Search Results (26)

Tetrodotoxin (TTX), widely known as pufferfish venom, is a low-molecular-weight guanidinium neurotoxin. It can accumulate to extremely high concentrations in certain animals, including pufferfish, blue-ringed octopuses, flatworms, and nemerteans. However, the origin of TTX and the mechanisms that enable such extreme accumulation in these animals remain poorly understood. In this study, using confocal laser scanning microscopy combined with electron immunocytochemistry and ultrastructural analysis, we demonstrate the presence of TTX-positive bacteria associated with specialized cellular structures—type II phagosomes of gut enterocytes—in the highly toxic nemertean Cephalothrix cf. simula. We hypothesize that TTX production in C. cf. simula results from interactions between the nemertean host and its endosymbionts. These findings clarify the origin and accumulation of the toxin in nemerteans and have broader implications for other TTX-bearing species. Full article

Cephalothrix cf. simula is a highly toxic ribbon worm of the class Palaeonemertea, known for its high concentrations of tetrodotoxin. Recent transcriptomic and proteomic studies across Nemertea have revealed that species from all classes possess a diverse array of protein and peptide toxins, which are associated with unicellular glands of the proboscis and the integument epithelium. Previous studies have identified a large number of putative toxins in the transcriptome of C. cf. simula; however, corresponding proteomic data have so far been lacking. This study presents the first comprehensive analysis of the mucus and proboscis proteome of C. cf. simula using high-performance liquid chromatography–tandem mass spectrometry. We identified three putative toxins in the proboscis and three in the mucus. Additionally, four cysteine-rich peptides with putative toxic activity were identified in the mucus and one in the proboscis. The expression of the corresponding genes in both tissues was quantified using quantitative real-time PCR. The toxin compositions of the proboscis and mucus showed clear signs of functional specialization, with no overlapping toxins and tissue-specific patterns of gene expression. Feeding experiments combined with transmission electron microscopy confirmed the involvement of specialized proboscis structures, pseudocnidae, in delivering toxins into the prey. Full article

Tetrodotoxin (TTX) is an extremely potent neurotoxin, a selective blocker of voltage-gated sodium (NaV) channels, produced by bacteria and accumulated across a wide range of taxa. Several TTX-bearing animals have developed molecular adaptations in their NaV channels that provide TTX resistance, making this toxin one of the factors of molecular evolution. However, the molecular basis of TTX resistance in NaV channels of a significant proportion of tetrodotoxic species remains poorly studied. Nemertea is a phylum of marine worms, comprising both TTX-bearing and non-TTX-bearing species. Here, we analyzed the amino acid sequences of the NaV1 channel regions responsible for TTX binding from 22 species of nemerteans. Substitutions previously characterized as conferring TTX resistance in other taxa were detected in sixteen nemerteans; local clustering was observed within several families. These findings suggest that TTX resistance in nemerteans evolved multiple times independently and may serve as either as an adaptation facilitating TTX accumulation for subsequent use for defense and predation, or as a mechanism allowing consumption of tetrodotoxic prey without toxin accumulation. Full article

Aphid control often relies on synthetic pesticides, but their overuse has raised concerns about resistance development and negative impact on wildlife and human health. Consequently, the search for new biopesticide agents has gained significant attention. Nemertide alpha-1, a peptide toxin from the marine nemertean worm Lineus longissimus (Gunnerus, 1770), is known for its pesticide activity but has less documented biological safety. This study investigates the aphid feeding deterrence and biological safety of the experimental biopesticide nemertide alpha-1. Nemertide alpha-1 demonstrated a clear dose-dependent repellent effect on the penetration behaviour of the green peach aphid (Myzus persicae, Sulzer). It also demonstrates bacteriostatic and bactericidal effects in an MIC (Minimum Inhibitory Concentration) assay, respectively, on E. coli (MIC: 112.5 µM) and S. aureus (MIC: 28.4 µM). In a bacterial liposome leakage assay, nemertide alpha-1 exhibits a less pronounced effect than the melittin control (20% maximum leakage at 100 µM), strengthening the hypothesis on the specificity of its neurotoxic mode of action. It is not toxic to mammalian cell U-937 GTB with only a slight decline in the percentage of survival at the highest concentration tested (80 µM). Finally, nemertide alpha-1 displays thermal stability over time for four weeks in three different conditions: cold (6 °C), room temperature (20–24 °C), and physiological temperature (37 °C). Nemertide alpha-1 deters green peach aphid feeding in the low micromolar range and exhibits low antimicrobial properties and very low toxicity to human cells. Its potential utility is further underscored by thermal stability over time. Full article

