Lessepsian migration and tetrodotoxin poisoning due to Lagocephalus sceleratus in the eastern Mediterranean

Background

The Suez Canal permits migration of fish from the Indo-Pacific Ocean to the Mediterranean Sea. This phenomenon (Lessepsian migration) has enabled poisonous fish species to colonize the Mediterranean Sea.

Objective

To report clinical tetrodotoxin poisoning after consumption of the Lessepsian immigrant fish Lagocephalus sceleratus caught on the Israeli coast of the eastern Mediterranean.

Case series

Thirteen patients aged 26-70 years were admitted after consuming L. sceleratus. Signs of toxicity appeared within 1 h. The main manifestations included vomiting, diarrhea, headache, paraesthesias, slurred speech, muscle weakness, dyspnea, hypertension, tachycardia, respiratory arrest, seizures and coma. Treatment was supportive, including mechanical ventilation (two patients). Patients recovered within 4 days. All fish were identified as L. sceleratus, a species known to contain tetrodotoxin.

Discussion

The diagnosis of tetrodotoxin poisoning was suggested by typical clinical manifestations together with temporal proximity to consumption of tetrodotoxin-containing fish. To the best of our knowledge, this is the first case series of tetrodotoxin poisoning reported from the eastern Mediterranean and due to L. sceleratus. Man made disruption of the ecological balance has resulted in the spread of tetrodotoxin-containing fish from the Indo-Pacific region to the Mediterranean Sea. Increased awareness is required to identify tetrodotoxin poisoning in an atypical fauna.     

Toxicity assessment of Amphidinium carterae, Coolia cfr. monotis and Ostreopsis cfr. ovata (Dinophyta) isolated from the northern Ionian Sea (Mediterranean Sea)

In many coastal areas the abundant proliferation of microalgae producing biotoxins determines the occurrence of Harmful Algal Blooms (HABs). Their presence in temperate waters is well documented and often associated with marine toxin-derived disease. The occurrence and toxicity of three harmful microalgae (Amphidinium carterae, Coolia cfr. monotis and Ostreopsis cfr. ovata) from the northern Ionian Sea (Mediterranean Sea) is hereby reported. The three dinoflagellates were sampled both on macroalgae and water and their morphology and occurrence were compared to those of other Mediterranean sites. The toxicity of the three cultured strains was tested by Artemia salina and hemolysis tests and their effects on the first stages of the sea urchin development was also evaluated. The contemporary presence of the three species inhibited the in vitro sea urchin embryonic development. But this action could be ascribed to the sole Ostreopsis as the addition of the single species to the sea urchins embryos evidenced no effects in presence of Amphidinium or Coolia cells, and an irregular segmentation in presence of Ostreopsis. In particular, this latter species exerted a cytotoxic effect in a dose-dependent manner, with a production of deformed embryos even at very low cell concentration (42 cells mL⁻¹). Nevertheless, when algal cell lysate was added, some effects on the sea urchin development was detected for each dinoflagellate, and also in this case Ostreopsis has proved to be the most toxic species. However, the lysate of Amphidinium and Ostreopsis strongly affects the A. salina nauplii vitality, while the hemolytic activity was very low for Amphidinium and Coolia lysate and very strong for Ostreopsis.Our results highlight the importance to monitoring the presence of these dinoflagellates whose effects may also be reflected on the early life stages of marine organisms, especially those species that are important from both an ecological and economic point of view, as the sea urchins are.     

First episode of shellfish contamination by palytoxin-like compounds from Ostreopsis species (Aegean Sea, Greece).

