New Pyridyl and Dihydroisoquinoline Alkaloids Isolated from the Chevron Nemertean Amphiporus angulatus.

Nemertean worms contain toxins that are used to paralyze their prey and to deter potential predators. Hoplonemerteans often contain pyridyl alkaloids like anabaseine that act through nicotinic acetylcholine receptors and crustacean chemoreceptors. The chemical reactivity of anabaseine, the first nemertean alkaloid to be identified, has been exploited to make drug candidates selective for alpha7 subtype nAChRs. GTS-21, a drug candidate based on the anabaseine scaffold, has pro-cognitive and anti-inflammatory actions in animal models. The circumpolar chevron hoplonemertean Amphiporus angulatus contains a multitude of pyridyl compounds with neurotoxic, anti-feeding, and anti-fouling activities. Here, we report the isolation and structural identification of five new compounds, doubling the number of pyridyl alkaloids known to occur in this species. One compound is an isomer of the tobacco alkaloid anatabine, another is a unique dihydroisoquinoline, and three are analogs of the tetrapyridyl nemertelline. The structural characteristics of these ten compounds suggest several possible pathways for their biosynthesis.

The localization of Toll and Imd pathway and complement system components and their response to Vibrio infection in the nemertean Lineus ruber.

BACKGROUND: Innate immunity is the first line of defense against pathogens. In animals, the Toll pathway, the Imd pathway, the complement system, and lectins are well-known mechanisms involved in innate immunity. Although these pathways and systems are well understood in vertebrates and arthropods, they are understudied in other invertebrates. RESULTS: To shed light on immunity in the nemertean Lineus ruber, we performed a transcriptomic survey and identified the main components of the Toll pathway (e.g., myD88, dorsal/dif/NFκB-p65), the Imd pathway (e.g., imd, relish/NFκB-p105/100), the complement system (e.g., C3, cfb), and some lectins (FreD-Cs and C-lectins). In situ hybridization showed that TLRß1, TLRß2, and imd are expressed in the nervous system; the complement gene C3-1 is expressed in the gut; and the lectins are expressed in the nervous system, the blood, and the gut. To reveal their potential role in defense mechanisms, we performed immune challenge experiments, in which Lineus ruber specimens were exposed to the gram-negative bacteria Vibrio diazotrophicus. Our results show the upregulation of specific components of the Toll pathway (TLRα3, TLRß1, and TLRß2), the complement system (C3-1), and lectins (c-lectin2 and fred-c5). CONCLUSIONS: Therefore, similarly to what occurs in other invertebrates, our study shows that components of the Toll pathway, the complement system, and lectins are involved in the immune response in the nemertean Lineus ruber. The presence of these pathways and systems in Lineus ruber, but also in other spiralians; in ecdysozoans; and in deuterostomes suggests that these pathways and systems were involved in the immune response in the stem species of Bilateria.

Proteo-Transcriptomic Analysis Identifies Potential Novel Toxins Secreted by the Predatory, Prey-Piercing Ribbon Worm Amphiporus lactifloreus.

Nemerteans (ribbon worms) employ toxins to subdue their prey, but research thus far has focused on the small-molecule components of mucus secretions and few protein toxins have been characterized. We carried out a preliminary proteotranscriptomic analysis of putative toxins produced by the hoplonemertean Amphiporus lactifloreus (Hoplonemertea, Amphiporidae). No variants were found of known nemertean-specific toxin proteins (neurotoxins, cytotoxins, parbolysins or nemertides) but several toxin-like transcripts were discovered, expressed strongly in the proboscis, including putative metalloproteinases and sequences resembling sea anemone actitoxins, crown-of-thorn sea star plancitoxins, and multiple classes of inhibitor cystine knot/knottin family proteins. Some of these products were also directly identified in the mucus proteome, supporting their preliminary identification as secreted toxin components. Two new nemertean-typical toxin candidates could be described and were named U-nemertotoxin-1 and U-nemertotoxin-2. Our findings provide insight into the largely overlooked venom system of nemerteans and support a hypothesis in which the nemertean proboscis evolved in several steps from a flesh-melting organ in scavenging nemerteans to a flesh-melting and toxin-secreting venom apparatus in hunting hoplonemerteans.

