The expression of a hunchback ortholog in the polychaete annelid Platynereis dumerilii suggests an ancestral role in mesoderm development and neurogenesis

Orthologs of the Drosophila gap gene hunchback have been isolated so far only in protostomes. Phylogenetic analysis of recently available genomic data allowed us to confirm that hunchback genes are widely found in protostomes (both lophotrochozoans and ecdysozoans). In contrast, no unequivocal hunchback gene can be found in the genomes of deuterostomes and non-bilaterians. We cloned hunchback in the marine polychaete annelid Platynereis dumerilii and analysed its expression during development. In this species, hunchback displays an expression pattern indicative of a role in mesoderm formation and neurogenesis, and similar to the expression found for hunchback genes in arthropods. These data suggest altogether that these functions are ancestral to protostomes.Pierre Kerner and Fabiola Zelada González contributed equally to this work.     

A novel fibrinolytic serine protease from the polychaete Nereis (Neanthes) virens (Sars): purification and characterization

A novel fibrinolytic serine protease has been identified and purified to homogeneity from the coelomic fluid of polychaete Nereis (Neanthes) virens (Sars), and named N-V protease. N-V protease is a 29kDa single chain protein with an isoelectric point of pH 4.5. It hydrolyzes Aalpha-chain of fibrinogen with a high efficiency, and the Bbeta- and gamma-chains (Aalpha>Bbeta>gamma) with a lower efficiency. The proteolytic activity peaks at pH 7.8 is 45 degrees C. The activity is completely inhibited by serine protease inhibitors DFP (I(50)=5.8 x 10(-4)M) and PMSF (I(50)=5.5 x 10(-2)M), and almost completely by TLCK (I(50)=7.7 x 10(-1) M). But aprotinin, elastinal, SBTI, benzamidine, PCMB, EDTA, EGTA, iodoacetate, E64, and beta-mercaptoethanol have no effect on the protease activity. Therefore, N-V protease is identified as a serine protease. The primary amino acid sequence of N-V protease was determined by mass spectrometry (N-V protease, No. P83433). According to the MALDI-TOF MS analysis, there is no existing protein in the NCBI Non-redundant Protein Sequence Database that matches the N-V protease sequence. Therefore, N-V protease is a novel and special protein in N. virens. Furthermore, we have successfully established an expression cDNA library from the whole body of N. virens (data not shown).     

A correlative study of experimentally changed first cleavage and Janus development in the trunk of Platynereis dumerilii (Annelida,Polychaeta)

Summary Among zygotes of Platynereis dumerilii treated with cytochalasin B (CCB) prior to first cleavage, a wide variety of developmental effects were observed. One effect is a delay in the first cleavage. Treated embryos may skip the first or even more than one cleavage cycle and become multinucleated. Once these eggs start cleaving their cleavage plane takes the same position as in synchronously fertilized controls. Accordingly, the first cleavage in embryos having skipped the first normal cleavage cycle is meridional and equal, but their second cleavage is equatorial as in the third cleavage in controls. None of the embryos that were observed to skip early cleavages showed normal organogenesis, but developed into vesicle-shaped embryos with little cytological differentiation. Another effect of CCB treatment is altered blastomere size in those embryos which begin cleaving in synchrony with controls. While the majority of treated embryos followed a normal cleavage pattern, i.e. they cleaved at the right time and inequally, some of them cleaved equally or almost equally (adequally). Most of these embryos showed cleavage defects in subsequent cleavage cycles and became abnormal vesicle-shaped embryos. However, some of these embryos cleaving on schedule and equally or adequally developed into juvenile worms showing complete duplication of urites and parapodial rows (0.3% of all treated eggs) and are described as Janus duplicitates. This means that the occurrence of duplicitates and geometrically altered first cleavage patterns are correlated phenomena. The character and origin of the duplications and the consequences for dorsoventral polarity are discussed.     

