Toxicity Equivalency Factors for Tetrodotoxin Analogues Determined with Automated Patch Clamp on Voltage-Gated Sodium Channels in Neuro-2a Cells.

Tetrodotoxin (TTX) is a potent marine neurotoxin, responsible for numerous poisoning incidents and some human fatalities. To date, more than 30 TTX analogues have been identified, but their individual toxicities and roles in poisoning remain largely unknown. In this work, the toxicity equivalency factors (TEFs) of five TTX analogues were determined by assessing the blockade of voltage-gated sodium channels in Neuro-2a cells using automated patch clamp (APC). All TTX analogues were less toxic than TTX. The derived TEFs were applied to the individual TTX analogues concentrations measured in pufferfish samples, using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). A comparison of these results with those obtained from APC analysis demonstrated that TEFs can be effectively used to translate LC-MS/MS analytical data into meaningful toxicological information. This is the first study to utilize APC device for the toxicological assessment of TTX analogues, highlighting its potential as a bioanalytical tool for seafood safety management and human health protection.

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.

Fluid-Solid Interfacial Properties and Drag-Reducing Characterization of the Flexible Conical Microstructured Film Inspired by the Streamlined Body Surface of the Pufferfish.

Given the challenges in accurately replicating the surface of the pufferfish, this study employed three-dimensional (3D) printing to create a model based on inverse modeling. The morphology of the pufferfish exhibits a streamlined configuration, characterized by a gradual widening from the anterior oral region to the central ocular area, followed by a progressive narrowing from the midabdominal region toward the caudal extremity. The RNG k-ε turbulence simulation results demonstrate that the streamlined body surface of the pufferfish diminishes differential pressure resistance. This enhancement promotes laminar flow formation, delays fluid separation, minimizes turbulence-induced vortices, and reduces frictional resistance. Moreover, the pufferfish's supple and uneven outer epidermis was simplified into a flexible, nonsmooth planar film to conduct fluid-solid coupling simulations. These revealed that the pufferfish's unique skin can absorb turbulent energy and minimize momentum transfer between the fluid and the solid film, lowering the fluid resistance during swimming. In summary, The high-efficiency swimming capacity of pufferfish stems not only from their streamlined body surface but also significantly from the unique structural characteristics and mechanical properties of their flexible skin. This research provides critical theoretical underpinnings for the design of functional bionic surfaces aimed at drag reduction.

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.

Lunar Age-Dependent Oscillations in Expression of the Genes for Kisspeptin, GnIH, and Their Receptors in the Grass Puffer during the Spawning Season.

Grass puffer is a semilunar-synchronized spawner: spawning occurs on beaches only for several days of spring tide around new moon (lunar age 0) and full moon (lunar age 15) every 2 weeks from spring to early summer. To investigate the role of kisspeptin and gonadotropin-inhibitory hormone (GnIH) in the semilunar-synchronized spawning, lunar age-dependent expression of the genes encoding kisspeptin (kiss2), kisspeptin receptor (kissr2), GnIH (gnih), GnIH receptor (gnihr), gonadotropin-releasing hormone 1 (GnRH1) (gnrh1), and three gonadotropin (GTH) subunits (gpa, fshb, lhb) was examined in the male grass puffer, which was kept in an aquarium under natural light condition in a lunar month during the spawning period. In the brain, both kiss2 and kissr2 showed lunar variations with a peak at lunar age 10, while both gnih and gnihr showed semilunar variations with two peaks at lunar age 0 and 20. On the other hand, gnrh1 showed semilunar variation with two peaks at lunar age 0 and 15. In the pituitary, kiss2, kissr2, gnih, and gnihr showed similar variations to those shown in the brain. The fshb and lhb mRNA levels showed semilunar variations with two peaks at lunar age 0 and 15. The present study shows lunar and semilunar oscillations of kiss2/kissr2 and gnih/gnihr expressions, respectively, with their peaks around spring tide in the brain and pituitary along with the semilunar expressions of gnrh1 and the pituitary GTH subunit genes. These results suggest that the lunar age-dependent expressions of the kisspeptin, GnIH, and their receptor genes may be primarily important in the control of the precisely timed semilunar spawning of the grass puffer.