New Invasive Nemertean Species (Cephalothrix Simula) in England with High Levels of Tetrodotoxin and a Microbiome Linked to Toxin Metabolism.

The marine nemertean Cephalothrix simula originates from the Pacific Ocean but in recent years has been discovered in northern Europe. The species has been associated with high levels of the marine neurotoxin Tetrodotoxin, traditionally associated with Pufferfish Poisoning. This study reports the first discovery of two organisms of C. simula in the UK, showing the geographical extent of this species is wider than originally described. Species identification was initially conducted morphologically, with confirmation by Cox 1 DNA sequencing. 16S gene sequencing enabled the taxonomic assignment of the microbiome, showing the prevalence of a large number of bacterial genera previously associated with TTX production including Alteromonas, Vibrio and Pseudomonas. LC-MS/MS analysis of the nemertean tissue revealed the presence of multiple analogues of TTX, dominated by the parent TTX, with a total toxin concentration quantified at 54 µg TTX per g of tissue. Pseudomonas luteola isolated from C. simula, together with Vibrio alginolyticus from the native nemertean Tubulanus annulatus, were cultured at low temperature and both found to contain TTX. Overall, this paper confirms the high toxicity of a newly discovered invasive nemertean species with links to toxin-producing marine bacteria and the potential risk to human safety. Further work is required to assess the geographical extent and toxicity range of C. simula along the UK coast in order to properly gauge the potential impacts on the environment and human safety.

The potential roles of c-Jun N-terminal kinase (JNK) during the maturation and aging of oocytes produced by a marine protostome worm.

Previous investigations have indicated that c-Jun N-terminal kinase (JNK) regulates the maturation and aging of oocytes produced by deuterostome animals. In order to assess the roles of this kinase in a protostome, oocytes of the marine nemertean worm Cerebratulus were stimulated to mature and subsequently aged before being probed with phospho-specific antibodies against active forms of JNK and maturation-promoting factor (MPF). Based on blots of maturing oocytes, a 40-kD putative JNK is normally activated during germinal vesicle breakdown (GVBD), which begins at 30 min post-stimulation with seawater, whereas treating immature oocytes with JNK inhibitors downregulates both the 40-kD JNK signal and GVBD, collectively suggesting a 40-kD JNK may facilitate oocyte maturation. Along with this JNK activity, mature oocytes also exhibit high levels of MPF at 2 h post-stimulation. However, by ~6-8 h post-GVBD, mature oocytes lose the 40-kD JNK signal, and at ~20-30 h of aging, an ~48-kD phospho-JNK band arises as oocytes deactivate MPF and begin to lyse during a necroptotic-like mode of death. Accordingly, JNK inhibitors reduce the aging-related 48-kD JNK phosphorylation while maintaining MPF activity and retarding oocyte degradation. Such findings suggest that a 48-kD JNK may help deactivate MPF and trigger death. Possible mechanisms by which JNK activation either together with, or independently of, protein neosynthesis might stimulate oocyte degradation are discussed.

Oocyte aging in a marine protostome worm: The roles of maturation-promoting factor and extracellular signal regulated kinase form of mitogen-activated protein kinase.

The roles of maturation-promoting factor (MPF) and an extracellular signal regulated kinase form of mitogen-activated protein kinase (ERK MAPK) are analyzed during oocyte aging in the marine protostome worm Cerebratulus. About a day after removal from the ovary, unfertilized metaphase-I-arrested oocytes of Cerebratulus begin to flatten and swell before eventually lysing, thereby exhibiting characteristics of a necroptotic mode of regulated cell death. Based on immunoblots probed with phospho-specific antibodies, MPF and ERK are initially active in freshly mature specimens. However, as oocytes age, both kinase activities decline, with ERK deactivation occurring well before MPF downregulation. Experiments using pharmacological modulators indicate that oocyte degradation is promoted by the maturation-initiated activation of ERK as well as by the deactivation of MPF that occurs in extensively aged specimens. The potential significance of these findings is discussed relative to previously published results for apoptotic eggs and oocytes of echinoderm and vertebrate deuterostomes.