In order to investigate the toxicity of Ostreopsis species present in Greek coastal waters, cultures of Ostreopsis sp. and Ostreopsis ovata, mixed Ostreopsis field populations and shellfish collected from coastal waters of North Aegean Sea during late summer and autumn periods of 2004, 2005 and 2006 were examined by both mouse bioassay (MBA) and hemolysis neutralization assay (HNA). MBA testing was based on two different extraction protocols, while HNA also included the use of ouabain, a known palytoxin (PLT) antagonist. Results indicated the presence of a compound in both Ostreopsis cells and shellfish tissues, which was strongly toxic to mice. This compound exhibited characteristic symptomatology in mice (death, numbness, waddling gait and blindness) to that of PLT, as well as delayed hemolytic activity, which was neutralized by ouabain. HNA indicated that Ostreopsis cells contained a PLT-like compound (putative PLT, p-PLT) at concentrations ranging between 0.4 and 0.9 pg/cell, whereas concentration in shellfish tissues was estimated to range from about 33.3 to 97.0 microg p-PLT/kg tissue. To our knowledge, this is the first report of p-PLT contamination of shellfish by natural Ostreopsis species populations in European coastal waters and possibly globally, and also the first evidence on Ostreopsis cells' toxicity in the Eastern Mediterranean Sea.     

First report on toxins in the Panamanian toads Atelopus limosus, A. glyphus and A. certus

Major toxins from skin extracts of 18 specimens of six Atelopus toad species collected in Panama were analyzed. Chiriquitoxin was identified using ¹H NMR in A. limosus and A. glyphus for the first time. Zetekitoxin in A. zeteki and tetrodotoxin in A. varius, A. chiriquiensis and A. zeteki were identified again. Furthermore, A. certus was suggested to contain a water-soluble toxin other than tetrodotoxin.     

Concentrations of Tetrodotoxin (TTX) and Its Analogue 4,9-Anhydro TTX in Different Tissues of the Silver-Cheeked Pufferfish (Lagocephalus sceleratus,Gmelin, 1789) Caught in the South-Eastern Mediterranean Sea,Lebanon

Pufferfishes are among the best-known marine organisms that accumulate marine biotoxins such as Tetrodotoxin (TTX). In the Mediterranean Sea, the silver-cheeked toadfish Lagocephalus sceleratus is the most reported TTX-bearer, causing many fatal and non-fatal cases. In Lebanon, no previous studies have measured TTX levels although the possibility of TTX-poisoning is high since L. sceleratus is caught in different sizes and can be mistaken with other small fishes. Hence, this study reports TTX and its analogue 4,9-anhydro TTX in L. sceleratus collected from Lebanese waters in the Eastern Mediterranean Sea. The results show that TTX concentrations in fish tissues varied between 0.10 and 252.97 µg/g, while those of 4,9-anhydro TTX oscillated between 0.01 and 43.01 µg/g. Internal organs of L. sceleratus were the most toxic parts of its body, with the highest TTX levels found in gonads (mainly ovaries) and liver, followed by the muscles and skin with concentrations always exceeding the safety level. Toxicity fluctuations of L. sceleratus, its expansion, ecological and economic effects were also elucidated. Based on the present findings, it has been confirmed that L. sceleratus constitutes a health, ecological and economic risks, and therefore its trade in seafood markets should be banned to avoid any potential intoxication.     

Difference in the localization of tetrodotoxin between the female and male pufferfish Takifugu niphobles, during spawning

In order to understand the sexual differences in TTX-usage in the pufferfish, Takifugu niphobles, localization of TTX and toxin amount in tissues of mature male and female specimens were investigated by immunohistochemical methods using anti-TTX antibody and LC/MS analysis. Subsequently, differences in the immunohistochemical signals were compared with the amount of TTX. The paraffin-embedded sections of the skin, muscle, liver, gonad and intestinal tract were subjected to anti-TTX monoclonal antibody based on the fluorescent immunohistochemical techniques. Immuno-positive reaction was observed in the skin and liver in males, and the skin and ovary in females. In the skin, TTX was localized at the epidermis, the basal cell layer, the mucous cells and the sacciform cells, and with intense immunoreaction at the flat epithelial cell layer and the sacciform cells. The signal from the liver cells was stronger in males than in females. The intensity of the signal from the tissues correlated with the toxin amounts therein. These results suggest that tissue distributions of TTX and toxin amount in the pufferfish were sex-dependent.     

