Wheat germ agglutinin affinity chromatography enrichment and glyco-proteomic characterization of tetrodotoxin-binding proteins from the plasma of cultured tiger pufferfish (Takifugu rubripes).

Efficient enrichment of tetrodotoxin (TTX)-binding proteins from the plasma of cultured tiger pufferfish (Takifugu rubripes) was achieved by ammonium sulfate fractionation and wheat germ agglutinin (WGA) affinity chromatography. The enrichment efficiency was validated by ultrafiltration-LC/MS-based TTX-binding assay and proteomics. Major proteins in the WGA-bound fraction were identified as isoform X1 (125 kDa) and X2 variants (88 and 79 kDa) derived from pufferfish saxitoxin and tetrodotoxin-binding protein (PSTBP) 1-like gene (LOC101075943). The 125-kDa X1 protein was found to be a novel member of the lipocalin family, having three tandemly repeated domains. X2 variants, X2α and X2ß, were estimated to have two domains, and X2ß is structurally related to Takifugu pardalis PSTBP2 in their domain type and arrangement. Among 11 potential N-glycosylation sites in the X2 precursor, 5 N-glycosylated Asn residues (N55, N89, N244, N308, and N449) were empirically determined. Structural relationships among PSTBP homologs and complexity of their proteoforms are discussed.

Trichloroethanol, an active metabolite of chloral hydrate, modulates tetrodotoxin-resistant Na+ channels in rat nociceptive neurons.

BACKGROUND: Chloral hydrate is a sedative-hypnotic drug widely used for relieving fear and anxiety in pediatric patients. However, mechanisms underlying the chloral hydrate-mediated analgesic action remain unexplored. Therefore, we investigated the effect of 2',2',2'-trichloroethanol (TCE), the active metabolite of chloral hydrate, on tetrodotoxin-resistant (TTX-R) Na+ channels expressed in nociceptive sensory neurons. METHODS: The TTX-R Na+ current (INa) was recorded from acutely isolated rat trigeminal ganglion neurons using the whole-cell patch-clamp technique. RESULTS: Trichloroethanol decreased the peak amplitude of transient TTX-R INa in a concentration-dependent manner and potently inhibited persistent components of transient TTX-R INa and slow voltage-ramp-induced INa at clinically relevant concentrations. Trichloroethanol exerted multiple effects on various properties of TTX-R Na+ channels; it (1) induced a hyperpolarizing shift on the steady-state fast inactivation relationship, (2) increased use-dependent inhibition, (3) accelerated the onset of inactivation, and (4) retarded the recovery of inactivated TTX-R Na+ channels. Under current-clamp conditions, TCE increased the threshold for the generation of action potentials, as well as decreased the number of action potentials elicited by depolarizing current stimuli. CONCLUSIONS: Our findings suggest that chloral hydrate, through its active metabolite TCE, inhibits TTX-R INa and modulates various properties of these channels, resulting in the decreased excitability of nociceptive neurons. These pharmacological characteristics provide novel insights into the analgesic efficacy exerted by chloral hydrate.

Functional expression of NaV1.7 channels in freshly dispersed mouse bronchial smooth muscle cells.

Isolated smooth muscle cells (SMCs) from mouse bronchus were studied using the whole cell patch-clamp technique at ∼21°C. Stepping from -100 mV to -20 mV evoked inward currents of mean amplitude -275 pA. These inactivated (tau = 1.1 ms) and were abolished when external Na+ was substituted with N-Methyl-d-glucamine. In current-voltage protocols, current peaked at -10 mV and reversed between +20 and +30 mV. The V1/2s of activation and inactivation were -25 and -86 mV, respectively. The current was highly sensitive to tetrodotoxin (IC50 = 1.5 nM) and the NaV1.7 subtype-selective blocker, PF-05089771 (IC50 = 8.6 nM), consistent with NaV1.7 as the underlying pore-forming α subunit. Two NaV1.7-selective antibodies caused membrane-delineated staining of isolated SMC, as did a nonselective pan-NaV antibody. RT-PCR, performed on groups of ∼15 isolated SMCs, revealed transcripts for NaV1.7 in 7/8 samples. Veratridine (30 µM), a nonselective NaV channel activator, reduced peak current evoked by depolarization but induced a sustained current of 40 pA. Both effects were reversed by tetrodotoxin (100 nM). In tension experiments, veratridine (10 µM) induced contractions that were entirely blocked by atropine (1 µM). However, in the presence of atropine, veratridine was able to modulate the pattern of activity induced by a combination of U-46619 (a thromboxane A2 mimetic) and PGE2 (prostaglandin E2), by eliminating bursts in favor of sustained phasic contractions. These effects were readily reversed to control-like activity by tetrodotoxin (100 nM). In conclusion, mouse bronchial SMCs functionally express NaV1.7 channels that are capable of modulating contractile activity, at least under experimental conditions.

