Geographical differences in the composition of tetrodotoxin and 5,6,11-trideoxytetrodotoxin in Japanese pufferfishes and their origins.

Tetrodotoxin (TTX)-bearing fish are thought to accumulate TTXs in their bodies through a food chain that begins with marine bacteria. However, the mechanism of TTXs transfer between prey and predators in the food chain remains unclear and the reasons for regional differences in pufferfish toxicity are also unknown. To investigate these matters, we collected juveniles of four species of pufferfish, Takifugu alboplumbeus, Takifugu flavipterus, Takifugu stictonotus, and Chelonodon patoca, from various locations in the Japanese Islands, and subjected them to liquid chromatography-tandem mass spectrometry analysis for TTX and its analog 5,6,11-trideoxyTTX (TDT). Concentrations of these substances tended to be higher in pufferfish juveniles collected from the Sanriku coastal area (Pacific coast of northern Japan) than in those from other locations. Juveniles had higher concentrations of TTX at all locations than of TDT. Mitochondrial cytochrome c oxidase subunit I (COI) sequences specific to the TTX-bearing flatworm, Planocera multitentaculata, were detected in the intestinal contents of up to 100% of pufferfish juveniles from various sampling sites, suggesting that P. multitentaculata was widely involved in the toxification of the juveniles in the coastal waters of Japan. A toxification experiment was conducted on three species of pufferfish juveniles (T. alboplumbeus, Takifugu rubripes and C. patoca) using TTX-bearing flatworm eggs harboring equal amounts of TTX and TDT. The TTX content of juveniles fed on flatworm eggs was found to be more than twice that of TDT, suggesting that pufferfish preferentially incorporate TTX compared to TDT.

Levels of Tetrodotoxins in Spawning Pufferfish, Takifugu alboplumbeus.

Tetrodotoxin (TTX), also known as pufferfish toxin, is an extremely potent neurotoxin thought to be used as a biological defense compound in organisms bearing it. Although TTX was thought to function as a chemical agent for defense and anti-predation and an attractant for TTX-bearing animals including pufferfish, it has recently been demonstrated that pufferfish were also attracted to 5,6,11-trideoxyTTX, a related compound, rather than TTX alone. In this study, we attempted to estimate the roles of TTXs (TTX and 5,6,11-trideoxyTTX) in the pufferfish, Takifugu alboplumbeus, through examining the location of TTXs in various tissues of spawning pufferfish from Enoshima and Kamogawa, Japan. TTXs levels in the Kamogawa population were higher than those in the Enoshima population, and there was no significant difference in the amount of TTXs between the sexes in either population. Individual differences were greater in females than in males. However, the location of both substances in tissues differed significantly between sexes: male pufferfish accumulated most of their TTX in the skin and liver and most of their 5,6,11-trideoxyTTX in the skin, whereas females accumulated most of their TTX and 5,6,11-trideoxyTTX in the ovaries and skin.

Tetrodotoxin Detection in Japanese Bivalves: Toxification Status of Scallops.

Tetrodotoxin (TTX), or pufferfish toxin, has been frequently detected in edible bivalves around the world during the last decade and is problematic in food hygiene and safety. It was reported recently that highly concentrated TTX was detected in the midgut gland of the akazara scallop Chlamys (Azumapecten) farreri subsp. akazara collected in coastal areas of the northern Japanese archipelago. The toxification of the bivalve was likely to involve the larvae of the flatworm, Planocera multitentaculata. However, the overall status of bivalve TTX toxification has not been elucidated. In this study, 14 species/subspecies of bivalves from various Japanese waters were subjected to LC-MS/MS analysis to reveal TTX toxification state, demonstrating that the Pectinidae, including C. farreri akazara, Chlamys farreri nipponensis, Chlamys (Mimachlamys) nobilis, and Mizuhopecten yessoensis, accumulated TTX in their midgut gland. Many individuals of C. farreri akazara and C. farreri nipponensis were found with high concentrations of TTX, while C. nobilis and M. yessoensis exhibited low concentrations. The extent of TTX accumulation in C. farreri akazara and C. farreri nipponensis varied widely by region and season. Curiously, no other bivalve species investigated in this study showed evidence of TTX. These results suggest that monitoring for TTX, like other shellfish toxins, is necessary to ensure that pectinid bivalves are a safe food resource.

