Local Differences in the Toxin Amount and Composition of Tetrodotoxin and Related Compounds in Pufferfish (Chelonodon patoca) and Toxic Goby (Yongeichthys criniger) Juveniles.

Tetrodotoxin (TTX)-bearing fish ingest TTX from their preys through the food chain and accumulate TTX in their bodies. Although a wide variety of TTX-bearing organisms have been reported, the missing link in the TTX supply chain has not been elucidated completely. Here, we investigated the composition of TTX and 5,6,11-trideoxyTTX in juveniles of the pufferfish, Chelonodon patoca, and toxic goby, Yongeichthys criniger, using LC-MS/MS, to resolve the missing link in the TTX supply chain. The TTX concentration varied among samples from different localities, sampling periods and fish species. In the samples from the same locality, the TTX concentration was significantly higher in the toxic goby juveniles than in the pufferfish juveniles. The concentration of TTX in all the pufferfish juveniles was significantly higher than that of 5,6,11-trideoxyTTX, whereas the compositional ratio of TTX and 5,6,11-trideoxyTTX in the goby was different among sampling localities. However, the TTX/5,6,11-trideoxyTTX ratio in the goby was not different among samples collected from the same locality at different periods. Based on a species-specific PCR, the detection rate of the toxic flatworm (Planocera multitentaculata)-specific sequence (cytochrome c oxidase subunit I) also varied between the intestinal contents of the pufferfish and toxic goby collected at different localities and periods. These results suggest that although the larvae of the toxic flatworm are likely to be responsible for the toxification of the pufferfish and toxic goby juveniles by TTX, these fish juveniles are also likely to feed on other TTX-bearing organisms depending on their habitat, and they also possess different accumulation mechanisms of TTX and 5,6,11-trideoxyTTX.

Fish Cytolysins in All Their Complexity.

The majority of the effects observed upon envenomation by scorpaenoid fish species can be reproduced by the cytolysins present in their venoms. Fish cytolysins are multifunctional proteins that elicit lethal, cytolytic, cardiovascular, inflammatory, nociceptive, and neuromuscular activities, representing a novel class of protein toxins. These large proteins (MW 150-320 kDa) are composed by two different subunits, termed α and ß, with about 700 amino acid residues each, being usually active in oligomeric form. There is a high degree of similarity between the primary sequences of cytolysins from different fish species. This suggests these molecules share similar mechanisms of action, which, at least regarding the cytolytic activity, has been proved to involve pore formation. Although the remaining components of fish venoms have interesting biological activities, fish cytolysins stand out because of their multifunctional nature and their ability to reproduce the main events of envenomation on their own. Considerable knowledge about fish cytolysins has been accumulated over the years, although there remains much to be unveiled. In this review, we compiled and compared the current information on the biochemical aspects and pharmacological activities of fish cytolysins, going over their structures, activities, mechanisms of action, and perspectives for the future.

The Geographic Distribution, Venom Components, Pathology and Treatments of Stonefish (Synanceia spp.) Venom.

Stonefish are regarded as one of the most venomous fish in the world. Research on stonefish venom has chiefly focused on the in vitro and in vivo neurological, cardiovascular, cytotoxic and nociceptive effects of the venom. The last literature review on stonefish venom was published over a decade ago, and much has changed in the field since. In this review, we have generated a global map of the current distribution of all stonefish (Synanceia) species, presented a table of clinical case reports and provided up-to-date information about the development of polyspecific stonefish antivenom. We have also presented an overview of recent advancements in the biomolecular composition of stonefish venom, including the analysis of transcriptomic and proteomic data from Synanceia horrida venom gland. Moreover, this review highlights the need for further research on the composition and properties of stonefish venom, which may reveal novel molecules for drug discovery, development or other novel physiological uses.

Combined proteomic and functional analysis reveals rich sources of protein diversity in skin mucus and venom from the Scorpaena plumieri fish.

