First metal and metalloid study assessment for the Black Triggerfish Melichthys niger (Bloch, 1786): Baseline data from a pristine South Atlantic oceanic island.

Metal and metalloid concentrations in Black Triggerfish, Melichthys niger (Tetraodontiformes, Balistidae), and sediments from Trindade Island, an isolated, pristine, and understudied environment in the Southwestern Atlantic Ocean were determined. Several elements were detected in sediment, including Rare Earth Elements (REE). Hg and the REE Nd were, however, not detected. Elements determined in triggerfish are indicative of systemic circulation and the presence of toxic elements indicates an exposure source. No significant associations were detected between elements in fish organs and total length, suggesting no bioaccumulation with age, nor between organs and sediment, indicating probable dietary origin. Biliary excretion indicated a local chronic source of As and Cd and non-quantifiable REE elimination. Hg concentrations were over thresholds that affect fish biochemistry processes. These baseline data are valuable concerning elemental contamination in remote oceanic islands, with the potential to be applied to future biomonitoring efforts and conservation measures for reef ecosystems worldwide.

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

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

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.

Multibiomarker approach in fish to assess a heavily polluted Brazilian estuary, Guanabara Bay.

Brazil's Guanabara Bay (GB), located in Rio De Janeiro, is a deeply contaminated, eutrophic waterbody that challenges the understanding of the effects of pollutants on the biota. This paper presents a strategy to evaluate the impact of contamination utilizing a multibiomarker approach in two fish species: corvine (Micropogonias furnieri) and burrfish (Chilomycterus spinosus). The strategy is comprised of a general biomarker of fish' physical condition, the condition factor (CF), and specific biomarkers of pollutant exposure such as acetylcholinesterase (AChE), metallothionein (MT) activity and biliary polycyclic aromatic hydrocarbons (PAH) metabolites. Our results indicate that fish from GB are greatly affected by environmental pollution. CF values were lower in fishes from GB than in the reference site indicating that these fishes were under higher environmental stress. Lower AChE activity level in both species showed vulnerability to the presence of pesticide residues. Higher levels of MT in both species in GB reflect the consequences of heavy metal exposure in the bay, in spite of their bioavailability being restricted specially by the high organic matter content of GB. The levels of PAHs were higher in GB for both fish species, indicating exposure to these substances. However, the fish species showed different behavior regarding the origin of the PAHs. The multibiomarker approach used in this study evidently depicted effects on the health of fish in a waterbody with a complex polluted situation and further categorized the effects of anthropogenic activities in this aquatic system.

Case Report of Tetrodotoxin Poisoning from Lagocephalus sceleratus in Lebanon.

The Indo-Pacific pufferfish Lagocephalus sceleratus is a tetrodotoxin-containing species believed to have entered the Mediterranean Sea through the Suez Canal. Tetrodotoxin (TTX) is primarily found in the liver, intestine, and ovaries of L. sceleratus. We report a case of a patient with TTX poisoning from L. sceleratus consumption in Lebanon. History of ingestion, clinical presentation, and exam findings were obtained during phone-based consultation with the patient and intensive care physician. A 46-year-old male presented to a hospital in Lebanon with perioral and extremity numbness as well as dizziness 1 h after ingestion of an L. sceleratus fish. He had caught and prepared the fish himself and had eaten a skinless piece of flesh. Over the following 6 h he also developed ataxia and generalized body numbness. His treatment included systemic hydrocortisone, antihistamine, activated charcoal, and fluids. He was admitted to the intensive care unit, where he developed self-limited, stable sinus bradycardia. He was discharged home on hospital day 5 with residual lightheadedness that improved over several days. This is one of the first reported cases of tetrodotoxin poisoning due to L. sceleratus in Lebanon. Public awareness regarding the toxicity of this species after any ingestion is essential to prevent toxicity and death.

Does Ocean Sunfish Mola spp. (Tetraodontiformes: Molidae) Represent a Risk for Tetrodotoxin Poisoning in the Portuguese Coast?