Changes in Tissue Distribution of Tetrodotoxin and Its Analogues in Association with Maturation in the Toxic Flatworm, Planocera multitentaculata.

The toxic flatworm, Planocera multitentaculata, possesses highly concentrated tetrodotoxin (TTX), also known as pufferfish toxin, throughout its life cycle, including the egg and larval stages. Additionally, TTX analogues, 5,6,11-trideoxyTTX and 11-norTTX-6(S)-ol, have also been detected in the flatworm. The high concentration of TTX in the eggs and larvae appears to be for protection against predation, and 11-norTTX-6(S)-ol in the pharyngeal tissue in the adults is likely used to sedate or kill prey during predation. However, information on the role of 5,6,11-trideoxyTTX, a potential important biosynthetic intermediate of TTX, in the toxic flatworm is lacking. Here, we aimed to determine the region of localization of TTX and its analogues in the flatworm body, understand their pharmacokinetics during maturation, and speculate on their function. Flatworm specimens in four stages of maturity, namely juvenile, mating, spawning, and late spawning, were subjected to LC-MS/MS analysis, using the pharyngeal tissue, oocytes in seminal receptacle, sperm, and tissue from 12 other sites. Although TTX was consistently high in the pharyngeal tissue throughout maturation, it was extremely high in the oocytes during the spawning period. Meanwhile, 5,6,11-trideoxyTTX was almost undetectable in the pharyngeal part throughout the maturation but was very abundant in the oocytes during spawning. 11-norTTX-6(S)-ol consistently localized in the pharyngeal tissue. Although the localization of TTX and its analogues was approximately consistent with the MS imaging data, TTX and 11-norTTX-6(S)-ol were found to be highly localized in the parenchyma surrounding the pharynx, which suggests the parenchyma is involved in the accumulation and production of TTXs.

Tetrodotoxins in the flatworm Planocera multitentaculata.

The marine polyclad flatworm Planocera multitentaculata is known to possess high levels of tetrodotoxin (TTX), but the presence of TTX analogues in the species has been unexplored. In this study, TTX and several analogues such as 5,6,11-trideoxyTTX, monodeoxyTTXs, dideoxyTTXs, and 11-norTTX-6(S)-ol were identified in three adults and egg plates of P. multitentaculata using high resolution liquid chromatography-mass spectrometry (HR-LC/MS) for the first time.

Local Differences in the Toxin Amount and Composition of Tetrodotoxin and Related Compounds in Pufferfish (Chelonodon patoca) and Toxic Goby (Yongeichthys criniger) Juveniles.

Tetrodotoxin (TTX)-bearing fish ingest TTX from their preys through the food chain and accumulate TTX in their bodies. Although a wide variety of TTX-bearing organisms have been reported, the missing link in the TTX supply chain has not been elucidated completely. Here, we investigated the composition of TTX and 5,6,11-trideoxyTTX in juveniles of the pufferfish, Chelonodon patoca, and toxic goby, Yongeichthys criniger, using LC-MS/MS, to resolve the missing link in the TTX supply chain. The TTX concentration varied among samples from different localities, sampling periods and fish species. In the samples from the same locality, the TTX concentration was significantly higher in the toxic goby juveniles than in the pufferfish juveniles. The concentration of TTX in all the pufferfish juveniles was significantly higher than that of 5,6,11-trideoxyTTX, whereas the compositional ratio of TTX and 5,6,11-trideoxyTTX in the goby was different among sampling localities. However, the TTX/5,6,11-trideoxyTTX ratio in the goby was not different among samples collected from the same locality at different periods. Based on a species-specific PCR, the detection rate of the toxic flatworm (Planocera multitentaculata)-specific sequence (cytochrome c oxidase subunit I) also varied between the intestinal contents of the pufferfish and toxic goby collected at different localities and periods. These results suggest that although the larvae of the toxic flatworm are likely to be responsible for the toxification of the pufferfish and toxic goby juveniles by TTX, these fish juveniles are also likely to feed on other TTX-bearing organisms depending on their habitat, and they also possess different accumulation mechanisms of TTX and 5,6,11-trideoxyTTX.