The biological activities observed upon envenomation by Scorpaena plumieri could be linked to both the venom and the skin mucus. Through a proteomic/functional approach we analyzed protein composition and biological activities of the venom and skin mucus. We identified 885 proteins: 722 in the Venomous Apparatus extracts (Sp-VAe) and 391 in the Skin Mucus extract (Sp-SMe), with 494 found exclusively in Sp-VAe, being named S. plumieri Venom Proteins (Sp-VP), while 228 were found in both extracts. The majority of the many proteins identified were not directly related to the biological activities reported here. Nevertheless, some were classified as toxins/potentially interesting molecules: lectins, proteases and protease inhibitors were detected in both extracts, while the pore-forming toxin and hyaluronidase were associated with Sp-VP. Proteolytic and anti-microbial activities were linked to both extracts, while the main toxic activities - cardiovascular, inflammatory, hemolytic and nociceptive - were elicited only by Sp-VAe. Our study provided a clear picture on the composition of the skin mucus and the venom. We also show that the classic effects observed upon envenomation are produced by molecules from the venomous gland. Our results add to the growing catalogue of scorpaeniform fish venoms and their skin mucus proteins. SIGNIFICANCE: In this study a large number of proteins - including classical and non-classical toxins - were identified in the venomous apparatus and the skin mucus extracts of the Scorpaena plumieri fish through shotgun proteomic approach. It was shown that the toxic effects observed upon envenomation are elicited by molecules originated from the venomous gland. These results add to the growing catalogue of scorpaeniform fish venoms and their skin mucus proteins - so scarcely explored when compared to the venoms and bioactive components of terrestrial animals. Data are available via ProteomeXchange with identifier PXD009983.

A review on the Scorpaena plumieri fish venom and its bioactive compounds

The most poisonous fish species found along the Brazilian coast is the spotted scorpionfish Scorpaena plumieri. Though hardly ever life-threatening to humans, envenomation by S. plumieri can be quite hazardous, provoking extreme pain and imposing significant socioeconomic costs, as the victims may require days to weeks to recover from their injuries. In this review we will walk the reader through the biological features that distinguish this species as well as the current epidemiological knowledge related to the envenomation and its consequences. But above all, we will discuss the challenges involved in the biochemical characterization of the S. plumieri venom and its compounds, focusing then on the successful isolation and pharmacological analysis of some of the bioactive molecules responsible for the effects observed upon envenomation as well as on experimental models. Despite the achievement of considerable progress, much remains to be done, particularly in relation to the non-proteinaceous components of the venom. Therefore, further studies are necessary in order to provide a more complete picture of the venoms chemical composition and physiological effects. Given that fish venoms remain considerably less studied when compared to terrestrial venoms, the exploration of their full potential opens a myriad of possibilities for the development of new drug leads and tools for elucidating the complex physiological processes.

Maturity-related changes in venom toxicity of the freshwater stingray Potamotrygon leopoldi.

Aquatic venomous animals such as stingrays represent a largely untapped source for venom-based drug development. However, the major challenge for a potential drug development pipeline is the high inter- and intraspecific variability in toxicity and venom composition. As of today, little is known about maturity-driven changes in these traits in stingrays. The present study investigates the differences in toxicity and venom composition in different maturity stages of the freshwater stingray Potamotrygon leopoldi. This species can be found in the Xingú River basin (Brazil), where it mainly feeds on invertebrates, while being predated by other stingrays or large catfishes. P. leopoldi, as commonly known for stingrays, can cause severe injuries with the venomous dentine spine located at its tails. The toxicity of tissue extracts of juvenile and mature specimens was recorded on a myoblast cell culture bioassay. Venom composition and bioactivity of compounds were analyzed with planar chromatography linked to an Aliivibrio fischeri bioassay. Results revealed a decrease in venom toxicity during maturation, but no changes in venom composition. These findings may indicate that toxicity in mature specimens becomes evolutionary less important, probably due to a decrease in predation pressure.

Systemic rhabdomyolysis induced by venom of freshwater stingrays Plesiotrygon iwamae and Potamotrygon motoro (Chondrichthyes-Potamotrygonidae) from the Amazon Basin.

Injuries caused by freshwater stingrays are characterized by intense pain and pathological changes at the lesion site, including oedema, erythema and, in most cases, necrosis. In this study, the systemic myotoxic activity induced by mucus extracts from the dorsal region and stinger of the stingrays Plesiotrygon iwamae and Potamotrygon motoro was described, analysed and quantified. Twenty-four hours after injection of 400 µg of the extracts into the gastrocnemius muscle of mice, the following effects were observed: coagulative necrosis of the muscle tissue, muscle fibre regeneration and the presence of inflammatory infiltrates, including neutrophils, macrophages, and a reduced number of eosinophils and lymphocytes. These changes were also observed, although to a lesser extent, in the gastrocnemius muscles of the contralateral limbs, demonstrating that the extracts from the two species could induce systemic rhabdomyolysis. Based on morphometric analysis, it was observed that the stinger extract of P. motoro was more potent in inducing local and systemic myotoxic activity, followed by the dorsal extract from P. motoro and stinger and dorsal extracts from P. iwamae, which induced similar effects.

