Nemertean Research Articles

Size‐Related Variation in the Diet of Juvenile Tautogs from Long Island Sound

AbstractJuvenile tautogs Tautoga onitis were collected from shallow‐water nursery areas along the Connecticut coast of Long Island Sound from June through September of 2000, 2001, and 2003. A total of 164 juveniles (8.9–165 mm standard length) were collected using several different methods. The digestive tracts of 152 (92.68%) juveniles each contained at least one identifiable prey item; 12 (7.32%) stomachs were empty. The number of different identifiable prey taxa in the digestive tracts ranged from one to four. Juvenile tautogs from the study area consumed 21 mutually exclusive prey taxa. Over all size‐classes, crustacean prey constituted a major component of the diet. Gammarid amphipods (present in 45.39% of the fish with food in their digestive tracts) and calanoid copepods (16.45%) were the predominant prey. Less commonly consumed prey included hermit crabs Pagurus spp., lady crabs Ovalipes ocellatus, mud crabs (Xanthidae), Asian shore crabs Hemigrapsus sanguineus, eggs of horseshoe crabs Limulus polyphemus, sevenspine bay shrimp Crangon septemspinosa, shore shrimp Palaemonetes spp., polychaete worms (Polychaeta), milky ribbon worms Cerebratulus lacteus, common Atlantic slipper shells Crepidula fornicata, eastern oysters Crassostrea virginica, and blue mussels Mytilus edulis. Hierarchical cluster analysis of prey consumed by young tautogs revealed three distinct size‐related feeding groups: (1) small fish (9–30 mm) that consumed calanoid copepods and gammarid amphipods, (2) midsized fish (31–70 mm) that were generalist feeders, concentrating on polychaete worms and crustaceans, and (3) large fish (81–165 mm) that preyed on mollusks, shrimps, and crabs.

Osmotic induction of stress proteins in nemerteans

Nemerteans can experience fluctuating salinity during exposure to low tides in intertidal mudflats. This study attempted to determine if stress proteins (SPs) can be induced in nemerteans exposed to fluctuating salinities under laboratory conditions. Paranemertes peregrina Coe 1901 were collected from intertidal mudflats. The ribbon worms were then acclimated at 34‰ for 3–4 days prior to the salinity stress experiments. For the hyperosmotic experiment, 39 and 44‰ were selected. Salinities of 24 and 29‰ were used for the hyposmotic experiment. Both experiments used 34‰ as the control. Three worms were sampled at 0, 2, 4, 6, 12, 18 and 24 h after incubation at each of the salinities. Weights were recorded before and after the sampling periods to determine water efflux and influx under hyperosmotic and hyposmotic conditions, respectively. SPs detected in the nemertean proteins were separated by SDS–PAGE electrophoresis and identified using Western immunoblots with specific antibodies. SP levels were then quantified by densitometry. Worms from the 39 and 44‰ treatments showed significant weight loss compared with control worms. Worms from the 24 and 29‰ treatments had significant weight gain compared with control worms. SP70 and SP90 were identified. Two‐way analysis of variance showed varying significant effects of salinity, time and the interaction between the two parameters on the induction of SP70 and SP90 in both the hyperosmotic and hyposmotic experiments. Detection of SPs demonstrates their importance in the physiological response of nemerteans to fluctuating salinities.

Delimiting species in the hoplonemertean genus Tetrastemma (phylum Nemertea): morphology is not concordant with phylogeny as evidenced from mtDNA sequences

The marine genus Tetrastemma contains monostiliferous hoplonemertean (phylum Nemertea) species which mostly undergo direct development with no free-swimming stages of larvae. The lack of a pelagic phase, and the fact that many benthic species lay eggs attached to the bottom substrate, are obvious restrictions on dispersal and gene flow. Nevertheless, some of the species, for example T. candidum and T. melanocephalum, are described as ubiquitous and are reported from waters all over the world. We studied genetic variation and evolutionary relationships in order to assess whether they are concordant with external characters in samples of nine morphologically distinct forms formally named as different Tetrastemma species, from different geographical localities. We estimated the phylogeny and species network based on 539 base pairs of the mitochondrial protein-coding gene cytochrome oxidase-1 (CO1) for 30 ingroup specimens. From this, we assessed the evolutionary history and phylogenetic relationships between these forms. We conclude that in most cases there was no correspondence between evolutionary lineage and morphotype. Our results thus indicate that morphological species delimitation in nemerteans may be questionable, and that this in turn may have a profound effect upon estimates of species diversity within the phylum. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 86, 201–212.

