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.

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.

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.

Development of a Proton-Enhanced ESI UPLC-MS/MS Method for the Determination of Tetrodotoxin.

Tetrodotoxin (TTX) is a kind of low-molecular-weight non-protein neurotoxin. It is one of the most potent neurotoxins found in nature, and it is found in puffer fish and various marine biota. The low sensitivity of previous analytical methods limited their application in puffer fish organ samples. This study established a method for the accurate and fast determination of TTX by reversed ultra-performance liquid chromatography coupled with proton-enhanced electron spray ionization-tandem mass spectrometry. The method yields good peak shapes, high sensitivity and low coeluted interferences. The method was successfully applied to determine TTX in puffer fish tissue samples of about 0.2 g.

[Tetrodotoxin Intoxications in Nagasaki, Japan: Symptoms of Patients, Tetrodotoxin Levels in Leftover Food and Clinical Urine and Serum Samples].

Incidents of food poisoning associated with tetrodotoxin (TTX) contamination occur every year throughout Japan. Here, we determined TTX levels in leftover foods and serum and urine samples from eight food poisoning incidents associated with TTX contamination in Nagasaki, Japan, from 2011 to 2017.Seven food samples associated with four of these food poisoning incidents were classified as weakly toxic (four samples), moderate-to-strongly toxic (two samples), and strongly toxic (one sample).In comparison with previous reports, TTX was detected at harmful levels in the urine samples, but the grade of poisoning symptoms varied.The patients' time to maximum serum TTX levels (Tmax) was estimated to be 12-24 h after ingestion of TTX-containing foods. Additionally, serum TTX levels of 19.5 ng/mL or higher within 24 h after ingestion indicated Grade 3 poisoning associated with respiratory abnormalities.These conditions were considered indicators of severe symptoms, while TTX levels of 1-3 ng/mL relate to the onset and disappearance of symptoms. A negative correlation was found between the logarithm of serum TTX concentration and the time after ingestion for two patients, indicating that the TTX serum levels decreased logarithmically. Furthermore, the TTX serum half-lives (T1/2) were 17.5 and 23.7 h.The results of this study enhance our understanding of TTX food safety and contribute to TTX risk assessment.

Expression dynamics of the genes for the hypothalamo-pituitary-gonadal axis in tiger puffer (Takifugu rubripes) at different reproductive stages.

Tiger puffer, Takifugu rubripes, a commercially important long-distance migratory fish, return to specific spawning grounds for reproduction. To clarify reproductive neuroendocrine system of the tiger puffer, the changes in the expression levels of the genes encoding three gonadotropin-releasing hormones (GnRHs), gonadotropin-inhibitory hormone (GnIH), GnIH receptor (GnIH-R), kisspeptin and kisspeptin receptor in the brain and gonadotropin (GTH) subunits, growth hormone (GH) and prolactin (PRL) in the pituitary were examined in the tiger puffer captured in the wild at different reproductive stages, namely immature and mature fish of both sexes, and post-ovulatory females that were obtained by hormonal treatment. The amounts of three gnrh mRNAs, gnih, gnih-r, fshb and lhb were substantially increased in the mature fish compared to the immature fish, especially in the females, and these augmented expressions were drastically decreased in the post-ovulatory females. gh expression showed a slight increase in the mature males. In contrast, kiss2, kiss2r and prl did not show significant changes in the males but significantly decreased in the post-ovulatory females. The present results demonstrate the expression dynamics of the hypothalamo-pituitary-gonadal axis genes associated with the reproductive conditions and the possible involvement of the GnRH/GnIH/GTH system in the regulation of the sexual maturation and spawning in the wild tiger puffer.

Puffer Fish Gut Microbiota Studies Revealed Unique Bacterial Co-Occurrence Patterns and New Insights on Tetrodotoxin Producers.

Tetrodotoxin (TTX) is a potent neurotoxin isolated mainly from toxic puffer fish. To date, the TTX biosynthetic mechanism inside its hosts remains unresolved. Here, we hypothesize the TTX synthesis relies on the host gut microbiota, including the neglected non-culturable bacteria. In these studies, we collected the gut contents from 5 puffer fish species of the genus Takifugu including one suspected hybrid species for gut microbiota study by 16S rRNA amplicon metagenomics approach. Their gut samples were divided into toxic and non-toxic groups based on the TTX concentrations in the livers detected by LC-MS/MS. Bacterial diversity studies showed that gut microbiota structures were significantly different between toxic and non-toxic species. Vibrio and Cyanobacteria centered at the gut bacterial co-occurrence network, suggesting their importance in TTX biosynthesis. The results of PICRUSt2 metagenomic prediction and gene set enrichment analysis provided new support of arginine-precursor required in TTX biosynthesis. This is the first study to profile the gut microbiota in toxic and non-toxic puffer fish species by 16S rRNA amplicon metagenomic approach, defining significant microbial co-occurrence patterns in their gut environment. Our data supported the proposed biosynthesis of TTX inside the hosts by their gut bacterial symbionts using arginine as a precursor.