Specialization of the sting venom and skin mucus of Cathorops spixii reveals functional diversification of the toxins.

Cathorops spixii is the most common venomous fish on the Brazilian coast. Apart from the involvement with defense against pathogens, the possible contribution of skin mucus components to the development of injuries caused by venomous fish species has not been investigated. Thus, the present study was conducted to gain a better understanding of the peptide and protein components of fish skin mucus and the sting venom from the catfish C. spixii. Our results show that sting venom and skin mucus have distinct constituents that distinguished them like structural proteins, chaperones, ion transport, carbohydrate metabolism, oxidoreductase, cell cycle and protein binding present in sting venom and like tropomyosin 3 isoform 2 and energy metabolim proteins in skin mucus. But in a group of common 13 proteins we identified and isolated a WAP65 protein. The peptide fractions caused more harmful effects, such as venular stasis, hemorrhage and changes in the arteriolar wall diameter, and the protein fractions produced a typical inflammatory process in post-capillary venules. And finally we showed for the first time the presence WAP65 in sting venom and skin mucus of C. spixii using LC/MS/MS and also we purified this protein in the sting venom. Wap65 shows inflammatory action, working at different doses inducing an increase in the number of leukocytes rolling and adhering to the endothelium.

Insights into the local pathogenesis induced by fish toxins: role of natterins and nattectin in the disruption of cell-cell and cell-extracellular matrix interactions and modulation of cell migration.

Combined proteomic and transcriptomic approaches to study the composition of the venom of Thalassophryne nattereri venomous fish revealed the primary structures of the major toxins as a family of proteases natterins, never described on venoms and a C-type lectin nattectin. To gain new insights into the mechanisms of venom pathogenesis and to further elucidate the role of its major toxins, the natterins and nattectin, we undertook in vitro investigations using these isolated toxins. Here we demonstrated the specific ability of the nattectin to bind types I and V collagen and natterins to bind and cleave type I collagen as well as type IV collagen, disrupting cell attachment and HeLa cells survival. Natterins have cytotoxic effect on both adherent cells or at in suspension, showing direct induction of necrosis that is followed by cell detachment. Nattectin improves integrin-mediated HeLa cell adhesion and resistance to apoptosis by its binding to RGD-dependent integrins, especially the ß1 subunit. Based on our studies we now report that extracellular matrix (ECM) components as well as the integrin ß1 subunit are targets for the natterins and nattectin. The ECM degradation or remodeling activities exerted by these toxins affect cell-cell and cell-ECM adhesion and survival and impair inflammatory cell migration into inflamed tissues.

Studies on pharmacological properties of mucus and sting venom ofPotamotrygon cf. henlei

Stingrays from the Potamotrygon cf. henlei species are widely distributed in high numbers throughout therivers of central-west Brazil, being the source of numerous envenomations occurring in the dry season, posinga serious public health problem even if not properly reported. The accidents usually involve fishermen andbathers, and to date there is no effective treatment for the injured. Considering these facts and limitations ofstudies aiming at understanding the effects induced by P. cf. henlei envenoming, this study aimed to describethe principal pharmacological and certain biochemical properties of the mucus and sting venom. We foundthat mucus and sting venom is toxic to mice having nociceptive, edematogenic and proteolysis activities. Ourresults also indicate that the inflammatory cellular influx observed could be triggered by the venom andmucus. Furthermore the venom and mucus were partially purified by solid-phase extraction tested forantimicrobial activity in which only the mucus presented activity. It could be inferred from the present studythat P. cf. henlei venom possesses a diverse mixture of peptides, enzymes and pharmacologically activecomponents.

