Marine Annelid Worm Research Articles

DNA methylation atlas and machinery in the developing and regenerating annelid Platynereis dumerilii

BackgroundMethylation of cytosines in DNA (5mC methylation) is a major epigenetic modification that modulates gene expression and constitutes the basis for mechanisms regulating multiple aspects of embryonic development and cell reprogramming in vertebrates. In mammals, 5mC methylation of promoter regions is linked to transcriptional repression. Transcription regulation by 5mC methylation notably involves the nucleosome remodeling and deacetylase complex (NuRD complex) which bridges DNA methylation and histone modifications. However, less is known about regulatory mechanisms involving 5mC methylation and their function in non-vertebrate animals. In this paper, we study 5mC methylation in the marine annelid worm Platynereis dumerilii, an emerging evolutionary and developmental biology model capable of regenerating the posterior part of its body post-amputation.ResultsUsing in silico and experimental approaches, we show that P. dumerilii displays a high level of DNA methylation comparable to that of mammalian somatic cells. 5mC methylation in P. dumerilii is dynamic along the life cycle of the animal and markedly decreases at the transition between larval to post-larval stages. We identify a full repertoire of mainly single-copy genes encoding the machinery associated with 5mC methylation or members of the NuRD complex in P. dumerilii and show that this repertoire is close to the one inferred for the last common ancestor of bilaterians. These genes are dynamically expressed during P. dumerilii development and regeneration. Treatment with the DNA hypomethylating agent Decitabine impairs P. dumerilii larval development and regeneration and has long-term effects on post-regenerative growth.ConclusionsOur data reveal high levels of 5mC methylation in the annelid P. dumerilii, highlighting that this feature is not specific to vertebrates in the bilaterian clade. Analysis of DNA methylation levels and machinery gene expression during development and regeneration, as well as the use of a chemical inhibitor of DNA methylation, suggest an involvement of 5mC methylation in P. dumerilii development and regeneration. We also present data indicating that P. dumerilii constitutes a promising model to study biological roles and mechanisms of DNA methylation in non-vertebrate bilaterians and to provide new knowledge about evolution of the functions of this key epigenetic modification in bilaterian animals.

Synthesis of the oxazolidinone fragment of thelepamide

Thelepamide, an unique ketide-amino acid isolated from a marine annelid worm Thelepus crispus, has a unique oxazolidinone ring derived from cysteine, glycine and valine. Rareness in nature as well as promising bioactive possibility make the oxazolidinone ring an attractive synthetic target. The hydroxy oxazolidinone fragment of thelepamide was prepared by acid-catalysed N,O-acetal formation between a ketoamide and formaldehyde. Lactone-carbonyl selective isopropyl addition to an oxazilidine–dione under Grignard conditions also forms the target compound.

Bioluminescence properties of Thelepus japonicus (Annelida: Terebelliformia).

Terebelliformia is a benthic group of marine annelid worms. The bioluminescence of several species has been reported in taxonomical and histological literature, but very little information is known about the biochemical aspects of this phenomenon. In this study, we examined the basic properties of the luminescence system using an extract of the Japanese terebelliform worm, Thelepus japonicus. The bioluminescence extract was soluble in water, and emitted blue-green light at λmax 508nm following the addition of divalent cations. This triggering action was highly specific to Fe2+ and addition of ATP, H2 O2 or coelenterazine did not enhance activity. The bioluminescence was inactivated by heat treatment and organic solvents, indicating the involvement of a protein component. These results suggested that Thelepus worm produces light using a novel system that differs from that in other known luminescent annelids.