The ribbon worms of the closely related species Cephalothrix simula, Cephalothrix cf. simula, and Cephalothrix mokievskii, representing the C. simula species complex, possess high concentrations of tetrodotoxin (TTX) and its analogues in all developmental stages from eggs to adults. It has recently been suggested that the eggs and larvae of these animals can be a source of tetrodotoxins (TTXs) for other aquatic organisms. In the current study, TTXs in mature and post-spawning individuals and in the eggs of C. mokievskii were identified using high-performance liquid chromatography–tandem mass spectrometry. For the first time, the quantity and profile of TTXs that these nemerteans released into the environment during spawning were estimated. We showed that the spawning C. mokievskii females released significant amounts of TTX and 5,6,11-trideoxyTTX with their eggs; these levels were sufficient for the potential toxification of marine bioresources. The issues surrounding the monitoring of TTXs in commercial marine animals, and collecting at the sites of the spawning of nemerteans from the C. simula species complex, are discussed. Full article

Nemertea is a phylum of bilaterally symmetrical, coelomate, unsegmented worms, also known as ribbon worms. Most species of the phylum Nemertea are marine predators that contain toxins in the single-celled glands of the proboscis and/or integument. Recent transcriptomic studies have shown that nemerteans from all taxonomic groups possess a wide range of putative protein and peptide toxins, while the proteomic data for these animals are highly limited. In this study, proteotranscriptomic analysis was used to investigate the major protein components of the poison of the nemertean Kulikovia alborostrata. We identified 146 transcripts of putative toxins in the transcriptome of K. alborostrata and five putative toxins among the secreted proteins and peptides of the mucus of the animal. The expression levels of cysteine-rich peptides found in the mucus with similarity to known toxins were evaluated in different parts of the body of the worm by quantitative real-time PCR. The high level of expression of investigated peptides in the integument indicate the protective function of these toxins. Overall, this supports the idea that the mucus of nemerteans is a valuable source of peptide and protein toxins. Full article

During fertilization, a series of reactions between the eggs and spermatozoa proceed predominantly in a species-specific manner. The molecules mediating these species-specific reactions remain unknown except in a few organisms. In this study, we focused on two species belonging to the phylum Nemertea, Kulikovia alborostrata and K. fulva, and explored molecules involved in species-specific interactions between gametes. Orthologs of molecules known to be involved in species-specific reactions were not expressed in the ovaries of these two species. In contrast, we identified six novel proteins, named NeZPL1–NeZPL6, containing a ZP module. Among these, we found that NeZPL6 is located on the surface of an unfertilized egg and is suggested to be involved in its interaction with spermatozoa. Furthermore, we found an indel of three amino acids in the EGF-like domain of NeZPL6, which possibly confers species specificity to this interaction. Our results suggested the existence of a novel system for species recognition in animal gametes. Full article

