Automated Patch Clamp for the Detection of Tetrodotoxin in Pufferfish Samples.

Tetrodotoxin (TTX) is a marine toxin responsible for many intoxications around the world. Its presence in some pufferfish species and, as recently reported, in shellfish, poses a serious health concern. Although TTX is not routinely monitored, there is a need for fast, sensitive, reliable, and simple methods for its detection and quantification. In this work, we describe the use of an automated patch clamp (APC) system with Neuro-2a cells for the determination of TTX contents in pufferfish samples. The cells showed an IC50 of 6.4 nM for TTX and were not affected by the presence of muscle, skin, liver, and gonad tissues of a Sphoeroides pachygaster specimen (TTX-free) when analysed at 10 mg/mL. The LOD achieved with this technique was 0.05 mg TTX equiv./kg, which is far below the Japanese regulatory limit of 2 mg TTX equiv./kg. The APC system was applied to the analysis of extracts of a Lagocephalus sceleratus specimen, showing TTX contents that followed the trend of gonads > liver > skin > muscle. The APC system, providing an in vitro toxicological approach, offers the advantages of being sensitive, rapid, and reliable for the detection of TTX-like compounds in seafood.

Microencapsulation of Lactobacillus plantarum with Improved Survivability Using Pufferfish Skin Gelatin-Based Wall Materials.

To improve the survivability of probiotics, Lactobacillus plantarum was microencapsulated using pufferfish skin gelatin (PSG)-based wall materials by spray-drying. This work investigated the protective effect of three different pH-dependent proteins (sodium caseinate (SC), soy protein isolate (SPI), and whey protein isolate (WPI)) combined with PSG on L. plantarum. The experimental results of spray-drying with an inlet temperature of 120 °C and an outlet temperature of 80 °C, storage at 4 °C for 6 months, simulated digestion, and turbidity indicated that PSG/SC had better stability and encapsulation effects and was more suitable to encapsulate L. plantarum than PSG/SPI and PSG/WPI. The optimum preparation conditions for L. plantarum microcapsules were a PSG/SC mass ratio of 2:1, an SC concentration of 20 g/L, and a cell concentration of 10 g/L. The encapsulation efficiency of the obtained microcapsules was 95.0%, and the survival rate was 94.2% in simulated gastric fluid for 2 h and 98.0% in simulated intestinal fluid for 2 h. Amino acid composition analysis exhibited that the imino acid and aspartic acid contents of PSG were 27.98 and 26.16 g/100 g protein, respectively, which was much higher than commercial bovine gelatin. This characteristic was favorable to the high encapsulation efficiency and stability of microcapsules. In vitro release experiments showed that the PSG/SC microcapsules did not disintegrate in simulated gastric fluid for 2 h but could completely release in simulated intestinal fluid for 2 h, which can maintain the high survivability of L. plantarum in simulated digestion. In general, this study demonstrated that microcapsules using PSG/SC as wall materials can effectively improve the survivability of probiotics and have great potential for application in probiotic products.

Phylogeny and ultrastructure of Elthusa raynaudii (Isopoda, Cymothoidae) firstly recorded from the invasive silver cheeked toadfish (Lagocephalus sceleratus) (Gmelin 1789) in eastern Mediterranean Sea coast.

With the opening of the Suez Canal in 1869, many changes have occurred in the Mediterranean Sea ecosystem so became a home to many invasive Lessepsian marine species that have migrated from the Red Sea. About 500 marine species including pufferfish have immigrated and rapidly established a population in the Mediterranean Sea causing significant impact on its ecosystem and fisheries sector. The parasitic fauna of these pufferfish has scarcely been studied in the Mediterranean Sea and also in their native habitat. During this surveillance study on the invasive pufferfish species from the Egyptian Mediterranean coast, the female cymothoid isopod Elthusa raynaudii was detected from the branchial cavity and also in the buccal cavity of 23.9% of the examined Lagocephalus sceleratus. The isolated isopod species was firstly identified and described through electron microscopy and molecular phylogeny based on the sequences of mitochondrial 16S rRNA gene. Additionally, the description of eggs, embryonic stage, and manca of E. raynaudii was firstly provided. The pathological impact on the infested fish tissues was investigated and revealed curling and loss of secondary gill lamellae in addition to mucous exudates in between the gill filaments and granuloma formation in the gill arch. The study provided the first report of L. sceleratus as a new host for the isopod E. raynaudii collected from the Egyptian Mediterranean coast as a new locality record. The role of the Lessepsian invasive pufferfish in transmitting parasites to the native fish species was discussed.