Transcriptome analysis revealed gene expression feminization of testis after exogenous tetrodotoxin administration in pufferfish Takifugu flavidus.

Tetrodotoxin (TTX) is a deadly neurotoxin and usually accumulates in large amounts in the ovaries but is non-toxic or low toxic in the testis of pufferfish. The molecular mechanism underlying sexual dimorphism accumulation of TTX in ovary and testis, and the relationship between TTX accumulation with sex related genes expression remain largely unknown. The present study investigated the effects of exogenous TTX treatment on Takifugu flavidus. The results demonstrated that exogenous TTX administration significantly incresed level of TTX concentration in kidney, cholecyst, skin, liver, heart, muscle, ovary and testis of the treatment group (TG) than that of the control group (CG). Transcriptome sequencing and analysis were performed to study differential expression profiles of mRNA and piRNA after TTX administration of the ovary and testis. The results showed that compared with female control group (FCG) and male control group (MCG), TTX administration resulted in 80 and 23 piRNAs, 126 and 223 genes up and down regulated expression in female TTX-treated group (FTG), meanwhile, 286 and 223 piRNAs, 2 and 443 genes up and down regulated expression in male TTX-treated group (MTG). The female dominant genes cyp19a1, gdf9 and foxl2 were found to be up-regulated in MTG. The cyp19a1, whose corresponding target piRNA uniq_554482 was identified as down-regulated in the MTG, indicating the gene expression feminization in testis after exogenous TTX administration. The KEGG enrichment analysis revealed that differentially expressed genes (DEGs) and piRNAs (DEpiRNAs) in MTG vs MCG group were more enriched in metabolism pathways, indicating that the testis produced more metabolic pathways in response to exogenous TTX, which might be a reason for the sexual dimorphism of TTX distribution in gonads. In addition, TdT-mediated dUTP-biotin nick end labeling staining showed that significant apoptosis was detected in the MTG testis, and the role of the cell apoptotic pathways was further confirmed. Overall, our research revealed that the response of the ovary and testis to TTX administration was largely different, the ovary is more tolerant whereas the testis is more sensitive to TTX. These data will deepen our understanding on the accumulation of TTX sexual dimorphism in Takifugu.

Total Syntheses of the Proposed Biosynthetic Intermediates of Tetrodotoxin Tb-210B, Tb-226, Tb-242C, and Tb-258.

The collective synthesis of the four spiro-cyclic guanidines Tb-210B, Tb-226, Tb-242C, and Tb-258, all of which have been isolated from puffer fish and are considered possible biosynthetic intermediates of tetrodotoxin, has been achieved. Our synthesis is based on the stepwise deoxygenation or hydroxylation of a common intermediate, prepared from a known oxazoline.

Inactivation of Prefrontal Cortex Attenuates Behavioral Arousal Induced by Stimulation of Basal Forebrain During Sevoflurane Anesthesia.

