Assessment of Human Health Impacts from Invasive Pufferfish (Attacks, Poisonings and Fatalities) across the Eastern Mediterranean.

The silver-cheeked toadfish Lagocephalus sceleratus (Gmelin 1789), and to a lesser degree the orange spotted toadfish Torquigener hypselogeneion (Bleeker, 1852), pose threats to human health from physical attacks and poisonings in the Eastern Mediterranean Sea. This study reviewed human health-related impacts resulting from these pufferfish, compiling and assessing records from online sources, the peer-reviewed literature, medical records, personal interviews, and observations across the Eastern Mediterranean in the years 2004 to 2023. A total of 198 events impacting human health were documented: 28 records of physical attacks, at least 144 non-lethal poisoning episodes, and 27 human fatalities resulting from consumption. The majority of the reported incidences occurred in Syria, Türkiye, and Lebanon. Most physical attacks occurred in summer, while most poisoning events occurred during winter. The number of recorded incidents greatly increased after 2019, especially with regard to poisonings, yet whether this is related to greater media attention, or to increased fish abundance is unclear. This is the first comprehensive study to collate findings on attacks, poisonings and fatalities caused by these pufferfish in the Mediterranean Sea, and may help in improving national health policies. We urge the continuation of national campaigns to caution residents and tourists of these species' high toxicities and potential aggressiveness.

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.

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.

Tandem spawning and development in the African freshwater pufferfish Tetraodon schoutedeni.

To advance breeding techniques for the African freshwater pufferfish Tetraodon schoutedeni and observe tandem spawning closely, we monitored the reproduction of captive individuals. Eight spawning sessions (stable water temperature 24-25°C; daily light period 07:00-19:00) occurred between May 2016 and November 2017. After 65-150 min of tandem swimming (the male biting and clinging to the female's abdomen), 3-50 spherical, weakly adhesive eggs were spawned, being scattered onto the sandy substrate or water plants. The removal of cohabitants (potentially eating spawned eggs) and provision of small initial food items, such as small-type Brachionus spp., for larval fish were essential for successful breeding.

Characterization of the complete mitochondrial genome of Takifugu pseudommus (Actinopterygii, Tetraodontidae).

The complete mitochondrial genome of Takifugu pseudommus was reported in the present study. It was 16,448 bp in length and consisted of 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and a non-coding control region. The nucleotide content of this genome was 29.91% for A, 29.09% for C, 15.20% for G, and 25.80% for T. The phylogenetic tree, constructed with complete mitochondrial genome, suggested T. pseudommus was closely related with T. chinensis, T. flavidus, and T. rubripes among Tetraodontidae species. This study could provide an important dataset for genetic diversity and species identification among the genus Takifugu.

Are pufferfishes a viable source of nutritional value for human consumption-an investigation on recently commercialized marine pufferfishes (Tetraodontidae: Tetraodontiformes).

Estimation of the nutritional profile of edible fishes is essential, and thus, we have evaluated nutritional composition and amino and fatty acids profiling of recently commercialized marine pufferfishes from the coast of Mandapam in the Gulf of Mannar. Proximate analyses including assessment of ash, moisture, proteins, carbohydrates, and lipids in the muscles of five edible marine pufferfishes were carried out. Major fatty acids profile of five fish species disclosed the presence of saturated fatty acids (SFA, 6.22-16.78%), monounsaturated fatty acids (MUFAs, 3.01-10.59%), and polyunsaturated fatty acids (PUFAs, 8.4-20.01%). The overall percentage of omega fatty acids ranged between ω3 and ω6 (5.63-14.01% and 2.77-6.1%), and the ratio of ω3/ω6 was 3.08-2.03% and ω6/ω3 was 0.32-0.49%. Moreover, n-3 PUFA was more than n-6 PUFA. In contrast, major amino acids in five fish species were glycine (4.77%), lysine (3.57%), methionine (2.91%), arginine (2.04%), and threonine (1.81%). The results obtained from this study can act as baseline data for evaluating the nutritional profiles of various fishes in the future. This is the first report of fatty and amino acids analysis on recently commercialized marine pufferfishes in the Gulf of Mannar region, and this finding may lead to new insights for further research.