Identification of the yolk receptor protein in oocytes of Nereis virens (Annelida,Polychaeta) and comparison with the locust vitellogenin receptor

Summary In oviparous animals large amounts of yolk proteins of extraovarian origin are accumulated by developing oocytes during vitellogenesis. The yolk protein precursors, the vitellogenins (VTG), are transported into the oocytes by receptor-mediated endocytosis. In oocytes of the polychaetous annelid, Nereis virens, the receptor protein for VTG was visualized by ligand blotting studies as a protein with an apparent molecular mass of 190 kDa under non-reducing conditions. Anti-Locusta VTG receptor antibodies recognize the Nereis VTG receptor protein. The Nereis VTG receptor protein binds Locusta and Schistocerca VTG; the VTG receptor proteins of both locust species bind the Nereis vitellin. These results indicate the conservation of structural elements important for internalization of VTG.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane-sulphonic acid - HBS HEPES-buffered saline - PAP peroxidase-anti-peroxidase - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - TRIS, TBS TRIS-buffered saline - VT vitellin - VTG vitellogenin     

Genotoxic damage in polychaetes: A study of species and cell-type sensitivities

The marine environment is becoming increasingly contaminated by environmental pollutants with the potential to damage DNA, with marine sediments acting as a sink for many of these contaminants. Understanding genotoxic responses in sediment-dwelling marine organisms, such as polychaetes, is therefore of increasing importance. This study is an exploration of species-specific and cell-specific differences in cell sensitivities to DNA-damaging agents in polychaete worms, aimed at increasing fundamental knowledge of their responses to genotoxic damage. The sensitivities of coelomocytes from three polychaetes species of high ecological relevance, i.e. the lugworm Arenicola marina, the harbour ragworm Nereis diversicolor and the king ragworm Nereis virens to genotoxic damage are compared, and differences in sensitivities of their different coelomic cell types determined by use of the comet assay. A. marina was found to be the most sensitive to genotoxic damage induced by the direct-acting mutagen methyl methanesulfonate (MMS), and showed dose-dependent responses to MMS and the polycyclic aromatic hydrocarbon benzo(a)pyrene. Significant differences in sensitivity were also measured for the different types of coelomocyte. Eleocytes were more sensitive to induction of DNA damage than amoebocytes in both N. virens and N. diversicolor. Spermatozoa from A. marina showed significant DNA damage following in vitro exposure to MMS, but were less sensitive to DNA damage than coelomocytes. This investigation has clearly demonstrated that different cell types within the same species and different species within the polychaetes show significantly different responses to genotoxic insult. These findings are discussed in terms of the relationship between cell function and sensitivity and their implications for the use of polychaetes in environmental genotoxicity studies.     

Detecting possibly saturated positions in 18S and 28S sequences and their influence on phylogenetic reconstruction of Annelida (Lophotrochozoa)

Phylogenetic reconstructions may be hampered by multiple substitutions in nucleotide positions obliterating signal, a phenomenon called saturation. Traditionally, plotting ti/tv ratios against genetic distances has been used to reveal saturation by assessing when ti/tv stabilizes at 1. However, interpretation of results and assessment of comparability between different data sets or partitions are rather subjective. Herein, we present the new C factor, which quantifies convergence of ti/tv ratios, thus allowing comparability. Furthermore, we introduce a comparative value for homoplasy, the O/E ratio, based on alterations of tree length. Simulation studies and an empirical example, based on annelid rRNA-gene sequences, show that the C factor correlates with noise, tree length and genetic distance and therefore is a proxy for saturation. The O/E ratio correlates with the C factor, which does not provide an intrinsic threshold of exclusion, and thus both together can objectively guide decisions to exclude saturated nucleotide positions. However, analyses also showed that, for reconstructing annelid phylogeny using Maximum Likelihood, an increase in numbers of positions improves tree reconstruction more than does the exclusion of saturated positions.     