The Bacterial (Vibrio alginolyticus) Production of Tetrodotoxin in the Ribbon Worm Lineus longissimus-Just a False Positive?

We test previous claims that the bacteria Vibrio alginolyticus produces tetrodotoxin (TTX) when living in symbiosis with the nemertean Lineus longissimus by a setup with bacteria cultivation for TTX production. Toxicity experiments on the shore crab, Carcinus maenas, demonstrated the presence of a paralytic toxin, but evidence from LC-MS and electrophysiological measurements of voltage-gated sodium channel-dependent nerve conductance in male Wistar rat tissue showed conclusively that this effect did not originate from TTX. However, a compound of similar molecular weight was found, albeit apparently non-toxic, and with different LC retention time and MS/MS fragmentation pattern than those of TTX. We conclude that C. maenas paralysis and death likely emanate from a compound <5 kDa, and via a different mechanism of action than that of TTX. The similarity in mass between TTX and the Vibrio-produced low-molecular-weight, non-toxic compound invokes that thorough analysis is required when assessing TTX production. Based on our findings, we suggest that re-examination of some published claims of TTX production may be warranted.

Preliminary Report of a Neurokinin-Like Receptor Gene Sequence for the Nemertean Paranemertes sp.

Tachykinins (TKs) are a family of neurotransmitters that function as signaling molecules for such processes as maintaining homeostasis, regulating stress response, and modulating pain. TKs require the expression of at least one of three receptor subtypes: Neurokinin Receptor-1 (NKR-1), Neurokinin Receptor-2 (NKR-2), or Neurokinin Receptor-3 (NKR-3). We have isolated and cloned a portion of a gene coding for a tachykinin-like receptor from the nemertean Paranemertes sp. This 488-bp portion contains a short 101-bp segment that shares 85% similarity to the mouse substance-K receptor in Mus musculus and 83% similarity to the moth neuropeptide receptor A24 in Bombyx mori. Translated homology analysis aligning the coding sequence with the initial cytoplasmic carboxyl terminus of numerous G-protein coupled neuropeptide receptors also revealed 73% similarity to B. mori neuropeptide receptor A24. Our finding is the first report of a sequence amplified from Paranemertes sp. that may code for a small portion of a G-protein-coupled neuropeptide receptor with significant similarity to the TKR family, particularly the NKR-3 receptor isoform. This novel finding may open new avenues into exploring the role of tachykinin and its receptor in nemertean neurophysiology.

Malacobdella arrokeana: Parasite or Commensal of the Giant Clam Panopea abbreviata?

We examined trophic relationship between the nemertean Malacobdella arrokeana and its host, the edible geoduck Panopea abbreviata by studying the diets of both species by direct (stomach contents) and indirect methods (stable-isotope analysis of C and N). In addition to these methods, the feeding behavior of M. arrokeana within the host and the morphology of its feeding organs were examined. The feeding behavior of M. arrokeana did not exhibit parasitic characteristics, and the proboscis morphology indicates it is unable to injure host tissues. Analysis of stomach contents revealed a diet consisting mainly of microalgae and diatoms. Panopea abbreviata and M. arrokeana shared similar trophic levels, presenting no differences in the spread of the isotopic niches and high overlap (SEAB overlapped 63%). Consistent with this, our results showed no differences in δ(15)N or δ(13)C values between the two species. The combination of direct and indirect approaches revealed that M. arrokeana has a diet similar to that of its host, confirming a commensal relationship.

Peptide Toxins from Antarctica: The Nemertean Predator and Scavenger Parborlasia corrugatus (McIntosh, 1876).