Toxicological studies of Karlodinium micrum (Dinophyceae) isolated from East China Sea

Karlodinium micrum (Strain NMBjah047) was isolated from the water samples of East China Sea (ECS). The hemolytic, ichthyotoxic, and cytotoxic activities of the algae was characterized. Embryotoxicity of both intra and extracellular extracts were also tested on a local sea urchin species. The algal intracellular hemolytic toxicity averaged about 87.5% at different algal growth phases. However, extracellular hemolytic activity depended on the population growth phase. The toxicity increased with the increase in the population size, reaching the highest hemolytic activity during the stationary phase, and maintained a relatively high activity even when the population declined. Time and density dependent ichthyotoxicity to Lateolabrax maculates juveniles was also detected. The LD₅₀ in 24 h was 1.1 × 10⁵ cells/mL. Inhibition of the fertilized egg hatching was also observed and estimated the IC₅₀ in 40 h with 3.5 × 10⁴ cells/mL. Extracellular extracts of K. micrum dense culture also showed significant cytotoxic activity on HUVEC (IC₅₀ = 70.8 μg/mL). A dose dependent acute toxicity to embryos of sea urchin was also determined. The algal intracellular and extracellular extracts delayed or even restricted the embryological development of the sea urchin, illustrating the potential toxicity of K. micrum not only to vertebrates, but also to marine invertebrates. The hemolytic compounds in the ECS strain were extracted and analyzed. At least two fractions had significant hemolytic activities. A lipid-like compound, named Digalactosyldiacylglycerol (DGDG), was suggested to be responsible for the hemolytic activity in one of these fractions. From the results of the present studies, this strain of K. micrum isolated from the East China Sea might be considered a toxic strain with hemolytic activity, ichthyotoxicity, cytotoxicity and embryotoxicity.     

Differential accumulation of paralytic shellfish toxins from Alexandrium minutum in the pearl oyster, Pinctada imbricata

To investigate the potential for differential accumulation of paralytic shellfish toxins (PSTs) in various tissues of the akoya pearl oyster, Pinctada imbricata, two feeding trials were carried out using the PST-producing dinoflagellate, Alexandrium minutum. When fed with A. minutum at concentrations between 100 and 1300 cells ml⁻¹, the maximum clearance by P. imbricata was shown to occur at a density of 300 cells ml⁻¹. When fed twice daily at this rate for up 12 days, P. imbricata accumulated analogues of gonyautoxins (GTXs): GTXs 1,4 and 2,3. The levels of GTXs in the viscera increased progressively on days 4, 8 and 12 to peak at 17.9 ± 4.47 μg STX-equivalent 100 g⁻¹ biomass. Following 12 days of depuration, in the absence of A. minutum, GTX levels fell by approximately 65% to 6.0 ± 2.20 μg STX-equivalent 100 g⁻¹ biomass. No GTX was found in the oysters at the start of the trial or in untreated controls. The accumulation of GTX was found to be tissue specific. No GTX was detected in the muscle tissue of P. imbricata during the feeding trial.     

First report of (homo)anatoxin-a and dog neurotoxicosis after ingestion of benthic cyanobacteria in The Netherlands

In April and May 2011, three dogs died and one dog became ill after swimming in Lake IJmeer (The Netherlands). At the time, the lake was infested with the benthic cyanobacterial species Phormidium. A Eurasian Coot (Fulica atra) and a Black-headed Gull (Chroicocephalus ridibundus) also died near Lake IJmeer in the same period. One of the dogs and both birds were subjected to a pathological investigation. Furthermore, the Phormidium mat; algal samples from the dikes; contents of the animals’ digestive systems and organ tissues were analysed for the following cyanobacterial toxins: (homo)anatoxin-a; (7-deoxy-)cylindrospermopsin; saxitoxins and gonyautoxins by LC-MS/MS. Samples were also analysed for the nontoxic (homo)anatoxin-a metabolites dihydro(homo)anatoxin-a and epoxy(homo)anatoxin-a. The dog necropsy results indicated neurotoxicosis and its stomach contained Phormidium filaments. Anatoxin-a was detected in the Phormidium mat (272 μg g⁻¹ dry weight, stdev 65, n = 3) and in the dog’s stomach contents (9.5 μg g⁻¹ dry weight, stdev 2.4, n = 3). Both samples also contained the anatoxin-a metabolite dihydroanatoxin-a, and a trace of homoanatoxin-a was detected in the Phormidium mat. The birds were in bad nutritive condition at the time of necropsy and their stomachs and intestines did not contain any cyanobacterial material. Furthermore, no cyanobacterial toxins were detected in their stomachs, intestines and organs and they both had lesions that are not associated with cyanobacterial intoxication. This is the first report of anatoxin-a and homoanatoxin-a occurrence in The Netherlands, these toxins have likely caused the deaths of three dogs. The birds probably died of other causes. Dutch recreational waters are at this moment only screened for pelagic cyanobacterial species, the current bathing water protocol therefore does not protect humans and animals from negative effects of blooms of benthic cyanobacteria.     