Ultrastructure of the nuchal organs in the polychaete Platynereis dumerilii (Annelida,Nereididae)

Abstract. We examined the nuchal organs of adults of the nereidid polychaete Platynereis dumerilii by means of scanning and transmission electron microscopy. The most prominent features of the nuchal organs are paired ciliary bands located dorsolaterally at the posterior margin of the prostomium. They are composed of primary sensory cells and multiciliated supporting cells, both covered by a thin cuticle. The supporting cells have motile cilia that penetrate the cuticle and are responsible for the movement of water. Subapically, they have a narrowed neck region; the spaces between the neck regions of these supporting cells comprise the olfactory chamber. The dendrites of the sensory cells give rise to a single modified cilium that crosses the olfactory chamber; numerous thin microvillus-like processes, presumably extending from the sensory cells, also traverse the olfactory chamber. At the periphery of the ciliated epithelium runs a large nervous process between the ciliated supporting cells. It consists of smaller bundles of sensory dendrites that unite to form the nuchal nerve, which leaves the ciliated epithelium basally and runs toward the posterior part of the brain, where the perikarya of the sensory cells are located in clusters. The ciliated epithelium of the nuchal organs is surrounded by non-ciliated, peripheral epidermal cells. Those immediately adjacent to the ciliated supporting cells have a granular cuticle; those further away have a smooth cuticle. The nuchal organs of epitokous individuals of P. dumerilii are similar to those described previously in other species of polychaetes and are a useful model for understanding the development of nuchal organs in polychaetes.     

Development and differentiation of the eye in Platynereis dumerilii (Annelida,Polychaeta)

The nereid polychaete, Platynereis dumerilii, possess two pairs of post-trochophoral eyes with one vitreous body each. The development of these eyes has first been observed in 2-day-old larvae. Whether the eye anlagen arise from stem cells or from undifferentiated ectodermal tissue was not determined. At first, the anlagen of the anterior and the posterior eyes adjoin each other. They separate in late 3-day-old larvae. The first separated eye complexes consist each of two supporting and two sensory cells. The supporting cells synthesize two different kinds of granules, the pigment granules of the pigment cup and the prospective tubules of the vitreous body. These tubules accumulate in the distal process of the supporting cell. The vitreous body is formed by compartments of the supporting cells filled with the osmiophilic vitreous body tubules. The short, bulbar photosensory processes bear microvilli that emerge into the ocular cavity. At the apex of each sensory cell process, a single cilium (or occasionally two) arises. The sensory cells contain a different kind of pigment granule within their necks at the level of the pigment cup. The rate of eye development and differentiation varies. New supporting cells are added to the rim of the eye cup. They contribute to the periphery of the vitreous body like onion skins, and sensory cells move between supporting cells. The older the individual compartments of the vitreous body are, the more densely packed is their content of vitreous body tubules. Elongation of the sensory and supporting cell processes of the older cells increases the volume of the eye. The eyespots of the trochophore are briefly described as of the two-celled rhabdomeric type with a single basal body with ciliary rootlet.     

The role of chemical signals in the spawning induction of polychaete worms and other marine invertebrates

Extracts of gonad, body fluid and spawning water (into which gametes have been released by the process of spawning) were tested for the presence of spawning-inducing activity (SIA) on both sexes of the polychaetes Nereis succinea (Frey and Leuckhart) and Platynereis dumerilii (Audouin and Milne-Edwards). Gonadal and body fluid extracts from Asterias rubens (L.) and Echinus esculentus (L.) (echinoderms), Arenicola marina (L.) (polychaete), Clupea harengus (L.) (teleost) and body fluid and spawning water of Nereis virens (Sars) (polychaete) all exhibit SIA on male N. succinea. C. harengus extract from male gonads also had SIA on male P. dumerilii. Phytoplankton monoculture extracts were also tested. The cryptomonad (Rhodomonas baltica) (Karsten) had SIA on male P. dumerilii, and the diatom Phaeodactylum tricornutum (Bohlin) had some activity on Mytilus edulis (L.). The active fraction of R. baltica is chromatographically similar to uric acid (the natural sperm-release pheromone produced by females of P. dumerilii) [Nature 382 (1996) 214], but has a different retention time and UV absorbance spectra on reverse phase HPLC. Conspecific extracts of spawning water and gonadal material did not have SIA on the same and opposite sex in A. marina and N. virens. The role of chemical signals in marine invertebrates is discussed in relation to their mode of spawning.     