Genus Tetrastemma Ehrenberg, 1831 (Phylum Nemertea)—A natural group? Phylogenetic relationships inferred from partial 18S rRNA sequences

We investigated the monophyletic status of the hoplonemertean taxon Tetrastemma by reconstructing the phylogeny for 22 specimens assigned to this genus, together with another 25 specimens from closely related hoplonemertean genera. The phylogeny was based on partial 18S rRNA sequences using Bayesian and maximum likelihood analyses. The included Tetrastemma-species formed a well-supported clade, although the within-taxon relationships were unsettled. We conclude that the name Tetrastemma refers to a monophyletic taxon, but that it cannot be defined by morphological synapomorphies, and our results do not imply that all the over 100 species assigned to this genus belong to it. The results furthermore indicate that the genera Amphiporus and Emplectonema are non-monophyletic.

Germinal vesicle breakdown is not fully dependent on MAPK activation in maturing oocytes of marine nemertean worms

Previously, it has been shown that oocytes of marine nemertean worms resume meiosis and undergo germinal vesicle breakdown (GVBD) following treatment with either natural seawater (NSW), or the neurohormone serotonin (5-hydroxytryptamine or 5-HT). In this investigation of the nemerteans Cerebratulus lacteus and Cerebratulus sp., immunoblots and kinase assays were used to compare the roles of two regulatory kinases: mitogen-activated protein kinase (MAPK) and Cdc2/cyclin B (referred to as maturation promoting factor or MPF). Based on such analyses, an ERK (extracellular signal regulated kinase) type of MAPK was found to be activated concurrently with Cdc2/cyclin B during NSW- and 5-HT-induced maturation. MAPK activation occurred prior to GVBD and seemed to be controlled primarily by phosphorylation rather than de novo protein synthesis. Inhibition of MAPK signaling by U0126 was capable of delaying but not permanently blocking Cdc2/cyclin B activation and GVBD in 5-HT treated oocytes and subsets of NSW-treated oocytes. Collectively such data indicated that GVBD is not fully dependent on MAPK activation, since Cdc2/cyclin B can apparently be activated by MAPK-independent mechanism(s) in maturing nemertean oocytes.

Usamaro Takakura (1867–1944), Japanese pioneer nemertean researcher

Usamaro Takakura (1867–1944), a pioneer Japanese specialist on nemerteans (phylum Nemertea), established 14 new species and one new genus, Coeia. This paper gives a brief outline of his life and publications, together with the list of his new taxa, the type materials for which are all lost.

Immunoenzymatic visualization of tetrodotoxin (TTX) in Cephalothrix species (Nemertea: Anopla: Palaeonemertea: Cephalotrichidae) and Planocera reticulata (Platyhelminthes: Turbellaria: Polycladida: Planoceridae)

Tetrodotoxin (TTX) was localized as brown color in different tissues of an undescribed species of the nemertean genus Cephalothrix (phylum Nemertea) and a turbellarian Planocera reticulata (phylum Platyhelminthes) on light microscopy by means of a monoclonal anti-TTX antibody. In the Cephalothrix sp., TTX was recognized in the vesicles apically arranged in the bacillary cells in the epidermis, basal lamina, the granular cells in the proboscis epithelium, rhynchocoel epithelium, and the vesicles in the basal portion of the intestinal wall near the blood vessels and rhynchocoel. The excretory system and the ovum also showed positive reaction of TTX antigen–antibody. On the other hand, the hermaphrodite flatworm P. reticulata exhibited TTX antigen–antibody complex only in their ovum. To our knowledge, this is the first experimental effort on micro-distribution of TTX in invertebrates.