Phylogenetic position of the Atlantic Gnomefish, Scombrops oculatus (Teleostei: Scombropidae), within the genus Scombrops, inferred from the sequences of complete mitochondrial genome and cytochrome c oxidase subunit I genes.

We determined the complete mitochondrial genome of the Atlantic Gnomefish, Scombrops oculatus (Scombropidae). The total length of mitochondrial DNA (mtDNA) was 16,515 bp and included 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and one control region. The gene arrangement of S. oculatus was identical to those of three Japanese scombropid species and those of other teleosts. The phylogenetic analysis using the whole mtDNA, excluding the control region, indicates the Atlantic species is distinct from the Japanese clade, whereas that using cytochrome c oxidase subunit I gene showed the Atlantic species is most closely related to the African species.

The role of toxic planocerid flatworm larvae on tetrodotoxin accumulation in marine bivalves.

Tetrodotoxin (TTX), also known as pufferfish toxin, has been detected in marine edible bivalves worldwide. In this study, several bivalve species, Azumapecten farreri subsp. akazara, Patinopecten yessoensis and Mytilus galloprovincialis, collected from the Pacific side of the northern Japanese Islands, were studied for the accumulation of TTX in the presence of toxic planocerid larvae. LC-MS/MS analysis demonstrated that TTX was detected only in the midgut gland of A. farreri subsp. akazara. Toxic flatworm-specific PCR and direct sequencing of the amplicons showed that the DNA fragments of the Planocera multitentaculata COI gene were detected in the gut contents of the toxified bivalves. The planocerid larvae were also detected in the environmental seawaters. Toxification experiments in the aquarium demonstrated that the mussel M. galloprovincialis was also toxified by feeding on the toxic flatworm larvae. These results suggest that the source of TTX accumulation in edible bivalves is toxic flatworm larvae.

First Detection of Tetrodotoxins in the Cotylean Flatworm Prosthiostomum trilineatum.

Several polyclad flatworm species are known to contain high levels of tetrodotoxin (TTX), but currently TTX-bearing flatworms seem to be restricted to specific Planocera 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 Prosthiostomum trilineatum, 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 Planocera multitentaculata. 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.

Evolutionary origin of the Atlantic Cabo Verde nibbler (Girella stuebeli), a member of a primarily Pacific Ocean family of antitropical herbivorous reef fishes.

Nibblers (family Girellidae) are reef fishes that are mostly distributed in the Indo-Pacific, with one exception: Girella stuebeli, which is found in the Cabo Verde Archipelago, in the Atlantic Ocean. We capitalized on this unusual distribution to study the evolutionary history of the girellids, and determine the relationship between G. stuebeli and the remaining nibbler taxa. Based on thousands of genomic markers (RAD sequences), we identified the closest relatives of G. stuebeli as being a clade of three species endemic to the northwestern Pacific, restricted to the Sea of Japan and vicinity. This clade diverged from G. stuebeli approximately 2.2 Mya. Two alternative potential routes of migration may explain this affinity: a western route, from the Tropical Eastern Pacific and the Tropical Western Atlantic, and an eastern route via the Indian Ocean and Southern Africa. The geological history and oceanography of the regions combined with molecular data presented here, suggest that the eastern route of invasion (via the Indian Ocean and Southern Africa) is a more likely scenario.

Genetic and Morphological Characteristics in the Local Population of the Landlocked Salmon Oncorhynchus masou Originally Distributed in Kanagawa Prefecture, Japan.

