Pufferfish Species Research Articles

Chelonodontops bengalensis Habib et al. 2018, a junior synonym of Tetrodon patoca Hamilton 1822, with a type species designation for Chelonodon Müller 1841

A comparison of the recent description of Chelonodontops bengalensis Habib, Neogi, Og, Lee & Kim 2018 with that of Tetrodon patoca Hamilton 1822 reveals that the former is a junior synonym of the latter. Chelonodon patoca appears to be restricted in its distribution to coastal brackish waters at the mouth of the Ganges/Brahmaputra/Meghna system in India and Bangladesh and further south to at least the mouth of the Mahanadi. The valid name for the widespread Indo-Pacific species previously confused with C. patoca is C. kappa Hamilton 1822. We show that Bleeker (1850), not Rüppell (1852), was the first author to include pufferfish species in the genus Chelonodon Müller 1841, and as first revisers we choose Tetrodon kappa Hamilton 1822 as the type species of Chelonodon.

Epidermal distribution of tetrodotoxin-rich cells in newly hatched larvae of Takifugu spp.

Pufferfish of the genus Takifugu possess tetrodotoxin (TTX), known as "pufferfish toxin" and it is believed that pufferfish eggs and newly hatched larvae utilize TTX as a defensive substance against predators. However, the mechanism for the placement of TTX to specific cells on the larval body surface during the developmental process remains unknown. In this study, we clarify the distribution and characteristics of TTX-rich cells. We performed whole-mount immunohistochemistry (IHC) using anti-TTX monoclonal antibody on larvae of two pufferfish species, Takifugu rubripes and Takifugu alboplumbeus, just after hatching. This allowed observation of the TTX location and compared it with those of wheat germ agglutinin (WGA)-positive (periodic acid-Schiff (PAS)-positive) cells for mucous cells and IHC using anti-Na+/K+-ATPase (NKA) monoclonal antibody for ionocytes. As a result, uniformly scattered localization of TTX-rich cells was commonly observed in the epidermis of the larvae of the two Takifugu species. TTX-rich cells were WGA-negative (PAS-negative) and structurally distinct from NKA-positive cells, suggesting that TTX-rich cells are unreported small cells unique to pufferfish skin, but not mucous cells nor ionocytes.

Length-Weight Relationship of Diamondback puffer Lagocephalus guentheri from the northeastern Mediterranean Sea

The length-weight relationship data is important basic information that helps to compare species between populations and habitats. In this study, we provide detailed information on the length-weight relationships and condition factors of Lagocephalus guentheri from the Iskenderun Bay, northeastern Mediterranean Sea. A total of 408 (262 Female, 146 Male) specimens were collected on commercial trawlers by local fishermen caught between December 2022 to December 2023 from Iskenderun Bay. The total length range was 11.0-36.9 cm and 11.2-36.7 cm for females and males, respectively. The total weight values of females and males ranged from 20.30-1080.50 g and 20.50-1012.36 g, respectively. The length-weight relationship of L. guentheri was calculated as W = 0.0098 × L3.2145 (R2 = 0.9960) for females, W = 0.009 × L3.2399 (R2 = 0.9963) for males, and W = 0.0095 × L3.2243 (R2 = 0.9962) both sexes. The Fulton’s condition factor was observed as 1.2384 for females, 1.2504 for males, and 1.2335 for both sexes. This study is the most recent reference on length-weight relationships and condition factors for L. guentheri after correction of species identification. It is very important to determine the population parameters of pufferfish species to shed light on the studies to be carried out to restore the ecological balance in the Mediterranean ecosystem. This study will provide basic information that will be useful for fisheries scientists and managers in the Mediterranean Sea.

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.

