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Butterflies of the genus Danaus are known to sequester toxic cardenolides from milkweed host plants (Apocynaceae). In particular, Danaus plexippus efficiently sequesters and stores these compounds, whereas D. chrysippus, is considered to poorly sequester cardenolides. To estimate its sequestration capability compared with that of D. plexippus, larvae of both species were jointly reared on Asclepias curassavica and the major cardenolides of the host plant, calotropin and calactin, were analyzed in adults sampled at different time intervals after eclosion. Both cardenolides were detected in body and wings of D. plexippus. Whereas the calotropin-concentration remained constant over a period of 24 days, that of calactin steadily decreased. In the body, but not in the wings of D. chrysippus, calactin only was detected in low amounts, which was then almost completely lost during the following 8 days after eclosion, suggesting that in contrast to D. plexippus, cardenolides seem to be less important for that butterfly's defence against predators., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Caterpillars of the monarch butterfly, Danaus plexippus, feed on milkweed plants, Asclepias spp. (Apocynaceae), and sequester their toxic cardenolides aimed at deterring predators. Nevertheless, Chinese praying mantids, Tenodera sinensis, consume these caterpillars after removing the midgut ("gutting") including its plant content. In the present study, monarch caterpillars raised on A. curassavica, and those of the death's-head hawkmoth, Acherontia atropos, raised on Atropa belladonna containing atropine, were fed to mantids, Hierodula membranacea, which removed the gut of both species discarding about 59% of cardenolides and more than 90% of atropine, respectively. The ingestion of these compounds produced no apparent ill effects in the mantids and both were excreted with faeces. On the other hand, when mantids were fed with larvae of two moth species, Amata mogadorensis and Brahmaea certia, raised on non-poisonous host plants, the mantids showed the same gutting behaviour, thereby discarding indigestible plant material. As polar compounds, e.g. cardenolides and atropine, are not absorbed from the mantids midgut and do not pass the gut membrane, this enables the mantids to feed on toxic prey., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Acid-sensing ion channels (ASICs) are proton-gated Na + channels that are expressed throughout the nervous system. ASICs have been implicated in several neuronal disorders, like ischemic stroke, neuronal inflammation, and pathological pain. Several toxins from venomous animals have been identified that target ASICs with high specificity and potency. These toxins are extremely useful in providing protein pharmacophores and to characterize function and structure of ASICs. Marine cone snails contain a high diversity of toxins in their venom such as conotoxins, which are short polypeptides stabilized by disulfide bonds, and conopeptides, which have no or only one disulfide bond. Whereas conotoxins selectively target specific neuronal proteins, mainly ion channels, the targets of conopeptides are less well known. Here, we perform an in vitro screen of venoms from 18 cone snail species to identify toxins targeting ASICs. We identified a small conopeptide of only four amino acids from the venom of Conus textile that strongly potentiated currents of ASIC3, which has a specific role in the pain pathway. This peptide, RPRFamide, belongs to the subgroup of cono-RFamides. Electrophysiological characterization of isolated dorsal root ganglion (DRG) neurons revealed that RPRFamide increases their excitability. Moreover, injection of the peptide into the gastrocnemius muscle strongly enhanced acid-induced muscle pain in mice that was abolished by genetic inactivation of ASIC3. In summary, we identified a conopeptide that targets the nociceptor-specific ion channel ASIC3., Competing Interests: The authors declare no conflict of interest.

The biosynthesis of tetrodotoxin (TTX), a potent neurotoxin consisting of a 2,4-dioxaadamantane skeleton and a guanidine moiety, is an unsolved problem in natural product chemistry. Recently, the first C5-C10 directly bonded TTX analogue, 4,9-anhydro-10-hemiketal-5-deoxyTTX, was obtained from toxic newts and its carbon skeleton suggested a possible monoterpene origin. On the basis of this hypothesis, screening of predicted biosynthetic intermediates of TTX was performed using two MS-guided methods. Herein, five novel cyclic guanidine compounds from toxic newts are reported which commonly contain a cis-fused bicyclic structure including a six-membered cyclic guanidine. These structures could be biosynthetically derived from geranyl guanidine through oxidation, cyclization, and/or isomerization steps. LC-MS analysis confirmed the widespread distribution of the five novel compounds in toxic newt species. These results support the hypothesis that TTX is derived from a monoterpene., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Animals in zoological gardens are at risk of severe and even lethal poisoning when they accidentally ingest toads. Here we report the case of an eleven month old male South American fur seal (Arctocephalus australis) which was found dead in its outdoor enclosure in the zoo of Dortmund, Germany. Autopsy revealed the presence of two adult, partly digested common toads (Bufo bufo) in the stomach. Toxicological analysis of the stomach content using high performance liquid chromatography coupled to time-of-flight mass spectrometry (LC-TOF MS) proved the presence of bufadienolides, the major cardiotoxic components of toad poisons. Using electrochemical luminescens immunoassay (ECLIA) compounds equivalent to digitoxin were detected in the blood sample confirming the absorption of toad poison components from the intestines into the circulation potentially leading to cardiac failure. In zoological gardens special precautions are necessary to protect non-native animals from encountering toads and the risk of poisoning, particularly in early spring, the spawning period of the toads.