Toxicity of cylindrospermopsin, and other apparent metabolites from Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum, to the zebrafish (Danio rerio) embryo

Cyanobacteria produce a diverse array of toxic or otherwise bioactive compounds that pose growing threats to human and environmental health. We utilized the zebrafish (Danio rerio) embryo, as a model of vertebrate development, to investigate the inhibition of development pathways (i.e. developmental toxicity) by the cyanobacterial toxin, cylindrospermopsin (CYN), as well as extracts from various isolates of Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum. CYN was toxic only when injected directly into embryos, but not by direct immersion at doses up to 50 μg/ml. Despite the dose dependency of toxicity observed following injection of CYN, no consistent patterns of developmental defects were observed, suggesting that toxic effects of CYN may not target specific developmental pathways. In contrast, direct immersion of embryos in all of the extracts resulted in both increased mortality and reproducible, consistent, developmental dysfunctions. Interestingly, there was no correlation of developmental toxicity observed for these extracts with the presence of CYN or with previously reported toxicity for these strains. These results suggest that CYN is lethal to zebrafish embryos, but apparently inhibits no specific developmental pathways, whereas other apparent metabolites from C. raciborskii and A. ovalisporum seem to reproducibly inhibit development in the zebrafish model. Continued investigation of these apparent, unknown metabolites is needed.     

Variability of tetrodotoxin and of its analogues in the red-spotted newt, Notophthalmus viridescens (Amphibia: Urodela: Salamandridae)

Efts and adult specimens (n = 142) of the red-spotted newt Notophthalmus viridescens from various locations in Canada and USA were analyzed for the presence of tetrodotoxin (TTX) and of its analogues 6-epitetrodotoxin and 11-oxotetrodotoxin. Considerable individual variations in toxin levels were found within and among populations from New Hampshire, New York, Pennsylvania, and Virginia ranging from non-detectable to 69 μg TTX per g newt. TTX and its analogues were absent in efts and adults from various locations in the Canadian province Nova Scotia, the northernmost distribution of the newt, and in adults from Florida. Newts kept in captivity for several years and reared on toxin-free diet lost their toxicity. Bayesian and maximum likelihood phylogenetic analysis of specimens from the various populations using three phylogenetic markers (COI, ND2 and 16S RNA) revealed that populations from the northern states of the USA and Canada are genetically homogenous, whereas the newts from Florida exhibited a much higher level of genetic divergence. An exogenous source of TTX in the newts either via the food chain or by synthesis of symbiotic bacteria is suggested to explain the high variability and lack of TTX in certain populations.     

Detection of tetrodotoxin from the grey side-gilled sea slug - Pleurobranchaea maculata, and associated dog neurotoxicosis on beaches adjacent to the Hauraki Gulf, Auckland, New Zealand

Investigations into a series of dog poisonings on beaches in Auckland, North Island, New Zealand, resulted in the identification of tetrodotoxin (TTX) in the grey side-gilled sea slug, Pleurobranchaea maculata. The levels of TTX in P. maculata, assayed by liquid chromatography-mass spectrometry (LC-MS) ranged from 91 to 850 mg kg^-1 with a median level of 365 mg kg^-1 (n = 12). In two of the dog poisoning cases, vomit and gastrointestinal contents were found to contain TTX. Adult P. maculata were maintained in aquaria for several weeks. Levels of TTX decreased only slightly with time. While in the aquaria, P. maculata spawned, with each individual producing 2-4 egg masses. The egg masses and 2-week old larvae also contained TTX. Tests for other marine toxins were negative and no other organisms from the area contained TTX. This is the first time TTX has been identified in New Zealand and the first detection of TTX in an opisthobranch.     