Effects of high and low temperature on expression of GnIH, GnIH receptor, GH and PRL genes in the male grass puffer during breeding season.

Gonadotropin-inhibitory hormone (GnIH) is a multifunctional hypophysiotropic neurohormone and has a stimulatory role in the control of reproduction in the grass puffer. To clarify the neuroendocrine mechanisms underlying the effect of changes in water temperature on reproduction in fish, we previously revealed that, in parallel to gonadal regression, both low and high temperature significantly decreased the expressions of the genes encoding kisspeptin (kiss2), kisspeptin receptor (kiss2r), gonadotropin-releasing hormone 1 (gnrh1) in the brain and gonadotropin (GTH) subunits (fshb and lhb) in the pituitary of sexually mature male grass puffer. In this study, we examined the changes in expression of gnih and GnIH receptor gene (gnihr) in the brain and pituitary along with the genes for growth hormone (gh) and prolactin (prl) in the pituitary of male grass puffer exposed to low temperature (14 °C), normal temperature (21 °C, as initial control) and high temperature (28 °C) conditions for 7 days. The levels of gnih and gnihr mRNAs were significantly decreased in both low and high temperature conditions compared to normal temperature in the brain and pituitary. Similarly, the gh mRNA levels were significantly decreased in both low and high temperature conditions. The prl mRNAs showed no significant changes at high temperature, whereas drastically decreased at low temperature possibly by dysfunctional cold stress. Taken together, the present results suggest that, in addition to the inhibitory effect of temperature changes on the Kiss2/GnRH1/GTH system, the suppression of GnIH/GH system may also be involved in the termination of reproduction by high temperature at the end of breeding season.

Comparison of Physicochemical Characteristics and Fibril Formation Ability of Collagens Extracted from the Skin of Farmed River Puffer (Takifugu obscurus) and Tiger Puffer (Takifugu rubripes).

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from the skin of river puffer (ASC-RP and PSC-RP) and tiger puffer (ASC-TP and PSC-TP) were extracted and physicochemically examined. Denaturation temperature (Td) for all the collagens was found to be 25.5-29.5 °C, which was lower than that of calf skin collagen (35.9 °C). Electrophoretic patterns indicated all four samples were type I collagen with molecular form of (α1)2α2. FTIR spectra confirmed the extracted collagens had a triple-helical structure, and that the degree of hydrogen bonding in ASC was higher than PSC. All the extracted collagens could aggregate into fibrils with D-periodicity. The fibril formation rate of ASC-RP and PSC-RP was slightly higher than ASC-TP and PSC-TP. Turbidity analysis revealed an increase in fibril formation rate when adding a low concentration of NaCl (less than 300 mM). The fibril formation ability was suppressed with further increasing of NaCl concentration, as illustrated by a reduction in the turbidity and formation degree. SEM analysis confirmed the well-formed interwoven structure of collagen fibrils after 24 h of incubation. Summarizing the experimental results suggested that the extracted collagens from the skin of river puffer and tiger puffer could be considered a viable substitute to mammalian-derived collagens for further use in biomaterial applications.

Peroxiredoxin 1 (Prx1) is a dual-function enzyme by possessing Cys-independent catalase-like activity.

Peroxiredoxin (Prx) was previously known as a Cys-dependent thioredoxin. However, we unexpectedly observed that Prx1 from the green spotted puffer fish Tetraodon nigroviridis (TnPrx1) was able to reduce H2O2 in a manner independent of Cys peroxidation and reductants. This study aimed to validate a novel function for Prx1, delineate the biochemical features and explore its antioxidant role in cells. We have confirmed that Prx1 from the puffer fish and humans truly possesses a catalase (CAT)-like activity that is independent of Cys residues and reductants, but dependent on iron. We have identified that the GVL motif was essential to the CAT-like activity of Prx1, but not to the Cys-dependent thioredoxin peroxidase (POX) activity, and generated mutants lacking POX and/or CAT-like activities for individual functional validation. We discovered that the TnPrx1 POX and CAT-like activities possessed different kinetic features in the reduction of H2O2 The overexpression of wild-type TnPrx1 and mutants differentially regulated the intracellular levels of reactive oxygen species (ROS) and the phosphorylation of p38 in HEK-293T cells treated with H2O2 Prx1 is a dual-function enzyme by acting as POX and CAT with varied affinities towards ROS. This study extends our knowledge on Prx1 and provides new opportunities to further study the biological roles of this family of antioxidants.

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.

LPXRFamide peptide stimulates growth hormone and prolactin gene expression during the spawning period in the grass puffer, a semi-lunar synchronized spawner.