Oncorhynchus masou, including subspecies of Oncorhynchus masou masou (yamame) and Oncorhynchus masou ishikawae (amago), is one of the salmonid groups impacted by human activity such as dam construction and release of non-native salmonids. In this study, we investigated the genetic structure of O. masou populations in the Sakawa and Sagami Rivers, Japan, by sequencing the mitochondrial control region. We hoped to identify genetically the O. masou populations specific to and originally native to Kanagawa Prefecture, where the two subspecies are thought to be present. The populations found in the upstream tributaries, where there has been no human impact and no upstream migration of fishes, were assumed to be descendants of the local O. masou populations in both river systems, and the morphological features seen here were similar to amago and yamame. However, both populations were genetically related to amago. In addition, only six haplotypes were detected in 315 individuals collected from 20 localities in the two river systems. Furthermore, haplotype diversity and nucleotide diversity of these populations were low, and high FST values were observed. These results suggest that the population size is restricted and genetic diversity is decreasing in the O. masou populations of the Sakawa and Sagami Rivers.

Taxonomic Distribution of Tetrodotoxin in Acotylean Flatworms (Polycladida: Platyhelminthes).

Tetrodotoxin (TTX), also known as pufferfish toxin, causes a respiratory disorder by blocking neurotransmission, with voltage-gated sodium channel inhibition on muscle and nerve tissues. The toxin is widely distributed across vertebrates, invertebrates and bacteria. Therefore, it is generally thought that TTX in pufferfish accumulates via the food webs, beginning with marine bacteria as a primary producer. Polyclad flatworms in the genus Planocera are also known to be highly toxic, TTX-bearing organisms. Unlike the case of pufferfish, the source of TTX in these flatworms is unknown. In this study, taxonomical distribution patterns of TTX were investigated for acotylean flatworms from coastal waters using molecular phylogenetic analysis and high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). A maximum likelihood tree based on the 28S rRNA gene sequence showed that the flatworms belonged to several different lineages among the genera Planocera, Stylochus, Paraplanocera, Discocelis, Notocomplana, Notoplana, Callioplana and Peudostylochus. After LC-MS/MS analysis, the distribution of TTX was mapped onto the molecular phylogenetic tree. TTX-bearing flatworm species were seen to be restricted to specific Planocera lineages, suggesting that the TTX-bearing flatworm species have common genes for TTX-accumulating mechanisms.

The planocerid flatworm is a main supplier of toxin to tetrodotoxin-bearing fish juveniles.

Tetrodotoxin (TTX), a potent neurotoxin, is found in various phylogenetically diverse taxa. In marine environments, the pufferfish is at the top of the food chain among TTX-bearing organisms. The accumulation of TTX in the body of pufferfish appears to be of the food web that begins with bacteria. It is known that toxic pufferfishes possess TTX from the larval/juvenile stage. However, the source of the TTX is unknown because the maternally sourced TTX is extremely small in quantity. Therefore, the TTX has to be obtained from other organisms or directly from the environment. Here, we report evidence that the source of TTX for toxic fish juveniles including the pufferfish (Chelonodon patoca) and the goby (Yongeichthys criniger) is in the food organisms, as seen in their gut contents. Next generation sequencing analysis for the mitochondrial COI gene showed that the majority of the sequence recovered from intestinal contents of these toxic fishes belonged to the flatworm Planocera multitentaculata, a polyclad flatworm containing highly concentrated TTX from the larval stage. PCR specific to P. multitentaculata also showed that DNA encoding the planocerid COI gene was strongly detected in the intestinal contents of the goby and pufferfish juveniles. Additionally, the planocerid specific COI sequence was detected in the environmental seawater collected from the water around the sampling locations for TTX-bearing fish. These results suggest that planocerid larvae are the major TTX supplier for juveniles of TTX-bearing fish species.

Heptavalinamide A, an Extensively N-Methylated Linear Nonapeptide from a Cyanobacterium Symploca sp. and Development of a Highly Sensitive Analysis of N,N-Dimethylvaline by LCMS.

An extensively N-methylated linear nonapeptide heptavalinamide A (1) was isolated from the marine cyanobacterium Symploca sp. collected at Kabira Reef of Ishigaki Island, Okinawa. The amino acid sequence of 1 was assigned by interpretation of 2D NMR and MS/MS data. The absolute configurations of the constituent amino acids were determined by the application of Marfey's method. A method to assign the configuration of N,N-dimethylvaline by LCMS is discussed.