The identification of novel tetrodotoxin (TTX, 1) analogues would significantly contribute to the elucidation of its biosynthetic pathway. In this study, the first C5-C10 directly bonded TTX analogues, 4,9-anhydro-10-hemiketal-5-deoxyTTX (2) and 4,9-anhydro-8-epi-10-hemiketal-5,6,11-trideoxyTTX (3), were found in the newt Cynops ensicauda popei by using a screening method involving HILIC-LC-MS/MS focused on the fragment ions of TTX analogues, and their structures were elucidated by spectroscopic methods. Compound 2 was detected in a wide range of newt species, and the 2 and TTX contents of 22 newt specimens were correlated (rs =0.88). Based on these results and its structural features, 2 was predicted to serve as a precursor of TTX that would be directly converted into 4,9-anhydroTTX (4) by Baeyer-Villiger-like oxidation or via 4,9-anhydro-5-deoxyTTX formed by cleavage of the C5-C10 bond. The bicyclic carbon skeletons of 2 and 3 suggested a possible monoterpene origin for TTX., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Jellyfish are distributed worldwide; they cause local skin injuries upon contact which are often followed by systemic signs of envenoming., Objectives: Which jellyfish species are of medical importance, which skin reactions and systemic symptoms occur, which first-aid measures and treatment options exist?, Methods: Review of the medical literature and discussion of first-aid and therapeutic options., Results: Jellyfish capable of causing skin injuries occur in almost all oceans. Several jellyfish species may cause severe, potentially lethal, systemic symptoms; they include the Portuguese man-of-war (Physalia physalis) and box jellyfish (Chironex fleckeri, Carukia barnesi, Chiropsalmus quadrigatus)., Conclusions: Among the injuries and envenoming symptoms caused by marine organisms, jellyfish dermatitis should not be underestimated. Skin reactions may not only a dermatological problem, but also be accompanied by complex systemic toxic symptoms which are a challenge for internists.

Frogs of the genus Phyllobates from Colombia are known to contain the highly toxic alkaloid batrachotoxin, but species from Central America exhibit only very low levels or are entirely free of this toxin. In the present study alcohol extracts from 101 specimens of Phyllobates lugubris and Phyllobates vittatus and 21 of three sympatric species (Dendrobates pumilio, Dendrobates auratus, Dendrobates granuliferus) from Costa Rica were analyzed by gas chromatography-mass spectrometry. Whereas the extracts of the Dendrobates species exhibited typical profiles of toxic alkaloids, those of the two Phyllobates species contained low levels of few alkaloids only, batrachotoxin was not detected. Although the feeding pattern of the Dendrobates and Phyllobates species are similar as revealed by examination of their stomach content (mainly ants and mites), the Phyllobates species are poorly sequestering alkaloids from their food source in contrast to the Dendrobates frogs., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Whereas interspecific associations receive considerable attention in evolutionary, behavioural and ecological literature, the proximate bases for these associations are usually unknown. This in particular applies to associations between vertebrates with invertebrates. The West-African savanna frog Phrynomantis microps lives in the underground nest of ponerine ants (Paltothyreus tarsatus). The ants usually react highly aggressively when disturbed by fiercely stinging, but the frog is not attacked and lives unharmed among the ants. Herein we examined the proximate mechanisms for this unusual association. Experiments with termites and mealworms covered with the skin secretion of the frog revealed that specific chemical compounds seem to prevent the ants from stinging. By HPLC-fractionation of an aqueous solution of the frogs' skin secretion, two peptides of 1,029 and 1,143 Da were isolated and found to inhibit the aggressive behaviour of the ants. By de novo sequencing using tandem mass spectrometry, the amino acid sequence of both peptides consisting of a chain of 9 and 11 residues, respectively, was elucidated. Both peptides were synthesized and tested, and exhibited the same inhibitory properties as the original frog secretions. These novel peptides most likely act as an appeasement allomone and may serve as models for taming insect aggression.

