Marine Worm Research Articles

Absorption spectroscopy and binding constants for first-row transition metal complexes of a DOPA-containing peptide

A diverse array of biological systems incorporate 3,4-dihydroxyphenlyalanine (DOPA) into proteins and small molecules for cross-linking and material generation. Marine worm eggshells, sea squirt wound plugs, and marine mussel adhesives may all be formed by combining DOPA-containing molecules with high levels of metals. In order to provide model systems for characterizing these biomaterials, we carried out a study on metal binding to a DOPA-containing peptide. Ultraviolet-visible absorption spectra are presented for the AdopaTP peptide binding to Fe3+, V3+, VO2+, Mn3+, Ti4+, Cu2+, Co2+, and Ni2+ in mono, bis, and where applicable, tris coordination modes. Association constants were determined for selected metal ions binding to the peptide. In general, the spectroscopic and binding properties of this DOPA-containing peptide were found to be similar to those of catechol.

Where is the difference between the genomes of humans and annelids?

The first systematic investigation of an annelid genome has revealed that the genes of the marine worm Platynereis dumerilii are more closely related to those of vertebrates than to those of insects or nematodes.

A β-galactose-specific lectin isolated from the marine worm Chaetopterus variopedatus possesses anti-HIV-1 activity

A 30 kDa β-galactose-specific lectin named CVL was isolated from the polychaete marine worm Chaetopterus variopedatus (Annelida) and its anti-HIV-1 activity in vitro was determined. Results showed that CVL inhibited cytopathic effect induced by HIV-1 and the production of viral p24 antigen. The EC 50 values were 0.0043 and 0.057 μM, respectively. Time-of-addition analysis of anti-HIV-1 activity indicated its action was at the early stage of virus replication. CVL could blocked the cell-to-cell fusion process of HIV infected and uninfected cells with an EC 50 of 0.073 μM. The inhibition of HIV-1 entry into host cells was demonstrated by using fluorescence-based real-time quantify PCR. At CVL concentration of 0.33 μM and 0.07 μM, 86% and 21% virus attachment were blocked, respectively. The anti-HIV-1 action of CVL might relate to blockade of HIV-1 entry into cells.

Bromophenols, both present in marine organisms and in industrial flame retardants, disturb cellular Ca 2+ signaling in neuroendocrine cells (PC12)

Bromophenols are present in polychaetes as well as in algae in marine environments including the North Sea. They are thought to cause the typical sea-like taste and flavour. The ecological function of brominated phenols is not clear yet, but they may play a role in chemical defence and deterrence [Kicklighter, C.E., Kubaneck, J., Hay, M.E., 2004. Do brominated natural products defend marine worms from consumers? Some do, most don’t. Limnol. Oceanogr. 49, 430–441]. Some brominated phenols are commercially used as industrial flame retardants as, e.g., 2,4,6-tribromophenol and are suspected to disrupt the humoral system by showing tyroid hormone-like activity [Legler, I., Brouwer, A., 2003. Are brominated flame retardants endocrine disruptors? Environ. Int. 29, 879–885]. In this study 2-bromophenol (2-BP), 4-bromophenol (4-BP), 2,4-dibromophenol (2,4-DBP), 2,6-dibromophenol (2,6-DBP) and 2,4,6-tribromophenol (2,4,6-TBP), all of which are present in marine organisms, were tested. Especially 2,4-DBP and 2,4,6-TBP showed a significant effect on the Ca 2+ homeostasis in endocrine cells (PC 12). The reduction of depolarization induced Ca 2+ elevations by 2,4-DBP and 2,4,6-TBP and the increase of intracellular Ca 2+ by both substances, partly released from intracellular stores, may suggest a link to the disrupting effect of endocrine systems by brominated phenols. 2,4-DBP was the most potent substance we tested in respect to inhibition of voltage dependent Ca 2+ currents as revealed in whole cell patch clamp experiments. Brominated phenols disturb cellular Ca 2+ signaling with differential efficacy, depending on the number and position of bromine.