Effects of copper and other metals on fertilization,embryo development,larval survival and settlement of the polychaete Nereis (Neanthes) virens

Summary

Nectochaete larvae of the ecologically and economically important ragworm, Nereis virens, were exposed to cadmium, chromium, copper, lead and zinc dissolved in seawater to nominal concentrations ranging from 0 to 5000 μg l^?1. Copper was the most toxic (mean LC50 of 76.5 μg l^?1 ± 95% CI 73.8–79.2 after 96 h exposure) and so was used for subsequent experiments. Exposure of gametes to greater than 500 μg l^?1 copper for 2 or 4 h at 10°C prior to fertilization, or a 10 min exposure during fertilization, significantly reduced embryo developmental success. The effect of copper on larval settlement was also assessed using sediment spiked to a range of concentrations (0, 50, 250, 500, 1000 mg kg!1 dry weight). Significantly fewer larvae were found in sediment of $250 mg kg!1 in comparison to the control or the 50 mg kg!1 treatment. Assessment of living larvae also confirmed a significant reduction in settlement, but in all treatments compared to the control, although the number of dead larvae also increased as the concentrations increased. These effects may have important implications for reproductive success and recruitment of N. virens to polluted sediments.     

Characterisation of two novel CYP4 genes from the marine polychaete Nereis virens and their involvement in pyrene hydroxylase activity

Cytochrome P450 enzymes (CYP enzymes) catalyse the initial step in biotransformation of xenobiotics like polycyclic aromatic hydrocarbons (PAHs). The marine polychaete Nereis virens has a high capacity for biotransformation of PAHs. In the present study, the complete cDNA sequences of two novel CYP genes isolated from N. virens gut tissue are reported. One named CYP342A1, the first member of a new family and the other named CYP4BB1, the first member of a new subfamily. This is the first investigation of specific CYP enzymes from marine polychaetes in which catalytic activity has been determined. Both CYP enzymes had monooxygenase activity and catalysed hydroxylation of pyrene to 1-hydroxypyrene. Based on the present results it is likely that both CYP4BB1 and CYP342A1 are involved in xenobiotic biotransformation. Furthermore, site-directed mutagenesis of the conserved cysteine residue of the heme binding domain resulted in complete loss of monooxygenase activity of both CYP enzymes, indicating that this cysteine residue is indispensable for monooxygenase activity of invertebrate CYP enzymes, as has been well documented in vertebrates. Considering the important role of CYP enzymes in biotransformation of xenobiotics and the presence of N. virens in estuarine environments that accumulates organic xenobiotics, our results are important in understanding the molecular mechanism of biotransformation in marine polychaetes.     

Cellular immunity in an annelid (Nereis diversicolor,Polychaeta): production of melanin by a subpopulation of granulocytes

Summary We attempted to identify the nature and origin of the pigment produced by the marine worm Nereis diversicolor in order to isolate, in inert brown capsules, foreign objects introduced into its body cavity. This brown pigment, characterized by cytochemical techniques, could be a melanin. The activity of the enzyme phenoloxidase responsible for melanin biosynthesis was detected by enzyme cytochemistry techniques in vacuoles and the Golgi apparatus of coelomocytes activated by the presence of foreign bodies. Morphological techniques combined with a monoclonal immunological probe enabled us to establish that the G₂ granulocytes contain both the precursor of the pigment in dense bodies and the capacity for phenoloxidase synthesis when activated to encapsulate foreign bodies. The G₂ granulocyte may therefore be compared to a melanocyte in which melanin is not stored as in mammals, but immediately extruded following synthesis in the form of a thick fluid.Abbreviations ACTH adrenocorticotropic hormone - Dopa L-3,4 dihydroxyphenylalanine - FITC fluorescein isothiocyanate - G ₁, G ₂, G ₃ granulocyte of types 1, 2, 3 - MSH melanocyte-stimulating hormone - proPo prophenoloxidase     