Tango waves in a bidomain model of fertilization calcium waves

Fertilization of an egg cell is marked by one or several Ca 2+ waves that travel across the intra-cellular space, called fertilization Ca 2+ waves. Patterns of Ca 2+ waves observed in mature or immature oocytes include traveling fronts and pulses as well as concentric and spiral waves. These patterns have been studied in other excitable media in physical, chemical, and biological systems. Here, we report the discovery of a new wave phenomenon in the numerical study of a bidomain model of fertilization Ca 2+ waves. This wave is a front that propagates in a back-and-forth manner that resembles the movement of tango dancers, thus is called a tango wave. When the medium is excitable, a forward-moving tango wave can generate traveling pulses that propagate down the space without reversal. The study shows that the occurrence of tango waves is related to spatial inhomogeneity in the local dynamics. This is tested and confirmed by simulating similar waves in a medium with stationary spatial inhomogeneity. Similar waves are also obtained in a FitzHugh–Nagumo system with a linear spatial ramp. In both the bidomain model of Ca 2+ waves and the FitzHugh–Nagumo system, the front is stable when the slope of a linear ramp is large. As the slope decreases beyond a critical value, front oscillations occur. The study shows that tango waves facilitate the dispersion of localized Ca 2+. Key features of the bidomain model underlying the occurrence of tango waves are revealed. These features are commonly found in egg cells of a variety of species. Thus, we predict that tango waves can occur in real egg cells provided that a slowly varying inhomogeneity does occur following the sperm entry. The observation of tango wave-like waves in nemertean worm and ascidian eggs seems to support such a prediction.

Endoplasmic reticulum reorganizations and Ca2+ signaling in maturing and fertilized oocytes of marine protostome worms: the roles of MAPKs and MPF.

Before a proper Ca(2+) response is produced at fertilization, oocytes typically undergo a maturation process during which their endoplasmic reticulum (ER) is restructured. In marine protostome worms belonging to the phylum Nemertea, the ER of maturing oocytes forms numerous distinct clusters that are about 5 micro m in diameter. After fertilization, mature oocytes with such aggregates generate a normal series of Ca(2+) oscillations and eventually disassemble their ER clusters at around the time that the oscillations cease. Immature oocytes, however, lack prominent ER clusters and fail to exhibit repetitive Ca(2+) oscillations upon insemination, collectively suggesting that cell cycle-related changes in ER structure may play a role in Ca(2+) signaling. To assess the effects of meiotic regulators on the morphology of the ER and the type of Ca(2+) response that is produced at fertilization, nemertean oocytes were treated with pharmacological modulators of mitogen-activated protein kinases (MAPKs) or maturation-promoting factor (MPF) prior to confocal microscopic analyses. Based on such imaging studies and correlative assays of kinase activities, MAPKs of the ERK1/2 type (extracellular signal regulated kinases 1/2) do not seem to be essential for either structural reorganizations of the ER or repetitive Ca(2+) signaling at fertilization. Conversely, MPF levels appear to modulate both ER structure and the capacity to produce normal Ca(2+) oscillations. The significance of these findings is discussed with respect to other reports on ER structure, MPF cycling and Ca(2+) signaling in oocytes of deuterostome animals.

