Abstract: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 origin… Show more
“…More recently, there have been reports of the presence of the TTX-producing bacteria Bacillus sp. 1839 in C. simula isolated from the Sea of Japan [ 55 ], fitting with the findings of other authors who have established that these species and other species of bacteria such as Vibrio alginolyticus may play an important role in either the production or accumulation of toxin within nemerteans [ 23 , 56 , 57 , 58 , 59 , 60 , 61 ].…”
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.
“…More recently, there have been reports of the presence of the TTX-producing bacteria Bacillus sp. 1839 in C. simula isolated from the Sea of Japan [ 55 ], fitting with the findings of other authors who have established that these species and other species of bacteria such as Vibrio alginolyticus may play an important role in either the production or accumulation of toxin within nemerteans [ 23 , 56 , 57 , 58 , 59 , 60 , 61 ].…”
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.
“…To deduce the biological function of α-nemertides we used an established model organism to study toxicity of nemertean mucus in vivo : the green crab Carcinus maenas 5,7 . This model has biological significance because crustaceans are both well-known preys and possible predators of nemerteans 13 .…”
Section: Resultsmentioning
confidence: 99%
“…L. longissimus first sparked our interest as a possible source of tetrodotoxin (TTX). However, TTX could never be identified, but the mucus still showed potent activity when injected into the branchial chamber of green crabs ( Carcinus maenas ) 7 . Instead, as shown in the current work, the activity is explained by the presence of a novel family of disulfide-rich peptide toxins.Figure 1The nemertean Lineus longissimus and schematic of its phylogeny.…”
Polypeptides from animal venoms have found important uses as drugs, pharmacological tools, and within biotechnological and agricultural applications. We here report a novel family of cystine knot peptides from nemertean worms, with potent activity on voltage-gated sodium channels. These toxins, named the α-nemertides, were discovered in the epidermal mucus of Lineus longissimus, the ‘bootlace worm’ known as the longest animal on earth. The most abundant peptide, the 31-residue long α-1, was isolated, synthesized, and its 3D NMR structure determined. Transcriptome analysis including 17 species revealed eight α-nemertides, mainly distributed in the genus Lineus. α-1 caused paralysis and death in green crabs (Carcinus maenas) at 1 µg/kg (~300 pmol/kg). It showed profound effect on invertebrate voltage-gated sodium channels (e.g. Blattella germanica Nav1) at low nanomolar concentrations. Strong selectivity for insect over human sodium channels indicates that α-nemertides can be promising candidates for development of bioinsecticidal agents.
“…It is worth noting that although the genus Vibrio, widespread in marine ecosystems, was commonly reported as the main bacterial TTX-producer, its ability to produce the toxin was questioned. Strand et al [32] showed that using liquid chromatography-mass spectrometry (LC-MS) and bioassay for TTX detection in bacterial and nemertean samples might lead to false-positive results and should be supplemented with tandem mass spectrometry (MS/MS). The authors revealed a compound produced by Vibrio alginolyticus isolated from the ribbon worm Lineus longissimus.…”
A potent marine toxin, tetrodotoxin (TTX), found in a great variety of marine and some terrestrial species, leaves intriguing questions about its origin and distribution in marine ecosystems. TTX-producing bacteria were found in the cultivable microflora of many TTX-bearing hosts, thereby providing strong support for the hypothesis that the toxin is of bacterial origin in these species. However, metagenomic studies of TTX-bearing animals addressing the whole microbial composition and estimating the contribution of TTX-producing bacteria to the overall toxicity of the host were not conducted. The present study is the first to characterize and compare the 16S rRNA gene data obtained from four TTX-bearing and four non-TTX-bearing species of marine ribbon worms. The statistical analysis showed that different nemertean species harbor distinct bacterial communities, while members of the same species mostly share more similar microbiomes. The bacterial species historically associated with TTX production were found in all studied samples but predominated in TTX-bearing nemertean species. This suggests that deeper knowledge of the microbiome of TTX-bearing animals is a key to understanding the origin of TTX in marine ecosystems.
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