CFD analysis of the complex japanese pufferfish nest

Existing research on the pufferfish species (Torquigener Albomaculosus) has shown that its nest structure has a certain impact on flow velocity. This study aims to investigate the nest-building behavior by using different materials. A 3D model will be created based on nest geometry, and 2D simulations will be conducted by using computational fluid dynamics (CFD). Furthermore, the study examines the nest’s fluid dynamics characteristics in its original state, with varied materials and heights. Results demonstrate that the nest reduces flow velocity, adapts to external flow conditions, and enhances shear strength with rough materials. Additionally, nest height affects internal circulation area and pressure. This unique structure protects the spawning area, improves oxygen transport, and strengthens the nest. The study further expands biomimetic engineering applications and suggests future interdisciplinary research directions.

CRISPR/Cas12a integrated electrochemiluminescence biosensor for pufferfish authenticity detection based on NiCo2O4 NCs@Au as a coreaction accelerator

Meat adulteration has brought economic losses, health risks, and religious concerns, making it a pressing global issue. Herein, combining the high amplification efficiency of polymerase chain reaction (PCR) and the accurate recognition of CRISPR/Cas12, a sensitive and reliable electrochemiluminescence (ECL) biosensor was developed for the detection of pufferfish authenticity using NiCo2O4 NCs@Au-ABEI as nanoemitters. In the presence of target DNA, the trans-cleavage activity of CRISPR/Cas12a is activated upon specific recognition by crRNA, and then it cleaves dopamine-modified single stranded DNA (ssDNA-DA), triggering the ECL signal from the “off” to “on” state. However, without target DNA, the trans-cleavage activity of CRISPR/Cas12a is silenced. By rationally designing corresponding primers and crRNA, the biosensor was applied to specific identification of four species of pufferfish. Furthermore, as low as 0.1 % (w/w) adulterate pufferfish in mixture samples could be detected. Overall, this work provides a simple, low-cost and sensitive approach to trace pufferfish adulteration.

DNA barcoding, identification, and validation of the pufferfish (Order: Tetraodontiformes) in China coastal waters.

The order Tetraodontiformes are one of the most unique groups of teleostean fish, exhibiting highly derived and greatly diversified phenotypes. It is a difficult task for both professionals and nonprofessionals to accurately identify these species only according to morphological characteristics. DNA barcoding can identify species at the molecular level to overcome the limitations of morphological classification. In this study, we collected 616 specimens of pufferfish from the coastal waters of China. According to the morphological characteristics, they were preliminarily identified as 50 species. Further analysis using DNA barcodes identified these specimens as 46 species, belonging to 23 genera, 6 families. According to the species classification results of DNA barcoding, the three species identified by morphology as Takifugu pseudommus, Takifugu chinensis, and Takifugu rubripes should be the same species. Similarly, Lagocephalus wheeleri is the synonym of Lagocephalus spadiceus. Another important discovery of DNA barcoding analysis is that there are closer interspecific genetic distances within the genus Takifugu. If T. rubripes, T. pseudommus, and T. chinensis are taken as one species, the average interspecific to intraspecific genetic distance ratio of Takifugu is only 6.21 times, which does not reach the DNA barcoding threshold of more than 10 times proposed previously. Although the interspecific genetic distance in the genus Takifugu is relatively small, each species can be clustered into independent clades in the NJ tree. In conclusion, this study not only found that there are synonymous phenomena in the order Tetraodontiformes but also provided molecular evidence for the valid species names of Takifugu rubripes and Lagocephalus Spadiceus. The results can provide reliable DNA barcoding information for the identification of pufferfish species, help solve the problem of classification confusion in this order, and provide technical support for the identification of the original components of related commodities on the aquatic product market.

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.Graphical

An in-depth study of the biology, trophic ecology and catchability of the invasive pufferfish Lagocephalus sceleratus from southern Turkey, eastern Mediterranean Sea