Peptide toxins from marine invertebrates have found use as drugs and in biotechnological applications. Many marine habitats, however, remain underexplored for natural products, and the Southern Ocean is among them. Here, we report toxins from one of the top predators in Antarctic waters: the nemertean worm Parborlasia corrugatus (McIntosh, 1876). Transcriptome mining revealed a total of ten putative toxins with a cysteine pattern similar to that of alpha nemertides, four nemertide-beta-type sequences, and two novel full-length parborlysins. Nemertean worms express toxins in the epidermal mucus. Here, the expression was determined by liquid chromatography combined with mass spectrometry. The findings include a new type of nemertide, 8750 Da, containing eight cysteines. In addition, we report the presence of six cysteine-containing peptides. The toxicity of tissue extracts and mucus fractions was tested in an Artemia assay. Notably, significant activity was observed both in tissue and the high-molecular-weight mucus fraction, as well as in a parborlysin fraction. Membrane permeabilization experiments display the membranolytic activity of some peptides, most prominently the parborlysin fraction, with an estimated EC50 of 70 nM.

Discovery of the Nicotinic Receptor Toxin Anabaseine in a Polystiliferan Nemertean.

Nemerteans (also called Nemertines) are a phylum of predominantly marine worms that use toxins to capture prey and to defend themselves against predators. Hoplonemerteans have a proboscis armed with one or more stylets used in prey capture and are taxonomically divided into Order Monostilifera, whose members possess a single large proboscis stylet, and Order Polystilifera, whose members have multiple small stylets. Many monostiliferans contain alkaloidal toxins, including anabaseine, that stimulate and then desensitize nicotinic acetylcholine receptors that are present in all animals. These compounds also interact with pyridyl chemoreceptors in crustaceans, reducing predation and larval settlement. Anabaseine has been a lead compound in the design of alpha7 nicotinic acetylcholine receptor agonists like GTS-21 (also called DMXBA) to treat disorders of cognition such as Alzheimer's disease and schizophrenia. These drug candidates also display anti-inflammatory activities of potential medical importance. Most polystiliferans live deep in open oceans and are relatively inaccessible. We fortunately obtained two live specimens of a large benthic polystiliferan, Paradrepanophorus crassus (Pc), from the coast of Spain. MS and NMR analyses of the Ehrlich's reagent derivative allowed identification of anabaseine. A spectrophotometric assay for anabaseine, also based on its reaction with Ehrlich's reagent, revealed high concentrations of anabaseine in the body and proboscis. Apparently, the biosynthetic mechanism for producing anabaseine was acquired early in the evolution of the Hoplonemertea, before the monostiliferan-polystiliferan divergence.

Investigation of Peptide Toxin Diversity in Ribbon Worms (Nemertea) Using a Transcriptomic Approach.

Nemertea is a phylum of nonsegmented worms (supraphylum: Spiralia), also known as ribbon worms. The members of this phylum contain various toxins, including peptide toxins. Here, we provide a transcriptomic analysis of peptide toxins in 14 nemertean species, including Cephalothrix cf. simula, which was sequenced in the current study. The summarized data show that the number of toxin transcripts in the studied nemerteans varied from 12 to 82. The most represented groups of toxins were enzymes and ion channel inhibitors, which, in total, reached a proportion of 72% in some species, and the least represented were pore-forming toxins and neurotoxins, the total proportion of which did not exceed 18%. The study revealed that nemerteans possess a much greater variety of toxins than previously thought and showed that these animals are a promising object for the investigation of venom diversity and evolution, and in the search for new peptide toxins.

Interaction Between a Gelsolin from Dendrorhynchus zhejiangensis with Three Gelsolin-Like Domains and Actin In Vitro.

