Pufferfish belong to the Tetraodontidae family, which consists of 28 genera and 184 species; however, there are limited studies on the species from East Java, especially Pasuruan. This indicates the need for the collection of more accurate morphological and genetic resources as an important step for its conservation. Therefore, this study was conducted to identify pufferfish species found in the Lekok Coastal Waters, Pasuruan District, through morphological characterization and molecular analysis using DNA barcoding of the COI gene. A total of 12 samples were collected from the Lekok Coastal Waters, Pasuruan Regency. Fin samples were stored in 96% absolute ethanol. DNA extraction, polymerase chain reaction (PCR), and sequence analysis were performed using bioinformatics tools, the BOLD system, and the Automatic Barcode Gap Discovery (ABGD) web platform. Phenetic taxonomy was further applied using Ntysc to enhance the robustness of genetic analysis. The results showed that there were identified five pufferfish species were identified: Lagocephalus spadiceus, Chelonodontops patoca, Cyclichthys orbicularis, Arothron stellatus, and Arothron reticularis. High haplotype diversity (Hd) of 1 and nucleotide diversity (π) of 0.1 indicate significant genetic variation. The frequency of parsimonious informative sites was 22.6%, with 13 polymorphic sites and an overall ts/tv ratio of 2.2. The phylogenetic tree showed unambiguous branching patterns among species. These findings are supported by morphological and molecular identification results, which indicate the presence of five species of pufferfish along the Lekok Coast, Pasuruan Regency.

Pufferfish are an economically important food in Asia despite the potential risk of tetrodotoxin (TTX) poisoning. To promote food safety by ensuring the correct identification of pufferfish species, we developed common and species-specific primer sets for four Lagocephalus species (Lagocephalus spadiceus, Lagocephalus cheesemanii, Lagocephalus wheeleri, and Lagocephalus inermis) based on analysis of mitochondrial DNA cytochrome c oxidase subunit I (COI) in various pufferfish species commonly distributed and/or legally sold in Korea. The common primers were developed based on complete sequence data acquired from GenBank. The total length of fragments amplified by the common primer set was 1280 bp. Then, species-specific multiplex polymerase chain reaction (PCR) amplification was conducted for the four target species, obtaining 980 bp for L. spadiceus, 859 bp for L. cheesemanii, 672 bp for L. wheeleri, and 563 bp for L. inermis. Multiplex PCR is an important tool for the simple, rapid, accurate, and simultaneous identification of target species. The newly developed primer sets will contribute to reducing the occurrence of TTX poisoning and protect consumer rights by eradicating the mislabeling or fraudulent use of pufferfish products.

Heavy metals in Lagocephalus spadiceus (Richardson, 1845) from the Beibu Gulf, China: occurrence and health risk assessment

To elucidate the influence of marine environmental factors on the distribution of Lagocephalus spadiceus, we analyzed fisheries and environmental survey data from the Beibu Gulf in April (spring) and August (autumn), 2022. We used the generalized additive model (GAM) to examine the relationship between the distribution of L. spadiceus and marine environmental factors, both before and after the implementation of a fishing moratorium. We showed that the density of L. spadiceus in the Beibu Gulf exhibited significant spatio-temporal variation, which concentrated in the central and northern parts of the gulf during spring and autumn, respectively. Such variation in resource density may be attributed to factors such as chlorophyll a content, salinity, pH, and latitude. The GAM accounted for 91.3% of the cumulative variance in resource density. The resource density of L. spadiceus increased with the increases of chlorophyll content, while it began to decrease after chlorophyll reaching 5 μg·L-1. Both salinity and pH demonstrated a nonlinear relationship with resource density, characterized by multiple peaks, while latitude was positively correlated with resource density. This research provides valuable scientific evidence for the conservation of L. spadiceus resources.

A new species of Heterobothrium (Monogenea: Diclidophoridae) from half-smooth golden pufferfish Lagocephalus spadiceus in the Gulf of Tonkin, Vietnam, with an identification key for the genus

The demersal fish populations constitute a valuable commercial resource in the Beibu Gulf, northern South China. However, our understanding of the ontogenetic dietary shifts of fish, particularly Lagocephalus spadiceus, which is the most widely distributed fish in the northern South China Sea, is lacking. Therefore, we conducted a study using high-throughput sequencing and stable isotope analyses to examine the diet of L. spadiceus. The individuals were collected during 2 bottom trawl surveys in April and August 2022. The length distribution showed that small L. spadiceus was more abundant in shallow water, whereas large fish were generally found in offshore water. High-throughput sequencing indicated that L. spadiceus mainly fed on crustaceans and fish. Ontogenetic changes in diet composition and stable isotope values were also observed. As L. spadiceus grew, they started preying on larger individuals, specifically fish. The 15N enrichment in large fish supported to small fish supports these findings. The trophic niche indicated a segregation between individuals in shallow waters (mostly small fish) and offshore waters (mostly large fish), which helps in reducing the competition within the species. The L. spadiceus occupied the role of mid-predator (trophic level ~ 3.66) and increased with body length and water depth. Consequently, both high-throughput sequencing and stable isotope analyses provided evidence of intraspecific dietary variation and trophic niche partitioning of L. spadiceus in the Beibu Gulf, South China Sea.