Japanese Planocerid Flatworms: Difference in Composition of Tetrodotoxin and Its Analogs and the Effects of Ingestion by Toxin-Bearing Fishes in the Ryukyu Islands, Japan.

Tetrodotoxin (TTX), known as pufferfish toxin, is a potent neurotoxin blocking sodium channels in muscle and nerve tissues. TTX has been detected in various taxa other than pufferfish, including marine polyclad flatworms, suggesting that pufferfish toxin accumulates in fish bodies via food webs. The composition of TTX and its analogs in the flatworm Planocera multitentaculata was identical to those in wild grass puffer Takifugu alboplumbeus. Previously, Planocera sp. from Okinawa Island, Japan, were reported to possess high level of TTX, but no information was available on TTX analogs in this species. Here we identified TTX and analogs in the planocerid flatworm using high-resolution liquid chromatography-mass spectrometry, and compared the composition of TTX and analogs with those of another toxic and non-toxic planocerid species. We show that the composition of TTX and several analogs, such as 5,6,11-trideoxyTTX, dideoxyTTXs, deoxyTTXs, and 11-norTTX-6(S)-ol, of Planocera sp. was identical to those of toxic species, but not to its non-toxic counterpart. The difference in the toxin composition was reflected in the phylogenetic relationship based on the mitochondrial genome sequence. A toxification experiment using predatory fish and egg plates of P. multitentaculata demonstrated that the composition of TTX and analogs in wild T. alboplumbeus juveniles was reproduced in artificially toxified pufferfish. Additionally, feeding on the flatworm egg plates enhanced the signal intensities of all TTX compounds in Chelonodon patoca and that of deoxyTTXs in Yongeichthys criniger.

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.

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.

Morphological and genetic analysis for the diversity conservation of rare species, Thamnaconus multilineatus (Tetraodontiformes: Monacanthidae).

Climate changes have altered biodiversity and ultimately induced community changes that have threatened the survival of certain aquatic organisms such as fish species. Obtaining biological and genetic information on endangered fish species is critical for ecological population management. Thamnaconus multilineatus, registered as an endangered species by the IUCN in 2019, is a Data Deficient (DD) species with a remarkably small number of habitats worldwide and no known information other than its habitat and external form. In this study, we characterized the external and osteological morphology of a T. multilineatus specimen collected from eastern Jeju Island, South Korea, in 2020. We also investigated the phylogenetic relationships among related fish species through complete mitochondrial DNA (mtDNA) analysis of the T. multilineatus specimen. The external and skeletal characteristics of T. multilineatus were similar to those of previous reports describing other fish of the genus Thamnaconus, making it difficult to classify T. multilineatus as a similar species based only on morphological characteristics. As a result of analyzing the complete mtDNA of T. multilineatus, the length of the mtDNA was determined to be 16,435 bp, and the mitochondrial genome was found to have 37 CDCs, including 13 PCGs, 22 tRNAs, and 2 rRNAs. In the phylogenetic analysis within the suborder Balistoidei, T. multilineatus mtDNA formed a cluster with fish of the genus Thamnaconus. This study is the first to report on the skeletal structure and complete mtDNA of T. multilineatus. Since the current research on T. multilineatus has only been reported on morphology, the results of this study will be utilized as important information for the management and restoration of T. multilineatus as an endangered species and significant fishery resource.

Sex determination by ultrasonography prior to artificial breeding of the pufferfish Arothron manilensis (Tetraodontiformes, Tetraodontidae).

