Seasonal Changes in the Tetrodotoxin Content of the Flatworm Planocera multitentaculata.
ABSTRACT: Tetrodotoxin (TTX) is a potent neurotoxin that acts specifically on voltage-gated sodium channels on excitable membranes of muscle and nerve tissues. The biosynthetic process for TTX is unclear, although marine bacteria are generally thought to be the primary producers. The marine flatworm Planocera multitentaculata is a known TTX-bearing organism, and is suspected to be a TTX supplier to pufferfish. In this study, flatworm specimens were collected from an intertidal zone in Hayama, Kanagawa, Japan, the TTX content of the flatworm was measured using liquid chromatography with tandem mass spectrometry (LC-MS/MS), and seasonal changes in TTX content were investigated. No significant difference in TTX concentration of the flatworm body was found between the spawning period and other periods. However, the TTX content in individual flatworms was significantly higher in the spawning period than at other times. The TTX content rose in association with an increase in the body weight of the flatworm.
Project description:The pufferfish Takifugu niphobles (at present Takifugu alboplumbeus) possesses highly concentrated tetrodotoxin (TTX), an extremely potent neurotoxin that provides effective protection from predators, at least at the larval stages. However, the source of the toxin has remained unclear. Recently, DNA from the toxic flatworm Planocera multitentaculata was detected in the intestinal contents of juveniles and young of the pufferfish, suggesting that the flatworm contributes to its toxification at various stages of its life. In this study, we describe the behavior of the pufferfish in the intertidal zone that appears to contribute to its toxification before and during its spawning period: pufferfish were found to aggregate and ingest flatworm egg plates by scraping them off the surface of rocks. DNA analysis based on 28S rRNA and cytochrome c oxidase subunit I (COI) genes identified the egg plates as those of P. multitentaculata. Liquid chromatography with tandem mass spectrometry analysis revealed that the egg plates contain highly concentrated TTX. The feeding behavior of the pufferfish on the flatworm egg plates was also observed in the aquarium. These results suggest that pufferfish feed on the flatworm egg plate, which enables them to acquire toxicity themselves while providing their offspring with the protective shield of TTX.
Project description:Several polyclad flatworm species are known to contain high levels of tetrodotoxin (TTX), but currently TTX-bearing flatworms seem to be restricted to specific <i>Planocera</i> lineages belonging to the suborder Acotylea. During our ongoing study of flatworm toxins, high concentrations of TTXs were detected for the first time in the flatworm <i>Prosthiostomum trilineatum</i>, suborder Cotylea, from the coastal area of Hayama, Kanagawa, Japan. Toxin levels were investigated by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS), revealing that this species contains comparable concentrations of toxins as seen in planocerid flatworms such as <i>Planocera multitentaculata</i>. This finding indicated that there may be other species with significant levels of TTXs. The distribution of TTXs among other flatworm species is thus of great interest.
Project description:Polyclad flatworms comprise a highly diverse and cosmopolitan group of marine turbellarians. Although some species of the genera Planocera and Stylochoplana are known to be tetrodotoxin (TTX)-bearing, there are few new reports. In this study, planocerid-like flatworm specimens were found in the sea bottom off the waters around the Ryukyu Islands, Japan. The bodies were translucent with brown reticulate mottle, contained two conical tentacles with eye spots clustered at the base, and had a slightly frilled-body margin. Each specimen was subjected to TTX extraction followed by liquid chromatography with tandem mass spectrometry analysis. Mass chromatograms were found to be identical to those of the TTX standards. The TTX amounts in the two flatworm specimens were calculated to be 468 and 3634 ?g. Their external morphology was found to be identical to that of Planocera heda. Phylogenetic analysis based on the sequences of the 28S rRNA gene and cytochrome-c oxidase subunit I gene also showed that both specimens clustered with the flatworms of the genus Planocera (Planocera multitentaculata and Planocera reticulata). This fact suggests that there might be other Planocera species that also possess highly concentrated TTX, contributing to the toxification of TTX-bearing organisms, including fish.
Project description:Beginning with the larval stages, marine pufferfish such as Takifugu niphobles contain tetrodotoxin (TTX), an extremely potent neurotoxin. Although highly concentrated TTX has been detected in adults and juveniles of these fish, the source of the toxin has remained unclear. Here we show that TTX in the flatworm Planocera multitentaculata contributes to the toxification of the pufferfish throughout the life cycle of the flatworm. A species-specific PCR method was developed for the flatworm, and the specific DNA fragment was detected in the digesta of wild pufferfish adults. Predation experiments showed that flatworm larvae were eaten by the pufferfish juveniles, and that the two-day postprandial TTX content in these pufferfish was 20-50??g/g. Predation experiments additionally showed flatworm adults were also eaten by pufferfish young, and after two days of feeding, TTX accumulated in the skin, liver and intestine of the pufferfish.