Cell lineage and gene expression in the development of polychaetes

The developmental strategies of embryos within the various taxa of polychaetes are designed to set up the fate of the cell lines. Some of these traits of pattern formation are considered to be ancestral, but we also find a number of derived developmental characteristics, some of which might be useful indicators for phylogenetic relationships. A combination of ooplasmic segregation and anisotropic cleavage rapidly establishes the fate of several larval cell lines in the polychaete embryo. The setting-up of the primary trochoblasts basically concerns the same cell line (la²–ld²) in polychaetes and even in molluscs. Such mechanisms may thus be regarded as ancestral. The determination of the mesoderm precursor occurs very late in both equally cleaving annelids and mollusks, indicating that an equal cleavage pattern may be regarded as an ancestral trait. Since both disproportionate cytoplasmic distribution (either by spindle shift or polar lobe formation) and cell cycle asynchronies appear to speed up the development of the mesoderm-forming cell line, these strategies represent derived traits. An analysis of these derived traits of early development is given and is discussed in the light of the phylogenetic relationships among the polychaetes. These data are extended by an analysis of some of the postlarval structures in polychaetes and the molecular developmenta1 circuitry involved.     

Nodulin gene expression during soybean (Glycine max) nodule development

In vitro translation products of total RNA isolated from soybean nodules at successive stages of nodule development were analyzed by two-dimensional gel electrophoresis. In that way the occurrence of over 20 mRNAs specifically transcribed from nodulin genes was detected. The nodulin genes could be divided into two classes according to the time of expression during nodule development. Class A comprises at least 4 nodulin mRNAs which are found when a globular meristem is present in the root cortex. These class A nodulin genes have a transient expression. Class B nodulin genes are expressed when the formation of a nodule structure has been completed. Bradyrhizobium japonicum nod ⁺ fix^-mutants, with large deletions spanning the nif H,DK region, still induced nodules showing normal expression of all nodulin genes, indicating that the nif H,DK region is not involved in the induction of nodulin genes. In nodules induced by Bradyrhizobium japonicum nod ⁺ fix^-mutant HS124 the bacteria are rarely released from the infection thread and the few infected cells appear to be collapsed. All class A and class B nodulin genes are expressed in HS124 nodules with the exception of 5 class B genes.     

The atokous-epitokous border is determined before the onset of heteronereid development in Platynereis dumerilii (Annelida,Polychaeta)

Summary In most nereids sexual maturation is accompanied by a dramatic reorganization of the body that enables swarming of the formerly benthic worms. However, a border exists between unchanged anterior (atokous) and metamorphosed posterior (epitokous) segments. The site of this atokous-epitokous border (a/e border) is different in sexually mature males and females of Platynereis dumerilii. There is no correlation between the total number of setigerous segments of a specimen and the location of the a/e border. The location of the a/e border and sexual development are affected neither by cutting off caudal segments of juveniles (including the prospective a/e border) nor by transecting the ventral nerve cord. When parapodia are transplanted from prospective epitokous regions to prospective atokous regions and vice versa, they maintain their original character during metamorphosis. The results presented here suggest that prospective atokous as well as epitokous characters are determined at or only very shortly after formation of the respective segments. Thus the a/e border is established well in advance of the onset of epitokous metamorphosis.     

Immunocytochemical demonstration of vertebrate neuropeptides in the earthworm Lumbricus terrestris (Annelida,Oligochaeta)