Insight into aquaculture's potential of marine annelid worms and ecological concerns: a review

AbstractPolychaetes are marine annelid worms that can contribute to aquaculture diversification. Its culture has been viable, and commercially attempted, but intensive production has progressed only in few countries around the world. In the countries with no production, marine polychaetes are imported or harvested. A strong and sustained research investment provided to a better understanding of the nutritional requirements and reproduction of some species. Recent studies showed new technical improvements, which can lead to an important progress in productivity and give a new impetus to the polychaete production. Some marine worm species were identified as good candidates for integrated multitrophic aquaculture. The development of cost‐effective aquaculture techniques for marine annelid worms is essential to ensure a balance between commercial interests and the preservation of ecosystems. The influence of polychaete aquaculture on the environment and vice versa raise important concerns related to ecological security and sustainability of this activity. This review focus on the main technical improvements and advances that have been made in areas as diverse as: aquaculture potential of polychaetes, reared species, main species used worldwide, and highlights biological and ecological concerns, important challenges and recommendations.

Whole-head recording of chemosensory activity in the marine annelid Platynereis dumerilii.

Chemical detection is key to various behaviours in both marine and terrestrial animals. Marine species, though highly diverse, have been underrepresented so far in studies on chemosensory systems, and our knowledge mostly concerns the detection of airborne cues. A broader comparative approach is therefore desirable. Marine annelid worms with their rich behavioural repertoire represent attractive models for chemosensation. Here, we study the marine worm Platynereis dumerilii to provide the first comprehensive investigation of head chemosensory organ physiology in an annelid. By combining microfluidics and calcium imaging, we record neuronal activity in the entire head of early juveniles upon chemical stimulation. We find that Platynereis uses four types of organs to detect stimuli such as alcohols, esters, amino acids and sugars. Antennae are the main chemosensory organs, compared to the more differentially responding nuchal organs or palps. We report chemically evoked activity in possible downstream brain regions including the mushroom bodies (MBs), which are anatomically and molecularly similar to insect MBs. We conclude that chemosensation is a major sensory modality for marine annelids and propose early Platynereis juveniles as a model to study annelid chemosensory systems.

Wnt/\u03b2-catenin signalling is necessary for gut differentiation in a marine annelid, Platynereis dumerilii

BackgroundWnt/β-catenin (or canonical) signalling pathway activity is necessary and used independently several times for specification of vegetal fate and endoderm, gut differentiation, maintenance of epithelium in adult intestine and the development of gut-derived organs in various vertebrate and non-vertebrate organisms. However, its conservation in later stages of digestive tract development still remains questionable due to the lack of detailed data, mainly from Spiralia.ResultsHere we characterize the Pdu-Tcf gene, a Tcf/LEF orthologue and a component of Wnt/β-catenin pathway from Platynereis dumerilii, a spiralian, marine annelid worm. Pdu-Tcf undergoes extensive alternative splicing in the C-terminal region of the gene generating as many as eight mRNA isoforms some of which differ in the presence or absence of a C-clamp domain which suggests a distinct DNA binding activity of individual protein variants. Pdu-Tcf is broadly expressed throughout development which is indicative of many functions. One of the most prominent domains that exhibits rather strong Pdu-Tcf expression is in the putative precursors of endodermal gut cells which are detected after 72 h post-fertilization (hpf). At day 5 post-fertilization (dpf), Pdu-Tcf is expressed in the hindgut and pharynx (foregut), whereas at 7 dpf stage, it is strongly transcribed in the now-cellularized midgut for the first time. In order to gain insight into the role of Wnt/β-catenin signalling, we disrupted its activity using pharmacological inhibitors between day 5 and 7 of development. The inhibition of Wnt/β-catenin signalling led to the loss of midgut marker genes Subtilisin-1, Subtilisin-2, α-Amylase and Otx along with a drop in β-catenin protein levels, Axin expression in the gut and nearly the complete loss of proliferative activity throughout the body of larva. At the same time, a hindgut marker gene Legumain was expanded to the midgut compartment under the same conditions.ConclusionsOur findings suggest that high Wnt/β-catenin signalling in the midgut might be necessary for proper differentiation of the endoderm to an epithelium capable of secreting digestive enzymes. Together, our data provide evidence for the role of Wnt/β-catenin signalling in gut differentiation in Platynereis.

Acquisition of a Novel Sulfur-Oxidizing Symbiont in the Gutless Marine Worm Inanidrilus exumae.