Gonadotropin-inhibitory hormone (GnIH) plays as a multifunctional neurohormone that controls reproduction in birds and mammals. LPXRFamide (LPXRFa) peptide, the fish ortholog of GnIH, has been shown to regulate the secretion of not only gonadotropin (GTH) but also growth hormone (GH) and prolactin (PRL), which are potentially important for gonadal function. To investigate the role of LPXRFa peptide on reproduction of the grass puffer, which spawns in semilunar cycles, we examined changes in the levels of gh and prl expression over the several months during the reproductive cycle, and the effects of goldfish LPXRFa peptide-1 (gfLPXRFa-1) on their expression were examined using primary pituitary cultures. The expression levels of both gh and prl showed significant changes during the reproductive cycle in both sexes with one peak in the spawning and pre-spawning periods for gh and prl, respectively. Particularly, gh showed substantial increase in expression in the spawning and post-spawning periods, indicative of its essentiality in the advanced stage of reproduction. gfLPXRFa-1 stimulated the expression of both gh and prl but there was a marked difference in response between them: gfLPXRFa-1 stimulated gh expression at a relatively low dose but little effect was observed on prl. Combined with the previous results of daily and circadian oscillations of lpxrfa expression, the present results suggest that LPXRFa peptide is important in the control of the cyclic reproduction by serving as a multifunctional hypophysiotropic factor that regulates the expression of gh and prl as well as GTH subunit genes.

Application of a puffer fish skin gelatin film containing Moringa oleifera Lam. leaf extract to the packaging of Gouda cheese.

This study aims to develop a puffer fish skin gelatin (PSG) film that contains Moringa oleifera Lam. leaf extract (ME) as a new biodegradable film. With the increase in ME concentration, the tensile strength and elongation at break of the PSG film increased, whereas the oxygen permeability and water vapor permeability decreased. In addition, the PSG film with ME exhibited antimicrobial activity against Listeria monocytogenes and antioxidant activity. To apply the ME-containing PSG film to food packaging, Gouda cheese was wrapped with the ME-containing PSG film. During storage, the cheese packaging with the ME-containing PSG film effectively inhibited the microbial growth and retarded the lipid oxidation of cheese compared with the control sample. Thus, the ME-containing PSG film can be used as an antimicrobial and antioxidative packaging material to improve the quality of food products.

Aptameric Recognition-Modulated Electroactivity of Poly(4-Styrenesolfonic Acid)-Doped Polyaniline Films for Single-Shot Detection of Tetrodotoxin.

The work being reported is the first electrochemical sensor for tetrodotoxin (TTX). It was developed on a glassy carbon electrodes (C) that was modified with poly(4-styrenesolfonic acid)-doped polyaniline film (PANI/PSSA). An amine-end functionalized TTX-binding aptamer, 5'-NH2-AAAAATTTCACACGGGTGCCTCGGCTGTCC-3' (NH2-Apt), was grafted via covalent glutaraldehyde (glu) cross-linking. The resulting aptasensor (C//PANI⁺/PSSA-glu-NH2-Apt) was interrogated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in sodium acetate buffer (NaOAc, pH 4.8) before and after 30 min incubation in standard TTX solutions. Both CV and EIS results confirmed that the binding of the analyte to the immobilized aptamer modulated the electrochemical properties of the sensor: particularly the charge transfer resistance (Rct) of the PANI⁺/PSSA film, which served as a signal reporter. Based on the Rct calibration curve of the TTX aptasensor, the values of the dynamic linear range (DLR), sensitivity and limit of detection (LOD) of the sensor were determined to be 0.23-1.07 ng·mL(-1) TTX, 134.88 ± 11.42 Ω·ng·mL(-1) and 0.199 ng·mL(-1), respectively. Further studies are being planned to improve the DLR as well as to evaluate selectivity and matrix effects in real samples.

Cell-penetrating peptide delivery of biologically active oct4 protein into cultured Takifugu rubripes spermary cells.

Continuous cell culture of a puffer fish Takifugu rubripes has been established for efficient delivery of exogenous genes or proteins to cultured fish cells. Transcription factor oct4 was chosen for transduction into cultured fish cells because of its conserved structure and function between fish and mammals. In this work, the T. rubripes oct4 gene was cloned and expressed in Escherichia coli as a recombinant protein by introducing cell-penetrating peptide (CPP) poly-arginine (11R) and 6His-tag at the C-terminus. After purification, recombinant proteins were added to the growth medium and incubated with T. rubripes spermary cells. Recombinant proteins that crossed the cell membrane were detected in the cytoplasm and nucleus by western blot and immunofluorescent observation. The function of transduced oct4 as a transcription factor in fish cells was confirmed by driving green fluorescent protein expression in the pEGFP-1 reporter construct with the conserved specific oct4-binding sequence from mouse Mus musculus. Taken together, 11R can be an efficient CPP in delivering fusion proteins to cultured fish cells.

