of numerous methane flares. One find was made in the estuary area of the Lena River at a depth of 25 m. A possible association of siboglinids with methane seepage areas is discussed.

Role of BMP signaling during early development of the annelid Capitella teleta.

The mechanisms regulating nervous system development are still unknown for a wide variety of taxa. In insects and vertebrates, bone morphogenetic protein (BMP) signaling plays a key role in establishing the dorsal-ventral (D-V) axis and limiting the neuroectoderm to one side of that axis, leading to speculation about the conserved evolution of centralized nervous systems. Studies outside of insects and vertebrates show a more diverse picture of what, if any role, BMP signaling plays in neural development across Bilateria. This is especially true in the morphologically diverse Spiralia (≈Lophotrochozoa). Despite several studies of D-V axis formation and neural induction in spiralians, there is no consensus for how these two processes are related, or whether BMP signaling may have played an ancestral role in either process. To determine the function of BMP signaling during early development of the spiralian annelid Capitella teleta, we incubated embryos and larvae in BMP4 protein for different amounts of time. Adding exogenous BMP protein to early-cleaving C. teleta embryos had a striking effect on formation of the brain, eyes, foregut, and ventral midline in a time-dependent manner. However, adding BMP did not block brain or VNC formation or majorly disrupt the D-V axis. We identified three key time windows of BMP activity. 1) BMP treatment around birth of the 3rd-quartet micromeres caused the loss of the eyes, radialization of the brain, and a reduction of the foregut, which we interpret as a loss of A- and C-quadrant identities with a possible trans-fate switch to a D-quadrant identity. 2) Treatment after the birth of micromere 4d induced formation of a third ectopic brain lobe, eye, and foregut lobe, which we interpret as a trans-fate switch of B-quadrant micromeres to a C-quadrant identity. 3) Continuous BMP treatment from late cleavage (4d â€‹+ â€‹12 â€‹h) through mid-larval stages resulted in a modest expansion of Ct-chrdl expression in the dorsal ectoderm and a concomitant loss of the ventral midline (neurotroch ciliary band). Loss of the ventral midline was accompanied by a collapse of the bilaterally-symmetric ventral nerve cord, although the total amount of neural tissue was not greatly affected. Our results compared with those from other annelids and molluscs suggest that BMP signaling was not ancestrally involved in delimiting neural tissue to one region of the D-V axis. However, the effects of ectopic BMP on quadrant-identity during cleavage stages may represent a non-axial organizing signal that was present in the last common ancestor of annelids and mollusks. Furthermore, in the last common ancestor of annelids, BMP signaling may have functioned in patterning ectodermal fates along the D-V axis in the trunk. Ultimately, studies on a wider range of spiralian taxa are needed to determine the role of BMP signaling during neural induction and neural patterning in the last common ancestor of this group. Ultimately, these comparisons will give us insight into the evolutionary origins of centralized nervous systems and body plans.

Segment number threshold determines juvenile onset of germline cluster expansion in Platynereis dumerilii.

Development of sexual characters and generation of gametes are tightly coupled with growth. Platynereis dumerilii is a marine annelid that has been used to study germline development and gametogenesis. P. dumerilii has germ cell clusters found across the body in the juvenile worms, and the clusters eventually form the gametes. Like other segmented worms, P. dumerilii grows by adding new segments at its posterior end. The number of segments reflect the growth state of the worms and therefore is a useful and measurable growth state metric to study the growth-reproduction crosstalk. To understand how growth correlates with progression of gametogenesis, we investigated germline development across several developmental stages. We discovered a distinct transition period when worms increase the number of germline clusters at a particular segment number threshold. Additionally, we found that keeping worms short in segment number, by manipulating environmental conditions or via amputations, supported a segment number threshold requirement for germline development. Finally, we asked if these clusters in P. dumerilii play a role in regeneration (as similar free-roaming cells are observed in Hydra and planarian regeneration) and found that the clusters were not required for regeneration in P. dumerilii, suggesting a strictly germline nature. Overall, these molecular analyses suggest a previously unidentified developmental transition dependent on the growth state of juvenile P. dumerilii leading to substantially increased germline expansion.

Distinct patterns of gene expression during regeneration and asexual reproduction in the annelid Pristina leidyi.