Project description:The deadly neurotoxin tetrodotoxin (TTX) is found in a variety of animal phyla and, because of its toxicity, is most often assumed to deter predation. On the tropical Pacific island of Guam, we found an undescribed flatworm (planocerid sp. 1) that contains high levels of TTX and its analogs. Through ecological experiments, we show that TTXs do not protect these flatworms from some predators but instead are used to capture mobile prey. TTX is known to have multiple ecological functions, which has probably led to its widespread presence among prokaryotes and at least 10 metazoan phyla.
Project description:The potent neurotoxin tetrodotoxin (TTX) is known from a diverse array of taxa, but is unknown in terrestrial invertebrates. Tetrodotoxin is a low molecular weight compound that acts by blocking voltage-gated sodium channels, inducing paralysis. However, the origins and ecological functions of TTX in most taxa remain mysterious. Here, we show that TTX is present in two species of terrestrial flatworm (Bipalium adventitium and Bipalium kewense) using a competitive inhibition enzymatic immunoassay to quantify the toxin and high phase liquid chromatography to confirm the presence. We also investigated the distribution of TTX throughout the bodies of the flatworms and provide evidence suggesting that TTX is used during predation to subdue large prey items. We also show that the egg capsules of B. adventitium have TTX, indicating a further role in defense. These data suggest a potential route for TTX bioaccumulation in terrestrial systems.
Project description: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.
Project description:Tetrodotoxin (TTX) is one of the most potent naturally occurring compounds and is responsible for many human intoxications worldwide. Paphies australis are endemic clams to New Zealand which contain varying concentrations of TTX. Research suggests that P. australis accumulate the toxin exogenously, but the source remains uncertain. The aim of this study was to identify potential bacterial TTX-producers by exploring differences in bacterial communities in two organs of P. australis: the siphon and digestive gland. Samples from the digestive glands of a non-toxic bivalve Austrovenus stutchburyi that lives amongst toxic P. australis populations were also analyzed. Bacterial communities were characterized using 16S ribosomal RNA gene metabarcoding in P. australis sourced monthly from the Hokianga Harbor, a site known to have TTX-bearing clams, for 1 year, from ten sites with varying TTX concentrations around New Zealand, and in A. stutchburyi from the Hokianga Harbor. Tetrodotoxin was detected in P. australis from sites all around New Zealand and in all P. australis collected monthly from the Hokianga Harbor. The toxin averaged 150 ?g kg-1 over the year of sampling in the Hokianga Harbor but no TTX was detected in the A. stutchburyi samples from the same site. Bacterial species diversity differed amongst sites (p < 0.001, F = 5.9) and the diversity in siphon samples was significantly higher than in digestive glands (p < 0.001, F = 65.8). Spirochaetaceae (4-60%) and Mycoplasmataceae (16-78%) were the most abundant families in the siphons and the digestive glands, respectively. The bacterial communities were compared between sites with the lowest TTX concentrations and the Hokianga Harbor (site with the highest TTX concentrations), and the core bacterial communities from TTX-bearing individuals were analyzed. The results from both spatial and temporal studies corroborate with previous hypotheses that Vibrio and Bacillus could be responsible for the source of TTX in bivalves. The results from this study also indicate that marine cyanobacteria, in particular picocyanobacteria (e.g., Cyanobium, Synechococcus, Pleurocapsa, and Prochlorococcus), should be investigated further as potential TTX producers.
Project description:Tetrodotoxin (TTX) is a potent neurotoxin found mainly in puffer fish and other marine and terrestrial animals. TTX blocks voltage-gated sodium channels (VGSCs) which are typically classified as TTX-sensitive or TTX-resistant channels. VGSCs play a key role in pain signaling and some TTX-sensitive VGSCs are highly expressed by adult primary sensory neurons. During pathological pain conditions, such as neuropathic pain, upregulation of some TTX-sensitive VGSCs, including the massive re-expression of the embryonic VGSC subtype Na<sub>V</sub>1.3 in adult primary sensory neurons, contribute to painful hypersensitization. In addition, people with loss-of-function mutations in the VGSC subtype Na<sub>V</sub>1.7 present congenital insensitive to pain. TTX displays a prominent analgesic effect in several models of neuropathic pain in rodents. According to this promising preclinical evidence, TTX is currently under clinical development for chemo-therapy-induced neuropathic pain and cancer-related pain. This review focuses primarily on the preclinical and clinical evidence that support a potential analgesic role for TTX in these pain states. In addition, we also analyze the main toxic effects that this neurotoxin produces when it is administered at therapeutic doses, and the therapeutic potential to alleviate neuropathic pain of other natural toxins that selectively block TTX-sensitive VGSCs.
Project description:Tetrodotoxin (TTX)-producing bacteria have attracted great interest as a model system for study of the TTX biosynthetic route. Here, we report the complete genome of the TTX-producing bacterium <i>Bacillus</i> sp. 1839. The genome of the strain <i>Bacillus</i> sp. 1839, previously isolated from the TTX-bearing marine ribbon worm <i>Cephalothrix</i> cf. <i>simula</i>, was obtained using second generation Illumina and third generation nanopore sequencing technologies. Phylogenetic analysis has classified this strain as <i>Cytobacillus gottheilii</i>.