ABSTRACTGutless phallodrilines are marine annelid worms without a mouth or gut, which live in an obligate association with multiple bacterial endosymbionts that supply them with nutrition. In this study, we discovered an unusual symbiont community in the gutless phallodriline Inanidrilus exumae that differs markedly from the microbiomes of all 22 of the other host species examined. Comparative 16S rRNA gene sequence analysis and fluorescence in situ hybridization revealed that I. exumae harbors cooccurring gamma-, alpha-, and deltaproteobacterial symbionts, while all other known host species harbor gamma- and either alpha- or deltaproteobacterial symbionts. Surprisingly, the primary chemoautotrophic sulfur oxidizer “Candidatus Thiosymbion” that occurs in all other gutless phallodriline hosts does not appear to be present in I. exumae. Instead, I. exumae harbors a bacterial endosymbiont that resembles “Ca. Thiosymbion” morphologically and metabolically but originates from a novel lineage within the class Gammaproteobacteria. This endosymbiont, named Gamma 4 symbiont here, had a 16S rRNA gene sequence that differed by at least 7% from those of other free-living and symbiotic bacteria and by 10% from that of “Ca. Thiosymbion.” Sulfur globules in the Gamma 4 symbiont cells, as well as the presence of genes characteristic for autotrophy (cbbL) and sulfur oxidation (aprA), indicate that this symbiont is a chemoautotrophic sulfur oxidizer. Our results suggest that a novel lineage of free-living bacteria was able to establish a stable and specific association with I. exumae and appears to have displaced the “Ca. Thiosymbion” symbionts originally associated with these hosts.IMPORTANCE All 22 gutless marine phallodriline species examined to date live in a highly specific association with endosymbiotic, chemoautotrophic sulfur oxidizers called “Ca. Thiosymbion.” These symbionts evolved from a single common ancestor and represent the ancestral trait for this host group. They are transmitted vertically and assumed to be in transition to becoming obligate endosymbionts. It is therefore surprising that despite this ancient, evolutionary relationship between phallodriline hosts and “Ca. Thiosymbion,” these symbionts are apparently no longer present in Inanidrilus exumae. They appear to have been displaced by a novel lineage of sulfur-oxidizing bacteria only very distantly related to “Ca. Thiosymbion.” Thus, this study highlights the remarkable plasticity of both animals and bacteria in establishing beneficial associations: the phallodriline hosts were able to acquire and maintain symbionts from two very different lineages of bacteria, while sulfur-oxidizing bacteria from two very distantly related lineages were able to independently establish symbiotic relationships with phallodriline hosts.

Advanced analytical method of nereistoxin using mixed-mode cationic exchange solid-phase extraction and GC/MS

Nereistoxin(NTX) was originated from a marine annelid worm Lumbriconereis heteropoda and its analogue pesticides including cartap, bensultap, thiocyclam and thiobensultap have been commonly used in agriculture, because of their low toxicity and high insecticidal activity. However, NTX has been reported about its inhibitory neuro toxicity in human and animal body, by blocking nicotinic acetylcholine receptor and it cause significant neuromuscular toxicity, resulting in respiratory failure. We developed a new method to determine NTX in biological fluid. The method involves mixed-mode cationic exchange based solid phase extraction and gas chromatography/mass spectrometry for final identification and quantitative analysis. The limit of detection and recovery were substantially better than those of other methods using liquid–liquid extraction or headspace solid phase microextraction. The good recoveries (97±14%) in blood samples were obtained and calibration curves over the range 0.05–20mg/L have R2 values greater than 0.99. The developed method was applied to a fatal case of cartap intoxication of 74 years old woman who ingested cartap hydrochloride for suicide. Cartap and NTX were detected from postmortem specimens and the cause of the death was ruled to be nereistoxin intoxication. The concentrations of NTX were 2.58mg/L, 3.36mg/L and 1479.7mg/L in heart, femoral blood and stomach liquid content, respectively. The heart blood/femoral blood ratio of NTX was 0.76.