Isolation and Purification of Protamine from the Cultured Takifugu flavidus and Its Physicochemical Properties.

Protamine is a cationic peptide derived from fish sperm and has several important functional properties: antibacterial properties, acting as a carrier for injectable insulin and as a heparin antagonist, combatting fatigue, etc. Thus, it has been widely used in medicinal applications and food products. Cultured Takifugu flavidus is a type of pufferfish with a delicious taste that is popular in China, and its production is increasing significantly. Therefore, protamine was extracted via acid extraction from the sperm of Takifugu flavidus and further isolated and purified via sephadex gel chromatography, ion exchange chromatography, and desalination chromatography. Furthermore, the physicochemical properties of protamine were investigated. The results showed that the sperm of the cultured T. flavidus were non-toxic, and the extracted and purified protamine had high contents of arginine (36.90%) and lysine (27.02%), respectively. The secondary structure of protamine was mainly ß-folded and irregularly curled. Additionally, protamine exhibited high thermal stability with a denaturation temperature of 176 °C. This study would provide a theoretical basis for the structural analysis, bioactivity, and resource development of pufferfish protamine and help to promote the development of the pufferfish industry.

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.

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.

Assessing the Toxicity of Lagocephalus sceleratus Pufferfish from the Southeastern Aegean Sea and the Relationship of Tetrodotoxin with Gonadal Hormones.

Given the dramatic increase in the L. sceleratus population in the southeastern Aegean Sea, there is growing interest in assessing the toxicity of this pufferfish and the factors controlling its tetrodotoxin (TTX) content. In the present study, liver, gonads, muscle and skin of 37 L. sceleratus specimens collected during May and June 2021 from the island of Rhodes, Greece, were subjected to multi-analyte profiling using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in order to quantitate TTX and evaluate whether this biotoxin interrelates with hormones. TTX and its analogues 4-epiTTX, 11-deoxyTTX, 11-norTTX-6-ol, 4,9-anhydroTTX and 5,11/6,11-dideoxyTTX were detected in all tissue types. Liver and gonads were the most toxic tissues, with the highest TTX concentrations being observed in the ovaries of female specimens. Only 22% of the analyzed muscle samples were non-toxic according to the Japanese toxicity threshold (2.2 µg TTX eq g-1), confirming the high poisoning risk from the inadvertent consumption of this species. Four steroid hormones (i.e., cortisol, testosterone, androstenedione and ß-estradiol) and the gonadotropin-releasing hormone (GnRH) were detected in the gonads. Androstenedione dominated in female specimens, while GnRH was more abundant in males. A positive correlation of TTX and its analogues with ß-estradiol was observed. However, a model incorporating sex rather than ß-estradiol as the independent variable proven to be more efficient in predicting TTX concentration, implying that other sex-related characteristics are more important than specific hormone-regulated processes.

[Tetrodotoxin Content of the Small Gastropod Nassarius sufflatus].

Severe tetrodotoxin (TTX) poisoning due to small gastropods has been documented in Japan. In this study, we investigated the TTX content of the muscles and viscera of Nassarius sufflatus collected off the coast of Futaoi Island, Shimonoseki, Yamaguchi Prefecture, Japan, to prevent the occurrence of TTX poisoning caused by this small gastropod. Live specimens were obtained, and their muscles and viscera were collected. Test solutions were prepared from tissues of specimens and analyzed for TTX by HPLC-fluorescence detection. TTX was detected in both tissues at concentrations ranging from <0.1 to 18.2 µg/g for muscle and <0.1 to 130.7 µg/g for viscera. These results suggested that N. sufflatus accumulates TTX not only in its viscera but also in its muscles, and that precautions should be taken to prevent food poisoning due to this gastropod.

Moraxella tetraodonis sp. nov., isolated from freshwater pufferfish (Tetraodon cutcutia) skin.

A novel aerobic bacterium, strain PS-22 of the genus Moraxella, was isolated from the skin of freshwater pufferfish (Tetraodon cutcutia). Cells were Gram stain negative, aerobic, non-motile, and coccoid. Optimum growth occurred at 28-30 °C and pH 6.5-7.5. The major cellular fatty acids were C18:1 ω9c, C10:0, C16:0, and C12:0 anteiso. The predominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phospholipid, amino lipid, and seven unknown lipids. The genome size is 2.68 Mbp, and the DNA G + C content was 43.3%. A gene ontology study revealed that the major fraction of genes were associated with biological processes (46.81%), followed by molecular function (34.27%) and cellular components (18.8%). Comparisons of 16S rRNA gene sequences revealed 99.11-90% sequence similarity with the closely related type strains of the genus Moraxella. The average nucleotide identity (ANI) and average amino acid identity (AAI) of strain PS-22 with reference type strains of the genus Moraxella were below 95-96%, and the corresponding in silico DNA-DNA hybridization (DDH) values were below 70%. A phylogenetic tree based on genome-wide core genes and 16S rRNA gene sequences revealed that strain PS-22 clustered with Moraxella osloensis CCUG350T in both the phylogenetic trees. Genotypic and phenotypic characteristics of strain PS-22 represent a novel species for which Moraxella tetraodonis sp. nov. is proposed. The type strain is PS-22T (= TBRC 15232T = NBRC 115236T).