Parasites and Symbionts of Crabs from Ubatuba Bay, São Paulo State, Brazil

During July 1999, 172 crabs from Ubatuba Bay, Sao Paulo, Brazil, were examined from 4 families as follows: Portunidae, Arenaeus cribrarius, Callinectes danae, Callinectes ornatus, Charybdis hellerii, Portunus spinimanus, and Portunus spinicarpus; Calappidae, Hepatus pudibundus; Majidae, Libinia ferreirae; Leucosiidae, Persephona mediterranea and Persephona lichtensteinii. One cephaline gregarine symbiont was found in the hindgut of a specimen of Cal. ornatus, and no positively identified, internal and multicelled symbionts were detected. Twenty-one specimens (14.3%) from 7 of the 10 species (all except H. pudibundus, P. mediterranea, and P. lichtensteinii) carried the nemertean worm, Carcinonemertes carcinophila ct. imminuta in either their egg masses or gills. This is the first report of this epibiont from the Ubatuba region. Two specimens of an undescribed polychaete worm were found in the gill of a specimen of L. ferreirae. A stalked barnacle, Octolasmis lowei, was found on the gills of 11 crabs (7.5%). Only 3 crab species (36.6% of Cal. danae, 9.0% of Cal. ornatus, and 7.7% of Po. spinimanus) carried the typical barnacle, Chelonibia patula, on their dorsal carapaces. The percentage of individuals of Cal. danae and Cal. ornatus hosting barnacles (prevalence) as well as the number of barnacles on individual crabs was positively correlated with crab size.

Paralytic toxicity in the ribbon worm Cephalothrix species (Nemertea) in Hiroshima Bay, Hiroshima Prefecture, Japan and the isolation of tetrodotoxin as a main component of its toxins

Paralytic toxicity of ribbon worms ‘himomushi’ in Japanese, identified as undescribed species of the genus Cephalothrix, found on the surface of the shells of cultured oysters in Hiroshima Bay, Hiroshima Prefecture was examined between April 1998 and December 2001. The toxicity study showed that all of specimens were found to contain toxins with strong paralytic action in mice; the highest toxicity (as tetrodotoxin, TTX) was 25,590 mouse units (MU) per gram for whole body throughout the monitoring period. The main toxic component of this himomushi toxin (HMT) was isolated from a pooled specimen (390 g; total toxicity 2,897,000 MU) by a method that consisted of treatment with activated charcoal, chromatography on Bio-Gel P-2 and Bio-Rex 70 (H + form), and finally crystallization from an acidified methanolic solution. The recrystallized toxin showed a specific toxicity of 3520 MU/mg. This toxin showed (M+H) + and (M+H–H 2O) + ion peaks at m/ z 320 and 302, respectively, by electrospray ionization–mass spectrometry (ESI–MS). The absorption band at 3353, 3235, 1666, 1612 and 1076 cm −1 were observed in infrared spectrum of this toxin. This spectrum was indistinguishable from that of TTX. The 1H-NMR spectrum for the recrystallized toxin was the same as that for TTX. The pair of doublets centered at 2.33 ( J=10.0 Hz) and 5.48 ppm ( J=10.0 Hz) which are characteristic of TTX, were shown to be coupled by double irradiation. Furthermore, by gas chromatography–mass spectrometry (GC–MS) of the alkali-hydrolyzate of this toxin indicated the presence of quinazoline skeleton (C9-base) specific to TTX.

The production of tetrodotoxin-like substances by nemertean worms in conjunction with bacteria

Evidence is presented which strongly indicates a relationship between the presence of Vibrio bacteria, probably Vibrio alginolyticus, and the synthesis of tetrodotoxin (TTX)-like chemicals in seven species of British nemerteans. The occurrence of these substances and associated Vibrio bacteria in these species was investigated by bacteriological, chromatographic, spectroscopic and ultraviolet spectrometric techniques. It is suggested that these toxins are utilised by the nemerteans as a chemical defence against potential predators.

The 109 Residue Nerve Tissue Minihemoglobin from Cerebratulus lacteus Highlights Striking Structural Plasticity of the α-Helical Globin Fold

A very short hemoglobin (CerHb; 109 amino acids) binds O 2 cooperatively in the nerve tissue of the nemertean worm Cerebratulus lacteus to sustain neural activity during anoxia. Sequence analysis suggests that CerHb tertiary structure may be unique among the known globin fold evolutionary variants. The X-ray structure of oxygenated CerHb (R factor 15.3%, at 1.5 Å resolution) displays deletion of the globin N-terminal A helix, an extended GH region, a very short H helix, and heme solvent shielding based on specific aromatic residues. The heme-bound O 2 is stabilized by hydrogen bonds to the distal TyrB10-GlnE7 pair. Ligand access to heme may take place through a wide protein matrix tunnel connecting the distal site to a surface cleft located between the E and H helices.