A Sperm Nuclear Basic Protein from the Sperm of the Marine WormChaetopterus variopedatuswith Sequence Similarity to the Arginine-Rich C-Termini of Chordate Protamine-Likes

The sperm nuclear basic proteins (SNBPs) of the marine annelid worm Chaetopterus variopedatus have been shown previously to consist of a mixture of two SNBPs: histone H1-like (CvH1) and C.variopedatus protamine-like (CvPL). Here, we report the structural characterization of CvPL. The protein has a molecular weight of 8370.5 Da, a K/R ratio of 0.34, and a secondary structure, which are intermediate between those of protamine (P) and protamine-like (PL) SNBPs. The N-terminal sequence of CvPL shows a high extent of similarity with the arginine-rich C-terminal domain of chordate PL-type SNBPs. Furthermore, the protein binds to DNA in a similar fashion as vertebrate PLs and their own CvH1, but in a way that is different from that of the lysine-rich somatic H1 histones. We have experimentally determined the molar ratio CvH1:CvPL to be ∼1:6 in C. variopedatus sperm. Based on all of these, a model is proposed for the organization of the sperm chromatin by CvH1 and CvPL.

Not whale-fall specialists, Osedax worms also consume fishbones

Marine annelid worms of the genus Osedax exploit sunken vertebrate bones for food. To date, the named species occur on whale or other mammalian bones, and it is argued that Osedax is a whale-fall specialist. To assess whether extant Osedax species could obtain nutrition from non-mammalian resources, we deployed teleost bones and calcified shark cartilage at approximately 1000 m depth for five months. Although the evidence from shark cartilage was inconclusive, the teleost bones hosted three species of Osedax, each of which also lives off whalebones. This suggests that rather than being a whale-fall specialist, Osedax has exploited and continues to exploit a variety of food sources. The ability of Osedax to colonize and to grow on fishbone lends credibility to a hypothesis that it might have split from its siboglinid relatives to assume the bone-eating lifestyle during the Cretaceous, well before the origin of marine mammals.

Bacterial Exopolysaccharides from Extreme Marine Habitats: Production, Characterization and Biological Activities

Many marine bacteria produce exopolysaccharides (EPS) as a strategy for growth, adhering to solid surfaces, and to survive adverse conditions. There is growing interest in isolating new EPS producing bacteria from marine environments, particularly from extreme marine environments such as deep-sea hydrothermal vents characterized by high pressure and temperature and heavy metal presence. Marine EPS-producing microorganisms have been also isolated from several extreme niches such as the cold marine environments typically of Arctic and Antarctic sea ice, characterized by low temperature and low nutrient concentration, and the hypersaline marine environment found in a wide variety of aquatic and terrestrial ecosystems such as salt lakes and salterns. Most of their EPSs are heteropolysaccharides containing three or four different monosaccharides arranged in groups of 10 or less to form the repeating units. These polymers are often linear with an average molecular weight ranging from 1 × 105 to 3 × 105 Da. Some EPS are neutral macromolecules, but the majority of them are polyanionic for the presence of uronic acids or ketal-linked pyruvate or inorganic residues such as phosphate or sulfate. EPSs, forming a layer surrounding the cell, provide an effective protection against high or low temperature and salinity, or against possible predators. By examining their structure and chemical-physical characteristics it is possible to gain insight into their commercial application, and they are employed in several industries. Indeed EPSs produced by microorganisms from extreme habitats show biotechnological promise ranging from pharmaceutical industries, for their immunomodulatory and antiviral effects, bone regeneration and cicatrizing capacity, to food-processing industries for their peculiar gelling and thickening properties. Moreover, some EPSs are employed as biosurfactants and in detoxification mechanisms of petrochemical oil-polluted areas. The aim of this paper is to give an overview of current knowledge on EPSs produced by marine bacteria including symbiotic marine EPS-producing bacteria isolated from some marine annelid worms that live in extreme niches.

