The odor of a nontoxic tetrodotoxin analog, 5,6,11-trideoxytetrodotoxin, is detected by specific olfactory sensory neurons of the green spotted puffers.

Toxic puffers accumulate tetrodotoxin (TTX), a well-known neurotoxin, by feeding on TTX-bearing organisms and using it to defend themselves from predators. Our previous studies have demonstrated that toxic puffers are attracted to 5,6,11-trideoxytetrodotoxin (TDT), a nontoxic TTX analog that is simultaneously accumulated with TTX in toxic puffers and their prey. In addition, activity labeling using immunohistochemistry targeting neuronal activity marker suggests that TDT activates crypt olfactory sensory neurons (OSN) of the green spotted puffer. However, it remains to be determined whether individual crypt OSNs can physiologically respond to TDT. By employing electroporation to express GCaMP6s in OSNs, we successfully identified a distinct group of oval OSNs that exhibited a specific calcium response when exposed to TDT in green spotted puffers. These oval OSNs showed no response to amino acids (AAs), which serve as food odor cues for teleosts. Furthermore, oval morphology and surface positioning of TDT-sensitive OSNs in the olfactory epithelium closely resemble that of crypt OSNs. These findings further substantiate that TDT is specifically detected by crypt OSNs in green spotted puffer. The TDT odor may act as a chemoattractant for finding conspecific toxic puffers and for feeding TTX-bearing organisms for effective toxification.

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.

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.

An almost nontoxic tetrodotoxin analog, 5,6,11-trideoxytetrodotoxin, as an odorant for the grass puffer.

Toxic puffers contain the potent neurotoxin, tetrodotoxin (TTX). Although TTX is considered to serve as a defense substance, previous behavioral studies have demonstrated that TTX acts as an attractive pheromone for some toxic puffers. To elucidate the physiological mechanism of putative pheromonal action of TTX, we examined whether grass puffers Takifugu alboplumbeus can detect TTX. Electroolfactogram (EOG) results suggest that the olfactory epithelium (OE) of grass puffers responded to a type of TTX analog (5,6,11-trideoxyTTX), although it did not respond to TTX. We also examined the attractive action of 5,6,11-trideoxyTTX on grass puffers by recording their swimming behavior under dark conditions. Grass puffers preferred to stay on the side of the aquarium where 5,6,11-trideoxyTTX was administered, and their swimming speed decreased. Additionally, odorant-induced labeling of olfactory sensory neurons by immunohistochemistry against neural activity marker (phosphorylated extracellular signal regulated kinase; pERK) revealed that labeled olfactory sensory neurons were localized in the region surrounding "islets" where there was considered as nonsensory epithelium. 5,6,11-trideoxyTTX has been known to accumulate in grass puffers, but its toxicity is much lower (almost nontoxic) than TTX. Our results suggest that toxic puffers may positively use this TTX analog, which has been present in their body with TTX but whose function was unknown, as an odorant for chemical communication or effective TTX accumulation.

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.

Green spotted puffers detect a nontoxic TTX analog odor using crypt olfactory sensory neurons.

Toxic puffers accumulate their defense substance (tetrodotoxin; TTX) through the food chain. Although the previous study suggests that 5,6,11-trideoxyTTX, a nontoxic TTX analog detected simultaneously with TTX in toxic puffers or their prey, acts as an olfactory chemoattractant for grass puffers, it is unclear whether toxic puffers are commonly attracted to 5,6,11-trideoxyTTX, and which types of olfactory sensory neurons (OSNs) detect 5,6,11-trideoxyTTX. Here, we demonstrated that green spotted puffer, a phylogenetically distant species from the grass puffer, is attracted to 5,6,11-trideoxyTTX. 5,6,11-TrideoxyTTX administration made green spotted puffers stay longer at the administered site, whereas a food odor (l-Arg) made them actively swim throughout the aquarium. Attractive responses were not observed when TTX or its vehicle was administered, nor when 5,6,11-trideoxyTTX was administered to anosmic fish. Furthermore, double immunohistochemistry with activity marker and crypt OSN marker antibodies labeled oval cells with apical invagination on the olfactory epithelium surface treated with 5,6,11-trideoxyTTX. These results suggest that 5,6,11-trideoxyTTX acts as an olfactory chemoattractant detected by crypt OSNs, and attraction to 5,6,11-trideoxyTTX odor appears to be a trait shared by toxic puffers for social communication or effective toxification.

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.

Characterization of a GH Family 20 Exo-ß-N-acetylhexosaminidase with Antifungal Activity from Streptomyces avermitilis.

We characterized SaHEX, which is a glycoside hydrolase (GH) family 20 exo-ß-N-acetylhexosaminidase found in Streptomyces avermitilis. SaHEX exolytically hydrolyzed chitin oligosaccharides from their non-reducing ends, and yielded N-acetylglucosamine (GlcNAc) as the end product. According to the initial rate of substrate hydrolysis, the rates of (GlcNAc)3 and (GlcNAc)5 hydrolysis were greater than the rates for the other oligosaccharides. The enzyme exhibited antifungal activity against Aspergillus niger, which was probably due to hydrolytic activity with regard to chitin in the hyphal tips. Therefore, SaHEX has potential for use in GlcNAc production and food preservation.