Characterization of the complete mitochondrial DNA sequence of the Lagocephalus gloveri (Tetraodontidae, Tetraodontiformes).

The complete mitochondrial genome of Lagocephalus gloveri is reported in the present study, which is 16,446 bp in length. It consists of 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes and a non-coding control region. The overall base composition of the genome is 27.58% for A, 25.07% for T, 30.83% for C and 16.52% for G. The phylogenetic tree, which is based on 12 protein coding gene sequences, suggested that L. gloveri was closest to L. lagocephalus. This study could give impetus to studies focused on population structure and molecular evolution of L. gloveri.

Enterovibrio baiacu sp. nov.

We report here the novel species to encompass the isolate A649T (=CBAS 716T = CBRVS P1061T) obtained from viscera of the healthy pufferfish Sphoeroides spengleri (Family Tetraodontidae). Genomic taxonomy analysis demonstrates that the novel strain A649T had < 95% average amino acid identity/average nucleotide identity (AAI/ANI) and < 70% similarity of genome-to-genome distance (GGDH) towards its closest neighbors which places A649T into a new Enterovibrio species (Enterovibrio baiacu sp nov.). In silico phenotyping disclosed several features that may be used to differentiate related Enterovibrio species. The nearly complete genome assembly of strain A649T consisted of 5.4 Mbp and 4826 coding genes.

Characterization of the complete mitochondrial DNA sequence of the Lagocephalus guentheri (Tetraodontidae, Tetraodontiformes).

The complete mitochondrial genome of Lagocephalus guentheri was reported in the present study, which was 16,461 bp in length. It consists of 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes and a non-coding control region. The overall base composition of the genome is 27.54% for A, 24.80% for T, 31.23% for C and 16.43% for G. The phylogenetic tree, which is based on 12 protein-coding gene sequences, suggested that L. guentheri was closest to L. spadiceus. This study could give impetus to studies focused on population structure and molecular evolution of L. guentheri.

Complete mitochondrial genome and the phylogenetic position of the stellate puffer (Arothron stellatus).

The complete mitochondrial genome of the stellate puffer (Arothron stellatus) was first determined in this study. It was a 16,475 bp circular molecule and consisted of 37 genes with typical gene order in vertebrate mitogenome. Its nucleotide content was 29.0% A, 32.0% C, 15.9% G and 23.1% T. This mitogenome had 30 bp short intergenic spaces located in 12 gene junctions and 28 bp overlaps located in 7 gene junctions. In the protein-coding genes, two start codons (GTG and ATG) and two stop codons (TAG and TAA/TA/T) were found. The 22 tRNA genes ranged from 64 bp (tRNA-Cys) to 74 bp (tRNA-Leu1 and tRNA-Lys). The phylogenetic result showed that Takifugu xanthopterus was most closely related to the reef-related A. stellatus among all Tetraodontiformes species.

A complex of the blood fluke genus Psettarium (Digenea: Aporocotylidae) infecting tetraodontiform fishes of east Queensland waters.