Intracellular domoic acid production in Pseudo-nitzschia multistriata isolated from the Gulf of Naples (Tyrrhenian Sea, Italy)

Twenty-six Pseudo-nitzschia multistriata cultures were tested for intracellular domoic acid production and fourteen were found to be toxic. Four suboptimal growth conditions were compared with conditions observed to be optimal to explore possible triggers for intracellular domoic acid production. Silica- and phosphate-limitation and low light treatment induced elevated toxin concentrations whereas high temperature appeared to suppress it. Inheritance of the toxin-production ability was investigated by measuring intracellular toxin content in a total of thirty-nine F₁ strains from two different crosses. Results showed radical differences in domoic acid levels among the F₁ offspring from the same parents.     

A review of the natural history, toxinology, diagnosis and clinical management of Nerium oleander (common oleander) and Thevetia peruviana (yellow oleander) poisoning

Nerium oleander (common oleander) and Thevetia peruviana (yellow oleander) are potentially lethal plants after ingestion. Poisoning by these plants is a common toxicological emergency in tropical and subtropical parts of the world and intentional self-harm using T. peruviana is prevalent in South Asian countries, especially India and Sri Lanka. All parts of these plants are toxic, and contain a variety of cardiac glycosides including neriifolin, thevetin A, thevetin B, and oleandrin. Ingestion of either oleander results in nausea, vomiting, abdominal pain, diarrhoea, dysrhythmias, and hyperkalemia. In most cases, clinical management of poisoning by either N. oleander or T. peruviana involves administration of activated charcoal and supportive care. Digoxin specific Fab fragments are an effective treatment of acute intoxication by either species. However, where limited economic resources restrict the use of such Fab fragments, treatment of severely poisoned patients is difficult. Data from case reports and clinical studies were reviewed to identify treatments supported by evidence for the management of poisoning by N. oleander and T. peruviana.     

First record of human envenomation by Atractaspis congica Peters, 1877 (Squamata: Atractaspididae)

We describe here the first known documented case of human envenomation by the burrowing asp Atractaspis congica. The patient received a single fang puncture in the index finger and suffered local pain, discolouration, blistering and swelling of the finger, hand and forearm. Analgesics were given, but no antivenom. Recovery was complete within 48 h, saves some moderate swelling, blistering and hypersensitivity.     

Anti-invasive and anti-adhesive activities of a recombinant disintegrin, r-viridistatin 2, derived from the Prairie rattlesnake (Crotalus viridis viridis)

Snake venom disintegrins inhibit platelet aggregation and have anti-cancer activities. In this study, we report the cloning, expression, and functional activities of a recombinant disintegrin, r-viridistatin 2 (GenBank ID: JQ071899), from the Prairie rattlesnake. r-Viridistatin 2 was tested for anti-invasive and anti-adhesive activities against six different cancer cell lines (human urinary bladder carcinoma (T24), human fibrosarcoma (HT-1080), human skin melanoma (SK-Mel-28), human colorectal adenocarcinoma (CaCo-2), human breast adenocarcinoma (MDA-MB-231) and murine skin melanoma (B16F10)). r-Viridistatin 2 shares 96% and 64% amino acid identity with two other Prairie rattlesnake medium-sized disintegrins, viridin and viridistatin, respectively. r-Viridistatin 2 was able to inhibit adhesion of T24, SK-MEL-28, HT-1080, CaCo-2 and MDA-MB-231 to various extracellular matrix proteins with different affinities. r-Viridistatin 2 decreased the ability of T24 and SK-MEL-28 cells to migrate by 62 and 96% respectively, after 24 h of incubation and the invasion of T24, SK-MEL-28, HT-1080 and MDA-MB-231 cells were inhibited by 80, 85, 65 and 64% respectively, through a reconstituted basement membrane using a modified Boyden chamber. Finally, r-viridistatin 2 effectively inhibited lung colonization of murine melanoma cells in BALB/c mice by 71%, suggesting that r-viridistatin 2 could be a potent anti-cancer agent in vivo.     

