Monthly Variation of Tetrodotoxin Levels in Pufferfish (Lagocephalus sceleratus) Caught from Antalya Bay, Mediterranean Sea.

The silver-cheeked toadfish (Lagocephalus sceleratus), an invasive alien pufferfish species that has rapidly settled throughout the Mediterranean region, poses significant threats not only to native marine species and fisheries but also to public health due to the tetrodotoxin (TTX) they harbor. In this study, TTX concentrations in L. sceleratus from Antalya Bay in the Northeastern Mediterranean Sea were investigated using Q-TOF-LC-MS on a monthly basis over a one-year period. Pufferfish were caught by angling from May 2018 to April 2019. The TTX levels in three different tissues (gonads, liver, and muscle) of 110 pufferfish in total were determined in both male and female individuals caught for 11 months. The highest TTX mean levels generally occurred in the gonads and the lowest in the muscle samples. As regards the maximum TTX contents, the highest concentrations determined were 68.2, 34.2, and 7.8 µg/g in the gonad, liver, and muscle tissues, respectively. The highest levels were generally observed in late autumn to winter (especially in November and December) in all tissues from both genders. Female individuals were generally found to be more toxic than male individuals. The TTX levels found confirm that the consumption of L. sceleratus from Antalya Bay remains dangerous throughout the year, and thus L. sceleratus constantly constitutes an important risk source for public health.

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.

Evaluation of Toxicity Equivalency Factors of Tetrodotoxin Analogues with a Neuro-2a Cell-Based Assay and Application to Puffer Fish from Greece.

Tetrodotoxin (TTX) is a potent marine neurotoxin involved in poisoning cases, especially through the consumption of puffer fish. Knowledge of the toxicity equivalency factors (TEFs) of TTX analogues is crucial in monitoring programs to estimate the toxicity of samples analyzed with instrumental analysis methods. In this work, TTX analogues were isolated from the liver of a Lagocephalus sceleratus individual caught on South Crete coasts. A cell-based assay (CBA) for TTXs was optimized and applied to the establishment of the TEFs of 5,11-dideoxyTTX, 11-norTTX-6(S)-ol, 11-deoxyTTX and 5,6,11-trideoxyTTX. Results showed that all TTX analogues were less toxic than the parent TTX, their TEFs being in the range of 0.75-0.011. Then, different tissues of three Lagocephalus sceleratus individuals were analyzed with CBA and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The obtained TEFs were applied to the TTX analogues' concentrations obtained by LC-MS/MS analysis, providing an indication of the overall toxicity of the sample. Information about the TEFs of TTX analogues is valuable for food safety control, allowing the estimation of the risk of fish products to consumers.

Evaluation of tetrodotoxins in puffer fish caught along the Mediterranean coast of Spain. Toxin profile of Lagocephalus sceleratus.

Although consumption of Tetraodontidae species is prohibited in the EU, intoxications are still reported. The evaluation of tetrodotoxins (TTXs) by mass spectrometry (LC-MS/MS and LC-HRMS) and a screening immunoassay (mELISA) in tetraodontid fishes caught along the Western Mediterranean Sea revealed high concentrations of TTXs in Lagocephalus sceleratus while no TTXs were identified in L. lagocephalus and Sphoeroides pachygaster individuals. The high TTXs content found in the L. sceleratus analysed herein demonstrate the occurrence of highly toxic puffer fish in the Western Mediterranean Sea. Being L. sceleratus a recent invasive species in the Mediterranean, surveillance, risk assessment and risk management measures are necessary. The strategy used within this research work could be a valuable tool for future food safety monitoring.

Keeping Lagocephalus sceleratus off the Table: Sources of Variation in the Quantity of TTX, TTX Analogues, and Risk of Tetrodotoxication.