Artificial breeding was induced in the pufferfish Arothron manilensis following ultrasonographic sex determination. Hormonal treatment of mature male and female specimens followed the collection (and measurement) of fully developed eggs by cannulation. Fertilized eggs (0.85 ± 0.02 mm diameter) were spherical, demersal and individually adhesive. Hatching occurred 5 days after fertilization, larvae being 2.23 ± 0.15 mm in total length and 2.08 ± 0.14 mm in notochord length. The larvae had all died within 14 days of hatching. To improve artificial breeding techniques for A. manilensis, it is necessary to determine more appropriate timing for hormone injection, as well as feeding nutrient-enhanced SS type Brachionus sp. to newly hatched larvae.

Effect of pH on the Structure, Functional Properties and Rheological Properties of Collagen from Greenfin Horse-Faced Filefish (Thamnaconus septentrionalis) Skin.

Collagen is an important biopolymer widely used in food, cosmetics and biomedical applications. Understanding the effect of pH on the structure and properties of collagen is beneficial for its further processing and exploitation. In this study, greenfin horse-faced filefish skin collagen (GHSC) was prepared and identified as a type I collagen. We systematically investigated the effect of pH on the structural, functional and rheological properties of GHSC. Scanning electron microscopy showed that the collagen morphology changed from an ordered stacked sheet structure to a rough silk-like structure as pH increased. Gaussian-fitted Fourier infrared spectroscopy results of the collagen revealed that it unfolded with increasing pH. Moreover, the ordered structure was reduced, and random coils became the dominant conformation. Its ß-sheet and random coil contents increased from 18.43 ± 0.08 and 33.62 ± 0.17 to 19.72 ± 0.02 and 39.53 ± 1.03%, respectively, with increasing pH. α-helices and ß-turns decreased from 35.00 ± 0.26 and 12.95 ± 0.01 to 29.39 ± 0.92 and 11.36 ± 0.10%, respectively. The increase in ß-sheets and random coils allowed the pI-treated collagen to exhibit maximum water contact angle. The emulsification and foaming properties decreased and then increased with increasing pH in a V-shape. The increased net surface charge and ß-sheets in collagen benefited its emulsification and foaming properties. The rheological results showed that the protoprotein exhibited shear-thinning properties in all pH ranges. The collagen solutions showed liquid-like behaviour in low-pH (2, 4) solutions and solid-like behaviour in high-pH (6, 7.83 and 10) solutions. Moreover, the frequency-dependent properties of the storage modulus (G') and loss modulus (G″) of the collagen solutions weakened with increasing pH. Collagen has considerable frequency-dependent properties of G' and G″ at low pH (2, 4). Thus, the importance of collagen raw material preparation for subsequent processing was emphasised, which may provide new insights into applying collagen-based materials in food, biomaterials and tissue engineering.

Multiple biomarkers in pufferfish as a proxy of environmental health in brazilian marine protected areas.

Marine Protected Areas (MPAs) are designed to conserve biodiversity and vulnerable ecosystems. This study aimed to assess the environmental quality of three Brazilian MPAs, based on the integrated analysis of biomarkers in pufferfish. The MPAs are differentiated by the degree of anthropogenic influences. The Barra do Una Estuary sustainable reserve (JUR) is a reference area due to its low levels of contamination and species diversity. The Cananéia Estuarine System (CAN) has been recognized as a biosphere reserve by UNESCO, as well as a Ramsar wetland. This MPA was influenced by upstream mining activities, resulting in the introduction of metals in the estuarine waters and the discharge of untreated urban sewage. The São Vicente estuary (SSV) lacks proper sanitation infrastructure. All collections were conducted during winter season, and, after collection, the animals were euthanized, their soft tissues were removed, and multiple biomarkers were analyzed in the gills and liver, as biometric, genotoxic, biochemical, and morphological. A one-factor multivariate analysis was applied to evaluate the differences between the data sets, and the matrices were analyzed using PERMANOVA to evaluate the "estuary" factor. The results were integrated using PCA with a 0.4 cut value and an Enhanced Integrated Biomarker Response (EIBR) was calculated. PCA was correlated with biochemical, genotoxic, and morphological biomarkers. In general, SVV differed from CAN and JUR as shown by both univariate and multivariate analyses. SVV also showed the highest EIBR, followed by CAN. Organisms from SSV showed greater gill pathology, elevated AChE activity and lipoperoxidation (LPO), and micronuclei frequency. CAN present intermediate EIBR, with severe pathologies in the liver. CAN seems to present an intermediate environmental quality between SSV and JUR indicating the importance of the existence of MPAs for environmental conservation and the need to monitor such areas, to maintain their suitable environmental quality.