The International Society on Toxinology (IST) was founded in 1962 with its main purpose to advance knowledge on venoms, poisons and toxins of animal, plant and microbial origin. World Congresses of the Society and Symposia of the IST-Sections (European, Pan-American, Asia-Pacific) are regularly organized to promote communication and exchange of results in toxinology research. The officers of the Society, the President and Secretary-Treasurer, are assisted by a Council and they all are elected by the membership. IST sponsors the Redi Award to recognize distinguished work in the field of toxinology. Toxicon is the official journal of the Society., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Venoms from cone snails (Conidae) have been extensively studied during the last decades, but those from other members of the suborder Toxoglossa, such as of Terebridae and Turridae superfamilies attracted less interest so far. Here, we report the effects of venom and gland extracts from three species of the superfamily Terebridae. By 2-electrode voltage-clamp technique the gland extracts were tested on Xenopus oocytes expressing nicotinic acetylcholine receptors (nAChRs) of rat neuronal (α3β2, α3β4, α4β2, α4β4, α7) and muscle subtypes (α1β1γδ), and expressing potassium (Kv1.2 and Kv1.3) and sodium channels (Nav1.2, 1.3, 1.4, 1.6). The extracts were shown to exhibit remarkably high inhibitory activities on almost all nAChRs tested, in particular on the α7 subtype suggesting the presence of peptides of the A-superfamily from the venom of Conus species. In contrast, no effects on the potassium and sodium channels tested were observed. The venoms of terebrid snails may offer an additional source of novel biologically active peptides.

We report on the screening of ethanolic extracts from 33 deep-sea Antarctic marine sponges for different biological activities. We monitored hemolysis, inhibition of acetylcholinesterase, cytotoxicity towards normal and transformed cells and growth inhibition of laboratory, commensal and clinically and ecologically relevant bacteria. The most prominent activities were associated with the extracts from sponges belonging to the genus Latrunculia, which show all of these activities. While most of these activities are associated to already known secondary metabolites, the extremely strong acetylcholinesterase inhibitory potential appears to be related to a compound unknown to date. Extracts from Tetilla leptoderma, Bathydorus cf. spinosus, Xestospongia sp., Rossella sp., Rossella cf. racovitzae and Halichondria osculum were hemolytic, with the last two also showing moderate cytotoxic potential. The antibacterial tests showed significantly greater activities of the extracts of these Antarctic sponges towards ecologically relevant bacteria from sea water and from Arctic ice. This indicates their ecological relevance for inhibition of bacterial microfouling.

History: A 59-year-old woman went into coma after she had taken a phytopharmacon from Vietnamese generally used as an antidiabetic drug to treat her skin disease., Investigations: CT-scans revealed signs of pneumonia and cerebral edema. Severe brain damage was diagnosed by MRI-investigation. By chemical-toxicological analysis of the drug glibenclamide was identified in a concentration of 1.1 mg/g., Diagnosis, Treatment and Course: The patient developed severe irreversible encephalopathy. By neurorehabilitative treatment her physical status slightly improved, but she died after 13 months without regaining consciousness., Conclusions: The cause of losing consciousness remained unclear, however, severe hypoglycaemia following the use of the drug may be taken into account. An urgent warning against the use of such preparations is highly recommended., (© Georg Thieme Verlag KG Stuttgart · New York.)

Tetrodotoxin (TTX) and its analogue 6-epiTTX had been detected in the red-spotted newt, Notophthalmus viridescens. Thirty specimens of a population from Pennsylvania, USA were histologically examined for the presence of intestinal parasites. More than 50% were found to be infected with nematodes (Trichocephalidae), trematodes or cestodes (Pseudophyllidae). The mean values of TTX and 6-epiTTX in parasitized and in non-parasitized newts were not significantly different. Using a monoclonal antibody-based immunoenzymatic technique, TTX was localized in the intestinal tissue as well as in the parasites indicating that they accumulate the toxin and suggesting that TTX and 6-epiTTX are not providing protection from parasites to the newts., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Cone snails are venomous predatory marine neogastropods that belong to the species-rich superfamily of the Conoidea. So far, the mitochondrial genomes of two cone snail species (Conus textile and Conus borgesi) have been described, and these feed on snails and worms, respectively. Here, we report the mitochondrial genome sequence of the fish-hunting cone snail Conus consors and describe a novel putative control region (CR) which seems to be absent in the mitochondrial DNA (mtDNA) of other cone snail species. This possible CR spans about 700 base pairs (bp) and is located between the genes encoding the transfer RNA for phenylalanine (tRNA-Phe, trnF) and cytochrome c oxidase subunit III (cox3). The novel putative CR contains several sequence motifs that suggest a role in mitochondrial replication and transcription.

Venoms of the marine cone snails (Conus spp.) consist of numerous proteins and peptides showing a wide variety of biological activities such as on ion-channels and receptors. Peptides acting on neuronal nicotinic acetylcholine receptors belong to several peptide superfamilies including the recently described αD-conopeptides which are homodimers of identical peptides with 47-49 amino acids. Among the venom glands of 27 Conus species analyzed by cDNA cloning, precursors of αD-conopeptides were identified in four species only: C. betulinus, C. capitaneus, C. mustelinus, and C. vexillum. Phylogenetic analysis of the relationships among the αD-conopeptides revealed that they belong to clades, which are characterized by an AVV- and EMM-motif in the signal peptide sequence.