The Antarctic nemertean Parborlasia corrugatus: an example of an extreme oxyconformer

The Antarctic nemertean worm, Parborlasia corrugatus, exhibits gigantism, reaching at least 100 g, yet lacks any specialised respiratory organs. The diffusion of oxygen into this worm occurs cutaneously. We examined the metabolic rate of P. corrugatus at -1degreesC in response to decreasing ambient PO2. As the PO2 of the water decreased. so did the metabolic rate of P. corrugatus, indicating that this nemertean worm is an extreme example of an oxyconformer. When the water PO2 decreased below about 120 mmHg, the normally short, round worms became elongated and extremely flattened. This behavioural mechanism would allow for an increase in surface area of the skin, thereby facilitating the diffusion of oxygen.

Comparative biology of oogenesis in nemertean worms

AbstractStricker, S. A., Smythe, T. L., Miller, L. and Norenburg, J. L. 2001. Comparative biology of oogenesis in nemertean worms. —Acta Zoologica (Stockholm) 82: 213–230In order to supplement previous analyses of oogenesis in nemertean worms, this study uses light and electron microscopy to compare the ovaries and oocytes in 16 species of nemerteans that represent various taxa within the phylum. Nemertean ovaries comprise serially repeated sacs with an ovarian wall that characteristically includes myofilament‐containing cells interspersed among the germinal epithelium. Each oocyte can attach to the germinal epithelium by a vegetally situated stalk and resides in the ovarian lumen without being surrounded by follicle cells. In the ovary, oocytes arrest at prophase I of meiosis and contain a hypertrophied nucleus (‘germinal vesicle’) that often possesses multiple nucleoli. Intraovarian growth apparently involves an autosynthetic mode of yolk formation in most nemerteans and generates oocytes that measure ~60 µm to 1 mm. When fully developed, oocytes can be discharged through a short gonoduct and are either spawned freely or deposited within egg cases. In most species, oocytes released from the ovary possess extracellular coats and resume maturation by undergoing germinal vesicle breakdown (GVBD). Such post‐GVBD specimens also form a punctate endoplasmic reticulum that may facilitate fertilization and development.

Crystallization and preliminary X-ray analysis of neural haemoglobin from the nemertean worm Cerebratulus lacteus.

The nemertean worm Cerebratulus lacteus neural tissue haemoglobin (109 amino acids, the shortest known haemoglobin) has been overexpressed in Escherichia coli, purified and crystallized. A highly redundant native data set has been collected at the Cu K(alpha) wavelength to 2.05 A resolution. The crystals belong to the orthorhombic P2(1)2(1)2(1) space group, with unit-cell parameters a = 42.5, b = 43.1, c = 60.2 A and one molecule per asymmetric unit. The anomalous difference Patterson map clearly reveals the position of the haem Fe atom, thus paving the way for MAD/SAD structure determination.

Phylogenetic Relationships among Higher Nemertean (Nemertea) Taxa Inferred from 18S rDNA Sequences

We estimated the phylogenetic relationships of 15 nemertean (phylum Nemertea) species from the four subclasses Hoplo-, Hetero-, Palaeo-, and Bdellonemertea with 18S rDNA sequence data. Three outgroup taxa were used for rooting: Annelida, Platyhelminthes, and Mollusca. Parsimony and maximum-likelihood analyses supported the monophyletic status of the Heteronemertea and a taxon consisting of hoplonemerteans and Bdellonemertea, while indicating that Palaeonemertea is paraphyletic. The monophyletic status of the two nemertean classes Anopla and Enopla is not supported by the data. The unambiguous clades are well supported, as assessed by a randomization test (bootstrapping) and branch support values.

5-HT causes an increase in cAMP that stimulates, rather than inhibits, oocyte maturation in marine nemertean worms.