Seven species of Psettarium (Digenea: Aporocotylidae), including four new species, are reported from tetraodontiform fishes from off coastal east Queensland. Psettarium pandora n. sp. infects the yellow boxfish, Ostracion cubicus (Ostraciidae), the first known aporocotylid to infect this family of fishes. Three new species are reported from pufferfishes of the genus Arothron (Tetraodontidae): Psettarium yoshidai n. sp. infects the map puffer (Arothron mappa), Psettarium hustoni n. sp. infects the black-spotted puffer (A. nigropunctatus) and Psettarium martini n. sp. infects the starry puffer (A. stellatus). We also report three species of Psettarium from Australian waters for the first time. Paracardicola hawaiensis Martin, 1960, the sole species of Paracardicola, is redescribed based on specimens collected from the type-host, the stars-and-stripes puffer, Arothron hispidus. Paracardicola is synonymised with Psettarium and P. hawaiensis is recombined as Psettarium hawaiiense (Martin, 1960) n. comb. Psettarium pulchellum Yong, Cutmore, Bray, Miller, Semarariana, Palm & Cribb, 2016, described from the narrow-lined puffer (Arothron manilensis) from off Bali, Indonesia, is reported from the same fish species at two locations on the Queensland coast, significantly extending the range of this species. Psettarium nolani (Bray, Cribb & Littlewood, 2013), originally described from French Polynesia, is reported from A. hispidus, A. manilensis and A. stellatus, representing both new host and locality records for this species. Molecular phylogenetic analysis shows these species to all be closely related, such that they cannot be considered to represent separate genera despite their differing morphology. Analysis of 28S sequence data for Psettarium anthicum Bullard & Overstreet, 2006, a non-tetraodontiform-infecting species, shows it to be distantly related to all other species of Psettarium for which sequence data are available. The species is re-assigned to a new genus, Cardallagium n. gen., as Cardallagium anthicum (Bullard & Overstreet, 2006) n. comb. We think it likely that the host range of species of Psettarium is limited to tetraodontiform fishes. We assessed the infection biology of two species, P. nolani and P. hawaiiense n. comb. infecting A. hispidus, using histology to assess the pathways of egg release for these species. Eggs of both species were observed in both circulatory and visceral organs of infected hosts, often in high numbers. Eggs were seen trapped in the mucosal layer of the intestine and, in rare instances, causing lesions in the laminar epithelium, providing the strongest evidence yet that they pass through the gut wall and escape the host via the faeces. Lastly, we discuss the biogeographical implications of our findings, noting that some Psettarium species now show very wide geographical distributions.

Revision of the Megaperidae Manter, 1934 n. comb. (Syn. Apocreadiidae Skrjabin, 1942) including a reorganization of the Schistorchiinae Yamaguti, 1942.

Modified and/or new keys to the four subfamilies now recognized within the Megaperidae Manter, 1934 n. comb. (Syn. Apocreadiidae Skrjabin, 1942) as well as the genera within each subfamily are presented. Two new genera, Paraschistorchis n. gen. and Plesioschistorchis n. gen., both within the Schistorchiinae Yamaguti, 1942, are erected and keys are provided to the species considered in both new genera-distinguished by possessing caeca that end either in separate ani or blindly. Plesioschistorchis callyodontis (Yamaguti, 1942) n. comb. and Plesioschistorchis haridis (Nagaty, 1957) n. comb. are re-described from new material collected from the common parrotfish, Scarus psittacus Forsskål (Perciformes: Scaridae), inhabiting the Red Sea off Egypt; S. psittacus represents a new host record for both species. The taxonomic status of Schistorchis sensu stricto Lühe, 1906 is examined and revised, a key to the four species we consider in this genus offered, and the monotypic genus Megacreadium Nagaty, 1956 declared a junior synonym of Schistorchis. Members of Schistorchis sensu stricto possess a unique "complex" (i.e. highly cellular/glandular) instead of "simple" (i.e. entirely muscular) type of oral sucker that is quite large in relation to body size; an elongate, somewhat sub-rectangular-shaped body; 5+ testes arranged in at least two rows; caeca that open via separate ani; a long post-testicular region; a median genital pore either at the anterior margin of or just anterior to the ventral sucker; and species of Schistorchis sensu stricto parasitize the intestine of marine fish within the Order Tetraodontiformes Berg. With the revision of this genus, we re-describe Schistorchis carneus Lühe, 1906 from the lower and mid-intestine of the white-spotted puffer, Arothron hispidus (Linnaeus) (Tetraodontiformes: Tetraodontidae), collected in the Red Sea off Egypt. Finally, a plea is made for further study of the Megaperidae n. comb. focusing, in particular, on the following: (1) obtaining new type/voucher materials of Plesioschistorchis manteri (Gupta & Tandon, 1984) n. comb. and Schistorchis paruchini Kurochkin, 1974; (2) elucidating the life histories (i.e. intermediate hosts) of members of the Postporinae Yamaguti, 1958 and Schistorchiinae; and (3) generating DNA sequence data for more species of megaperids to help future workers produce increasingly accurate taxonomic classifications that better reflect phylogenetic relationships within this ecologically diverse group of digeneans.