Cellular Resolution Expression Profiling Using Confocal Detection of NBT/BCIP Precipitate by Reflection Microscopy

The determination of gene expression patterns in three dimensions with cellular resolution is an important goal in developmental biology. However the most sensitive, efficient, and widely used staining technique for whole-mount in situ hybridization (WMISH), nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) precipitation by alkaline phosphatase, could not yet be combined with the most precise, high-resolution detection technique, confocal laser-scanning microscopy (CLSM). Here we report the efficient visualization of the NBT/BCIP precipitate using confocal reflection microscopy for WMISH samples of Drosophila, zebrafish, and the marine annelid worm, Platynereis dumerilii. In our simple WMISH protocol for reflection CLSM, NBT/BCIP staining can be combined with fluorescent WMISH, immunostainings, or transgenic green fluorescent protein (GFP) marker lines, allowing double labeling of cell types or of embryological structures of interest. Whole-mount reflection CLSM will thus greatly facilitate large-scale cellular resolution expression profiling in vertebrate and invertebrate model organisms.

Preface

A necessary limitation of any compilation on eye development is precisely its strength: the field is so broad and intensively studied that it is not possible to do it justice in one issue. Eyes are widely distributed and diverse. Simple organelle eyespots exist in protozoa and unicellular alga, and highly simplified eyes are present in larvae of sponges and jellyfish. Adult cubomedusan jellyfish have sophisticated eyes (ocelli) with ciliated photoreceptors (like vertebrates) and cellular lenses as well as lens-less ocelli; ancient flatworms, marine annelid worms and the protochordate ascidian larva have complex eyes with a rudimentary lens, photoreceptors and pigment cells. Insects have both lens-containing ocelli like the jellyfish and compound eyes that comprise hundreds of separate facets, the ommatidia, each with their own few rhabdomeric photoreceptors. Some shrimps have compound eyes to detect polarizing signals from each other, while other crustacea use specialized eyes to capture stray photons created by thermal vents in the depths of the ocean. Scallops use rows of mirrored, camera-type eyes with lenses along the mantle that cast focussed images on double retinas using separate phototransducing pathways to feed or evade prey. Salmon have specialized cones in their retina that appear transiently in geometric patterns to orient polarized light for the fresh water stint of their extensive migrations. The eyes of the 'four-eyed' surface fish, Anableps, are divided into a dorsal half used in air above the water surface and a ventral half submerged in water; each half uses the same lens but has a separate cornea and retina. Mice see shades of grey and ultraviolet; eagles and butterflies see brilliant color. The human eye serves us well, but we lack the resolution of the eagle, or the night vision of the owl, or the ultraviolet vision of the mouse and insects. These are but a sprinkling of eyes populating our planet. This Special Issue of The International Journal of Developmental Biology touches on the development of some of these remarkable eyes, regrettably few, but we hope it kindles your interest to learn more.

Characterization of a new variant DNA (cytosine-5)-methyltransferase unable to methylate double stranded DNA isolated from the marine annelid worm Chaetopterus variopedatus

The enzyme S-adenosylmethionine-DNA (cytosine-5)-methyltransferase has been identified, first time for invertebrates, in embryos of the marine polychaete annelid worm Chaetopterus variopedatus. The molecule has been isolated from embryos at 15 h of development. It is a single peptide of about 200 kDa molecular weight, cross-reacting with antibodies against sea urchin DNA methyltransferase. The enzymatic properties of the molecule are similar to those of Dnmt1 methyltransferases isolated from other organisms, but with the peculiarity to be unable to make ‘de novo’ methylation on double stranded DNA.

Genome methylation of the marine annelid worm Chaetopterus variopedatus: methylation of a CpG in an expressed H1 histone gene

Hydrolysis by methylation-dependent restriction enzymes shows that the genomic DNA of the polychaete annelid worm Chaetopterus variopedatus is methylated. Electrophoretic analyses of the digestion products indicate that the degree of methylation is lower in adult tissues than in sperm and embryonic DNA. 5-Methylcytosine was identified by HPLC, absorption spectroscopy and mass spectrometry analyses of free bases obtained by acid hydrolysis of the DNA. An average value of 1.6% methylated cytosines was determined in sperm DNA. Partial methylation was also found in an actively expressed H1 histone gene. This is the first time that genomic DNA methylation is demonstrated to occur in a worm.