The gelsolin family proteins are best known for involvement in cytoskeletal rearrangement by controlling actin organization during a variety of cellular processes. Previously, a 1962 bp cDNA encoding a 41.7 kDa protein with three gelsolin-like domains (G domains) from Dendrorhynchus zhejiangensis was identified and named as DzGSN. In this study, the sequence and function of a novel member of the gelsolin family proteins from D. zhejiangensis have been analyzed. Sequence alignment indicates that DzGSN is highly homologous to human gelsolin (35% identity) and human CapG (36% identity). The important functional motifs and critical amino acids were identified. The nucleating- and severing-actin activities of recombinant DzGSN (rDzGSN) were then investigated by using atomic force microscopy in vitro. After incubation with rDzGSN in the presence of Ca2+, global actin (G-actin) was observed to aggregate into a ring structure, while filament actin (F-actin) was observed to be shortened. Additionally, the yeast two-hybrid system also verified that DzGSN can interact with actin. The results provide new insight into functional diversity and evolution of gelsolin family proteins.

DNA barcoding supports identification of Malacobdella species (Nemertea: Hoplonemertea).

BACKGROUND: Nemerteans of the genus Malacobdella live inside of the mantle cavity of marine bivalves. The genus currently contains only six species, five of which are host-specific and usually found in a single host species, while the sixth species, M. grossa, has a wide host range and has been found in 27 different bivalve species to date. The main challenge of Malacobdella species identification resides in the similarity of the external morphology between species (terminal sucker, gut undulations number, anus position and gonad colouration), and thus, the illustrations provided in the original descriptions do not allow reliable identification. In this article, we analyse the relationships amongthree species of Malacobdella:M.arrokeana,M.japonica andM.grossa,adding new data for the M.grossa and reporting the first for M. japonica, analysing 658 base pairs of the mitochondrial cytochrome c oxidase subunit I gene(COI).Based on these analyses, we present and discuss the potential of DNA barcoding for Malacobdellaspecies identification. RESULTS: Sixty-four DNA barcoding fragments of the mitochondrial COI gene from three different Malacobdella species (M. arrokeana, M. japonica and M. grossa) are analysed (24 of them newly sequenced for this study, along with four outgroup specimens) and used to delineate species. Divergences, measured as uncorrected differences, between the three species were M.arrokeana-M. grossa11.73%,M.arrokeana-M.japonica 10.62%and M.grossa-M. japonica 10.97%. The mean intraspecific divergence within the ingroup species showed a patent gap with respect to the interspecific ones: 0.18% for M.arrokeana,0.13% for M.grossa and0.02% for M.japonica (rangesfrom 0 to 0.91%). CONCLUSIONS: We conclude that there is a clear correspondence between the molecular data and distinguishing morphological characters. Our results thus indicate that some morphological characters are useful for species identification and support the potential of DNA barcoding for species identification in a taxonomic group with subtle morphological external differences.

Recombinant expression and predicted structure of parborlysin, a cytolytic protein from the Antarctic heteronemertine Parborlasia corrugatus.

The heteronemertine Parborlasia corrugatus contains a cytolytic protein, parborlysin, which after extensive purification was found by Edman sequencing to be a mixture of several homologues. To investigate this microheterogeneity and enable the analysis of single toxins, we have obtained seven parborlysin isoform genes from P. corrugatus collected in Antarctica. Total RNA was isolated from the homogenized head region and parborlysin genes were identified from a cDNA library using degenerate primers. The translated sequences reveal that the isoforms are ∼ 10 kDa basic (pI ∼ 10) proteins of which all but one harbour six cysteine residues. We generated a model of the three dimensional structure of parborlysins, which suggests that they are composed of five alpha-helical segments that include large, exposed hydrophobic surfaces. Finally, we constructed plasmids and inserted them into Escherichia coli to obtain overexpressed amino- or carboxy-terminal polyhistidine-tagged parborlysin isoforms fused to the third domain of the E. coli periplasmic-protein TolA to facilitate toxin isolation. One of the isoforms adversely affected growth in the E. coli expressing it. Although we succeeded in isolating one of the recombinant parborlysin constructs, it lacked haemolytic activity.