BACKGROUND: Cholinergic stimulation of prefrontal cortex (PFC) can reverse anesthesia. Conversely, inactivation of PFC can delay emergence from anesthesia. PFC receives cholinergic projections from basal forebrain, which contains wake-promoting neurons. However, the role of basal forebrain cholinergic neurons in arousal from the anesthetized state requires refinement, and it is currently unknown whether the arousal-promoting effect of basal forebrain is mediated through PFC. To address these gaps in knowledge, we implemented a novel approach to the use of chemogenetic stimulation and tested the role of basal forebrain cholinergic neurons in behavioral arousal during sevoflurane anesthesia. Next, we investigated the effect of tetrodotoxin-mediated inactivation of PFC on behavioral arousal produced by electrical stimulation of basal forebrain during sevoflurane anesthesia. METHODS: Adult male and female transgenic rats (Long-Evans-Tg [ChAT-Cre]5.1 Deis; n = 22) were surgically prepared for expression of excitatory hM3D(Gq) receptors or mCherry in basal forebrain cholinergic neurons, and activation of these neurons by local delivery of compound 21, an agonist for hM3D(Gq) receptors. The transgenic rats were fitted with microdialysis probes for agonist delivery into basal forebrain and simultaneous prefrontal acetylcholine measurement. Adult male and female Sprague Dawley rats were surgically prepared for bilateral electrical stimulation of basal forebrain and tetrodotoxin infusion (156 µM and 500 nL) into PFC (n = 9) or bilateral electrical stimulation of piriform cortex (n = 9) as an anatomical control. All rats were implanted with electrodes to monitor the electroencephalogram. Heart and respiration rates were monitored using noninvasive sensors. A 6-point scale was used to score behavioral arousal (0 = no arousal and 5 = return of righting reflex). RESULTS: Compound 21 delivery into basal forebrain of rats with hM3D(Gq) receptors during sevoflurane anesthesia produced increases in arousal score (P < .001; confidence interval [CI], 1.80-4.35), heart rate (P < .001; CI, 36.19-85.32), respiration rate (P < .001; CI, 22.81-58.78), theta/delta ratio (P = .008; CI, 0.028-0.16), and prefrontal acetylcholine (P < .001; CI, 1.73-7.46). Electrical stimulation of basal forebrain also produced increases in arousal score (P < .001; CI, 1.85-4.08), heart rate (P = .018; CI, 9.38-98.04), respiration rate (P < .001; CI, 24.15-53.82), and theta/delta ratio (P = .020; CI, 0.019-0.22), which were attenuated by tetrodotoxin-mediated inactivation of PFC. CONCLUSIONS: This study validates the role of basal forebrain cholinergic neurons in behavioral arousal and demonstrates that the arousal-promoting effects of basal forebrain are mediated in part through PFC.

The chemistry and biology of guanidine secondary metabolites.

Covering: 2017-2019Guanidine natural products isolated from microorganisms, marine invertebrates and terrestrial plants, amphibians and spiders, represented by non-ribosomal peptides, guanidine-bearing polyketides, alkaloids, terpenoids and shikimic acid derived, are the subject of this review. The topics include the discovery of new metabolites, total synthesis of natural guanidine compounds, biological activity and mechanism-of-action, biosynthesis and ecological functions.

Molecular Characterization of the Von Willebrand Factor Type D Domain of Vitellogenin from Takifugu flavidus.

The von Willebrand factor type D (VWD) domain in vitellogenin has recently been found to bind tetrodotoxin. The way in which this protein domain associates with tetrodotoxin and participates in transporting tetrodotoxin in vivo remains unclear. A cDNA fragment of the vitellogenin gene containing the VWD domain from pufferfish (Takifugu flavidus) (TfVWD) was cloned. Using in silico structural and docking analyses of the predicted protein, we determined that key amino acids (namely, Val115, ASP116, Val117, and Lys122) in TfVWD mediate its binding to tetrodotoxin, which was supported by in vitro surface plasmon resonance analysis. Moreover, incubating recombinant rTfVWD together with tetrodotoxin attenuated its toxicity in vivo, further supporting protein-toxin binding and indicating associated toxicity-neutralizing effects. Finally, the expression profiling of TfVWD across different tissues and developmental stages indicated that its distribution patterns mirrored those of tetrodotoxin, suggesting that TfVWD may be involved in tetrodotoxin transport in pufferfish. For the first time, this study reveals the amino acids that mediate the binding of TfVWD to tetrodotoxin and provides a basis for further exploration of the molecular mechanisms underlying the enrichment and transfer of tetrodotoxin in pufferfish.

A Microencapsulation Method for Delivering Tetrodotoxin to Bivalves to Investigate Uptake and Accumulation.