Poisoning after eating puffer fish containing highly lethal tetrodotoxin (TTX) is widespread in Asia. In 2008, naïve inland populations in Bangladesh were exposed to cheap puffer fish sold on markets. In three outbreaks, 141 patients with history of puffer fish consumption were hospitalized. Symptoms of poisoning included perioral paraesthesia, tingling over the entire body, nausea and vomiting, dizziness, headache, abdominal pain and muscular paralysis of the limbs. Seventeen patients (12%) died from rapidly developing respiratory arrest. Blood and urine samples from 38 patients were analyzed using a TTX-specific enzyme-linked immunoassay (ELISA). Medium to high TTX levels were detected (1.7-13.7 ng/ml) in the blood of 27 patients. TTX was below detection level (< 1.6 ng/ml) in 11 blood samples but the toxin was detected in urine. Ten patients had blood levels above 9 ng/ml and developed paralysis; seven of these died. The remaining patients recovered with supportive treatment. High concentrations of TTX and its analogues 4-epiTTX and 4,9-anhydroTTX were also found in cooked puffer fish by post-column liquid chromatography-fluorescence detection. To prevent future instances of puffer fish poisoning of this magnitude, measures should be implemented to increase awareness, to control markets and to establish toxicological testing. To improve the management of this and other poisoning in Bangladesh, facilities for life-saving assisted ventilation and related training of healthcare personnel are urgently needed at all levels of the health system., (Copyright © 2010 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.)

From six Conus species (Conus coronatus, Conus lividus, Conus mozambicus f. lautus, Conus pictus, Conus sazanka, Conus tinianus) collected off the eastern coast of South Africa the venoms were analyzed using MALDI-TOF mass spectrometry. Between 56 and 151 molecular masses most in a range of 1000 to 2500 Da, were identified. Among the six venoms, between 0 and 27% (C. coronatus versus C. sazanka) of the peptide masses were found to be similar. In a study on venoms from 6 Conus species collected in the Philippines, the percentage of identical masses was between none and 9% only. The venoms from the South African Conus species antagonized the rat neuronal nicotinic acetylcholine receptors (nAChRs) α3β2, α4β2, and α7, except for C. coronatus venom that blocked the α4β2 and α7 nAChRs only. HPLC-fractionation of C. tinianus venom led to the isolation of a peptide that is active on all three receptor subtypes. It consists of 16 amino acid residues cross-linked by two disulfide bridges as revealed by de novo sequencing using tandem mass spectrometry: GGCCSHPACQNNPDYC. Posttranslational modifications include C-terminal amidation and tyrosine sulfation. The new peptide is a member of the α-conotoxin family that are competitive antagonists of nAChRs. Phylogenetic analysis of the 16S RNA from numerous Conus species has clarified the evolutionary position of endemic South African Conus species and provided the first evidence for their close genetic relationships., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

High levels of tetrodotoxin (TTX) and of its analogue 6-epi-tetrodotoxin have been detected in skin and liver extracts of the red-spotted newt Notophthalmus viridescens assayed by a post-column fluorescent-HPLC system. Using a monoclonal antibody-based immunoenzymatic technique, TTX was localized in the granular glands of the epidermis as well as in the muscle layer between the muscle fibres, but also in most organs suggesting that TTX is stored in the whole body of the newt and is secreted by skin glands., (2010 Elsevier Ltd. All rights reserved.)

We report on screening tests of 66 extracts obtained from 35 marine sponge species from the Caribbean Sea (Curaçao) and from eight species from the Great Barrier Reef (Lizard Island). Extracts were prepared in aqueous and organic solvents and were tested for hemolytic, hemagglutinating, antibacterial and anti-acetylcholinesterase (AChE) activities, as well as their ability to inhibit or activate cell protein phosphatase 1 (PP1). The most interesting activities were obtained from extracts of Ircinia felix, Pandaros acanthifolium, Topsentia ophiraphidites, Verongula rigida and Neofibularia nolitangere. Aqueous and organic extracts of I. felix and V. rigida showed strong antibacterial activity. Topsentia aqueous and some organic extracts were strongly hemolytic, as were all organic extracts from I. felix. The strongest hemolytic activity was observed in aqueous extracts from P. acanthifolium. Organic extracts of N. nolitangere and I. felix inhibited PP1. The aqueous extract from Myrmekioderma styx possessed the strongest hemagglutinating activity, whilst AChE inhibiting activity was found only in a few sponges and was generally weak, except in the methanolic extract of T. ophiraphidites.

The venom from the solitary bee Osmia rufa (Hymenoptera: Megachilidae) was analyzed using mass spectrometry (MS)-based techniques. Sensitive proteomic methods such as on-line LC-ESI-MS and nanoESI-MS analyses revealed more than 50 different compounds with molecular masses ranging from 400 to 4000Da. The major component has a monoisotopic molecular mass of 1924.20Da and its amino acid sequence was elucidated by de novo sequencing using tandem mass spectrometry and Edman degradation. This 17-residue cysteine-free peptide, named osmin, shows some similarities with the mast cell degranulation (MCD) peptide family. Free acid and C-terminally amidated osmins were chemically synthesized and tested for antimicrobial and haemolytic activities. The synthetic C-amidated peptide (native osmin) was found to be about three times more haemolytic than its free acid counterpart, but both peptides are much less lytic than melittin from social bee venom. Preliminary antimicrobial and antifungal tests indicate that both peptides are able to inhibit bacterial and fungal growth at micromolar concentrations.