The silver-cheeked toadfish (Lagocephalus sceleratus) is an invasive species of highest concern. Its population must be controlled to mitigate its negative impacts on marine ecosystems, fishers, fisheries and human health. This study thoroughly investigates the biology, diet and catchability of the L. sceleratus stock from Finike, Turkey from March 2017 to February 2018 in order to better manage its invasion. A total of 751 specimens were sampled for this study with a M/F ratio of 1.25/1. The species becomes sexually mature at three and a half years of age, and Lm50 was 41.39 cm for males and 42.08 cm for females. Its spawning season in this region was from June to August, peaking in July. Its diet was mostly crustaceans in spring, fish in summer and both fish and crustaceans in winter. This species consumed a large amount of other pufferfish species, resulting in over a quarter of its fish diet. The trophic level of L. sceleratus was 4.41, demonstrating that it is indeed a top predator carnivore in the eastern Mediterranean Sea. Ingested fishing gear parts such as net pieces and hooks were found in about 10% of the fish. A slight modification of longlines using steel branch lines and a swivel hook resulted in double the catch per unit effort than standard longlines, so this technique can be used to target and control more of this invasive species, which is a national priority.

Development and validation of an LC–MS/MS method for determination of tetrodotoxin and its analogues in various pufferfish tissues and its application in Korean pufferfish

A robust method was developed and validated for analyzing tetrodotoxin and its analogues (TTXs) in high- and low-fat tissues of pufferfish using LC–MS/MS. The tissue distribution of TTXs in four Takifugu pufferfish species and their phenotypic variants was determined using the validated method. A significant matrix effect was shown, and the matrix-matched TTX calibration curves (r2 > 0.999) were used for the analysis of TTX. The method detection limit for TTX ranged from 0.006 to 0.009 μg/g. Recoveries from TTX-fortified tissue samples (0.1–2.0 µg/g) were in the range of 96.1–107.3 % with RSDs less than 7.8 %. TTX levels in the pufferfish tissues reached a maximum of 1.32 × 104 µg/g. The developed method was used for the relative quantification of TTX analogues. The major TTX analogues were deoxyTTXs (i.e., trideoxy-, dideoxy-, and deoxyTTX). The ovary and liver of pufferfish showed higher concentrations of TTXs compared to other tissues. The developed method was successfully validated and applied to Korean pufferfish samples.

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.

Development and validation of novel SNP markers for the rapid identification of natural hybrids of the 11 closely related pufferfish species (Takifugu spp.) distributed in Japan

Accurate identification of interspecific hybrids is vital for breeding programs in hybridizing taxa, as well as for the use of hybrids in aquaculture and fisheries. The use of species-specific single nucleotide polymorphisms (SNPs) has become the standard for the molecular identification of hybrids. However, it remains challenging to use these SNPs to identify hybrids involving multiple candidate parental species. Here, we developed and validated SNP markers for the identification of hybrids of the 11 closely related pufferfish species (genus Takifugu) distributed in Japan; these species have different aquacultural characteristics, such as growth rate, physiology, behavior, and toxicity. In total, 1232 species-specific SNPs were identified through double-digest restriction-site-associated DNA sequencing. The species had 11 (T. flavipterus) to 295 (T. chrysops) species-specific SNPs. We validated 33 genome-wide species-specific SNPs using TaqMan® genotyping assays for the 11 species and their hybrids, which were identified using amplified fragment length polymorphism analyses. No false negative or positive assignments were observed in 77 and 30 specimens of pure species and F1 hybrids, respectively. This marker combination allows rapid and reliable identification of natural hybrids, which will be useful for pufferfish aquaculture and will enhance our understanding of the natural hybridization between various Takifugu species.

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.

First Toxicological Analysis of the Pufferfish Sphoeroides pachygaster Collected in Italian Waters (Strait of Sicily): Role of Citizens Science in Monitoring Toxic Marine Species.

Pufferfish (Tetraodontidae) inhabiting the Mediterranean Sea may represent an emerging public health risk due to the possible accumulation of marine neurotoxins such as tetrodotoxin (TTXs) and saxitoxin (STXs) in their tissues. In this study, the presence of pufferfish species in the Strait of Sicily (Lampedusa Island, Italy) was investigated using a citizen science (CS) approach, involving local fishermen. Samples (liver, intestine, gonads, muscle, skin) from 20 specimens were sent to the National Reference Laboratory on Marine Biotoxins for TTXs detection using a validated HILIC-MS/MS method on fish tissue. The presence of STXs was also screened in part of the specimens. Overall, 56 specimens identified as Sphoeroides pachygaster (Müller &Troschel, 1848) were collected. Data on their total length, body weight, fishing method and catch area (with relative depth temperature and salinity) were analyzed and compared with the S. pachygaster records reported in literature which were updated to 2022. All the analysed tissues were found to be negative for both TTXs and STXs. CS played an essential role in monitoring potentially toxic marine species in this investigation. Outcomes from this study, which is the first investigating S. pachygaster toxicity in Italian waters, may provide useful data for the proper assessment of this emerging risk.