Difference in tetrodotoxin content between two sympatric planocerid flatworms, Planocera multitentaculata and Planocera reticulata.

Planocerid flatworms and the related species (Platyhelminthes: polycladida) are known as tetrodotoxin (TTX)-bearing organisms, and they contribute to toxification of marine organisms at higher trophic levels, such as pufferfish and sea slugs. However, little is known of their biology or ecology. In this study, we therefore investigated the occurrence and toxicity of two sympatric planocerids, Planocera multitentaculata and Planocera reticulata, in intertidal zones of the central region of mainland Honshu, Japanese Islands. Planocera multitentaculata was much more abundant than P. reticulata. Body weight was greater in P. multitentaculata than in P. reticulata. Although a significant difference in TTX concentration was not observed between the two species, total TTX content per individual was greater in P. multitentaculata.

Toxic Flatworm Egg Plates Serve as a Possible Source of Tetrodotoxin for Pufferfish.

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.

Including planocerid flatworms in the diet effectively toxifies the pufferfish, Takifugu niphobles.

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 µg/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.

Role of maternal tetrodotoxin in survival of larval pufferfish.

It is known that tetrodotoxin (TTX), also known as pufferfish toxin, is an extremely potent neurotoxin and had been detected in various taxa. However, the exact function of the toxin in TTX-bearing organisms has remained unclear. In Takifugu pufferfish species, it has been suggested that TTX is utilized to protect larvae from predators but no experimental proof exists. In the present study, we used pufferfish Takifugu alboplumbeus larvae from wild and cultured parents to determine the effects of the maternal TTX on the survival of toxic and non-toxic pufferfish larvae, respectively. TTX contents in the larval pufferfish differed between the larvae derived from wild and cultured parents (1.23 ±â€¯0.20 ng/individual vs. undetectable levels, respectively). Immunohistochemical staining with anti-TTX monoclonal antibody demonstrated that the TTX-specific signals were primarily observed at the body surface of the larvae of wild parents, but not of cultured parents. Predation experiments demonstrated that the juveniles of Girella punctata and Chaenogobius gulosus, used as predator fish, ingested the pufferfish larvae derived from either type of parents, but instantly spat out those from wild parents only. These results indicate that larvae, which are at the most vulnerable stage in the life of pufferfish, are protected by maternal TTX.

Species composition of the genus Scombrops (Teleostei, Scombropidae) in the waters around the Japanese Archipelago: detection of a cryptic species.

Current literature states that family Scombropidae consists of a single genus Scombrops comprising three species worldwide, with two of them, Scombrops boops and Scombrops gilberti, distributed in the waters around the Japanese Archipelago. Although these two scombropids are commercially important species, little is known about the ecology of these fishes. It is difficult to discriminate between these two species based on external characteristics because of their morphological similarity. Here, we report two different morphotypes characterized by the relative growth between the otolith size and the standard length (SL) of the scombropid specimens caught in southern waters off Kyushu Island, Japan, and show the genetic relationship between the morphotypes by means of phylogenetic analyses using complete DNA sequences of the cytochrome b gene. The relationship between otolith weight and SL was significantly different between specimens < 505 mm SL and those > 550 mm SL. Phylogenetic analysis demonstrated that the sequences from these scombropid specimens formed three clades: two corresponded to S. boops and S. gilberti, while the third did not correspond to any sequence recorded in databases, suggesting that these specimens are undescribed scombropid species. Almost all the specimens with SL < 505 mm (n = 76) were identified as S. boops, and only nine as S. gilberti. On the other hand, almost all the specimens with SL > 550 mm (n = 41) fell in the unidentified group except for four specimens, whose sequences were identical to that of S. boops.

TTX-Bearing Planocerid Flatworm (Platyhelminthes: Acotylea) in the Ryukyu Islands, Japan.

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 µg. 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.