Fish species of the genera Amphiprion and Premnas (Perciformes: Pomacentridae) as well as various crustaceans seek protection from predators among the tentacles of sea anemones, where they live essentially unharmed from stinging by the host's nematocysts. The mucous coats of anemonefish and crustaceans have been suggested as mechanisms that prevent the discharge of the nematocysts upon contact. Whereas some fish species seem to produce their own protecting mucous coating, others may acquire mucus from the sea anemone during an acclimation period, as crustaceans do. Whether the natural or acquired mucus layers contain components that inhibit nematocyst discharge or simply lack compounds stimulating the stinging cell's exocytosis is still unknown.

Palytoxin (PTX), one of the most potent and chemically complex marine toxins, is predominantly found in zoanthid corals and sporadically in dinoflagellates. Its biosynthesis and metabolic pathways are largely unknown. However, the widespread occurrence of the toxin in phylogenetically distinct marine organisms is consistent with its production by microorganisms and subsequent accumulation in the food chain. To investigate a possible microbial origin, bacteria from two zoanthid corals (Palythoa caribaeorum, Zoanthus pulchellus) and one sponge (Neofibularia nolitangere) were isolated. More than 250 bacteria were screened for hemolysis using a newly developed PTX-screening assay of which 7% showed PTX-like hemolytic activity. 16S rRNA gene sequencing revealed that these bacterial isolates belonged to strains of Bacillus cereus group (n = 11) as well as the genera Brevibacterium (n = 4) and Acinetobacter (n = 2). The results indicate the presence of Na+/K+-ATPase toxins and possibly PTX in hemolytic bacteria from P. caribaeorum.

The venom of cone snails (Conus spp.) is a rich source of peptides exhibiting a wide variety of biological activities. Several of these conopeptides are neuronal nicotinic acetylcholine receptor (nAChR) antagonists and belong to the A-, M-, S-, C and the recently described D-superfamily (alphaD-conopeptides). Here we describe the discovery and characterization of two alphaD-conopeptides isolated from the venom of Conus mustelinus and Conus capitaneus. Their primary structure was determined by Edman degradation, MS/MS analysis and by a PCR based approach. These peptides show close structural homology to the alphaD-VxXIIA, -B and -C conopeptides from the venom of Conus vexillum and are dimers (about 11kDa) of similar or identical peptides with 49 amino acid residues and a characteristic arrangement of ten conserved cysteine residues. These novel types of conopeptides specifically block neuronal nAChRs of the alpha7, alpha3beta2 and alpha4beta2 subtypes in nanomolar concentrations. Due to their high affinity, these new ligands may provide a tool to decipher the localisation and function of the various neuronal nAChRs.

In two species of meloid beetles, Hycleus oculatus and Hycleus tinctus, from southern Africa, cantharidin and demethylcantharidin (palasonin) were assayed quantitatively. For cantharidin the mean value per specimen was about 1 mg for H. oculatus and 0.2 mg for H. tinctus, the mean palasonin concentration was 20 (H. oculatus) and 12 times (H. tinctus) lower, respectively. However, considerable individual variation in the cantharidin concentration was observed and values of more than 6 mg of this compound per beetle were measured pointing to the high risk of severe and even fatal poisoning when ingesting these insects.

A case of human poisoning by palytoxin after contact with zoanthid corals (Parazoanthus sp.) in an aquarium through skin injuries on fingers is reported. The clinical symptoms include swelling, paraesthesia and numbness around the site of the injury spreading over the arm, but also signs of systemic poisoning such as dizziness, general weakness and myalgia, irregularities in the ECG and indications of rhabdomyolysis. Symptomatic treatment consisted of infusion of physiological fluids. The patient recovered within 3 days. Analysis of the zoanthid coral involved revealed extremely high concentrations of palytoxin (between 2 and 3 mg/g).