In recent decades, hypoxic areas have rapidly expanded worldwide in estuaries and coastal zones. The Pearl River Estuary (PRE), one of China's largest estuaries, experiences frequent seasonal hypoxia due to intense human activities and eutrophication. However, the ecological effects of hypoxia in the PRE, particularly on fish communities, remain unclear. To explore these effects, we collected fish community and environmental data in July 2021 during the summer hypoxia development period. The results revealed that bottom‐layer dissolved oxygen (DO) in the PRE ranged from 0.08 to 5.71 mg/L, with extensive hypoxic zones (DO ≤ 2 mg/L) observed. Hypoxia has varied effects on fish community composition, distribution, species, and functional diversity in the PRE. A total of 104 fish species were collected in this study, with approximately 30 species (28.6%) exclusively found in hypoxic areas. Species responses to hypoxia varied: species such as Sardinella zunasi, Coilia mystus, and Nuchequula nuchalis were sensitive, while Decapterus maruadsi, Siganus fuscescens, and Lagocephalus spadiceus showed higher tolerance. Within the hypoxia area, dissolved oxygen was the main limiting factor for fish community diversity. Functional diversity (FDiv) decreased with higher dissolved oxygen levels, indicating a potential shift in the functional traits and ecological roles of fish species in response to changing oxygen conditions. Further analysis demonstrated that dissolved oxygen had a significantly stronger effect on fish community structure at hypoxic sites than in the whole PRE. Moreover, other environmental variables also had significant effects on the fish community structure and interacted with dissolved oxygen in the hypoxia area. These findings suggest that maintaining sufficient dissolved oxygen levels is essential for sustaining fish communities and ecosystem health in the PRE. This study provides novel insights into the effects of hypoxia on fish communities in estuarine ecosystems and has significant implications for the ecological health and management of the PRE.

The climate fluctuations during the Late Pleistocene significantly influenced the phylogeographic structure and historical dynamics of marine fishes in the marginal seas of the western Pacific Ocean. The puffer fish, Lagocephalus spadiceus, holds substantial nutritional and economic value in the South China Sea. To investigate the demographic history and population structure of the L. spadiceus, the mitochondrial DNA COI and Cyt b gene datasets from 300 individuals across eight populations in the South China Sea were sequenced. Our findings revealed high haplotype diversity (0.874 ± 0.013) and low nucleotide diversity (0.00075 ± 0.00058). The phylogenetic tree and haplotype networks revealed no significant genetic differentiation along the northern coast of South China Sea. Neutrality tests, mismatch distribution analyses, and Bayesian skyline plots suggested that L. spadiceus underwent population expansion during the Late Pleistocene. Both ocean currents and climate change significantly influenced the geographical distribution and genetic population structure of L. spadiceus.

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

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

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

The brain of the wild toxic marine pufferfishes accumulates tetrodotoxin

Intensive mechanized fishing has induced marked alterations in coastal marine ecosystems resulting in proliferation of low-value, nuisance species in marine fish catches. Assessment of 75 shrimp trawl catches taken in the near-shore waters off Goa, west coast of India during active fishing periods from 2006 – 2008 revealed that the demersal pufferfish, Lagocephalus spadiceus constituted ≈ 27 % by weight of the trawl catch during January – March, and its catch was inversely related to prey and predator species. Sub-tidal rock reefs and submerged rocky patches in the study area offer suitable substrates for spawning, and the predominance of adults during these months suggesting spawning migration. Comparison of the present data with published literature indicates a significant reduction in puffer predators (catfishes). Excessive removal of high-value predators favours the proliferation of pufferfish and its establishment as a meso-predator, probably triggering a potential trophic cascade. The paper discusses the role of removal of High Trophic Level (HTL) species in the proliferation of a meso-predator and its impact on ecosystem function.