Spikebot: A Multigait Tensegrity Robot with Linearly Extending Struts.

Numerous recent research efforts have leveraged networks of rigid struts and flexible cables, called tensegrity structures, to create highly resilient and packable mobile robots. However, the locomotion of existing tensegrity robots is limited in terms of both speed and number of distinct locomotion modes, restricting the environments that a robot is capable of exploring. In this study, we present a tensegrity robot inspired by the volumetric expansion of Tetraodontidae. The robot, referred to herein as Spikebot, employs pneumatically actuated rigid struts to expand its global structure and produce diverse gaits. Spikebot is composed of linear actuators that dually serve as rigid struts linked by elastic cables for stability. The linearly actuating struts can selectively protrude to initiate thrust- and instability-driven locomotion primitives. Such motion primitives allow Spikebot to reliably locomote, achieving rolling, lifting, and jumping. To highlight Spikebot's potential for robotic exploration, we demonstrate how it achieves multi-dimensional locomotion over varied terrestrial conditions.

Effect of plasma-activated lactic acid on microbiota composition and quality of puffer fish (Takifugu obscurus) fillets during chilled storage.

Fresh puffer fish (Takifugu obscurus) are susceptible to microbial contamination and have a very short shelf-life of chilled storage. Hence, this study aimed to evaluate the effects of plasma-activated lactic acid (PALA) on microbiota composition and quality attributes of puffer fish fillets during chilled storage. The results showed that PALA treatment effectively reduced the growth of bacteria and attenuated changes in physicochemical indicators (total volatile basic nitrogen, pH value, K value, and biogenic amines) of puffer fish fillets. Additionally, insignificant changes were observed in lipid oxidation during the first 8 days (p > 0.05). Illumina-MiSeq high-throughput sequencing revealed that PALA effectively inhibited the growth of Pseudomonas in puffer fish fillets and maintained the diverse characteristics of the microbial community. In combination with sensory analysis, PALA extended the shelf life of puffer fish fillets for 4 days, suggesting that PALA could be considered a potential fish fillet preservation method.

Improving photovoltaic cell parameter calculations through a puffer fish inspired optimization technique.

The precise estimation of solar PV cell parameters has become increasingly important as solar energy deployment expands. Due to the intricate and nonlinear characteristics of solar PV cells, meta-heuristic algorithms show greater promise than traditional ones for parameter estimation. This study utilizes the Puffer Fish (PF) meta-heuristic optimization method, inspired by male puffer fish's circular structures, to estimate parameters of a modified four-diode PV cell. The PF algorithm's performance is assessed against ten benchmark test functions, with results presented as mean and standard deviation for validation. Comparative analysis with Particle Swarm Optimization (PSO), Grey Wolf Optimization (GWO), Rat Search Algorithm (RAT), Heap Based Optimizer (HBO), and Cuckoo Search (CS) algorithms highlights PF's superior performance, achieving optimal solutions with minimal error of 7.8947E-08. Statistical tests, including Friedman Ranking (1st) and Wilcoxon's rank sum (3.8108E-07), confirm PF's superiority. The circular structures of male puffer fish serve as an effective model for optimization algorithms, enhancing parameter estimation. Benchmark tests and statistical analysis consistently underscore PF's superiority over other meta-heuristic algorithms. Future research should explore PF's potential applications in solar energy and beyond.

A Case of Puffer Fish Poisoning from United Arab Emirates.

Puffer fish (Lagocephalus sceleratus) is a well-known source of deadly food poisoning due to a neurotoxin, called tetrodotoxin. Cases of tetrodotoxin poisoning are common along the coasts of East Asian countries but rare in the Arabian Gulf region. Here, we report a case of a 19-year-old man who presented with signs and symptoms suggestive of puffer fish poisoning. Dietary history was the key to diagnosis though laboratory investigations and imaging yielded normal results. Early diagnosis and proper supportive management are essential for survival.

Toxicity of cultured bullseye puffer fish Sphoeroides annulatus.

The toxin content in various life cycle stages of tank-cultivated bullseye puffer (Sphoeroides annulatus) were analyzed by mouse bioassay and ESI-MS spectrometry analysis. The presence of toxin content was determined in extracts of sperm, eggs, embryo, larvae, post-larvae, juvenile, pre-adult, and adult fish, as well as in food items used during the cultivation of the species. Our findings show that only the muscle of juveniles, the viscera of pre-adults, and muscle, liver, and gonad of adult specimens were slightly toxic (<1 mouse unit). Thus, cultivated S. annulatus, as occurs with other cultivated puffer fish species, does not represent a food safety risk to consumers. This is the first report of toxin analysis covering the complete life stages of a puffer fish under controlled conditions.