Insights into the local pathogenesis induced by fish toxins: Role ofnatterins and nattectin in the disruption of cell–cell and cell–extracellularmatrix interactions and modulation of cell migration

Combined proteomic and transcriptomic approaches to study the composition of the venom of Thalassophryne nattereri venomous fish revealed the primary structures of the major toxins as a family of proteases natterins, never described on venoms and a C-type lectin nattectin. To gain new insights into the mechanisms of venom pathogenesis and to further elucidate the role of its major toxins, the natterins and nattectin, we undertookin vitro investigations using these isolated toxins. Here we demonstrated the specific ability of the nattectin to bind types I and V collagen and natterins to bind and cleave type I collagen as well as type IV collagen, disrupting cell attachment and HeLa cells survival. Natterins have cytotoxic effect on both adherent cells or at in suspension, showing direct induction of necrosis that is followed by cell detachment. Nattectin improves integrinmediated HeLa cell adhesion and resistance to apoptosis by its binding to RGD dependent integrins, especially the b1 subunit. Based on our studies we now report that extracellular matrix (ECM) components as well as the integrin b1 subunit are targets for the natterins and nattectin. The ECM degradation or remodeling activities exerted by these toxins affect cell–cell and cell–ECM adhesion and survival and impair inflammatory cell migration into inflamed tissues.

A novel RGD-toxin protein, Lj-RGD3, from the buccal gland secretion of Lampetra japonica impacts diverse biological activities.

RGD (Arg-Gly-Asp) motif toxin proteins from snake venoms, saliva glands secretion of leech or tick have typical characteristics of inhibiting platelet aggregation, angiogenesis, and tumor growth. Here we report cloning and characterization of a novel RGD-toxin protein from the buccal gland of Lampetra japonica. In an attempt to study the activities of anticoagulant in the buccal gland secretion of L. japonica, we established buccal gland cDNA library and identified a gene encoding a predicted protein of 118 amino acids with 3 RGD motifs. The predicted protein was named Lj-RGD3. We generated the cDNA of Lj-RGD3 and obtained the recombinant protein rLj-RGD3. The polyclonal antibodies against rLj-RGD3 recognized the native Lj-RGD3 protein in buccal gland secretion in Western blot analyses. The biological function studies reveal that rLj-RGD3 inhibited human platelet aggregation in a dose-dependent manner with IC(50) value at 5.277 µM. In addition, rLj-RGD3 repressed bFGF-induced angiogenesis in the chick chorioallantoic membrane model. rLj-RGD3 also inhibited the adhesion of ECV304 cells to vitronectin. Furthermore, rLj-RGD3 induced apoptosis and significantly inhibited proliferation, migration, and invasion evoked by bFGF in ECV304 cells. Taken together, these results suggested that rLj-RGD3 is a novel RGD-toxin protein possessing typical functions of the RGD-toxin protein.

Pardaxin, a fish toxin peptide interaction with a biomimetic phospholipid/polydiacetylene membrane assay.

Pardaxin is a fish toxin belonging to the alpha-helical, pore-forming peptide family, used in toxicological and biophysical research to study toxin-cell and -lipid-artificial membranes interactions. We investigated the membrane interaction of two pardaxin analogues using a colorimetric phospholipid/polydiacetylene biomimetic assay. In this assay, polydiacetylene undergoes visible, concentration dependent, blue-red transformation induced through interactions of pardaxins with the vesicle membrane. Pardaxins P4 and P5, are composed of 33 amino acids, but differ in a single amino acid substitution at the carboxy-terminal (G31 to D31, respectively) known to decrease the pore forming activity. Addition of pardaxins in the colorimetric assay induced dose-dependent color transitions with different kinetics. The colorimetric analysis could distinguish between different pardaxins-membrane interaction profiles, suggesting bilayer surface association for P4 and vesicle membrane penetration for P5. The colorimetric assay could distinguish between pardaxins membrane interaction profiles although circular dichroism spectra of vesicle-interacting pardaxins did not indicate a significant difference in the secondary structure between these two toxin analogues. The colorimetric platform utilized in the present report represents a useful assay with general applications for studying membrane interactions of peptides in general and pore-forming toxins in particular, and may become an important tool for evaluating quantitative toxin structure-activity relationship.

Detection of diarrhoetic shellfish toxins in mussels from Italy by ionspray liquid chromatography-mass spectrometry.