An Updated Review of Tetrodotoxin and Its Peculiarities.

Tetrodotoxin (TTX) is a crystalline, weakly basic, colorless organic substance and is one of the most potent marine toxins known. Although TTX was first isolated from pufferfish, it has been found in numerous other marine organisms and a few terrestrial species. Moreover, tetrodotoxication is still an important health problem today, as TTX has no known antidote. TTX poisonings were most commonly reported from Japan, Thailand, and China, but today the risk of TTX poisoning is spreading around the world. Recent studies have shown that TTX-containing fish are being found in other regions of the Pacific and in the Indian Ocean, as well as the Mediterranean Sea. This review aims to summarize pertinent information available to date on the structure, origin, distribution, mechanism of action of TTX and analytical methods used for the detection of TTX, as well as on TTX-containing organisms, symptoms of TTX poisoning, and incidence worldwide.

[Detection of Tetrodotoxin in Vomit and Urine from a Patient Suffering from Pufferfish Poisoning].

We experienced a pufferfish poisoning case where no food residue was available to detect a causative agent. However, tetrodotoxin (TTX) was detected in vomit and urine samples from a patient using LC-MS/MS. Furthermore, we found a significant matrix effect in this analysis, indicating that the retention time of vomit and urine was not identical to the TTX standard solution and measured values multiplied by the dilution factors were not constant. Elimination of this matrix effect was attained by dilution of samples based on the retention time of the TTX standard solution, i.e., 10-time dilution of vomit test sample for LC-MS/MS analysis or 100-200-time dilution of urine one. Further research on urine analytical methods revealed that when TTX concentrations were too low to identify its peak on a chromatogram, TTX could be identified through a dilution procedure. It also showed that the application of the matrix-added TTX standard solution was effective for quantitative analysis under the influence of the matrix.

Description of Acinetobacter kanungonis sp. nov., based on phylogenomic analysis.

A novel strain of a member of the genus Acinetobacter, strain PS-1T, was isolated from the skin of fresh water pufferfish (Tetraodon cutcutia) collected from Mahanadi River, India. Cells were Gram-stain-negative, aerobic, coccoid and non-motile. The predominant polar lipids were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and phospholipid (PL) and the cell wall sugars were glucose, galactose and ribose. The major cellular fatty acids of PS-1T were C18 : 1ω9c (30.67 %), C16 : 1ω7c (19.54 %), C16 : 0 (15.87 %), C12 : 0 (7.35 %) and C12 : 0 3-OH (6.77 %). The genome size was 3.5 Mbp and the DNA G+C content was 41.97 %. Gene ontology study revealed that the major fraction of genes were associated with biological processes (53.99 %) followed by molecular function (30.42 %) and cellular components (15.58 %). Comparisons of 16S rRNA gene sequences revealed 97.94-97.05 % sequence similarity with the closely related type strains of species of the genus Acinetobacter. The average nucleotide identity (ANI) and average amino acid identity (AAI) of PS-1T with reference strains of species of the genus Acinetobacter with validly published names were bellow 95-96 and the corresponding in-silico DNA-DNA hybridization (DDH) values were below 70 %. A phylogenomic tree based on core genome analysis supported these results. Genotypic and phenotypic characteristics of PS-1T indicate that the strain represents a novel species of the genus Acinetobacter and the name Acinetobacter kanungonis sp. nov. is proposed. The type strain is PS-1T (=JCM 34131T=NCIMB 15260T).

Voltage-Gated Sodium Channels: A Prominent Target of Marine Toxins.