First report on distribution of heavy metals and proximate analysis in marine edible puffer fishes collected from Gulf of Mannar Marine Biosphere Reserve, South India.

In the present study, the heavy metal concentration in different organs (skin, tissue, liver, kidney, gill, intestine, and ovary) and muscle proximate composition were studied in marine edible puffer fishes Takifugu oblongus, Lagocephalus guentheri, Arothron hispidus, Chelonodon patoca and Arothron immaculatus collected from Mandapam fish landing centre, South east coast of India. Heavy metals (Cd, Cu, Pb & Zn) were analyzed in different organs for the above mentioned species. The heavy metals concentration ranges in fish organs of all the five species were Cu (0.42 -6.31 mg/kg), Cd (0.01-0.79 mg/kg), Pb (5.80-19.87 mg/kg), and Zn (6.75-65.08 mg/kg). Zn was detected higher in all the samples followed by Pb, Cu and Cd. The proximate composition was determined in edible muscle tissues of all the five species. The highest and lowest protein contents were observed in T. oblongus (20.6 ± 0.6%) and C. patoca (17.9 ± 0.3%). In the present study, heavy metal concentrations were found very high in all the internal organs when compared to muscle tissues. Further, this is the first report on distribution of heavy metals and proximate compositions of commercialized important edible puffer fishes from Mandapam coast of Gulf of Mannar, Southeast coast of India.

Pufferfish mortality associated with novel polar marine toxins in Hawaii.

Fish die-offs are important signals in tropical marine ecosystems. In 2010, a mass mortality of pufferfish in Hawaii (USA) was dominated by Arothron hispidus showing aberrant neurological behaviors. Using pathology, toxinology, and field surveys, we implicated a series of novel, polar, marine toxins as a likely cause of this mass mortality. Our findings are striking in that (1) a marine toxin was associated with a kill of a fish species that is itself toxic; (2) we provide a plausible mechanism to explain clinical signs of affected fish; and (3) this epizootic likely depleted puffer populations. Whilst our data are compelling, we did not synthesize the toxin de novo, and we were unable to categorically prove that the polar toxins caused mortality or that they were metabolites of an undefined parent compound. However, our approach does provide a template for marine fish kill investigations associated with marine toxins and inherent limitations of existing methods. Our study also highlights the need for more rapid and cost-effective tools to identify new marine toxins, particularly small, highly polar molecules.

Complete mitochondrial DNA sequence of globe fish Tetraodon lineatus (Linnaeus, 1758) and the phylogenetic analysis of tetraodontidae.

Globe fish, Tetraodon lineatus (Linnaeus, 1758) is an ornamental freshwater fish in China. In this study, the complete mitochondrial genome of globe fish was first determined. The entire mitochondrial DNA sequence (mtDNA) sequence was 164595bp in length and consisted of 13 protein-coding genes, 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs) and a control region (CR). Its mitochondrial genome had the common features with those of other bony fishes with respect to gene arrangement, base composition, and tRNA structures.

TaqMan qPCR for detection and quantification of mitochondrial DNA from toxic pufferfish species.

Outbreaks of pufferfish food poisoning have been reported worldwide, most were from unsuitable food preparation and adulteration. In order to rapidly detect pufferfish adulterant in processed foods, we developed and proposed the use of a TaqMan probe-based quantitative PCR. The designed detection oligos targeted a unique region in 16S rDNA of toxic marine pufferfish in Tetraodontidae Family and gave a positive signal at ≥1.75 pg of genomic DNA. Non-target DNA samples from other fish, chicken, and beef were negative.

Developmental stages and fecundity of Lepeophtheirus simplex (Copepoda: Caligidae) parasitic on bullseye puffer fish (Sphoeroides annulatus).