Direct detection of okadaic acid (OA), dinophysistoxin-1 (DTX-1) and some of their related compounds in toxic mussels (Mytilus galloprovincialis) is reported using ionspray liquid chromatography-mass spectrometry (LC-ISP-MS). This was employed to analyse diarrhoetic shellfish poisoning (DSP) toxins in mussels collected from coastal areas of the northern and southern Adriatic Sea. DTX-1 was found in some samples from both the northern and southern Adriatic and this is the first report of the unambiguous identification of this toxin in Italian mussels. The low levels found indicate that this toxin did not play a significant role in toxicity in these samples. Okadaic acid was found in all the mussels examined, although its concentration was not always sufficient to account for DSP toxicity. Furthermore, two related compounds of OA were detected in all the samples and one related DTX-1 compound was observed in some samples from the northern Adriatic. All three compounds are still to be identified, but it is possible that these substances are involved in mussel DSP toxicity in the Adriatic Sea.

[Fatal ichthyosarcotoxism after eating shark meat. Implications of two new marine toxins]. / Ichtyosarcotoxisme mortel après consommation de requin. Mise en cause de deux nouvelles toxines marines.

A fish poisoning involving 188 hospitalizations occurred in November 1993, in Manakara, a middle-sized town on the south-east coast of Madagascar, following the ingestion of shark. A single shark was involved in this poisoning and was identified as Carcharinus leucas. There was no unusual characteristic of this shark or its meat. The attack rate was about 100%. First clinical signs appeared within five to ten hours after ingestion. The patients presented neurological symptoms almost exclusively, the most prominent being a constant, severe ataxia. Gastrointestinal troubles, like diarrhoea and vomiting were rare. The overall case-fatality ratio was close to 30% among the 200 poisoned inhabitants. Search for similar poisoning previously reported in this area was negative, and fishermen in Manakara usually eat that kind of shark without mistrust. Bacteriological and chemical origins were eliminated. Two liposoluble toxins were isolated from the shark liver and tentatively named carchatoxin-A and -B respectively. They were distinct from ciguatoxin in chromatographic properties.

Detection of a cytolytic toxin in the venom of the stonefish (Synanceia trachynis).

The venom of the stonefish, Synanceia trachynis, contains a cytolytic toxin which is antigenic and ammonium sulfate-precipitable, and has a pI of ca 5.7 and an Mr of ca 158,000. The toxin is a potent but narrow-spectrum cytolysin which is lytic in vitro for rabbit, dog, rat, and guinea pig erythrocytes, in that order, but is largely or completely inactive against sheep, cow, human, monkey, mouse, goat, horse, burro and cat erythrocytes. Fractionation of the venom by molecular sieve fast protein liquid chromatography and isoelectric focusing did not separate the haemolytic activity from the venom's lethal and vascular permeability-increasing activities. Also, the three activities were destroyed by heat, proteases, Congo red, potassium permanganate and stonefish antivenoms. The results suggest that the haemolytic, lethal and vascular permeability-increasing activities of stonefish venom are properties of the same molecule, a previously unrecognized, membrane-damaging protein toxin.

Purification of a lethal fraction from the venom of the weever fish, Trachinus vipera C.V.

We report the purification of a lethal fraction of Weever fish venom using preparative electrophoresis. It was found to be composed of four identical subunits with an overall mol. wt of approximately 324,000. A 20 g mouse was killed instantly by an i.v. injection of 2 micrograms.

Occurrence of palytoxin in the trigger fish Melichtys vidua.

The trigger fish Melichtys vidua, collected at Pingelap Atoll in Ponape in The Federated States of Micronesia, contained a potent toxin in the viscera, flesh, and digestive tracts with contents. The toxic principle appeared identical to palytoxin on the basis of toxicity and chromatographic properties.

Occurrence of pahutoxin and homopahutoxin in the mucus secretion of the Japanese boxfish.

The mucus secreted by the Japanese boxfish O. immaculatus was extracted with n-butanol. The extract was fractionated by silica gel open column chromatography and reversed phase high performance liquid chromatography to give two hemolytic components. The major hemolysin was identical with pahutoxin which was previously isolated from the Hawaiian boxfish O. lentiginosus, while the other hemolysin was a new choline chloride ester, named homopahutoxin.

Hemolytic, lethal and edema-forming activities of the skin secretion from the oriental catfish (Plotosus lineatus).

The crude extract from the skin secretion of the oriental catfish, Plotosus lineatus, contained at least one hemolysin, two lethal factors and two edema-forming factors; the lethal and edema-forming factors seem to be identical. The molecular weights of the hemolysin and the lethal factors (edema-forming factors) were estimated to be 180,000 and 12,000, respectively. Peculiar secretory cells, which resembled the venom glandular cells of the dorsal and pectoral stings, were observed in the epidermis.