Real-time qPCR for the detection of puffer fish components from Lagocephalus in food: L. inermis, L. lagocephalus, L. gloveri, L. lunaris, and L. spadiceus.

Puffer fish is a type of precious high-end aquatic product, is widely popular in Asia, especially in China and Japan, even though it naturally harbors a neurotoxin known as tetrodotoxin (TTX) that is poisonous to humans and causes food poisoning. With the increasing trade demand, which frequently exceeds existing supply capacities, fostering fraudulent practices, such as adulteration of processed products with non-certified farmed wild puffer fish species. To determine the authenticity of puffer fish processed food, we developed a real-time qPCR method to detect five common puffer fish species in aquatic products: Lagocephalus inermis, Lagocephalus lagocephalus, Lagocephalus gloveri, Lagocephalus lunaris, and Lagocephalus spadiceus. The specificity, cross-reactivity, detection limit, efficiency, and robustness of the primers and probes created for five species of puffer fish using TaqMan technology have been determined. No cross-reactivity was detected in the DNA of non-target sample materials, and no false-positive signal was detected; the aquatic products containing 0.1% of a small amount of wild puffer fish materials without certification can be reliably tracked; the statistical p-value for each method's Ct value was greater than 0.05. The developed qPCR method was sensitive, highly specific, robust, and reproducibility, which could be used to validate the authenticity of wild puffer fish in aquatic products sold for commercial purposes.

Parasites of pufferfish, Lagocephalus spp. and Torquigener flavimaculosus of the Israeli Mediterranean: A new case of Lessepsian endoparasites.

With the opening of the Suez Canal as a link between the Red Sea and the Mediterranean Sea in 1869, the biogeographical event of the Lessepsian migration has been starting. Aided by beneficial conditions in the new habitat, almost 500 marine species have immigrated and often established themselves in the Mediterranean Sea, including several pufferfish species, with all of them extending their range and becoming important components of the local fauna. The parasitic fauna of these pufferfish has scarcely been examined in the Mediterranean Sea or in their native range, which provides the opportunity to study host-parasite interaction in a new habitat. The present study describes the parasitic fauna in four alien invasive pufferfish species (Lagocephalus guentheri, L. sceleratus, L. suezensis, and Torquigener flavimaculosus) of various sizes and ages on the Israeli Mediterranean coast. The parasite fauna of these species was diverse (Maculifer dayawanensis Digenea; Calliterarhynchus gracilis, Nybelinia africana and Tetraphyllidea larvae Cestoda; Hysterothylacium reliquens, Hysterothylacium sp. and Raphidascaris sp. Nematoda; Trachellobdella lubrica Hirudinea and Caligus fugu and Taeniacanthus lagocephali Copepoda) and consisted of mostly generalist species, most likely acquired in the new habitat, and specialist copepod ectoparasites, having co-invaded with the pufferfish. Additionally, the oioxenic opecoelid digenean Maculifer dayawanensis was found in two pufferfish species. The genus was previously only known from the Indo-Pacific Ocean, representing the eighth reported case of a Lessepsian endoparasite so far. Our results suggest a change in parasite fauna to native Mediterranean species in the pufferfish like previously reported in other Lessepsian migrant predatory fish species and a wider spread of co-invasion of fish endoparasites to the Mediterranean Sea than previously assumed. The study also provides several new host records and the first report for parasites in T. flavimaculosus.

CRISPR-Based Colorimetric Nucleic Acid Tests for Visual Readout of DNA Barcode for Food Authenticity.

