For the first time, tetrodotoxin (TTX) was detected in a bacterial strain after five years of cultivation in laboratory conditions since its isolation from the animal host. A reliable method suitable for bacterial samples, high-performance liquid chromatography with tandem mass spectrometry, was used for toxin detection in spore and vegetative cultures of Bacillus sp. 1839. TTX was detected in a spore culture of the strain.

Some nemertean species from the genus Cephalothrix accumulate tetrodotoxin (TTX) in extremely high concentrations. The current study is the first to provide high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) data on tetrodotoxin and its analogues (TTXs) profile and concentration in different regions and organs of Cephalothrix cf. simula, and its secretions produced in response to stimulation. Different specimens of C. cf. simula possessed 7–11 analogues, including nine previously found in this species and two new for nemerteans—4,9-anhydro-8-epi-5,6,11-trideoxyTTX and 1-hydroxy-8-epi-5,6,11-trideoxyTTX. The study of the toxins’ distribution in different regions and organs of nemerteans revealed the same qualitative composition of TTXs throughout the body but differences in the total concentration of the toxins. The total concentration of TTXs was highest in the anterior region of the body and decreased towards the posterior; the ratio of the analogues also differed between regions. The data obtained suggest a pathway of TTXs uptake in C. cf. simula and the role of toxins in the life activity of nemerteans.

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.

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.

Nemertea is a phylum of marine worms whose members bear various toxins, including tetrodotoxin (TTX) and its analogues. Despite the more than 30 years of studying TTXs in nemerteans, many questions regarding their functions and the mechanisms ensuring their accumulation and usage remain unclear. In the nemertean Kulikovia alborostrata, we studied TTX and 5,6,11-trideoxyTTX concentrations in body extracts and in released mucus, as well as various aspects of the TTX-positive-cell excretion system and voltage-gated sodium (Nav1) channel subtype 1 mutations contributing to the toxins’ accumulation. For TTX detection, an immunohistological study with an anti-TTX antibody and HPLC-MS/MS were conducted. For Nav1 mutation searching, PCR amplification with specific primers, followed by Sanger sequencing, was used. The investigation revealed that, in response to an external stimulus, subepidermal TTX-positive cells released secretions actively to the body surface. The post-release toxin recovery in these cells was low for TTX and high for 5,6,11-trideoxyTTX in captivity. According to the data obtained, there is low probability of the targeted usage of TTX as a repellent, and targeted 5,6,11-trideoxyTTX secretion by TTX-bearing nemerteans was suggested as a possibility. The Sanger sequencing revealed identical sequences of the P-loop regions of Nav1 domains I–IV in all 17 studied individuals. Mutations comprising amino acid substitutions, probably contributing to nemertean channel resistance to TTX, were shown.

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.