Sodium Nitroprusside Potentiates Hydrogen-Sulfide-Induced Contractions in Body Wall Muscle From a Marine Worm

Hydrogen sulfide (H2S) at concentrations of about 0.05 to 1 mmol.l(-1) appears to function as a gasotransmitter in vertebrates, analogous to nitric oxide (NO) and carbon monoxide, but the actions of H2S in invertebrate tissue have not been well studied. In this study, we investigated the role of H2S in modulating body wall muscle tone in the marine echiuran worm Urechis caupo (Echiuridae). We first determined that U. caupo body wall homogenates produce H2S upon addition of L-cysteine and pyridoxal-5'-phosphate (PLP), and that the rate is increased by addition of 2-mercaptoethanol, suggesting the presence of an activated L-serine sulfhydrase pathway. We then measured the contractile response of U. caupo body wall circular muscle strips to sodium hydrosulfide (NaHS)--which produces H2S in solution--and the NO donor sodium nitroprusside (SNP), both with and without subsequent application of acetylcholine (ACh). We found that NaHS alone stimulated contraction in muscle strips equivalent to about one-third the force of ACh alone, whereas SNP alone had no effect on muscle tone. However, simultaneous addition of NaHS with SNP elicited a much stronger contraction, reaching more than twice that of ACh alone, which could be increased further by subsequent application of ACh.

Biomimetic Extraction ofBacillus thuringiensisInsecticidal Crystal Proteins from Soil Based on Invertebrate Gut Fluid Chemistry

Quantitative monitoring of Bacillus thuringiensis (Bt) insecticidal crystal proteins in soil has been hampered by the lack of efficient extraction/detection methods. A novel approach for simple and effective Bt protein extraction was explored by evaluating extraction solutions from invertebrate gut fluids. Marine worm gut fluids were identified as promising for extracting Bt protein from soil. An artificial gut fluid based on these marine worm gut fluids was developed using commercially available chemicals and was evaluated for its ability to extract Bt proteins from soil. On the basis of experiments with Cry1 proteins, the artificial gut fluid in combination with ELISA was highly effective for protein extraction and analysis in a variety of soil types and was well-correlated with bioassay results. Coupling of immunoassay with this extraction method provides, for the first time, an efficient, accurate, and quantitative assay for routine measurement of Bt protein residues in soil.

The changes of three components in coelomic fluid ofUrechis unicinctus exposed to different concentrations of sulfide

The changes in heme (associated with hemoglobin), hemoglobin and hematin in the coelomic fluid of marine worm,Urechis unicinctus, exposed to different concentrations of sulfide, were investigated using biochemical techniques. When exposed to different sulfide concentrations for up to 96 h, the relative amounts of the three components changed in a regular pattern suggesting that the coelomocytes play an important role in the worm's tolerance to sulfide. The possible roles of heme compounds in sulfide tolerance of this species are discussed on the basis of our experimental data.

Toxicity of tributyltin in the marine mollusc Mytilus edulis

Our previous studies have demonstrated that tributyltin (TBT) is genotoxic to the early life stages of marine mussels and worms. Here, the toxicity of TBT to adult organisms was determined using a suite of biomarkers designed to detect cytotoxic, immunotoxic and genotoxic effects. Exposure of adult mussels, Mytilus edulis, to environmentally realistic concentrations of TBTO for 7 days resulted in a statistically significant decrease in cell viability at concentrations of 0.5 μg/l and above. TBT had no effect on phagocytic activity or antioxidant capacity (FRAP assay). There was a statistically significant increase in DNA damage detected using the comet and micronucleus assays between the controls and 0.5, 1 and 5 μg/l of TBTO ( P > 0.0005). Furthermore there was a strong correlation between DNA strand breaks (comet assay) and formation of micronuclei ( P = 0.0005; R 2 = 61.5%). Possible mechanisms by which TBT could damage DNA either directly or indirectly are discussed including the possibility that TBT is genotoxic due to its ability to disrupt calcium homeostasis.