Nemerteans, or ribbon worms, possess tetrodotoxin and its analogues (TTXs), neurotoxins of bacterial origin, which they presumably use for capturing prey and self-defense. Most TTXs-containing nemertean species have low levels of these toxins and, therefore, have usually been neglected in studies of TTXs functions and accumulation. In the present study, Kulikovia alborostrata and K. manchenkoi, two closely related species, were analyzed for TTXs distribution in the body using the HPLC–MS/MS and fluorescence microscopy methods. The abundance of TTXs-positive cells was determined in the proboscis, integument, and digestive system epithelium. As a result, six TTXs-positive cell types were identified in each species; however, only four were common. Moreover, the proportions of the toxins in different body parts were estimated. According to the HPLC–MS/MS analysis, the TTXs concentrations in K. alborostrata varied from 0.91 ng/g in the proboscis to 5.52 ng/g in the precerebral region; in K. manchenkoi, the concentrations ranged from 7.47 ng/g in the proboscis to 72.32 ng/g in the posterior body region. The differences observed between the two nemerteans in the distribution of the TTXs were consistent with the differences in the localization of TTXs-positive cells. In addition, TTXs-positive glandular cell types were found in the intestine and characterized for the first time. TTXs in the new cell types were assumed to play a unique physiological role for nemerteans. Full article

Tetrodotoxin (TTX), an extremely potent low-molecular-weight neurotoxin, is widespread among marine animals including ribbon worms (Nemertea). Previously, studies on the highly toxic palaeonemertean Cephalothrix cf. simula showed that toxin-positive structures are present all over its body and are mainly associated with glandular cells and epithelial tissues. The highest TTXs concentrations were detected in a total extract from the intestine of the anterior part of the body and also in a total extract from the proboscis. However, many questions as to the TTXs distribution in the organs of the anterior part of the worm’s body and the functions of the toxins in these organs are still unanswered. In the present report, we provide additional results of a detailed and comprehensive analysis of TTXs distribution in the nemertean’s proboscis, buccal cavity, and cephalic gland using an integrated approach including high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS), confocal laser scanning microscopy with anti-TTX antibodies, light and electron microscopies, and observations of feeding behavior. For the proboscis, we have found a TTXs profile different from that characteristic of other organs and tissues. We have also shown for the first time that the major amount of TTXs is localized in the anterior part of the proboscis that is mainly involved in hunting. TTX-containing glandular cells, which can be involved in the prey immobilization, have been found in the buccal cavities of the nemerteans. A significant contribution of the cephalic gland to the toxicity of this animal has been shown for the first time, and the role of the gland is hypothesized to be involved not only in protection against potential enemies but also in immobilizing prey. The data obtained have made it possible to extend the understanding of the role and features of the use of TTXs in the organs of the anterior part of nemertean’s body. Full article

Some, probably most and perhaps all, members of the phylum Nemertea are poisonous, documented so far from marine and benthic specimens. Although the toxicity of these animals has been long known, systematic studies on the characterization of toxins, mechanisms of toxicity, and toxin evolution for this group are scarce. Here, we present the first investigation of the molecular evolution of toxins in Nemertea. Using a proteo-transcriptomic approach, we described toxins in the body and poisonous mucus of the pilidiophoran Lineus sanguineus and the hoplonemertean Nemertopsis pamelaroeae. Using these new and publicly available transcriptomes, we investigated the molecular evolution of six selected toxin gene families. In addition, we also characterized in silico the toxin genes found in the interstitial hoplonemertean, Ototyphlonemertes erneba, a meiofaunal taxa. We successfully identified over 200 toxin transcripts in each of these species. Evidence of positive selection and gene duplication was observed in all investigated toxin genes. We hypothesized that the increased rates of gene duplications observed for Pilidiophora could be involved with the expansion of toxin genes. Studies concerning the natural history of Nemertea are still needed to understand the evolution of their toxins. Nevertheless, our results show evolutionary mechanisms similar to other venomous groups. Full article