Tetrodotoxin (TTX) is a potent neurotoxin naturally occurring in terrestrial and marine organisms such as pufferfish. Due to the risk of TTX poisoning, fish of Tetraodontidae family and other puffer-related species must not be placed in the EU markets. This restriction applies to fish of the family Molidae even though no data on toxins' occurrence is available. In this study, the presence of TTX and its analogues was investigated in the main edible tissue (the white muscle) and the main xenobiotics storage organ (the liver) of ocean sunfish Mola spp. (n = 13) from the South Portuguese coast. HILIC-MS/MS analyses did not reveal TTX in the analyzed samples, suggesting an inexistent or very limited risk of TTX poisoning.

Vibrio fluminensis sp. nov. isolated from the skin of Southern Atlantic sharpnose-pufferfish (Canthigaster figueiredoi).

This manuscript provides the description of the bacterial strain A621T characterized by Gram negative motile rods, presenting green circular colonies on TCBS. It was obtained from the skin of the sharpnose pufferfish Canthigaster figueredoi (Tetraodontidae Family), collected in Arraial do Cabo, located in the Rio de Janeiro region, Brazil. Optimum growth occurs at 20-28 °C in the presence of 3% NaCl. The Genome sequence of the novel isolate consisted of 4.224 Mb, 4431 coding genes and G + C content of 44.5%. Genomic taxonomy analysis based on average amino acid (AAI), genome-to-genome-distance (GGDH) and phylogenetic reconstruction placed (A621T= CBAS 741T = CAIM 1945T = CCMR 150T) into a new species of the genus Vibrio (Vibrio fluminensis sp. nov). The genome of the novel species contains four gene clusters (~ 56.17 Kbp in total) coding for different types of bioactive compounds that hint to several possible ecological roles in the sharpnose pufferfish host.

Tetrodotoxin and Its Analogues in Food: Recent Updates on Sample Preparation and Analytical Methods Since 2012.

Tetrodotoxin (TTX), found in various organisms including pufferfish, is an extremely potent marine toxin responsible for numerous food poisoning accidents. Due to its serious toxicity and public health threat, detecting TTX and its analogues in diverse food matrices with a simple, fast, efficient method has become a worldwide concern. This review summarizes the advances in sample preparation and analytical methods for the determination of TTX and its analogues, focusing on the latest development over the past five years. Current state-of-the-art technologies, such as solid-phase microextraction, online technology, novel injection technology, two-dimensional liquid chromatography, high-resolution mass spectrometry, newly developed lateral flow immunochromatographic strips, immunosensors, dual-mode aptasensors, and nanomaterials-based approaches, are thoroughly discussed. The advantages and limitations of different techniques, critical comments, and future perspectives are also proposed. This review is expected to provide rewarding insights to the future development and broad application of pretreatment and detection methods for TTX and its analogues.

A new species of Huffmanela (Nematoda: Trichosomoididae) parasitizing Colomesus psittacus (Tetraodontiformes) from Marajó Island, Pará State, Brazil.

The puffer fish Colomesus psittacus, is not commercialized on Marajó Island. They are captured as bycacth and discarded dead in the environment in artisanal fisheries that occur in the estuaries of northern Brazil. In this sense, the objective was to identify the parasites present in the gills and to evaluate the histopathological alterations caused by these nematodes of the genus Huffmanela. Fifty-five fish were analyzed, and thirty-five specimens showed the parasite in the gills. Morphological characteristics suggest that it is a new species of the genus Huffmanela, and the histopathological exams showed an edematous inflammation in the secondary lamella and the presence of eggs of this nematode, which is the first record of this parasite in C. psittacus in Brazil.

Isolation and Biological Activity of 9-epiTetrodotoxin and Isolation of Tb-242B, Possible Biosynthetic Shunt Products of Tetrodotoxin from Pufferfish.