Most marine biotoxins are produced by microalgae. The neurotoxin tetrodotoxin (TTX) has been reported in many seafood species worldwide but its source is unknown, making accumulation and depuration studies in shellfish difficult. Tetrodotoxin is a water-soluble toxin and cannot be directly ingested by shellfish. In the present study, a method was developed which involved binding TTX to solid particles of humic acid and encapsulating them in agar-gelatin capsules. A controlled quantity of TTX-containing microcapsules (size range 20-280 µm) was fed to Paphies australis, a bivalve known to accumulate TTX in the wild. The TTX-containing microcapsules were fed to P. australis every second day for 13 days. Ten P. australis (including five controls fed non-toxic microalgae) were harvested after 7 days and ten after 13 days. Paphies australis accumulated TTX, reaching concentrations of up to 103 µg kg-1 by day 13, exceeding the European Food Safety Authority recommended concentration of 44 µg kg-1 in shellfish. This novel method will allow future studies to explore the effects, accumulation and depuration rates of TTX in different animals and document how it is transferred through food webs.

Synthesis of C12-Keto Saxitoxin Derivatives with Unusual Inhibitory Activity Against Voltage-Gated Sodium Channels.

A novel series of C12-keto-type saxitoxin (STX) derivatives bearing an unusual nonhydrated form of the ketone at C12 has been synthesized, and their NaV -inhibitory activity has been evaluated in a cell-based assay as well as whole-cell patch-clamp recording. Among these compounds, 11-benzylidene STX (3 a) showed potent inhibitory activity against neuroblastoma Neuro 2A in both cell-based and electrophysiological analyses, with EC50 and IC50 values of 8.5 and 30.7 nm, respectively. Interestingly, the compound showed potent inhibitory activity against tetrodotoxin-resistant subtype of NaV 1.5, with an IC50 value of 94.1 nm. Derivatives 3 a-d and 3 f showed low recovery rates from NaV 1.2 subtype (ca 45-79 %) compared to natural dcSTX (2), strongly suggesting an irreversible mode of interaction. We propose an interaction model for the C12-keto derivatives with NaV in which the enone moiety in the STX derivatives 3 works as Michael acceptor for the carboxylate of Asp1717 .

Puffer Fish Gut Microbiota Studies Revealed Unique Bacterial Co-Occurrence Patterns and New Insights on Tetrodotoxin Producers.

Tetrodotoxin (TTX) is a potent neurotoxin isolated mainly from toxic puffer fish. To date, the TTX biosynthetic mechanism inside its hosts remains unresolved. Here, we hypothesize the TTX synthesis relies on the host gut microbiota, including the neglected non-culturable bacteria. In these studies, we collected the gut contents from 5 puffer fish species of the genus Takifugu including one suspected hybrid species for gut microbiota study by 16S rRNA amplicon metagenomics approach. Their gut samples were divided into toxic and non-toxic groups based on the TTX concentrations in the livers detected by LC-MS/MS. Bacterial diversity studies showed that gut microbiota structures were significantly different between toxic and non-toxic species. Vibrio and Cyanobacteria centered at the gut bacterial co-occurrence network, suggesting their importance in TTX biosynthesis. The results of PICRUSt2 metagenomic prediction and gene set enrichment analysis provided new support of arginine-precursor required in TTX biosynthesis. This is the first study to profile the gut microbiota in toxic and non-toxic puffer fish species by 16S rRNA amplicon metagenomic approach, defining significant microbial co-occurrence patterns in their gut environment. Our data supported the proposed biosynthesis of TTX inside the hosts by their gut bacterial symbionts using arginine as a precursor.

The Microbial Community of Tetrodotoxin-Bearing and Non-Tetrodotoxin-Bearing Ribbon Worms (Nemertea) from the Sea of Japan.

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.

Optogenetic analysis of respiratory neuronal networks in the ventral medulla of neonatal rats producing channelrhodopsin in Phox2b-positive cells.