Lessons learned from inshore and deep-sea lobsters concerning alkylphenols

Summary In earlier studies it was found that metamorphosis-inducing activity brings about the settlement of swimming trochophore larvae of the marine worm Capitella capitata. Such activity was attributable to alkylphenols, which are present in marine sediments as well as in hemolymph and embryos of the lobster Homarus americanus. These samples were taken from Long Island Sound and Vineyard Sound. Little or none of this activity was found in offshore lobsters taken from the edge of the continental shelf far from human influence. A comprehensive analytical method based on organic solvent microwave-assisted extraction, followed by solid phase extraction and gas chromatography-mass spectrometry was developed for the simultaneous identification and quantification of bioactive compounds. These compounds include the following four alkylphenols: #1: 2-t-butyl-4-(dimethylbenzyl)phenol; #2: 2,6-bis-(t-butyl)-4-(dimethylbenzyl)phenol; #3: 2,4-bis-(dimethylbenzyl)phenol; and #4: 2,4-bis-(dimethlybenzyl)-6-t-butylphenol. It is reported that 95 (36%) of 262 inshore lobsters contained one or more alkylphenols with average values as follows: compound #1, 11.2 ± 55.9 ng/mL; #2, 23.3 ± 355.9 ng/mL; #3, 116.6 ± 434.2 ng/mL; and #4, 107.1±1167.9 ng/mL, respectively. Only one of 15 offshore lobsters (6.7%), had detectable alkylphenols. Three of five (60%) of deep-sea lobsters had embryos that were carrying higher levels of alkylphenols. These results suggest that inshore lobsters at higher water temperatures are exposed to alkylphenol contamination. Offshore lobsters at lower temperatures appear to be remediated and have lower alkylphenol levels in their blood by having remained offshore, presumably in less contaminated waters. The embryos were most likely formed during inshore reproductive maturation because offshore temperatures are too low for ovarian and egg maturation. These embryos remained contaminated because of their isolation within a relatively impervious shell.

Isolation, characterization, and cDNA-derived amino acid sequence of glycocyamine kinase from the tropical marine worm Namalycastis sp.

We isolated cytoplasmic glycocyamine kinase (GK) and creatine kinase (CK) from the tropical marine worm Namalycastis sp. by ammonium sulfate fractionation, gel filtration on Sephacryl S-200, and DEAE-5PW chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the isolated GK is highly purified and appears to be a heterodimer of two distinct subunits, α and β, with molecular masses of ∼40 kDa. The complete nucleotide sequences of the cDNAs for Namalycastis GKα and GKβ were 1527 (encoding 374 amino acids) and 1579 bp (encoding 390 amino acids), respectively. The predicted amino acid sequences differ only in the N-terminal 50 residues. This is consistent with the characteristics of Neanthes GKα and GKβ chains, which we have previously shown to be generated by alternative splicing. The recombinant enzymes GKα, GKβ, and CK from Namalycastis were successfully expressed in Escherichia coli as maltose-binding protein fusion proteins. In contrast to the stable GKβ enzyme, GKα was quite unstable, and its activity decreased remarkably with time. Thus, the N-terminal 50 residues appear to play a key role in enzyme stability. The kinetic parameters for the native GK heterodimer were similar to GKβ, suggesting that GKα would have an activity similar to GKβ if part of a heterodimer. This is the first report of precise kinetic parameters for GK. Finally, based on our results, we present a model for pluriphosphagen function in Namalycastis wherein cytoplasmic GK and CK and mitochondrial CK function together with phosphocreatine and phosphoglycocyamine to enable cells to respond quickly to a sudden large energy requirement.

Worm's Light-Sensing Proteins Suggest Eye's Single Origin

New data reported on page 869 showing unexpected similarities between photoreceptors of a marine worm and humans suggest that even the earliest animals had the makings of both vertebrate and invertebrate visual systems.

Marine worm sports two kinds of 'eyes'

Marine worm sports two kinds of 'eyes'

Cleavage in Nemertoderma westbladi (Nemertodermatida) and its phylogenetic significance

Recent phylogenetic analyses of ribosomal and protein coding nuclear genes place the marine worms within the Nemertodermatida as one of the oldest lineages among the bilaterian animals. We studied the early embryonic cleavage in Nemertoderma westbladi to provide the first account of nemertodermatid early development. Live embryos were studied with interference microscopy and fixed embryos were either sectioned or studied with confocal laser scanning microscopy. Initially the divisions in the embryo are radial, but then micromeres are shifted clockwise generating a spiral pattern. The four-cell stage is characterized by duets of macromeres and micromeres and thus resembles the duet cleavage reported from members of the Acoela. However, subsequent stages differ from the acoel duet pattern and also from quartet spiral cleavage. The optimization of the cleavage pattern on current phylogenetic hypotheses with Nemertodermatida and Acoela as early bilaterian branches is discussed.