Doubly Uniparental Inheritance (DUI) is considered one of the major exceptions to the common rules of eukaryotic cell biology and germline formation. DUI is known in bivalves, which belong to the phylum Mollusca; conversely, no DUI evidence was found in some gastropod species. Investigating the presence of DUI in Nemertea is of particular interest given the fact that these spiralian animals are suggested to be a sister group of Neotrochozoa (i.e., Mollusca and Annelida). DUI species are normally detected as having two sex-associated mtDNAs, which can be highly divergent. In this work, the presence of sex-associated mitochondrial genomes was investigated in the nemertean species Notospermus geniculatus and no evidence was found for DUI. Even if these are preliminary results, negative evidence is still interesting because of the high importance of the DUI phenomenon in many research fields, where it plays a pivotal role in understanding eukaryotic evolution. For this reason, further research on DUI species detection should be highly encouraged, as well as the publication of negative results beside positive ones, as is the case for the present study, improving the knowledge on the biology and ecology of a broad spectrum of marine species. Full article

To date, a total of 23 valid species of heteronemerteans belonging to 15 genera have been recorded in Antarctic and Subantarctic waters. The ribbon worm Heteronemertes longifissa (Hubrecht, 1887) is the only heteronemertean species reported to have bipolar distribution, but this statement is doubtful. The phylogenetic relationships of H. longifissa to other heteronemerteans remain uncertain. A genetic analysis of specimens from Antarctica has shown that the name H. longifissa refers to two sibling species with an uncorrected p-distance of 5.3% in COI. These species differ in body color: one is whitish, and the other is grayish-pink. The species with the whitish body has been reliably identified from off the Norway coast (as Cerebratulus sp. NemBar1383 (BOLD: ACM5920)), i.e., it has a bipolar distribution. A molecular phylogenetic analysis of Lineidae based on five gene markers (COI, 16S, 18S, 28S, and histone H3) has shown the genus Heteronemertes to belong to Lineage D of Clade 2 sensu Kajihara et al., 2022 (crown Lineidae). The phylogenetic positions of four more species of unidentified lineids are currently under discussion. Full article

Tetrodotoxin, an extremely potent low-molecular-weight neurotoxin, and its analogues (TTXs) are widely distributed in aquatic and terrestrial ecosystems. Most investigations concerning TTXs have been conducted mainly on puffer fish, octopus, and mollusks, without paying due attention to various non-edible animals including nemerteans, a small group of marine worms, several species of which have been shown to possess high amounts of TTXs. In this study, for the first time, variations in TTX and its analogues, in 32 specimens of Cephalothrix cf. simula and 36 specimens of Kulikovia alborostrata, from Peter the Great Bay Sea of Japan were investigated, which may contribute to elucidation of TTXs migration pathways in ecosystems. Using high performance liquid chromatography with tandem mass spectrometry (HPLC–MS/MS), it was found that the total TTXs concentrations within both species vary by one to several orders of magnitude, 85.75–7108.26 µg/g and 0.35–8.11 ng/g in C. cf. simula and K. alborostrata, respectively. The intra- and interspecies similarities in proportions of TTXs in both species were observed; based on the results, a possible way of their toxification was discussed. Full article

Many Antarctic marine benthic macroinvertebrates are chemically protected against predation by marine natural products of different types. Antarctic potential predators mostly include sea stars (macropredators) and amphipod crustaceans (micropredators) living in the same areas (sympatric). Recently, alien species (allopatric) have been reported to reach the Antarctic coasts, while deep-water crabs are suggested to be more often present in shallower waters. We decided to investigate the effect of the chemical defenses of 29 representative Antarctic marine benthic macroinvertebrates from seven different phyla against predation by using non-native allopatric generalist predators as a proxy for potential alien species. The Antarctic species tested included 14 Porifera, two Cnidaria, two Annelida, one Nemertea, two Bryozooa, three Echinodermata, and five Chordata (Tunicata). Most of these Antarctic marine benthic macroinvertebrates were chemically protected against an allopatric generalist amphipod but not against an allopatric generalist crab from temperate waters. Therefore, both a possible recolonization of large crabs from deep waters or an invasion of non-native generalist crab species could potentially alter the fundamental nature of these communities forever since chemical defenses would not be effective against them. This, together with the increasing temperatures that elevate the probability of alien species surviving, is a huge threat to Antarctic marine benthos. Full article

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. Full article