Voltage-gated sodium channels (VGSCs) are considered to be one of the most important ion channels given their remarkable physiological role. VGSCs constitute a family of large transmembrane proteins that allow transmission, generation, and propagation of action potentials. This occurs by conducting Na+ ions through the membrane, supporting cell excitability and communication signals in various systems. As a result, a wide range of coordination and physiological functions, from locomotion to cognition, can be accomplished. Drugs that target and alter the molecular mechanism of VGSCs' function have highly contributed to the discovery and perception of the function and the structure of this channel. Among those drugs are various marine toxins produced by harmful microorganisms or venomous animals. These toxins have played a key role in understanding the mode of action of VGSCs and in mapping their various allosteric binding sites. Furthermore, marine toxins appear to be an emerging source of therapeutic tools that can relieve pain or treat VGSC-related human channelopathies. Several studies documented the effect of marine toxins on VGSCs as well as their pharmaceutical applications, but none of them underlined the principal marine toxins and their effect on VGSCs. Therefore, this review aims to highlight the neurotoxins produced by marine animals such as pufferfish, shellfish, sea anemone, and cone snail that are active on VGSCs and discuss their pharmaceutical values.

[Toxicity of Takifugu exascurusCollected from the Sea of Kumano].

Marine pufferfish Takifugu exascurus is not approved for human consumption due to the lack of information on its toxicity. To clarify the toxicity of T. exascurus, ten live specimens were collected from the Sea of Kumano, Japan, and the toxicity and tetrodotoxin (TTX) concentration were determined using mouse bioassay and high performance liquid chromatography-fluorescence detection (HPLC-FLD), respectively. Toxicity was observed in the skin, liver, and ovaries, but the testes and muscle were non-toxic (<10 MU/g). On the other hand, HPLC-FLD revealed that TTX was detected in the muscle in two of the 10 specimens (1.4 and 1.5 MU/g). Based on the results, TTX is the main toxic component contributing to toxicity in T. exascurus.

Mystery pufferfish create elaborate circular nests at mesophotic depths in Australia.

In 2011, the enigma of "mystery circles," small but complex underwater structures first observed by divers from southern Japan in 1995, was solved when a new species of pufferfish, white-spotted pufferfish (Torquigener albomaculosus Matsuura 2014), was identified as the responsible agent. To date these circles have been described only from Japan, where they are formed on a sandy seafloor in water depths less than 30 m. A survey of oil field infrastructure on the North West Shelf of Western Australia in 2018 using a remotely operated vehicle and hybrid autonomous underwater vehicle (HAUV) recorded a high-resolution video and bathymetric data of 21 circular formations with similar features to those described in Japan. The circles display dimensions and morphology like those described from Japan, but were observed in water depths between 129 and 137 m. The HAUV also recorded high-resolution photographs which captured a Torquigener sp. fish in the immediate vicinity of the circles. An additional circle and Torquigener sp. were observed in images collected by baited remote underwater stereo-video in a nearby location in 129 m depth. These circles are the first to be found in Australia. The pufferfish species responsible cannot be identified from images collected. Such a discovery not only generates intrigue and wonder among scientists and the general public but also provides an insight into the reproductive behaviour and evolution of pufferfish globally.

Molecular characterization and expression analysis of galectins in Japanese pufferfish (Takifugu rubripes) in response to Vibrio harveyi infection.

Galectins are a family of proteins with conserved carbohydrate recognition domains (CRDs) that bind to specific glycans, including the glycans on the surface of pathogens, and therefore play a role in cytokine secretion, cell activation, migration, adhesion and apoptosis. Currently, galectins have been extensively studied in mammalian species but rarely studied in teleost fish species. In this study, a total of 12 galectin genes were characterized to understand the molecular mechanisms of galectin function in Japanese pufferfish (Takifugu rubripes). Phylogenetic analyses and syntenic analyses confirmed their correct annotation and suggested the strongest relationships to tetraodon. Furthermore, expression analyses were conducted in healthy tissues of Japanese pufferfish and after infection with Vibrio harveyi in the intestine, liver and spleen. The results showed that galectin genes were widely expressed in all examined tissues; however, most of the galectin genes were highly expressed in mucosal tissues (skin, gill and intestine). Moreover, majority of the galectin genes were significantly regulated after V. harveyi infection in the intestine, liver and spleen, suggesting that galectins were involved in the immune response to V. harveyi infection in Japanese pufferfish. This study established the foundation for future studies of galectin gene functions.

Muscle extract of Arothron immaculatus regulates the blood glucose level and the antioxidant system in high-fat diet and streptozotocin induced diabetic rats.