Paired-like homeobox gene Phox2b is predominantly expressed in pre-inspiratory neurons in the parafacial respiratory group (pFRG) in newborn rat rostral ventrolateral medulla. To analyse detailed local networks of the respiratory centre using optogenetics, the effects of selective activation of Phox2b-positive neurons in the ventral medulla on respiratory rhythm generation were examined in brainstem-spinal cord preparations isolated from transgenic newborn rats with Phox2b-positive cells expressing channelrhodopsin variant ChRFR(C167A). Photostimulation up to 43 s increased the respiratory rate > 200% of control, whereas short photostimulation (1.5 s) of the rostral pFRG reset the respiratory rhythm. At the cellular level, photostimulation depolarised Phox2b-positive pre-inspiratory, inspiratory and respiratory-modulated tonic neurons and Phox2b-negative pre-inspiratory neurons. In contrast, changes in membrane potential of Phox2b-negative inspiratory and expiratory neurons varied depending on characteristics of ongoing synaptic connections in local respiratory networks in the rostral medulla. In the presence of tetrodotoxin, photostimulation depolarised Phox2b-positive cells, but caused no significant changes in membrane potential of Phox2b-negative cells. We concluded that depolarisation of Phox2b-positive neurons was due to cell-autonomous photo-activation and summation of excitatory postsynaptic potentials, whereas membrane potential changes of Phox2b-negative neurons depended on the network configuration. Our findings shed further light on local networks among respiratory-related neurons in the rostral ventrolateral medulla and emphasise the important role of pre-inspiratory neurons in respiratory rhythm generation in the neonatal rat en bloc preparation.

Prolonged Duration Local Anesthesia by Combined Delivery of Capsaicin- and Tetrodotoxin-Loaded Liposomes.

BACKGROUND: Capsaicin, the active component of chili peppers, can produce sensory-selective peripheral nerve blockade. Coadministration of capsaicin and tetrodotoxin, a site-1 sodium channel blocker, can achieve a synergistic effect on duration of nerve blocks. However, capsaicin can be neurotoxic, and tetrodotoxin can cause systemic toxicity. We evaluated whether codelivery of capsaicin and tetrodotoxin liposomes can achieve prolonged local anesthesia without local or systemic toxicity. METHODS: Capsaicin- and tetrodotoxin-loaded liposomes were developed. Male Sprague-Dawley rats were injected at the sciatic nerve with free capsaicin, capsaicin liposomes, free tetrodotoxin, tetrodotoxin liposomes, and blank liposomes, singly or in combination. Sensory and motor nerve blocks were assessed by a modified hotplate test and a weight-bearing test, respectively. Local toxicity was assessed by histologic scoring of tissues at the injection sites and transmission electron microscopic examination of the sciatic nerves. Systemic toxicity was assessed by rates of contralateral nerve deficits and/or mortality. RESULTS: The combination of capsaicin liposomes and tetrodotoxin liposomes achieved a mean duration of sensory block of 18.2 hours (3.8 hours) [mean (SD)], far longer than that from capsaicin liposomes [0.4 hours (0.5 hours)] (P < .001) or tetrodotoxin liposomes [0.4 hours (0.7 hours)] (P < .001) given separately with or without the second drug in free solution. This combination caused minimal myotoxicity and muscle inflammation, and there were no changes in the percentage or diameter of unmyelinated axons. There was no systemic toxicity. CONCLUSIONS: The combination of encapsulated tetrodotoxin and capsaicin achieved marked prolongation of nerve block. This combination did not cause detectable local or systemic toxicity. Capsaicin may be useful for its synergistic effects on other formulations even when used in very small, safe quantities.

Stable Tetrodotoxin Production by Bacillus sp. Strain 1839.

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.

ß-adrenergic regulation of late Na+ current during cardiac action potential is mediated by both PKA and CaMKII.

Late Na+ current (INaL) significantly contributes to shaping cardiac action potentials (APs) and increased INaL is associated with cardiac arrhythmias. ß-adrenergic receptor (ßAR) stimulation and its downstream signaling via protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) pathways are known to regulate INaL. However, it remains unclear how each of these pathways regulates INaL during the AP under physiological conditions. Here we performed AP-clamp experiments in rabbit ventricular myocytes to delineate the impact of each signaling pathway on INaL at different AP phases to understand the arrhythmogenic potential. During the physiological AP (2 Hz, 37 °C) we found that INaL had a basal level current independent of PKA, but partially dependent on CaMKII. ßAR activation (10 nM isoproterenol, ISO) further enhanced INaL via both PKA and CaMKII pathways. However, PKA predominantly increased INaL early during the AP plateau, whereas CaMKII mainly increased INaL later in the plateau and during rapid repolarization. We also tested the role of key signaling pathways through exchange protein activated by cAMP (Epac), nitric oxide synthase (NOS) and reactive oxygen species (ROS). Direct Epac stimulation enhanced INaL similar to the ßAR-induced CaMKII effect, while NOS inhibition prevented the ßAR-induced CaMKII-dependent INaL enhancement. ROS generated by NADPH oxidase 2 (NOX2) also contributed to the ISO-induced INaL activation early in the AP. Taken together, our data reveal differential modulations of INaL by PKA and CaMKII signaling pathways at different AP phases. This nuanced and comprehensive view on the changes in INaL during AP deepens our understanding of the important role of INaL in reshaping the cardiac AP and arrhythmogenic potential under elevated sympathetic stimulation, which is relevant for designing therapeutic treatment of arrhythmias under pathological conditions.