Toxicity of Bothrops neuwiedi complex (“yarará chica”) venom from different regions of Argentina (Serpentes, Viperidae)

We report a study of toxic and enzymatic activities of Bothrops neuwiedi complex venoms collected from specimens of different regions of Argentina and a pool of these same venoms. Were determined lethal, hemorrhagic and pro-coagulant (plasma and fibrinogen) doses and the neutralization of these activities by a bivalent antivenom. The electrophoretic pattern of different regions venom was studied by SDS-PAGE. All samples exhibited lethal potencies, hemorrhagic and coagulant (plasma and fibrinogen) activities with potencies concordant with previous studies. The only conspicuous difference in the toxicological pattern of Bothrops diporus venoms was the low-thrombin-like activity found in one sample. The antivenom used in this study could neutralize all the toxic activities tested and the neutralizing potency of the antivenom was comparable for all samples. Despite the wide distribution of B. neuwiedi complex throughout Argentina and the evident morphological variation between B. diporus (B. neuwiedi complex), this study establishes a remarkably similar toxicity profile throughout its range. This is the first systematic study on the regional variation of enzymatic and toxic activities of venom from species belonging to the B. neuwiedi complex, one of the snakes of highest sanitary importance in South America and their neutralization by the type of antivenom most commonly used in the South of South America.     

中国南海群海绵Agelas mauritiana的化学成分研究

目的 对采自南中国海海域的群海绵(Agelas mauritiana)的化学成分进行研究。方法采用硅胶色谱柱、Sephadex LH-20凝胶色谱柱、高效液相色谱等色谱方法,对Agelas mauritiana正丁醇萃取物进行分离纯化;应用现代波谱技术对化合物进行化学结构鉴定;用MTT法对化合物进行体外人肺癌细胞株A549细胞生长抑制活性测试。结果 共分离得到8个化合物,分别鉴定为:agelasine A(1)、agelasine B(2)、epi-agelasine C(3)、(-)agelasine D(4)、agelasine E(5)、agelasine F(6)、(-)ageloxime D(7)、aurantiamide acetate(8)。体外活性筛选中,这些化合物对A549显示出不同程度的生长抑制活性,化合物1~3的活性与阳性对照阿霉素相近。结论 化合物1、2、4、5、6、8为首次从该种海绵中分离得到。首次选用A549对化合物1~7的活性进行评价,化合物2、3的显著生长抑制活性为进一步深入研究提供了依据。     

Antioxidant activities of the essential oils and methanol extracts from myrtle (Myrtus communis var. italica L.) leaf,stem and flower

This study was designed to examine the chemical composition and antioxidant activity of the essential oils and methanol extracts of Myrtus communis var. italica L. leaf, stem and flower. Myrtle leaf and flower were the valuable organs for the essential oil production representing a yield of 0.61% and 0.30% (w/w), respectively. The essential oil composition of myrtle leaf and flower was characterized by high proportions of α-pinene, the main compound of monoterpene hydrocarbon class, with 58.05% for leaf and 17.53% for flower. Stem was rich in oxygenated monoterpenes, largely due to 1,8-cineole with 32.84%. The total phenol contents varied between different myrtle parts; leaf extract had higher total phenol content (33.67 mg GAE/g) than flower (15.70 mg GAE/g) and stem (11.11 mg GAE/g) extracts. Significant differences were also found in total tannin contents among different myrtle parts, representing 26.55 mg GAE/g in leaf, 11.95 mg GAE/g in flower, 3.33 mg GAE/g in stem. The highest contents of total flavonoids and condensed tannins were observed in stem (5.17 and 1.99 mg CE/g, respectively) and leaf (3 and 1.22 mg CE/g, respectively) extracts. The HPLC analysis indicated that the main phenolic class was hydrolysable tannins (gallotannins) in leaf (79.39%, 8.90 mg/g) and flower (60.00%, 3.50 mg/g) while the stem was characterized by the predominance of flavonoid class (61.38%, 1.86 mg/g) due to the high presence of catechin (36.91%, 1.12 mg/g). Antioxidant activities of the essential oil and the methanolic extract from different myrtle parts were evaluated by using DPPH radical scavenging, β-carotene-linoleic acid bleaching, reducing power and metal chelating activity assays. In all tests, methanolic extracts of different myrtle parts showed better antioxidant activity than essential oils.