Tetrodotoxin (TTX, 1) is a potent voltage-gated sodium channel blocker detected in certain marine and terrestrial organisms. We report here a new TTX analogue, 9-epiTTX (2), and a TTX-related compound, Tb-242B (4), isolated from the pufferfish Takifugu flavipterus and Dichotomyctere ocellatus, respectively. NMR analysis suggested that 2 exists as a mixture of hemilactal and 10,8-lactone forms, whereas other reported TTX analogues are commonly present as an equilibrium mixture of hemilactal and 10,7-lactone forms. Compound 2 and TTX were confirmed not to convert to each other by incubation under neutral and acidic conditions at 37 °C for 24 h. Compound 4 was identified as the 9-epimer of Tb-242A (3), previously reported as a possible biosynthetic precursor of TTX. Compound 4 was partially converted to 3 by incubation in a neutral buffer at 37 °C for 7 days, whereas 3 was not converted to 4 under this condition. Compound 2 was detected in several TTX-containing marine animals and a newt. Mice injected with 600 ng of 2 by intraperitoneal injection did not show any adverse symptoms, suggesting that the C-9 configuration in TTX is critical for its biological activity. Based on the structures, 2 and 4 were predicted to be shunt products for TTX biosynthesis.

Fine-scale movement of northern Gulf of Mexico red snapper and gray triggerfish estimated with three-dimensional acoustic telemetry.

Red snapper and gray triggerfish are ecologically, economically, and culturally important reef fishes in the northern Gulf of Mexico (nGOM). Scientists and managers have sought to understand the effects of artificial reefs on reef fish ecology by focusing on fish residency and movement at artificial reefs with less attention paid to broader spatial and temporal patterns in reef fish movements among a seascape of artificial reefs and other natural habitats. We used novel large-scale (> 15 km2) geopositioning acoustic telemetry arrays to track the 3-dimensional movements of tagged red snapper (n = 59) and gray triggerfish (n = 15) among multiple nGOM artificial reefs up to 333 days. Tagged fish moved frequently among artificial reefs and had shorter residence times at the release reef (43 days for red snapper and 3 days for gray triggerfish) than reported in previous studies. Both species displayed high individual variability in movement dynamics, as well as seasonally variable diel patterns of habitat use, height above bottom, and distance to reefs, which may have been driven by dynamic influences of predation risk, physiological constraints, or foraging over time and space. The wider seascape view revealed in this study demonstrates the importance of including multiple artificial reefs over long timescales to capture individual, spatial, and temporal variability in reef fish movement.

The brain of the wild toxic marine pufferfishes accumulates tetrodotoxin.

We have previously detected tetrodotoxin (TTX) in the brain of the wild toxic torafugu Takifugu rubripes by immunohistochemistry and LC/MS analysis. We have also indicated that TTX is a stress-relieving substance in the brain and reduces agonistic interactions in torafugu juveniles. Although the toxicity of marine pufferfish in the Japanese waters has been extensively examined for food hygiene, whether wild toxic pufferfish generally possess TTX in the brain has not been investigated. In the present study, we examined the presence of TTX in the brain of several wild toxic marine pufferfishes such as kusafugu T. alboplumbeus, komonfugu T. flavipterus, shosaifugu T. snyderi, okinawafugu Chelonodontops patoca, and in wild non-toxic pufferfishes such as shirosabafugu Lagocephalus spadiceus and yoritofugu Sphoeroides pachygaster. We also examined tsumugihaze Yongeichthys criniger, known to possess TTX in the skin, viscera, and gonad. TTX was extracted from the brain, liver, skin, and muscle and was analyzed by LC/MS. TTX was detected in the brain as well as in the liver, skin, and muscle in kusafugu, komonfugu, shosaifugu, okinawafugu, and tsumugihaze. In shirosabafugu, low level of TTX (0.8 mouse unit/g-brain) was detected in the brain in 1 out of 3 individuals. In yoritofugu, no TTX was detected in any of the tissues. We conclude that the brain is also an organ that contains TTX in the wild toxic marine pufferfishes.

Coupled Bionic Drag-Reducing Surface Covered by Conical Protrusions and Elastic Layer Inspired from Pufferfish Skin.