Regeneration, the ability to replace lost body parts, is a widespread phenomenon in the animal kingdom often connected to asexual reproduction or fission, since the only difference between the two appears to be the stimulus that triggers them. Both developmental processes have largely been characterized; however, the molecular toolkit and genetic mechanisms underlying these events remain poorly unexplored. Annelids, in particular the oligochaete Pristina leidyi, provide a good model system to investigate these processes as they show diverse ways to regenerate, and can reproduce asexually through fission under laboratory conditions. Here, we used a comparative transcriptomics approach based on RNA-sequencing and differential gene expression analyses to understand the molecular mechanisms involved in anterior regeneration and asexual reproduction. We found 291 genes upregulated during anterior regeneration, including several regeneration-related genes previously reported in other annelids such as frizzled, paics, and vdra. On the other hand, during asexual reproduction, 130 genes were found upregulated, and unexpectedly, many of them were related to germline development during sexual reproduction. We also found important differences between anterior regeneration and asexual reproduction, with the latter showing a gene expression profile more similar to that of control individuals. Nevertheless, we identified 35 genes that were upregulated in both conditions, many of them related to cell pluripotency, stem cells, and cell proliferation. Overall, our results shed light on the molecular mechanisms that control anterior regeneration and asexual reproduction in annelids and reveal similarities with other animals, suggesting that the genetic machinery controlling these processes is conserved across metazoans.

Endogenous Fluorescent Proteins in the Mucus of an Intertidal Polychaeta: Clues for Biotechnology.

The vast ocean holds many unexplored organisms with unique adaptive features that enable them to thrive in their environment. The secretion of fluorescent proteins is one of them, with reports on the presence of such compounds in marine annelids being scarce. The intertidal Eulalia sp. is an example. The worm secretes copious amounts of mucus, that when purified and concentrated extracts, yield strong fluorescence under UV light. Emission has two main maxima, at 400 nm and at 500 nm, with the latter responsible for the blue-greenish fluorescence. Combining proteomics and transcriptomics techniques, we identified ubiquitin, peroxiredoxin, and 14-3-3 protein as key elements in the mucus. Fluorescence was found to be mainly modulated by redox status and pH, being consistently upheld in extracts prepared in Tris-HCl buffer with reducing agent at pH 7 and excited at 330 nm. One of the proteins associated with the fluorescent signal was localized in secretory cells in the pharynx. The results indicate that the secretion of fluorescent proteinaceous complexes can be an important defense against UV for this dweller. Additionally, the internalization of fluorescent complexes by ovarian cancer cells and modulation of fluorescence of redox status bears important considerations for biotechnological application of mucus components as markers.

New Data on Distribution of Nereilinum murmanicum (Annelida, Siboglinidae) in the Barents Sea.

The article presents a report on the findings of representatives of frenulate pogonophorans Nereilinum murmanicum in the northern and central parts of the Barents Sea, which significantly expands the range of this species and provides guidance on its distribution in this basin. Here we present the coordinates of new finds with an indication of the depth. Find points were associated with data on known and potential hydrocarbon deposits.

Symbiosis in the Cambrian: enteropneust tubes from the Burgess Shale co-inhabited by commensal polychaetes.

The in situ preservation of animal behaviour in the fossil record is exceedingly rare, but can lead to unique macroecological and macroevolutionary insights, especially regarding early representatives of major animal clades. We describe a new complex ecological relationship from the middle Cambrian Burgess Shale (Raymond Quarry, Canada). More than 30 organic tubes were recorded with multiple enteropneust and polychaete worms preserved within them. Based on the tubicolous nature of fossil enteropneusts, we suggest that they were the tube builders while the co-preserved polychaetes were commensals. These findings mark, to our knowledge, the first record of commensalism within Annelida and Hemichordata in the entire fossil record. The finding of multiple enteropneusts sharing common tubes suggests that either the tubes represent reproductive structures built by larger adults, and the enteropneusts commonly preserved within are juveniles, or these enteropneusts were living as a pseudo-colony without obligate attachment to each other, and the tube was built collaboratively. While neither hypothesis can be ruled out, gregarious behaviour was clearly an early trait of both hemichordates and annelids. Further, commensal symbioses in the Cambrian may be more common than currently recognized.

Have the eyes of bioluminescent scale worms adapted to see their own light? A comparative study of eyes and vision in Harmothoe imbricata and Lepidonotus squamatus.