Lepeophtheirus simplex Ho, Gómez et Fajer-Avila, 2001 is a parasite of Sphoeroides annulatus (Jenyns), an economically important fish species, with potential for aquaculture, in northwestern Mexico. The goal of this study was to describe the developmental stages under experimental conditions and seasonal fecundity of this parasite on wild fish. There are two naupliar, one copepodid, two chalimus and two pre-adult stages preceding the adult of L. simplex. The results support previous findings, which point out that the life cycle of the caligid copepods includes only six post-naupliar stages. The generation time from egg extrusion to adult for L. simplex was approximately 10 days at 22 °C. The body length of the ovigerous females ranged between 2.2 and 4.1 mm, and its fecundity between 12 and 36 eggs per string. Fecundity was negatively correlated with the egg size and positively correlated with the egg string length. Our data did not reveal significant differences in fecundity among sampling months, but ovigerous females were significantly larger in March (when water temperature was 22 °C) than in June and July (when water temperature was 30 °C). To some extent, our fecundity results contrast with those found in species of sea lice from higher latitudes. Undoubtedly, biological information on different species of sea lice from different environmental conditions will enhance our understanding of their infection strategies and will be valuable, given the increasing interest in marine fish farming in Mexico.

Characterization and evolutionary analysis of tributyltin-binding protein and pufferfish saxitoxin and tetrodotoxin-binding protein genes in toxic and nontoxic pufferfishes.

Understanding the evolutionary mechanisms of toxin accumulation in pufferfishes has been long-standing problem in toxicology and evolutionary biology. Pufferfish saxitoxin and tetrodotoxin-binding protein (PSTBP) is involved in the transport and accumulation of tetrodotoxin and is one of the most intriguing proteins related to the toxicity of pufferfishes. PSTBPs are fusion proteins consisting of two tandem repeated tributyltin-binding protein type 2 (TBT-bp2) domains. In this study, we examined the evolutionary dynamics of TBT-bp2 and PSTBP genes to understand the evolution of toxin accumulation in pufferfishes. Database searches and/or PCR-based cDNA cloning in nine pufferfish species (6 toxic and 3 nontoxic) revealed that all species possessed one or more TBT-bp2 genes, but PSTBP genes were found only in 5 toxic species belonging to genus Takifugu. These toxic Takifugu species possessed two or three copies of PSTBP genes. Phylogenetic analysis of TBT-bp2 and PSTBP genes suggested that PSTBPs evolved in the common ancestor of Takifugu species by repeated duplications and fusions of TBT-bp2 genes. In addition, a detailed comparison of Takifugu TBT-bp2 and PSTBP gene sequences detected a signature of positive selection under the pressure of gene conversion. The complicated evolutionary dynamics of TBT-bp2 and PSTBP genes may reflect the diversity of toxicity in pufferfishes.

The gas bladder of puffers and porcupinefishes (Acanthomorpha: Tetraodontiformes): phylogenetic interpretations.

The anatomy of the gas bladder of Diodontidae (porcupinefishes) and Tetraodontidae (pufferfishes) was studied on the basis of dissections and magnetic resonance imaging. Among the examined taxa of Tetraodontiformes, only puffers and porcupinefishes possess a thick walled and dorsally U-shaped or crescent-moon-shaped gas bladder. In the tetraodontid genus Lagocephalus the gas bladder is reduced to a rudiment. The species belonging to the genera Canthigaster, Arothron, and some species of Tetraodon differ in the positioning of their crescent-moon-shaped gas bladder. These observations confirm the close relationship of: (i) Diodontidae and Tetraodontidae and (ii) Canthigaster, Arothron, and some species of Tetraodon. The heterogeneity of the genus Tetraodon is supported by the gas bladder morphology, as previously suggested by molecular studies.

Envenenamento fatal por baiacu (Tetrodontidae): relato de um caso em criança / Fatal poisoning caused by puffer fish (Tetrodontidae): report of a case involving a child

É descrito um envenenamento pela ingestão de vísceras de um baiacu-pintado (Sphoeroides testudineus) por uma criança de dois anos, que apresentou sudorese fria, fraqueza muscular progressiva, parada cardiorrespiratória e morte. São discutidos os riscos do consumo da carne e vísceras de baiacus, fato comum em certas regiões do Brasil.

A case of poisoning resulting from ingestion of viscera from a spotted puffer fish (Sphoeroides testudineus) by a two-year-old child is described. The child presented cold sweating, progressive muscle weakness, cardiorespiratory arrest and death. The risks of consuming the meat and viscera of puffer fish, which is a common occurrence in certain regions of Brazil, are discussed.