Food authenticity is a critical issue associated with the economy, religion, and food safety. Herein, we report a label-free and colorimetric nucleic acid assay for detecting DNA barcodes, enabling the determination of food authenticity with the naked eye. This method, termed the CRISPR-based colorimetric DNA barcoding (Cricba) assay, utilizes CRISPR/Cas12a (CRISPR = clustered regularly interspaced short palindromic repeats; Cas = CRISPR associated protein) to specifically recognize the polymerase chain reaction (PCR) products for further trans-cleavaging the peroxidase-mimicking G-quadruplex DNAzyme. Based on this principle, the presence of the cytochrome oxidase subunit I gene could be directly observed with the naked eye via the color change of 3,3',5,5'-tetramethylbenzidine sulfate (TMB). The whole detection process, including PCR amplification and TMB colorimetric analysis, can be completed within 90 min. The proposed assay can detect pufferfish concentrations diluted to 0.1% (w/w) in a raw pufferfish mixture, making it one of the most sensitive methods for food authenticity. The robustness of the assay was verified by testing four common species of pufferfish, including Lagocephalus inermis, Lagocephalus spadiceus, Takifugu bimaculatus, and Takifugu alboplumbeus. The assay is advantageous in easy signal readout, high sensitivity, and general applicability and thus could be a competitive candidate for food authenticity.

LP-55 Phylogeny and Toxin Profile of Two Cambodian Freshwater Pufferfish Species of the Genus Pao

LP-55 Phylogeny and Toxin Profile of Two Cambodian Freshwater Pufferfish Species of the Genus Pao

Estimation of CPUE and CPUA of pufferfish (Tetraodontidae) caught by the Bottom Trawl Fishery in the eastern Mediterranean Coasts

Two indicators of pufferfish communities, catch per unit effort (CPUE) and catch per unit area (CPUA), were used with surveys of bottom trawl fishery to elucidate the level of the catch of each pufferfish species in the Mediterranean coasts of Turkey, comprising the Iskenderun Bay, Mersin Bay, and Antalya Bay. The surveys were conducted seasonally in 2019. The average rate of CPUE for Lagocephalus sceleratus, L. suezensis, L. spadiceus, and Torquigener flavimaculosus were 2.2, 0.8, 0.5, and 0.2 kg km-2, respectively that the highest CPUE values were in winter (8.0 kg km-2) for L. sceleratus and also lowest was in winter (0.08 kg km-2) for L. spadiceus. The highest and lowest CPUE values were at the depth of 20-50 m (2.7 kg km-2) for L. sceleratus and 50-80 m (0.08 kg km-2) for T. flavimaculosus, respectively. The highest CPUE values (3.5 kg km-2) were found at the sandy bottom for L. sceleratus, and the lowest one (0.01 kg km-2) was at the hardy-ground for T. flavimaculosus. The average annual CPUA of pufferfish species at the Iskenderun, Mersin, and Antalya bays was estimated as 15.6, 28.4, and 6.81 kg, respectively, with a mean value of 17.3 kg/day/boat. In multiple correspondence analysis, L. sceleratus showed a great contribution to CPUE and CPUA data. However, T. flavimaculosus revealed contribution, especially in catch numbers which were positively affected by precipitation. L. spadiceus, L. suezensis, and T. flavimaculosus were positively affected by depth, temperature, month, season, and bottom structure.

Tetrodotoxin and analogues in two local pufferfish species from Inhaca Island – South of Mozambique: First report in the Mozambican coast

Tetrodotoxins (TTXs) were investigated in two local pufferfish species, Diodon hystrix and Arothron hispidus, from Mozambican coast. TTX and analogues 4-epiTTX, 4,9-anhydroTTX, 11-deoxyTTX, and 11-norTTX-6-(R/S)-ol were found in both species and high level of TTX was found in A. hispidus (9522.0 μg TTX kg−1) than in D. hystrix (350.9 μg TTX kg−1). The distribution of TTX and their analogues in A. hispidus was intestine > liver > skin ≫ muscle > gonads. This is the first report of TTXs in Mozambican coast.