The venom of Zhaoermia mangshanensis, encountered solely in Mt Mang in China's Hunan Province, exhibits coagulant, phosphodiesterase, l-amino acid oxidase, kallikrein, phospholipase A2 and myotoxic activities. The catalytically inactive PLA2 homolog referred to as zhaoermiatoxin is highly myotoxic and displays high myonecrotic and edema activities. Zhaoermiatoxin possesses a molecular weight of 13,972Da, consists of 121 amino-acid residues cross-linked by seven disulfide bridges and shares high sequence homology with Lys49-PLA2s from the distantly related Asian pitvipers. However, zhaoermiatoxin possesses an arginine residue at position 49 instead of a lysine, thereby suggesting a secondary Lys49-->Arg substitution which results in a catalytically inactive protein. We have determined the first crystal structure of zhaoermiatoxin, an Arg49-PLA2, from Zhaoermia mangshanensis venom at 2.05 angstroms resolution, which represents a novel member of phospholipase A2 family. In this structure, unlike the Lys49 PLA2s, the C-terminus is well ordered and an unexpected non-polarized state of the putative calcium-binding loop due to the flip of Lys122 towards the bulk solvent is observed. The orientation of the Arg-49 side chain results in a similar binding mode to that observed in the Lys49 PLA2s; however, the guadinidium group is tri-coordinated by carbonyl oxygen atoms of the putative calcium-binding loop, whereas the Nzeta atom of lysine is tetra-coordinated as a result of the different conformation adopted by the putative calcium-binding loop.

Certain amphibians provide themselves with a chemical defense by accumulating lipophilic alkaloids into skin glands from dietary arthropods. Examples of such alkaloids are pumiliotoxins (PTXs). In general, PTXs are known as positive modulators of voltage-gated sodium channels (VGSCs). Unlike other PTXs, PTX 251D does not share this characteristic. However, mice and insect studies showed that PTX 251D is highly toxic and to date the basis of its toxicity remains unknown. In this work, we searched for the possible target of PTX 251D. The toxin was therefore made synthetically and tested on four VGSCs (mammalian rNa(v)1.2/beta(1), rNa(v)1.4/beta(1), hNa(v)1.5/beta(1) and insect Para/tipE) and five voltage-gated potassium channels (VGPCs) (mammalian rK(v)1.1-1.2, hK(v)1.3, hK(v)11.1 (hERG) and insect Shaker IR) expressed heterologously in Xenopus laevis oocytes, using the two-electrode voltage clamp technique. PTX 251D not only inhibited the Na(+) influx through the mammalian VGSCs but also affected the steady-state activation and inactivation. Interestingly, in the insect ortholog, the inactivation process was dramatically affected. Additionally, PTX 251D inhibited the K(+) efflux through all five tested VGPCs and slowed down the deactivation kinetics of the mammalian VGPCs. hK(v)1.3 was the most sensitive channel, with an IC(50) value 10.8+/-0.5 microM. To the best of our knowledge this is the first report of a PTX affecting VGPCs.

Tetrodotoxin (TTX) and its analogue 6-epitetrodotoxin (6-epiTTX) were quantitatively assayed in 59 newts representing four Triturus species (Triturus alpestris, Triturus cristatus, Triturus helveticus, Triturus vulgaris) from southern Germany by a post-column fluorescent-HPLC system. Both toxins were detected in only 15 specimens of the four species. The toxins levels varied considerably among individuals (TTX: 0.11-9.0microg/g; 6-epiTTX: 0.05-17.0microg/g). 6-epiTTX was found to be the major component.

In whole animal ethanolic extracts from adult specimens of Melanophryniscus atroluteus (27 specimens) and M. devincenzii (16 specimens) as well as of two egg clutches and four tadpole samples from the latter species, the major alkaloid pumiliotoxin (PTX) 251D and hydroquinone were assayed quantitatively by gas chromatography/mass spectrometry. All toad extracts contained high concentrations of PTX 251D and hydroquinone and exhibited considerable variation in the content of these compounds among individual specimens. The extracts of the eggs and tadpoles were entirely free of alkaloids as well as hydroquinone, pointing to a dietary origin of these compounds.

Zhaoermiatoxin, an Arg49 phospholipase A2 homologue from Zhaoermia mangshanensis (formerly Trimeresurus mangshanensis, Ermia mangshanensis) venom is a novel member of the PLA2-homologue family that possesses an arginine residue at position 49, probably arising from a secondary Lys49-->Arg substitution that does not alter the catalytic inactivity towards phospholipids. Like other Lys49 PLA2 homologues, zhaoermiatoxin induces oedema and strong myonecrosis without detectable PLA2 catalytic activity. A single crystal with maximum dimensions of 0.2 x 0.2 x 0.5 mm was used for X-ray diffraction data collection to a resolution of 2.05 A using synchrotron radiation and the diffraction pattern was indexed in the hexagonal space group P6(4), with unit-cell parameters a = 72.9, b = 72.9, c = 93.9 A.

Tetrodotoxin (TTX) was quantitatively assayed in six specimens of semi-adult blue-ringed octopus, Hapalochlaena maculosa, by a post-column fluorescent-HPLC system. TTX was found to be present in all body parts, e.g. in high concentrations in the arms followed by the abdomen and cephalothorax. The toxin is not associated exclusively with the posterior salivary gland.