The Bangladeshi reef fish species have not been studied thoroughly. An attempt has been made for taxonomic identification of coral-associated fish through morphological studies. Mostly dead and few live fishes were collected from local fishermen, fish landing zone, and fish markets in St. Martin’s Island, Bay of Bengal, Bangladesh, from October 2015 to July 2016, visiting seven times. Samples were also obtained from local boys who collected fishes by using hook and line. The study recorded the first occurrence of 16 coral-associated fish species such as Caesio cuning, Lethrinus ornatus, Upeneus suahelicus, Upeneus asymmetricus, Scarus taeniopterus, Scarus zufar, Bodianus neilli, Parapercis clathrata, Parapercis diplospilus, Pomadasys furcatus, Siganus fuscescens, Acanthopagrus berda, Gerres erythrourus, Lagocephalus spadiceus, Psettodes bennettii, and Heteroconger perissodon. Among them, Parapercis clathrata, Parapercis diplospilus belongs to the family Pinguipedidae before, no species was recorded under this family from Bangladesh and a rare and endemic species, Scarus zufar, of the Arabian Sea off Dhofar (Zufar), Oman in the Western Indian Ocean, was recorded for the first time in the Northern Indian Ocean in the northeastern part of Bay of Bengal. This study increased the number of coral-associated fish from 240 to 256 species and the number of families from 54 to 55 in the coral reef ecosystem of the Bay of Bengal, Bangladesh.
 Bangladesh J. Zool. 48(2): 263-288, 2020

Examination of the dorsal spinule patch in Lagocephalus spadiceus specimens revealed the existence of two intraspecific forms, the “wheeleri”-form, characterized by an elliptical patch, and the “spadiceus”-form, with a rhomboidal patch with a continuous or intermittent posterior extension. The forms were not indicative of sexual dimorphism, although the appearance of a posterior extension of the dorsal patch was apparently growth-related. Ontogenetic studies of both forms, including genetic analyses and cross breeding experiments, should be future considerations.

Human-induced biological introductions pose a major threat to global biodiversity, and this is especially frequent in the eastern Mediterranean region, which is a globally important biodiversity hotspot area of high conservation value. To predict at which level introduced species in this region might become invasive under current and projected climate conditions, 232 non-native aquatic organisms were screened using the Aquatic Species Invasiveness Screening Kit. Based on receiver operative characteristic curve analysis, thresholds were identified to distinguish between low, medium and high risk species. The “top invasive” (very high risk) species identified were: brown bullhead Ameiurus nebulosus, blue crab Callinectes sapidus, gibel carp Carassius gibelio, Philippine catfish Clarias batrachus, Chinese mitten crab Eriocheir sinensis, bluespotted cornetfish Fistularia commersonii, silver carp Hypophthalmichthys molitrix, silver-cheeked toadfish Lagocephalus sceleratus, half-smooth golden pufferfish Lagocephalus spadiceus, Suez pufferfish Lagocephalus suezensis, signal crayfish Pacifastacus leniusculus, fathead minnow Pimephales promelas, channeled applesnail Pomacea canaliculata, red swamp crayfish Procambarus clarkii, devil firefish Pterois miles and European catfish Silurus glanis. The risk of being invasive of more than half of the screened species increased after taking global warming predictions into account, and several species considered to be globally invasive (cf. high risk) were classified as posing only a medium risk for the eastern Mediterranean region. Region-specific risk screenings, as implemented in this study, are therefore essential for setting priorities in preventative management for the conservation of key biodiversity hotspots and the optimal allocation of resources in view of full risk assessment for the species identified as (very) high risk.

Background. The population of Lagocephalus spadiceus (Richardson, 1845), one of the Lessepsian species that migrated from the Red Sea to the Mediterranean Sea through the Suez Canal, has been growing rapidly in Turkey in recent years. This situation poses a serious danger to fisheries and human health. There is little information about the biology of this species. The presently reported study permits a greater understanding of the age and growth characteristics of the pufferfish L. spadiceus from the Gulf of Iskenderun, north-eastern Mediterranean. Materials and methods. Samples were captured by commercial bottom trawlers at depths of 24–50 m during 9 trawling operations conducted during the fishing seasons between 2012 and 2013 in the Gulf of Iskenderun, north-eastern Mediterranean. Age determinations were carried out using vertebrae. Images of vertebrae suitable for age determination were obtained with a dissecting microscope. The index of the mean percentage error (IMPE) was calculated to assess the precision of the age determinations between two independent readers. Growth parameters for all individuals were then determined by fitting observed and length-at-age data using the von Bertalanffy growth equation. In addition, the condition factor (CF) was determined. Results. A total of 1116 specimens (529 females and 587 males) were sampled, ranging from 6.7 to 34.0 cm in total length and from 4.30 to 557.64 g in weight. The female-to-male ratio was 1:1.1. Age determination was conducted using vertebral band counts. The age of the examined specimens ranged from 0 to 10 years. One female individual was determined to be 17 years of age (45.6 cm and 1269.0 g). The von Bertalanffy growth equations were Lt = 47.21[1 – e–0.116(t + 2.04)] and Wt = 1296.18 [1 – e–0.116(t + 2.04)]2.846 for females, Lt = 46.36 [1 – e–0.133(t + 1.76)] and Wt = 1381.86 [1 – e–0.133(t + 1.76)]2.817 for males and Lt = 46.90 [1 – e–0.120(t + 1.97)] and Wt = 1345.67 [1 – e–0.120(t + 1.97)]2.831 for all individuals. The growth performance index (Ф') value was calculated as 2.421 for all individuals. The condition factor ranged from 0.890 to 2.768 for females and from 0.899 to 2.767 for males. Conclusion. It is very important to investigate L. spadiceus, an invasive species, in order to prevent its harmful effects on fisheries and human health. As this study is the first study on the age and growth of L. spadiceus in the north-eastern Mediterranean, it will significantly contribute to future ecosystem conservation and management strategies.