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.

Genome analysis deciphered Chryseobacterium indicum is a distinct species associated with freshwater pufferfish.

A bacterium, strain PS-8T of the genus Chryseobacterium, was isolated from the skin of freshwater pufferfish (Tetraodon cutcutia). Strain PS-8T is a Gram-negative, aerobic, non-motile, and rod-shaped bacterium. Colonies appear in yellowish-orange colors. The major cellular fatty acids were C15:0 iso, C17:0 iso 3OH, C15:0 iso 3OH, and C11:0 anteiso. The predominant polar lipids were phosphatidylethanolamine and amino lipids. The genome size is 4.83 Mb. The G + C content was 35.6%. The in silico dDDH homology, ANI, and AAI were below the cutoff value, 70% and 95% to 96%, respectively, suggesting that strain PS-8T represents a defined species. The phylogenetic tree based on core and the non-recombinant genes showed the strain PS-8T clustered with Chryseobacterium gambrini DSM 18014T. Genome-wide analysis decodes several virulence factors of the genus Chryseobacterium, including genes for adherence, biofilm and stability, proliferation, resistance to immune response, and host-defense evasion system. The cladogram of the virulence genes showed a phylogenetic relationship among the Chryseobacterium species. Knowledge of the association of Chryseobacterium with freshwater pufferfish adds a new ecological niche to this bacterium.

Exploring foraging preference of local fish species towards non-indigenous fouling communities near marinas: Insights from Remote Video Foraging System (RVFS) trials.

Non-indigenous species (NIS) spread from marinas to natural environments is influenced by niche availability, habitat suitability, and local biotic resistance. This study explores the effect of indigenous fish feeding behaviour on NIS proliferation using fouling communities, pre-grown on settlement plates, as two distinct, representative models: one from NIS-rich marinas and the other from areas outside marinas with fewer NIS. These plates were mounted on a Remote Video Foraging System (RVFS) near three marinas on Madeira Island. After 24-h, NIS abundance was reduced by 3.5 %. Canthigaster capistrata's preference for marinas plates suggests potential biotic resistance. However, Sparisoma cretense showed equal biting frequencies for both plate types. The cryptogenic ascidian Trididemnum cereum was the preferred target for the fish. Our study introduces a global framework using RVFS for in-situ experiments, replicable across divers contexts (e.g., feeding behaviour, biotic resistance), which can be complemented by metabarcoding and isotopic analysis to confirm consumption patterns.

Differential blood cells associated with parasitism in the wild puffer fish Lagocephalus laevigatus (Tetraodontiformes) of the Campeche Coast, southern Mexico.