Inspired by the drag-reducing properties of the cone-like spines and elastic layer covering the pufferfish skin, important efforts are underway to establish rational multiple drag-reducing strategies for the development of new marine engineering materials. In the present work, a new drag-reducing surface (CPES) covered by conical protrusions (sparse "k-type" with rough height k+ = 13-15) and an elastic layer are constructed on copper substrate via a hybrid method, combining the sintering and coating processes. The drag-reducing feature of the prepared CPES biomimetic surface is achieved by rheometer and particle image velocimetry (PIV) experiments. To comprehensively investigate its drag reduction mechanism, the porous copper substrate (PCS), copper substrate (CS), conical protrusion resin substrate (CPRS), and conical protrusion porous copper substrate (CPPCS) were used for a comparative analysis. In laminar flow, we discovered that the conical protrusion structure and wettability of the elastic surface coupling affect the CPES sample's drag-reducing performance (7-8%) and that the interface produced slip to reduce the viscous drag. In turbulent flow, the CPES biomimetic surface exhibits an 11.5-17.5% drag-reducing performance. Such behavior was enabled by two concurrent mechanisms: (i) The conical protrusions as vortex generators enhance the number of vortices and the wake effect, enabling faster movement of downstream strips, reducing viscous drag; (ii) The conical protrusion elements break and lift large-scale vortices to produce numerous small-scale vortices with low energy, effectively weakening perturbations and momentum exchange. Additionally, the elastic layer shows high adhesion and stability on copper substrate after sandpaper abrasion and water-flow erosion tests. The copper substrate surface formed by the sintering method is also covered with dense porous structures, which gives the elastic layer and conical protrusions excellent combined robustness. Our findings not only shed new light on the design of robust drag-reducing surfaces but also provide new avenues for underwater drag reduction in the field of marine applications.

Reproductive biology of the bullseye puffer Sphoeroides annulatus: Gonadosomatic index and its suitability for estimating length at maturity.

This study aimed to describe updated information on the basic reproductive biology of Sphoeroides annulatus females and potential use of the gonadosomatic index I G as input data to assess length at 50% maturity ( L 50 ) and identify the breeding season. From April 2014 to December 2015, 767 females were analysed along the north-west coast of the Mexican Pacific Ocean. Both immature and mature females were represented in the total length ( L T ), which ranged from 81 to 440 mm, and the smallest mature female was 92 mm (new record for the species). Gonadal maturity phases were assigned through histological analysis. A I G threshold value I cut was established using the lower whisker value for maturity phase spawning capable (2.06%). The monthly variation of I G supported the annual spawning seasonality, and the presence of reproductively active mature females I G ≥ I cut coincided with the observed periods with histological data and the peaks for both seasons 2014 and 2015. Also the presence of reproductively inactive I G < I cut mature females confirmed the nonreproductive season observed from January to April 2015. Estimates of L 50 were based on the cumulative proportion of reproductively active mature females: both datasets showed a 'bumpy trajectory'. Considering the functional form of data and models, a second objective was to verify if each dataset presented a simple or double sigmoid trajectory and select the most suitable model through the Akaike information criterion. For both datasets, the best fitted model was the double sigmoidal M DS , while the single sigmoidal M SS had low performance and did not have statistical support. The M DS provided a L 50 value ca. 254 mm L T for both data sets. A trade-off between the model's correct fit and a reliable L 50 value was found, but this fact is not always considered when calculating the maturity, resulting in biased parameters. Correct data fitting may seem trivial, but the consequences for the conservation of immature individuals could be dramatic, affecting the population recruitment. In conclusion, the histologically calibrated I cut was suitable for classifying females of S. annulatus into reproductively inactive or active types, with an agreement percentage of 99%. The I cut  = 2.06% can be useful for subsequent studies for this species.

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 â‰« muscle > gonads. This is the first report of TTXs in Mozambican coast.

The impact of paleoclimatic changes on body size evolution in marine fishes.