Annelids constitute a diverse phylum with more than 19,000 species, which exhibit greatly varying morphologies and lifestyles ranging from sessile detritivores to fast swimming active predators. The lifestyle of an animal is closely linked to its sensory systems, not least the visual equipment. Interestingly, many errantian annelid species from different families, such as the scale worms (Polynoidae), have two pairs of eyes on their prostomium. These eyes are typically 100-200 µm in diameter and structurally similar judged from their gross morphology. The polynoids Harmothoe imbricata and Lepidonotus squamatus from the North Atlantic are both benthic predators preying on small invertebrates but only H. imbricata can produce bioluminescence in its scales. Here, we examined the eye morphology, photoreceptor physiology and light-guided behaviour in these two scale worms to assess their visual capacity and visual ecology. The structure and physiology of the two pairs of eyes are remarkably similar within each species, with the only difference being the gaze direction. The photoreceptor physiology, however, differs between species. Both species express a single opsin in their eyes, but in H. imbricata the peak sensitivity is green shifted and the temporal resolution is lower, suggesting that the eyes of H. imbricata are adapted to detect their own bioluminescence. The behavioural experiments showed that both species are strictly night active but yielded no support for the hypothesis that H. imbricata is repelled by its own bioluminescence.

The contributions of enchytraeids and earthworms to the soil mineralization process in soils with fungicide.

Pesticides may harm soil organisms such as earthworms and enchytraeids, but knowledge is lacking on their relative sensitivity to these chemicals and the consequences on soil functions. The aim of this study was to assess the impact of exposure to a commercial fungicide formulation (Swing® Gold, containing dimoxystrobin and epoxiconazole) on the function of earthworms (Aporrectodea caliginosa) and enchytraeids (Enchytraeus buchholzi) in soil organic matter (SOM) mineralization. The soil organisms were incubated alone and together in a 26-day laboratory experiment. At the recommended field rate, the fungicide induced a decrease in the SOM mineralization and a delay in the maximum daily CO2 emissions compared to the control soil without fungicide. Soil fauna also influenced SOM mineralization with a higher cumulated CO2 release after 26 days in the control soil with earthworms (by 21%) than without fauna. When both earthworms and enchytraeids were present, SOM mineralization did not increase, and there was a negative effect on earthworm weight gain. Finally, an alteration of fauna influence with treatment was observed from day 19, meaning that the effect of fauna on SOM mineralization changed with fungicide treatment. Earthworms no longer promoted SOM mineralization when fungicide was present at three-fold the recommended field rate. The effects of enchytraeids on SOM mineralization were similar with and without fungicide exposure. This study underlines the importance of considering the relative sensitivity of soil organisms to environmental factors and interactions between them when assessing soil functioning.

A Transcriptomic Approach to the Metabolism of Tetrapyrrolic Photosensitizers in a Marine Annelid.

The past decade has seen growing interest in marine natural pigments for biotechnological applications. One of the most abundant classes of biological pigments is the tetrapyrroles, which are prized targets due their photodynamic properties; porphyrins are the best known examples of this group. Many animal porphyrinoids and other tetrapyrroles are produced through heme metabolic pathways, the best known of which are the bile pigments biliverdin and bilirubin. Eulalia is a marine Polychaeta characterized by its bright green coloration resulting from a remarkably wide range of greenish and yellowish tetrapyrroles, some of which have promising photodynamic properties. The present study combined metabolomics based on HPLC-DAD with RNA-seq transcriptomics to investigate the molecular pathways of porphyrinoid metabolism by comparing the worm's proboscis and epidermis, which display distinct pigmentation patterns. The results showed that pigments are endogenous and seemingly heme-derived. The worm possesses homologs in both organs for genes encoding enzymes involved in heme metabolism such as ALAD, FECH, UROS, and PPOX. However, the findings also indicate that variants of the canonical enzymes of the heme biosynthesis pathway can be species- and organ-specific. These differences between molecular networks contribute to explain not only the differential pigmentation patterns between organs, but also the worm's variety of novel endogenous tetrapyrrolic compounds.

Concentric Inclusions in the Intestinal Epithelium of Echiura Bonellia viridis: Structure and Possible Function.