The invasion of the tetrodotoxin (TTX)-bearing silver-cheeked toadfish and potential poisoning due to its consumption (tetrodotoxication) threatens public safety in the Mediterranean Sea. In this study, TTX and TTX analogues of Lagocephalus sceleratus (Gmelin, 1789) were measured using liquid chromatography tandem mass spectrometry (LC-MS/MS) in fish collected off the island of Crete (Southern Mediterranean). We tested the synergistic effect of a suite of factors potentially affecting toxins' levels and tetrodotoxication risk using general and generalized linear models, respectively. The type of tissue, geographic origin (Cretan Sea, Libyan Sea), sex, and fish maturity stage were significant predictors of toxin concentrations. Mean TTX was higher in gonads and lower in muscles, higher in the Libyan Sea and in female fish, and lower in juvenile (virgin) fish. The concentration of TTX was also significantly and positively correlated with the concentration of several TTX analogues (4-epiTTX, 4,9-anhydroTTX, 11-deoxyTTX, 5,11/6,11-dideoxyTTX, 5,6,11-trideoxyTTX, 11-norTTX-6-ol). The analysis showed that fish originating from the Libyan Sea had significantly higher probability to cause tetrodotoxication in case of consumption. The variability explained by the models developed in this study was relatively low, indicating that toxin levels are hard to predict and the consumption of L. sceleratus should therefore be avoided.

Determination of tetrodotoxin (TTX) levels in various tissues of the silver cheeked puffer fish (Lagocephalus sceleratus (Gmelin, 1789)) in Northern Cyprus Sea (Eastern Mediterranean).

Lagocephalus sceleratus from the family Tetraodontidae, which is originated from the Red Sea, started to migrate to Mediterranean waters after the opening of the Suez Channel in 1869. Since they do not have any predators in the Mediterranean Sea, their population increased in number very fast. Different tissues of L. sceleratus have tetrodotoxin (TTX). TTX is a non-protein, heat resistant molecule which binds to voltage-gated sodium channels of musculatory and nervous system. Ingestion of the fish can cause a wide variety of effects from mild toxications to death. This study is the first toxicity report of L. sceleratus for Cyprus. Due to the lack of data on toxicity levels of L. sceleratus, and some cases of poising after the ingestion of the fish were the reasons for the start of this work. 24 L. sceleratus were sampled from 3 different areas to represent Northern Cyprus, during one year period. 16 fish samples selected and grouped into 4 fishes per season for toxicity assay. TTX levels from liver, gonad, intestine, muscle and skin tissue homogenate were analyzed with TTX ELISA. Totally 80 tissues were analyzed, 40% of them were above the toxic limit (2.2 µg/g). Most toxic tissue, according to the seasonal average, was found in the summer season and was determined as; Liver = 13.48 µg/g. Most toxic three fishes were found to be female with a length of 45-60 cm. TTX distribution among tissues have similarities with previously published studies from different regions.

Detection of tetrodotoxins in juvenile pufferfish Lagocephalus sceleratus (Gmelin, 1789) from the North Aegean Sea (Greece) by an electrochemical magnetic bead-based immunosensing tool.

Two small Lagocephalus sceleratus juveniles were captured in picarel targeting catches from North Aegean Sea (Greece) in the autumn of 2017. An electrochemical immunosensing tool using magnetic beads as immobilisation support was developed and applied to the rapid screening of tetrodotoxins (TTXs), potent neurotoxins that constitute a food safety hazard when present in seafood. This tool revealed the presence of TTXs in both individuals. Results were compared with those provided by mELISA and LC-HRMS, the latter confirming the presence of TTX. Some of the tissues contained TTX contents close to or above 2 mg/kg. L. sceleratus juveniles had been considered as non-toxic and, to our knowledge, this is the first report of high TTX levels in small L. sceleratus individuals. Such specimens can be mistaken with other edible species, posing a threat to consumers. The availability of low-cost and user-friendly tools for TTXs detection will contribute to guarantee seafood safety.

The complete mitochondrial genome of Lagocephalus sceleratus (Tetraodontiformes; Tetraodontidae) and phylogenetic studies of Tetraodontidae.

The complete mitochondrial genome sequence of Lagocephalus sceleratus was determined. The complete mitochondrial genome was 16,444 bp in length and contained 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and two non-coding region (the control region and the origin of light strand replication). The overall base composition was A 27.59%, C 31.43%, G 17.12%, T 23.85%. All protein-coding genes started with an ATG initiation codon, except COI used GTG. With the exception of ND6 and eight tRNA genes, all other genes were encoded on the heavy strand. Additionally, the phylogenetic relationship of 37 Tetraodontidae species based on the complete genome was analyzed, and the result showed that L. sceleratus was clustered with other Lagocephalus species. These results would be useful for the investigation of phylogenetic relationship, taxonomic classification and phylogeography of the Tetraodontidae.