Summary The localisation and distribution of 10 vertebrate-derived neuropeptides in the earthworm, Lumbricus terrestris, have been determined by an indirect immunofluorescence technique. The peptides are pancreatic polypeptide (PP), peptide tyrosine tyrosine (PYY), neuropeptide Y (NPY), glucagon (C-terminal), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), gastrinreleasing peptide (GRP), calcitonin gene-related peptide (CGRP), neurotensin (NT), and met-enkephalin. For 6 of the peptides — PYY, NPY, PHI, glucagon, GRP and CGRP — this is the first demonstration of their presence in any annelid, and NT has not previously been described in an oligochaete. Cell bodies and nerve fibres immunoreactive to the 10 peptides occur throughout the CNS. In the PNS, epidermal sensory cells displayed immunoreactivities to PP and PYY, and PP-, PYY-, NPY-, PHI- and GRP-like immunoreactivities occurred in nerve fibres supplying the main body muscles. Nerve fibres immunoreactive to PP and PYY are also associated with the innervation of the gut (pharynx, oesophageal glands, and mid and posterior regions of the intestine). No endocrine cells immunoreactive for any of the antisera tested could be identified in the gut epithelium, suggesting that dual location of peptides in the brain and gut epithelium is a phenomenon that occurred at a later stage in evolution. No immunoreactive elements were detected in any of the organs and ducts of the reproductive and excretory systems.     

Phylogenetic relationships of Serpulidae (Annelida: Polychaeta) based on 18S rDNA sequence data, and implications for opercular evolution

Phylogenetic relationships of (19) serpulid taxa (including Spirorbinae) were reconstructed based on 18S rRNA gene sequence data. Maximum likelihood, Bayesian inference, and maximum parsimony methods were used in phylogenetic reconstruction. Regardless of the method used, monophyly of Serpulidae is confirmed and four monophyletic, well-supported major clades are recovered: the Spirorbinae and three groups hitherto referred to as the Protula-, Serpula-, and Pomatoceros-group. Contrary to the taxonomic literature and the hypothesis of opercular evolution, the Protula-clade contains non-operculate (Protula, Salmacina) and operculate taxa both with pinnulate and non-pinnulate peduncle (Filograna vs. Vermiliopsis), and most likely is the sister group to Spirorbinae. Operculate Serpulinae and poorly or non-operculate Filograninae are paraphyletic. It is likely that lack of opercula in some serpulid genera is not a plesiomorphic character state, but reflects a special adaptation.     

Synchronous and early activation of planarian Hox genes and the re-specification of body axes during regeneration in Dugesia (G.) tigrina (Turbellaria; Tricladida)

Seven Hox cluster-related genes (Dthox-A to -G) have been isolated from the freshwater triclad Dugesia (G.) tigrina, their sequence compared to other Hox genes and their expression in intact and regenerating organisms analyzed by whole mount in situ hybridization. Sequence comparison analyses show high similarities of D. tigrina Hox genes to anterior and medial groups of coelomate Hox genes. Expression analyses show very early, synchronous, and overlapping expression of Dthox -A, -E, -G and -F in anterior, posterior and lateral regenerative tissues. At one hour of regeneration all Dthox genes studied showed a neat, clear expression at the wound boundary. Later, as the blastema grows, the expression area expands to more proximal regions covering the blastema and the distal postblastema regions. Blastemas formed by intercalary regeneration also show a synchronous expression of the same Hox genes though the onset of activation is much delayed. The finding that the same set of Hox genes is synchronously activated in anterior, posterior, intercalary and lateral regeneration is in sharp contrast to its well established role in specifying antero-posterior pattern during embryonic development. The implications of these results as regards ancestral versus co-opted roles of Hox genes in development and regeneration are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Reproduction and larval development of two sympatric Pseudopolydora species (Annelida: Spionidae) in Japan

Pseudopolydora cf. kempi and Pseudopolydora cf. reticulata, which are morphologically similar, are sympatric in Gamo Lagoon, near Sendai on the Pacific eastern coast of Honshu, Japan. Reproduction, larval development, and larval morphology of these two species were compared. The larval developmental pattern of P. cf. kempi was determined to be short-term planktonic with lecithotrophy and adelphophagy, whereas that of P. cf. reticulata was long-term planktonic with planktotrophy. The number of embryos per capsule differed between the two species: 0–16 for P. cf. kempi and 62–405 for P. cf. reticulata. Nurse eggs were only found for P. cf. kempi. The results of this study suggest that some previous studies attributed to P. kempi may have been to P. cf. reticulata or to another species in this complex.