The study of the anatomy and fine structure of Echiura is of great importance for understanding the biology of these animals, which lead a secretive life and dominate in various benthic communities. The first data on the organization of the siphonal part of the midgut of female Bonellia viridis were obtained by the methods of scanning and transmission electron microscopy. Unusual concentric inclusions similar in the ultrastructure to those described in other animals, e.g., in the gut of many nematode species and in the tegument of some cestodes, were first found in the cells of the midgut. It is known that, in these animals, the concentric inclusions play an important role in the binding of chemical agents inherent in redox environments. Interestingly, the individuals of B. viridis studied were found on the surface of a substrate devoid of redox environment signs. New results indicate the presence in B. viridis and, possibly, in all spoon worms, of preadaptations to life in redox environments. New data on the structure and composition of concentric inclusions will shed light on their origin and function.

The First Discovery of Pogonophora (Annelida, Siboglinidae) in the East Siberian Sea Coincides with the Areas of Methane Seeps.

Pogonophora or siboglinid tubeworms (Annelida, Siboglinidae) have been found in the East Siberian Sea for the first time. On the basis of the results of molecular phylogenetic analysis, the found specimens are presumably assigned to the genus Oligobrachia. The stations where the siboglinid tubeworms have been found are located in the area of methane seeps. This confirms the previously stated hypothesis about relationship of siboglinid tubeworm distribution with the areas of underwater methane seeps.

Morphological, cellular and molecular characterization of posterior regeneration in the marine annelid Platynereis dumerilii.

Regeneration, the ability to restore body parts after an injury or an amputation, is a widespread but highly variable and complex phenomenon in animals. While having fascinated scientists for centuries, fundamental questions about the cellular basis of animal regeneration as well as its evolutionary history remain largely unanswered. Here, we present a study of regeneration of the marine annelid Platynereis dumerilii, an emerging comparative developmental biology model, which, like many other annelids, displays important regenerative abilities. When P. dumerilii worms are amputated, they are able to regenerate the posteriormost differentiated part of their body and a stem cell-rich growth zone that allows the production of new segments replacing the amputated ones. We show that posterior regeneration is a rapid process that follows a well reproducible path and timeline, going through specific stages that we thoroughly defined. Wound healing is achieved one day after amputation and a regeneration blastema forms one day later. At this time point, some tissue specification already occurs, and a functional posterior growth zone is re-established as early as three days after amputation. Regeneration timing is only influenced, in a minor manner, by worm size. Comparable regenerative abilities are found for amputations performed at different positions along the antero-posterior axis of the worm, except when amputation planes are very close to the pharynx. Regenerative abilities persist upon repeated amputations without important alterations of the process. We also show that intense cell proliferation occurs during regeneration and that cell divisions are required for regeneration to proceed normally. Finally, 5-ethynyl-2'-deoxyuridine (EdU) pulse and chase experiments suggest that blastemal cells mostly derive from the segment immediately abutting the amputation plane. The detailed characterization of P. dumerilii posterior body regeneration presented in this article provides the foundation for future mechanistic and comparative studies of regeneration in this species.

The development of early pioneer neurons in the annelid Malacoceros fuliginosus.

BACKGROUND: Nervous system development is an interplay of many processes: the formation of individual neurons, which depends on whole-body and local patterning processes, and the coordinated growth of neurites and synapse formation. While knowledge of neural patterning in several animal groups is increasing, data on pioneer neurons that create the early axonal scaffold are scarce. Here we studied the first steps of nervous system development in the annelid Malacoceros fuliginosus. RESULTS: We performed a dense expression profiling of a broad set of neural genes. We found that SoxB expression begins at 4 h postfertilization, and shortly later, the neuronal progenitors can be identified at the anterior and the posterior pole by the transient and dynamic expression of proneural genes. At 9 hpf, the first neuronal cells start differentiating, and we provide a detailed description of axonal outgrowth of the pioneer neurons that create the primary neuronal scaffold. Tracing back the clonal origin of the ventral nerve cord pioneer neuron revealed that it is a descendant of the blastomere 2d (2d221), which after 7 cleavages starts expressing Neurogenin, Acheate-Scute and NeuroD. CONCLUSIONS: We propose that an anterior and posterior origin of the nervous system is ancestral in annelids. We suggest that closer examination of the first pioneer neurons will be valuable in better understanding of nervous system development in spirally cleaving animals, to determine the potential role of cell-intrinsic properties in neuronal specification and to resolve the evolution of nervous systems.