In aquaculture conditions, severe parasitic infections cause negative impacts on fish health and economic losses. The parasite load has been associated with anemia, which reduces the number of erythrocytes in fish. Therefore, the evaluation of hematological parameters as a feasible tool for diagnosing and monitoring fish health allows us to determine the indirect effect of parasites on the health status of fish. Our aim was to evaluate changes in the blood cell parameters of Lagocephalus laevigatus associated with parasitism. A total of 99 puffer fish were collected from the coast of Seybaplaya, Campeche. Each fish had 20 µl of peripheral blood drawn, and blood smears were performed in triplicate. The smears were stained with Giemsa stain, and a quantitative analysis of blood cells (erythrocytes, leukocytes, and monocytes) was obtained with an optical microscope at 100 ×. The parasites recovered from each fish were fixed and identified, and the infection parameters were calculated. Through generalized additive model analysis (GAMLSS), we observed that the infection intensity of puffer fish influenced changes in hematological parameters, principally in erythrocytes, neutrophils, thrombocytes, the total fish length, and the condition factor of the fish. In conclusion, this is the first study that provides baseline data on the hematological parameter variations in uninfected and infected L. laevigatus, the tropical wild puffer fish, as well as the possible effects on fish health. It is necessary to establish reference hematological patterns in wild populations for diagnosis and timely management with emphasis on aquaculture fish.

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.

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.

Interrelationships and biogeography of the New World pufferfish genus Sphoeroides (Tetraodontiformes: Tetraodontidae) inferred using ultra-conserved DNA elements.

Colonization of the New World by marine taxa has been hypothesized to have occurred through the Tethys Sea or by crossing the East Pacific Barrier. To better understand patterns and timing of diversification, geological events can be coupled with time calibrated phylogenetic hypotheses to infer major drivers of diversification. Phylogenetic relationships among members of Sphoeroides, a genus of four toothed pufferfishes (Tetraodontiformes: Tetraodontidae) which are found nearly exclusively in the New World (eastern Pacific and western Atlantic), were reconstructed using sequences from ultra-conserved DNA elements, nuclear markers with clear homology among many vertebrate taxa. Hypotheses derived from concatenated maximum-likelihood and species tree summary methods support a paraphyletic Sphoeroides, with Colomesus deeply nested within the genus. Analyses also revealed S. pachygaster, a pelagic species with a cosmopolitan distribution, as the sister taxon to the remainder of Sphoeroides and recovered distinct lineages within S. pachygaster, indicating that this cosmopolitan species may represent a species complex. Ancestral range reconstruction may suggest the genus colonized the New World through the eastern Pacific before diversifying in the western Atlantic, though date estimates for these events are uncertain due to the lack of reliable fossil record for the genus.

Histological and ultrastructural characterization of the dorso-ventral skin of the juvenile and the adult starry puffer fish (Arothron stellatus, Anonymous 1798).

BACKGROUND: The starry puffer fish (Arothron stellatus, Anonymous, 1798) is a poisonous tetradontidae fish inhabiting the Red sea. The skin constitutes an important defense against any external effects. The study aims to characterize the dorso-ventral skin of the juvenile and the adult starry puffer fish using light and scanning electron microscopies. Twenty specimens of juvenile and adult fresh fishes were used. RESULTS: The scanning electron microarchitecture of the skin of the juvenile and adult fish showed delicate irregular-shaped protrusions, and well-defined bricks-like elevations on the dorsal side and interrupted folds as well as irregular-shaped protrusions on the ventral side. In adult fish, the patterned microridges of the superficial and deep epithelial cells (keratinocytes) were larger and well-defined in the dorsal skin than in the ventral side, the contrary was seen in the juvenile fish. The microridges were arranged in a fingerprint or honeycomb patterns. The openings of the mucous cells were more numerous in the dorsal skin in both age stages but more noticeable in adult. Furthermore, the sensory cells were more dominant in the juveniles than the adults. The odontic spines were only seen in adult. Histologically, few taste buds were observed in the epidermis of the dorsal skin surface of the adult fish. Both mucous and club cells were embedded in the epidermis of the juvenile and adult fish with different shapes and sizes. Melanophores were observed at the dorsal skin of both juvenile and adult fishes while fewer numbers were noticed at the ventral surfaces. Several dermal bony plates with different shapes and sizes were demonstrated in the skin of both adult and juvenile fishes. CONCLUSION: The structural variations of skin of the juvenile and adult fishes may reflect the various environmental difficulties that they confront.