The South-American red bellied toads (Melanophryniscus spp.) belonging to the Bufonidae family contain toxic alkaloids in their skin, predominantly of the pumiliotoxin group. Whole animal methanolic extracts of individual specimens of three species (Melanophryniscus atroluteus, M. devincenzii, and M. montevidensis) were analyzed for the presence of toad specific bufadienolides and indolalkylamines (serotonin derivatives) by HPLC-electrospray (ESI)-MS-TOF. No bufadienolides, but few bufotenines, mainly dehydrobufotenine, were detected in the extracts in variable amounts. The concentration of the dehydrobufotenine in the extracts seems to be species specific. Whereas M. atroluteus and M. montevidensis contain very low or trace amounts, M. devincenzii specimens exhibit high concentrations of this indolalkylamine. In comparison, analysis of extracts from Bufo arenarum (Uruguay) and from B. bufo (Germany) confirmed the presence of bufadienolides as well as of bufotenine derivatives. Tadpoles of both species exhibited a different pattern: extracts from B. arenarum tadpoles contained only dehydrobufotenine, but those from B. bufo tadpoles bufotoxin and two alkylamines. Melanophryniscus toads appear not to be able to compensate the high variability of toxic skin alkaloids by producing defensive bufadienolides.

Most cases of poisoning by animals are caused by snakes, spiders or scorpions. In addition to"first aid" such as calming the victim and immobilization of the bitten limb, other measures include monitoring vital functions and rapid transportation to hospital as dictated by the symptoms presenting, and, where indicated, injection of an antiserum (identification of the animal concerned). On no account should the bite wound be cut or attempts made to suck out the venom. While the sting of a scorpion or a spider bite often do not lead to severe complications in adults, a brush with a poisonous snake may be much more serious.

Zhaoermia mangshanensis (formerly Trimeresurus mangshanensis, Ermia mangshanensis) represents a monotypic genus of pitviper known only from Mt Mang in China's Hunan Province, and is among the largest and most spectacular of Asian venomous snakes. The venom of Zhaoermia exhibits high coagulant activity on bovine and human fibrinogen and human plasma, high phosphodiesterase and arginine ester hydrolytic activity, and moderate to low l-amino acid oxidase, kallikrein, caseinolytic, phospholipase A(2) (PLA(2)), haemorrhagic and myotoxic activities. The approximate i.p. LD(50) of the venom in mice was estimated to be 4 mg/kg. We purified the major toxin of Zhaoermia venom by gel-filtration, cation-exchange chromatography and HPLC. The toxin, a homodimer with an experimental monomeric mass of 13,972 Da, induced edema and myonecrosis in mice, but was devoid of detectable PLA(2) catalytic activity. Its complete amino acid sequence is composed of 121 amino acid residues cross-linked by seven disulfide bridges, and shows more than 80% identity to two Lys49-PLA(2)s from distantly related Asian pitvipers, Protobothrops mucrosquamatus and Calloselasma rhodostoma. Phylogenetic analysis of the novel toxin, zhaoermiatoxin, confirmed that it is rooted within a comprehensive sample of Lys49-PLA(2)s despite having an arginine residue in position 49, suggesting a secondary Lys49-->Arg substitution which did not alter the catalytic inactivity of the molecule.

There is a general consensus that most of today's nonvenomous snakes are descendants of venomous snakes that lost their venomous capabilities secondarily. This implies that the evolutionary history of venomous snakes and their venom apparatus should be older than the current evidence from the fossil record. We compared some of the oldest-known fossil snake fangs from the Lower Miocene of Germany with those of modern viperids and elapids and found their morphology to be indistinguishable from the modern forms. The primary function of recent elapid and viperid snake fangs is to facilitate the extremely rapid, stab-like application of highly toxic venoms. Our findings therefore indicate that the other components of the venom-delivery system of Early Miocene vipers and elapids were also highly developed, and that these snakes used their venom in the same way as their modern relatives. Thus, the fossil record supports the view that snakes used their venoms to rapidly subdue prey long before the mid-Tertiary onset of the global environmental changes that seem to have supported the successful radiation of venomous snakes.

Toads belonging to the genus Melanophryniscus contain toxic alkaloids in their skin. From six locations in south-eastern Uruguay 81 specimens of Melanophryniscusmontevidensis were collected. In whole animal methanolic extracts of individual specimens, alkaloids of the pumiliotoxin (PTX) group and indolizidines were identified by gas chromatography/mass spectrometry; the predominant component PTX 251D was assayed quantitatively. The PTX-content of the various toad populations was found to be highly variable among individual specimens as well as among the populations. Very high levels of PTX 251D were detected in toads of the western part of the collection area, whereas very low levels of this alkaloid were assayed in toads near the Brazilian border. Remarkably high concentrations of the non-alkaloid hydroquinone were found to be present in all toads. The analysis of extracts from 125 arthropod samples (Arachnida and Insecta, including termites, ants and beetles), which may represent a potential food source, revealed no alkaloids of the PTX group.