Parallel lives of symbionts and hosts: chemical mutualism in marine animals.

Covering: up to 2018 Symbiotic microbes interact with animals, often by producing natural products (specialized metabolites; secondary metabolites) that exert a biological role. A major goal is to determine which microbes produce biologically important compounds, a deceptively challenging task that often rests on correlative results, rather than hypothesis testing. Here, we examine the challenges and successes from the perspective of marine animal-bacterial mutualisms. These animals have historically provided a useful model because of their technical accessibility. By comparing biological systems, we suggest a common framework for establishing chemical interactions between animals and microbes.

Of poisons and parasites-the defensive role of tetrodotoxin against infections in newts.

Classical research on animal toxicity has focused on the role of toxins in protection against predators, but recent studies suggest these same compounds can offer a powerful defense against parasites and infectious diseases. Newts in the genus Taricha are brightly coloured and contain the potent neurotoxin, tetrodotoxin (TTX), which is hypothesized to have evolved as a defense against vertebrate predators such as garter snakes. However, newt populations often vary dramatically in toxicity, which is only partially explained by predation pressure. The primary aim of this study was to evaluate the relationships between TTX concentration and infection by parasites. By systematically assessing micro- and macroparasite infections among 345 adult newts (sympatric populations of Taricha granulosa and T. torosa), we detected 18 unique taxa of helminths, fungi, viruses and protozoans. For both newt species, per-host concentrations of TTX, which varied from undetectable to >60 µg/cm2 skin, negatively predicted overall parasite richness as well as the likelihood of infection by the chytrid fungus, Batrachochytrium dendrobatidis, and ranavirus. No such effect was found on infection load among infected hosts. Despite commonly occurring at the same wetlands, T. torosa supported higher parasite richness and average infection load than T. granulosa. Host body size and sex (females > males) tended to positively predict infection levels in both species. For hosts in which we quantified leucocyte profiles, total white blood cell count correlated positively with both parasite richness and total infection load. By coupling data on host toxicity and infection by a broad range of micro- and macroparasites, these results suggest that-alongside its effects on predators-tetrodotoxin may help protect newts against parasitic infections, highlighting the importance of integrative research on animal chemistry, immunological defenses and natural enemy ecology.

Difference in Uptake of Tetrodotoxin and Saxitoxins into Liver Tissue Slices among Pufferfish, Boxfish and Porcupinefish.

Although pufferfish of the family Tetraodontidae contain high levels of tetrodotoxin (TTX) mainly in the liver, some species of pufferfish, boxfish of the family Ostraciidae, and porcupinefish of the family Diodontidae do not. To clarify the mechanisms, uptake of TTX and saxitoxins (STXs) into liver tissue slices of pufferfish, boxfish and porcupinefish was examined. Liver tissue slices of the pufferfish (toxic species Takifugu rubripes and non-toxic species Lagocephalus spadiceus, L. cheesemanii and Sphoeroides pachygaster) incubated with 50 µM TTX accumulated TTX (0.99-1.55 µg TTX/mg protein) after 8 h, regardless of the toxicity of the species. In contrast, in liver tissue slices of boxfish (Ostracion immaculatus) and porcupinefish (Diodon holocanthus, D. liturosus, D. hystrix and Chilomycterus reticulatus), TTX content did not increase with incubation time, and was about 0.1 µg TTX/mg protein. When liver tissue slices were incubated with 50 µM STXs for 8 h, the STXs content was <0.1 µg STXs/mg protein, irrespective of the fish species. These findings indicate that, like the toxic species of pufferfish T. rubripes, non-toxic species such as L. spadiceus, L. cheesemanii and S. pachygaster, potentially take up TTX into the liver, while non-toxic boxfish and porcupinefish do not take up either TTX or STXs.

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.