SummaryThe effects of season and gender on the proximate composition and fatty acid profiles of the most widespread pufferfish species in the Mediterranean Sea (Lagocephalus sceleratus, Lagocephalus spadiceus, Lagocephalus suezensis and Torquigener flavimaculosus) caught in Mersin Bay, in the north‐eastern Mediterranean during four seasons were investigated. The results showed that the crude protein, lipid, moisture and ash level in pufferfish were in the range of 18.7%–21.7%, 0.61%–1.93%, 76.1%–78.6% and 1.08%–1.87%, respectively. All of the pufferfish had significant levels of polyunsaturated fatty acid (PUFA). However, the consumption of L. sceleratus, L. suezensis and T. flavimaculosus may be risky due to the possibility of having tetrodotoxin in their muscles. Nevertheless, L. spadiceus is a pufferfish species that is popularly consumed in East Asian countries, and is of nutritional interest and commercial potential in the Mediterranean region as well so its proximate composition and fatty acid profile are of interest.

This study aimed at comparing the elemental levels of muscle and liver tissues of four pufferfish (Lagocephalus sceleratus, Lagocephalus spadiceus, Lagocephalus suezensis, and Torquigener flavimaculosus), which are common in the Northeastern Mediterranean Sea (Mersin Bay), in terms of sex and seasonal variables. Na, Mg, P, K, and Ca levels in the muscle tissues of pufferfish range from 1848.4 to 16495.7, 824.4 to 2684.5, 6936.2 to 20486.6, 13019.3 to 36165.5, and 276.4 to 5566.5μgg-1, respectively. Na, Mg, P, K, and Ca levels in the liver tissues of pufferfish range from 531.8 to 6136.1, 63.8 to 899.1, 773.1 to 6677.5, 1151.4 to 10165.4, and 47.4 to 1607.7μgg-1, respectively. The levels of macroelements in the muscle and liver tissues can be represented as K > P ≥ Na > Ca ≥ Mg and K ≥ P ≥ Na > Ca ≥ Mg, respectively. Pufferfish muscle tissue has more abundant macroelement levels than liver tissue. Fe, Cu, and Zn levels in pufferfish muscle tissues range from 21.53 to 219.42, 0.73 to 6.54, and 25.38 to 100.47μgg-1, respectively. Fe, Cu, and Zn levels in pufferfish liver tissues range from 157.8 to 1368.6, 0.86 to 11.23, and 23.17 to 507.75μgg-1, respectively. Levels of trace elements in the muscle and liver tissues can be represented as Zn ≥ Fe > Cu and Fe ≥ Zn > Cu, respectively. The pufferfish liver tissue is richer than muscle tissue in terms of trace element levels. Cd, Pb, As, and Cr levels in pufferfish muscle tissues range from 0.66 to 6.30, 2.17 to 20.76, 41.90 to 258.37, and 0.34 to 3.70μgg-1, respectively. Cd, Pb, As, and Cr levels in the liver tissues of pufferfish range from 0.22 to 2.34, 0.17 to 2.27, 9.35 to 61.93, and 0.14 to 2.10μgg-1, respectively. Levels of metal in the muscle and liver tissues can be represented as As > Pb > Cd ≥ Cr and As > Cd ≥ Pb ≥ Cr, respectively. Pufferfish muscle tissue has a higher association with liver tissue due to metal level accumulation. All pufferfish are contaminated by Cd, Pb, As, Cr, Fe, and Zn metals.

Tetrodotoxin levels of three pufferfish species (Lagocephalus sp.) caught in the North-Eastern Mediterranean sea