Immunohistochemical demonstration of a CCK-like peptide in the nervous system of a marine annelid worm, Nereis diversicolor O.F. Müller.

Perikarya and nerve fibers containing a substance immunologically related to CCK-8 were detected in the nervous system of Nereis, a marine annelid worm. The most noteworthy immunostaining was seen in cell bodies, localized at the periphery of the brain, within nuclei 5, 6, 7, 9, 10, 13, 14, 15, 17, 20, 23-24. Immunoreactive fibers were also found in the neuropile without any particular grouping. Numerous other "positive" perikarya occur in the medioventral portion of the ventral nerve cord, and in the ventral and dorsal parts of the suboesophageal ganglion. In addition to the cell bodies in the cerebral external layer, immunoreactive axons were abundantly observed in the connectives between the ganglia. Moreover, our results demonstrate CCK-like staining in neurons showing variations in size and shape, and in affinity for paraldehyde fuchsin. The present results support the hypothesis that this peptide may exert a role as neurotransmitter or neuromodulator in annelids.

Current status of marine biotoxicology--an overview.

Marine biotoxicology is the science that deals with the poisons produced by marine plants and animals. This subject is briefly reviewed in an attempt to provide an overview of the subject as it pertains to analytical toxicology, and to provide a brief résumé of the chemical structure, toxicity, and mechanism of action of these poisons. Subjects discussed include marine phytotoxicology, protozoan, sponge, coelenterate, echinoderm, mollusk, nemertine, annelid worm, crab, stingray, weeverfish, scorpionfish, ciguatoxin, tetrodotoxin, scombrotoxin, sea snake, turtle, and mammal poisons. Knowledge of the therapeutics of marine biotoxications is still quite meager. Knowledge of the nature and mechanism of action of these biotoxins is essential to a more scientific approach to the treatment of their intoxications.

Porphyrin Pigmentation in Deep-sea Medusae

THE photosensitizing effect of some porphyrins prevents their accumulation in the epidermal layers of animals living in well-illuminated environments, unless the porphyrins are accompanied by some other screening pigment or the animal can retreat into a dark micro-environment1–4. Thus some shallow water marine annelid worms contain considerable quantities of porphyrins in their tissues, but are tubicolous or burrowing in habit5. They are difficult to maintain in the light in the laboratory, often exhibiting symptoms which could be said to resemble those of human porphyria6. Beneath the immediate surface layers of the ocean, however, light intensity is rapidly reduced to very low values, and the deep sea is in almost total darkness. Kennedy7 has noted that the low light intensities in which many marine animals live would prevent the appearance of porphyria symptoms leading to illness and death in the human subject. In these conditions, the accumulation of porphyrins is no longer hazardous, and they may be used like any other pigment for purposes of tissue coloration. The identification of free porphyrins as the source of the general body coloration of certain deep-sea medusae supports this view.

Structure of the haemoglobin of the marine annelid worm, Glycera dibranchiata, at 5.5 A resolution.

THE wide, albeit sporadic, occurrence of haemoglobin among the phyla presents the opportunity to compare the structures of proteins with like functions but from dissimilar biological sources. Among the mammals myoglobin from the sperm whale1 and the seal2, and the subunits of horse3 and human4 haemoglobin, all have essentially the same tertiary structure. It has also been reported5 that haemoglobin from the fly, Chironomus thummi, has a similar structure.

Some fluorescent substances contained in the marine “blood worm” ( Thoracophelia mucronata)

Extracts of two large samples (10–11 kg.) of the marine annelid worm Thoracophelia mucronata were examined, by means of Chromatographic fractionation in ultraviolet light, for fluorescing substances. A great variety and variability of such compounds were found to be present in the worms, collected at different times from the same habitat.