Chaetognaths: a useful model for studying heat shock proteins. Effect of wound healing

The pattern of expression of Heat Shock Protein 70 (Hsp70), a highly conserved cellular protein chaperone, was investigated in Chaetognatha, a very important phylum of marine worms, which play a major role in marine food webs. The in toto distribution of Hsp70-like immunoreactivity was assessed in both intact and experimentally wounded specimens of Spadella cephaloptera Busch, 1851 that have been cut transversally just above the seminal vesicles. In intact animals, the ciliary organs, the corona ciliata, the coronal nerve and the spermatocytes express Hsp70 proteins. The kinetic of the expression pattern has been followed during the wound healing from the lesion to 5 days after. The Hsp70 immunoreactivity was observed according to time in the cerebral and suboesophageal ganglia and in the plexuses of the hood, the mouth, the neck and the tail region. At the wound level, the time-dependent Hsp70 expression was detected in the epidermal cells and along numerous muscle fibres of the tail region. Five days after the tail section, two Hsp70 immunoreactive areas were observed on both sides of the healed wound that correspond to the sites of formation of two new seminal vesicles. It is suggested that, in Chaetognaths, Hsp70 may be implicated in the regulation of several cellular processes especially at the level of the spermatocytes in intact and wounded specimens, and of the nervous system and muscular apparatus during the wound healing. It appears that chaetognaths are a good model as indicator of stress responses for experimental studies at the level of a whole organism.

High sensitivity to chronically elevated CO 2 levels in a eurybathic marine sipunculid

CO 2 levels are expected to rise (a) in surface waters of the oceans as atmospheric accumulation continues or (b) in the deep sea, once industrial CO 2 dumping is implemented. These scenarios suggest that CO 2 will become a general stress factor in aquatic environments. The mechanisms of sensitivity to CO 2 as well as adaptation capacity of marine animals are insufficiently understood. Here, we present data obtained in Sipunculus nudus, a sediment-dwelling marine worm that is able to undergo drastic metabolic depression to survive regular exposure to elevated CO 2 levels within its natural habitat. We investigated animal survival and the proximate biochemical body composition during long-term CO 2 exposure. Results indicate an unexpected and pronounced sensitivity characterized by the delayed onset of enhanced mortality at CO 2 levels within the natural range of concentrations. Therefore, the present study contrasts the previously assumed high-CO 2 tolerance of animals adapted to temporary hypercapnia. As a consequence, we expect future loss of species and, thereby, detrimental effects on marine benthic ecosystems with as yet poorly defined critical thresholds of long-term tolerance to CO 2.

Biography of Juan Carlos Castilla.

Chile boasts almost 4,500 kilometers of shoreline with hundreds of unique species, an exclusive ecology seen nowhere else. Along with distinct families of shellfish, marine worms, and tunicates, humans manipulate and dominate the environment. Before the last two decades, researchers had not investigated the impact of human activity on the Chilean coastline. However, in 1985, Juan Carlos Castilla published a pioneering article based on a study where humans were excluded from a small segment of the shore (1–3). His findings formed the basis for conservation practices and environmental laws, leading to important changes in the Chilean fishing industry.

The Sipuncula of sublittoral New Zealand, with a key to all New Zealand species

Fourteen species of the unsegmented marine worms known as Sipuncula are reported from sublittoral waters from recent collections of 519 specimens around New Zealand. Five are new to the New Zealand Exclusive Economic Zone (EEZ). A taxonomic key to all 26 known New Zealand species is provided. All six families and nine of the 17 genera of Sipuncula are represented.

C-terminal lysines determine phospholipid interaction of sarcomeric mitochondrial creatine kinase.