In the present study pufferfish, Arothron immaculatus muscle methanol extract (AIME) was used to evaluate the antidiabetic activity against the high-fat diet (HFD) in streptozotocin (STZ) induced diabetic rat models. Initially, the In vitro antioxidant activity of the different muscle extract was evaluated which showed that AIME has higher efficiency to scavenge the free radicals. The animal study results revealed that the AIME could decrease the blood glucose level after 14 days of oral treatment and recover the animal from the severe progression of the disease. The LC-ESI/MS analysis of AIME extract revealed the presence of compounds such as docosahexaenoic acid, adrenic acid, docosanol, codeine and metoprolol. Among these compounds, docosahexaenoic acid, adrenic acid and docosanol are reported for its antidiabetic studies. Hence, the muscle is recommended to consume by humans as natural food in order to overcome the development of diabetes. This is the first study on the muscle extract of marine pufferfish which is used as antidiabetic agent to treat the diabetes-induced in the animal model.

The Incidence of Tetrodotoxin and Its Analogs in the Indian Ocean and the Red Sea.

Tetrodotoxin (TTX) is a potent marine neurotoxin with bacterial origin. To date, around 28 analogs of TTX are known, but only 12 were detected in marine organisms, namely TTX, 11-oxoTTX, 11-deoxyTTX, 11-norTTX-6(R)-ol, 11-norTTX-6(S)-ol, 4-epiTTX, 4,9-anhydroTTX, 5,6,11-trideoxyTTX, 4-CysTTX, 5-deoxyTTX, 5,11-dideoxyTTX, and 6,11-dideoxyTTX. TTX and its derivatives are involved in many cases of seafood poisoning in many parts of the world due to their occurrence in different marine species of human consumption such as fish, gastropods, and bivalves. Currently, this neurotoxin group is not monitored in many parts of the world including in the Indian Ocean area, even with reported outbreaks of seafood poisoning involving puffer fish, which is one of the principal TTX vectors know since Egyptian times. Thus, the main objective of this review was to assess the incidence of TTXs in seafood and associated seafood poisonings in the Indian Ocean and the Red Sea. Most reported data in this geographical area are associated with seafood poisoning caused by different species of puffer fish through the recognition of TTX poisoning symptoms and not by TTX detection techniques. This scenario shows the need of data regarding TTX prevalence, geographical distribution, and its vectors in this area to better assess human health risk and build effective monitoring programs to protect the health of consumers in Indian Ocean area.

Studies of the mechanism of fatty liver formation in Takifugu fasciatus following copper exposure.

Fatty liver is widely observed during Takifugu fasciatus production, but the mechanisms underlying fatty liver formation remain unknown. The present study was conducted to determine the potential effects of copper (Cu) on hepatic lipid deposition and metabolism in T. fasciatus after 21 days of exposure to Cu (levels: 0, 20 and 100 µg/L). Copper exposure decreased the weight gain rate (WG) in T. fasciatus, but increased the values of the viscerosomatic index (VSI) and hepatosomatic index (HSI) compared with the control. The time-dependent Cu accumulation in tissues increased as the Cu concentration increased. The order of Cu accumulation was liver > intestine > muscle. The lipid content, triglyceride (TG) content and lipoprotein lipase (LPL) activity increased after Cu exposure compared with the control. In addition, more lipid droplets and greater vacuolization were observed in the liver after exposure to 20 µg/L Cu than after 100 µg/L Cu. The expression of genes involved in lipogenesis (g6pd, 6pgd, lpl, fas and acc), lipolysis (hsl and cpt 1) and transcription (ppar α and ppar ©) was dependent on Cu. An analysis of the intestinal microbiome community showed that the highest values of the Chao 1 index, ACE, Shannon index and Simpson index were obtained in fish exposed to 20 µg/L Cu, whereas the lowest values were obtained after the 100 µg/L Cu treatment. The Principal Coordinates Analysis (PCoA) plots of the data revealed structural differences in the groups treated with Cu compared with the control group. At the phylum level, the intestinal microbiota in the Cu-treated and control fish were dominated by Proteobacteria and Bacteroidetes. The higher Firmicutes to Bacteroidetes ratio was observed in fish treated with 20 µg/L Cu compared with other groups, while the lowest ratio was observed in fish exposed to 100 µg/L Cu. Our study revealed the mechanisms by which Cu exposure altered (i) lipid deposition in the body and (ii) the intestinal microbiome, which may contribute to maintain the health status of T. fasciatus for the aquaculture.

Demographics of Lagocephalus inermis in the Arabian Sea unveils complex conservation challenges.

The first assessment of the demographics of Lagocephalus inermis, a species associated with pufferfish bites and fishing down the food web in the Arabian Sea, south-west coast of India, was performed based on length structured population dynamics of 1601 individuals caught in commercial fisheries. Analysis revealed that the current level of exploitation is 90% of the predicted exploitation producing maximum relative yield per recruit (Emax ), suggesting high levels of exploitation with potential for significant negative consequences for trophic cascades.