Tetrodotoxin levels in pufferfish (Lagocephalus sceleratus) caught in the Northeastern Mediterranean Sea.

The toxicity of tetrodotoxin (TTX) in pufferfish (Lagocephalus sceleratus) from Mersin Bay in the Northeastern Mediterranean Sea was measured using a mouse bioassay (MBA) and LC-MS-MS. Pufferfish were caught by trawl fishing, longlining and fishing line from December 2012 to October 2013. Changes in the levels of TTX in the gonads, livers, intestines, skins and muscles as a function of season and sex were determined. The gonads of female fish were toxic in all seasons (>2µg/g), whereas the gonads of male fish were only toxic in the spring and autumn. The highest TTX levels in gonads, livers, intestines and skins of female fish were 52.1, 46.2, 7.64 and 3.43µg/g, respectively, using LC-MS-MS. The TTX level in the muscle of female fish in winter was 2.83µg/g but was otherwise below the toxic limit. Consequently, it can be dangerous to consume pufferfish, including the edible muscle, from the Eastern Mediterranean Sea.

Morphological Studies on the Gills of Puffer Fish (Lagocephalus sceleratus, Gmelin, 1789) / Estudios Morfológicos en Branquias del Pez Globo (Lagocephalus sceleratus, Gmelin, 1789)

In the present study, we focused on the morphology of L. sceleratus gills using gross anatomy, scanning electron microscopy as well as light microscopy. Results of this study revealed that the gill openings appeared as simple slits anterior to the pectoral fin without distinct opercular cover. The gill system consisted of three pairs of gill arches carrying two rows of gill rakers on its concave border and gill filaments on its convex border. SEM showed that all surfaces of the gill arch were characterized by the presence of the longitudinal ridges with many taste buds in addition to many spines around the rakers. Histologically, the gill arch was composed of curved bar of hyaline cartilage with slightly elevated area corresponding to the sites of gill rakers. Each filament was formed of a thin central cartilaginous core surrounded by peripheral cartilaginous matrix and covered by primary epithelial layer with abundant mucous cells. The chloride cells appeared mainly near to the base of secondary lamellae. Each gill filament gave rise to a very large number of secondary lamellae on both sides. The epithelial lining of the secondary lamellae comprised epithelial pavement cells, few mucous cells and pillar cells. The latter interposed the enriched blood capillaries. These findings suggest that L. sceleratus gills have characteristic morphological features that are related to adaptive functions for feeding habits, osmoregulation and respiratory mechanism with in their living environment.

El objetivo fue estudiar la morfología de las branquias de Sceleratus L. desde la anatomía macroscópica, microscopía electrónica de barrido, así como la microscopía de luz. Los resultados revelaron que las aberturas branquiales aparecían como simples rendijas por delante de la aleta pectoral sin una cubierta opercular distinta. El sistema branquial consistió en tres pares de arcos branquiales con dos filas de branquiespinas en sus filamentos branquiales frontales y cóncavos en el margen. La microscopía de barrido mostró que todas las superficies del arco branquial se caracterizaron por la presencia de crestas longitudinales con muchas papilas gustativas, además de una cantidad importante de espinas alrededor de los rastrillos. Histológicamente, el arco branquial se compone de una barra curva de cartílago hialino con una zona ligeramente elevada, correspondiente a los sitios de branquiespinas. Cada filamento se formó por un delgado núcleo central cartilaginoso rodeado de matriz cartilaginosa periférica y cubierto por una capa epitelial primaria con abundantes células mucosas. Las células de cloruro aparecieron principalmente cerca a la base de laminillas secundaria. Cada filamento de las branquias en ambos lados dio origen a un gran número de laminillas secundarias. El revestimiento epitelial de laminillas secundarias estaba formado de células epiteliales, algunas células mucosas y células sostenedoras. Estos hallazgos sugieren que las branquias de L. sceleratus tienen características morfológicas que están relacionadas con las funciones de adaptación de los hábitos de alimentación, la osmorregulación y el mecanismo respiratorio de acuerdo a su entorno.