High affinity interaction between octameric mitochondrial creatine kinase (MtCK) and the phospholipid cardiolipin in the inner mitochondrial membrane plays an important role in metabolite channeling between MtCK and inner membrane adenylate translocator, which itself is tightly bound to cardiolipin. Three C-terminal basic residues revealed as putative cardiolipin anchors in the x-ray structures of MtCK and corresponding to lysines in human sarcomeric MtCK (sMtCK) were exchanged by in vitro mutagenesis (K369A/E, K379Q/A/E, K380Q/A/E) to yield double and triple mutants. sMtCK proteins were bacterially expressed, purified to homogeneity, and verified for structural integrity by enzymatic activity, gel filtration chromatography, and CD spectroscopy. Interaction with cardiolipin and other acidic phospholipids was quantitatively analyzed by light scattering, surface plasmon resonance, and fluorescence spectroscopy. All mutant sMtCKs showed a strong decrease in vesicle cross-linking, membrane affinity, binding capacity, membrane ordering capability, and binding-induced changes in protein structure as compared with wild type. These effects did not depend on the nature of the replacing amino acid but on the number of exchanged lysines. They were moderate for Lys-379/Lys-380 double mutants but pronounced for triple mutants, with a 30-fold lower membrane affinity and an entire lack of alterations in protein structure compared with wild-type sMtCK. However, even triple mutants partially maintained an increased order of cardiolipin-containing membranes. Thus, the three C-terminal lysines determine high affinity sMtCK/cardiolipin interaction and its effects on MtCK structure, whereas low level binding and some effect on membrane fluidity depend on other structural components. These results are discussed in regard to MtCK microcompartments and evolution.

Diversity of folds in animal toxins acting on ion channels.

Animal toxins acting on ion channels of excitable cells are principally highly potent short peptides that are present in limited amounts in the venoms of various unrelated species, such as scorpions, snakes, sea anemones, spiders, insects, marine cone snails and worms. These toxins have been used extensively as invaluable biochemical and pharmacological tools to characterize and discriminate between the various ion channel types that differ in ionic selectivity, structure and/or cell function. Alongside the huge molecular and functional diversity of ion channels, a no less impressive structural diversity of animal toxins has been indicated by the discovery of an increasing number of polypeptide folds that are able to target these ion channels. Indeed, it appears that these peptide toxins have evolved over time on the basis of clearly distinct architectural motifs, in order to adapt to different ion channel modulating strategies (pore blockers compared with gating modifiers). Herein, we provide an up-to-date overview of the various types of fold from animal toxins that act on ion channels selective for K+, Na+, Ca2+ or Cl- ions, with special emphasis on disulphide bridge frameworks and structural motifs associated with these peptide folds.

Do brominated natural products defend marine worms from consumers? Some do, most don’t

Worms and other marine invertebrates living in soft sediments commonly produce brominated natural products that have been hypothesized to function as defenses against consumers, but this hypothesis has not been tested directly. When 16 species of worms from a Georgia mud flat were fed to two sympatric fishes (Fundulus heteroclitus, Leiostomus xanthurus) and a crab (Callinectes similis), 15 species (94×;) were palatable to all three predators. Only the hemichordate Saccoglossus kowalevskii was unpalatable to both fishes, but even it was readily consumed by the crab. Bioassay‐guided chemical investigations demonstrated that Saccoglossus kowalevskii was rejected by fishes because it contained 2,3,4‐tribromopyrrole at 0.2×; of worm dry mass. This is the first direct test of brominated worm metabolites as defenses against sympatric consumers. The deterrence of 2,3,4‐tribromopyrrole in S. kowalevskii may explain why densities of this worm increase 40‐fold during seasons when predation is high and densities of palatable worms decline sharply. To more broadly examine the effects of brominated metabolites on worm palatability, we collected from North Carolina, Georgia, and Florida 14 species reported to produce brominated metabolites. These species were fed to sympatric consumers to assess palatability and also analyzed for brominated metabolites by gas chromatography mass spectrometry (GC/MS). Nine of the fourteen species contained brominated metabolites, but only two were unpalatable. In a final test, five additional brominated metabolites produced by marine worms were added to palatable foods at natural concentrations and at up to 20× natural concentrations. None deterred feeding at natural concentrations, one was deterrent at 5−15× natural concentration, and four had no effect at even 20× natural concentration. Thus, while one worm was defended by a brominated compound, most worms containing brominated metabolites were palatable, and brominated natural products seldom functioned as chemical defenses against consumers.