Body size is an important species trait, correlating with life span, fecundity, and other ecological factors. Over Earth's geological history, climate shifts have occurred, potentially shaping body size evolution in many clades. General rules attempting to summarize body size evolution include Bergmann's rule, which states that species reach larger sizes in cooler environments and smaller sizes in warmer environments, and Cope's rule, which poses that lineages tend to increase in size over evolutionary time. Tetraodontiform fishes (including pufferfishes, boxfishes, and ocean sunfishes) provide an extraordinary clade to test these rules in ectotherms owing to their exemplary fossil record and the great disparity in body size observed among extant and fossil species. We examined Bergmann's and Cope's rules in this group by combining phylogenomic data (1,103 exon loci from 185 extant species) with 210 anatomical characters coded from both fossil and extant species. We aggregated data layers on paleoclimate and body size from the species examined, and inferred a set of time-calibrated phylogenies using tip-dating approaches for downstream comparative analyses of body size evolution by implementing models that incorporate paleoclimatic information. We found strong support for a temperature-driven model in which increasing body size over time is correlated with decreasing oceanic temperatures. On average, extant tetraodontiforms are two to three times larger than their fossil counterparts, which otherwise evolved during periods of warmer ocean temperatures. These results provide strong support for both Bergmann's and Cope's rules, trends that are less studied in marine fishes compared to terrestrial vertebrates and marine invertebrates.

3D model of the geometric nest structure, the "mystery circle," constructed by pufferfish.

A small pufferfish, Torquigener albomaculosus, is known to construct an elaborate geometric circular structure, which has been referred to as a "mystery circle," with a diameter of ~2 m in the sand of the seabed. We reconstructed a 3D model of this structure for the first time using a "structure from motion" (SfM) algorithm. The mystery circle constructed by the pufferfish may have potential applications for biomimetics similar to the structures constructed by termites and prairie dogs. To support the significance of its structural characteristics, it was observed that the water passing through the valley upstream always gathers in the center of the structure, regardless of the direction of water flow. Furthermore, it has the function of extracting fine-grained sand particles from the valleys and directing these to the center. Computational fluid analysis can be performed immediately using the quantified 3D data, and the structural features of the mystery circle is expected to be applied in a wide range of fields, such as architecture and engineering, via biomimetics.

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

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

Phoenixin-14 alters transcriptome and steroid profiles in female green-spotted puffer (Dichotomyctere nigroviridis).

Phoenixin (PNX) is a highly conserved, novel hormone with diverse functions, including hypothalamic control of reproduction, appetite modulation, and regulation of energy metabolism and inflammation. While some functions appear conserved across vertebrates, additional research is required to fully characterize these complex pleiotropic effects. For instance, very little is known about transcriptome level changes associated with PNX exposure, including responses in the hypothalamic-pituitary-gonadal (HPG) axis, which is critical in vertebrate reproduction. In addition, the PNX system may be especially complex in fish, where an additional receptor is likely present in some species. The purpose of this study was to assess hypothalamic and ovarian transcriptomes after PNX-14 administration in female vitellogenic green-spotted puffer (Dichotomyctere nigroviridis). Steroid-related changes were also assessed in the liver and blood plasma. Hypothalamic responses included pro-inflammatory signals such as interleukin 1ß, possibly related to gut-brain axis functions, as well as suppression of cell proliferation. Ovarian responses were more widely downregulated across all identified pathways, which may reflect progression to a less transcriptionally active state in oocytes. Both organs shared regulation in transforming growth factor-ß and extracellular matrix remodeling (periostin) pathways. Reproductive processes were in general downregulated, but both inhibiting (bone morphogenetic protein 15 and follistatin) and promoting (17-hydroxyprogesterone) factors for oocyte maturation were identified. Select genes involved in reproduction (vitellogenins, estrogen receptors) in the liver were unresponsive to PNX-14 and higher doses may be needed to induce reproductive effects in D. nigroviridis. These results reinforce the complexity of PNX actions in diverse tissues and highlight important roles for this hormone in regulating the immune response, energy metabolism, and cell growth.