In the nemertean worms Cerebratulus lacteus and Micrura alaskensis, 5-HT (=5-hydroxytryptamine, or serotonin) causes prophase-arrested oocytes to mature and complete germinal vesicle breakdown (GVBD). To identify the intracellular pathway that mediates 5-HT stimulation, follicle-free oocytes of nemerteans were assessed for GVBD rates in the presence or absence of 5-HT after being treated with various modulators of cAMP, a well known transducer of 5-HT signaling and an important regulator of hormone-induced maturation in general. Unlike in many animals where high levels of intra-oocytic cAMP block maturation, treatment of follicle-free nemertean oocytes with agents that elevate cAMP (8-bromo-cAMP, forskolin or inhibitors of phosphodiesterases) triggered GVBD in the absence of added 5-HT. Similarly, 5-HT caused a substantial cAMP increase prior to GVBD in nemertean oocytes that had been pre-injected with a cAMP fluorosensor. Such a rise in cAMP seemed to involve G-protein-mediated signaling and protein kinase A (PKA) stimulation, based on the inhibition of 5-HT-induced GVBD by specific antagonists of these transduction steps. Although the downstream targets of activated PKA remain unknown, neither the synthesis of new proteins nor the activation of MAPKs (mitogen-activated protein kinases) appeared to be required for GVBD after 5-HT stimulation. Alternatively, pre-incubation in roscovitine, an inhibitor of maturation-promoting factor (MPF), prevented GVBD, indicating that maturing oocytes eventually need to elevate their MPF levels, as has been documented for other animals. Collectively, this study demonstrates for the first time that 5-HT can cause immature oocytes to undergo an increase in cAMP that stimulates, rather than inhibits, meiotic maturation. The possible relationship between such a form of oocyte maturation and that observed in other animals is discussed.

OVICIDES JULIEAE N. GEN., N. SP. (NEMERTEA: CARCINONEMERTIDAE) ON XANTHID CRABS FROM THE GREAT BARRIER REEF, AUSTRALIA

A new genus and species of symbiotic, semi-parasitic nemertean worm, Ovicides julieae n. gen., n. sp., is described from the xanthid crabs Chlorodiella nigra and C. xishaensis. The new genus possesses accessory stylet pouches and exhibits simultaneous hermaphroditism, features new to the family Carcinonemertidae. The new species has a relatively small anterior proboscis chamber and small basis, yet it possesses the largest stylet to basis ratio (up to 0.650) reported for the family. It also has a flame cell pattern of at least 2[(3+3)], which is different from those reported for two other species. The worm occurred in a lightly granulated sheath on the abdominal sterna, pleopods, and clutches of its host, but few adult worms were found. The species exhibited a high host specificity and was more frequently found on C. nigra than on C. xishaensis (7.5 vs. 0.4%, overall prevalence, respectively). Worms were only found on female crabs, and worm intensity showed a modest but significantly positive correlation with host size. Ovicides julieae represents the second genus in the Carcinonemertidae, the fifth described carcinonemertid species from Australia, and the eleventh described species in the family.

Preliminary study of heat shock proteins in nemerteans

Nemerteans experience varying environmental temperatures during low tide exposures. Inducible heat shock proteins (hsps) have been reported for most organisms following both artificial heat stress and natural environmental temperature variations. This preliminary study reports the presence of hsps in the phylum Nemertea. A lethal temperature of 36 °C was determined for Paranemertes peregrina Coe, 1901. The nemerteans were exposed to a temperature of 34 °C for 2 h. After a 2 h recovery time, the worms were then analyzed for hsps by SDS–PAGE and western immunoblot protocols. Control worms were allowed to acclimate to ambient temperatures (13–15 °C) before hsp analysis. Hsp70 and hsp90 were detected in both the control and heat-shocked worms in highly variable concentrations, and the latter group had significantly elevated hsp70 levels. In addition, the analysis detected different isoforms of hsp70. The detection of hsps indicates a possible role in nemertean physiology during response to thermal stress, and potentially to other environmental challenges.