Ten venoms from marine cone snails were tested for ichthyotoxic effects on zebra fish (Brachydanio rerio) when added to the water. Only two venoms, from Conus capitaneus and Conus episcopatus, produced lethal effects at high concentrations (50-300 microg/ml) within 20-90 min. No sedative or hypnotic symptoms were observed. The experiments confirm that Conus venoms exert a quick and prompt activity only by parenteral injection into the prey as it is performed by the snail.

Butterflies of the Danaidae family are considered to be toxic or distasteful due to the presence of cardiac glycosides sequestered from their larval food plants. Alcoholic extracts of specimens of Danaus chrysippus aegyptius and Amauris ochlea ochlea from southern Africa (Namibia, S.-Africa, Mozambique) were analyzed by thin-layer chromatography for these cardenolides. But only 4 of 75 specimens of D. chrysippus aegyptius contained trace amounts, all others including 13 specimens of A. ochlea ochlea were negative. Genetic analysis of the ouabain binding site of the Na(+), K(+)-ATPase revealed that both species do not present an amino acid replacement at the position 122, which otherwise makes the enzyme insensitive to cardenolides suggesting that other strategies of toxin tolerance must have been developed.

The two major alkaloids, samandarine and samandarone, were identified in the skin secretion of individual specimens from two populations of the European fire salamander (Salamandra salamandra terrestris) by gas chromatography/mass spectrometry. High intraspecific variability in the ratio of both alkaloids was observed, but also in individual specimens over a period of 4 months suggesting separate metabolic pathways of the compounds. Alkaloid synthesis appears to take place also in liver, testes and ovaries, whereas the larvae of the salamanders are entirely free of alkaloids.

Conotoxins from the venom of marine cone snails (genus Conus) represent large families of proteins exhibiting a similar precursor organization, but highly diverse pharmacological activities. A directed PCR-based approach using primers according to the conserved signal sequence was applied to investigate the diversity of conotoxins from the O-superfamily. Using 3' RACE, cDNA sequences encoding precursor peptides were identified in five Conus species (Conus capitaneus, Conus imperialis, Conusstriatus, Conus vexillum and Conus virgo). In all cases, the sequence of the signal region exhibited high conservancy, whereas the sequence of the mature peptides was either almost identical or highly divergent among the five species. These findings demonstrate that beside a common genetic pattern divergent evolution of toxins occurred in a highly mutating peptide family.

King brown snakes or mulga snakes (Pseudechis australis) are the largest and among the most dangerous and wide-ranging venomous snakes in Australia and New Guinea. They occur in diverse habitats, are important predators, and exhibit considerable morphological variation. We infer the relationships and historical biogeography of P. australis based on phylogenetic analysis of 1,249 base pairs from the mitochondrial cytochrome b, NADH dehydrogenase subunit 4 and three adjacent tRNA genes using Bayesian, maximum-likelihood, and maximum-parsimony methods. All methods reveal deep phylogenetic structure with four strongly supported clades comprising snakes from New Guinea (I), localities all over Australia (II), the Kimberleys of Western Australia (III), and north-central Australia (IV), suggesting a much more ancient radiation than previously believed. This conclusion is robust to different molecular clock estimations indicating divergence in Pliocene or Late Miocene, after landbridge dispersal to New Guinea had occurred. While members of clades I, III and IV are medium-sized, slender snakes, those of clade II attain large sizes and a robust build, rendering them top predators in their ecosystems. Genetic differentiation within clade II is low and haplotype distribution largely incongruent with geography or colour morphs, suggesting Pleistocene dispersal and recent ecomorph evolution. Significant haplotype diversity exists in clades III and IV, implying that clade IV comprises two species. Members of clade II are broadly sympatric with members of both northern Australian clades. Thus, our data support the recognition of at least five species from within P. australis (auct.) under various criteria. We discuss biogeographical, ecological and medical implications of our findings.

Marine organisms have attracted much attention as a source of pharmacological tools or potential drugs. We have produced and screened a library of sponge extracts in search of biologically active compounds that may contain useful pharmaceutical lead structures. Sponges were collected from various locations and their aqueous extracts were freeze dried. Murine right and left atria were used to screen 75 extracts for putative cardiac effects. Among seven extracts with a positive inotropic and chronotropic effect the extract C47 from Ectyoplasia ferox proved to be the most active and was chosen for further analysis. C47 also produced a beta-adrenoceptor-independent, propranolol-resistant positive inotropic effect in human atrial trabeculae. To elucidate one possible mode of action the effects of C47 on L-type Ca(2+) current (I(Ca,L)) were measured with a standard patch-clamp technique. In isolated human atrial myocytes exposure to C47 increased peak amplitude of I(Ca,L) in a concentration-dependent manner. The threshold concentration was 15 microg/ml. In addition, voltage dependency of activation and steady-state inactivation were shifted to more negative potentials. C47 slowed the initial phase of time-dependent current inactivation and the recovery from inactivation. In cell-attached patches of HEK 293 cells expressing human Ca(v)1.2 addition of C47 to the bath solution did not affect gating properties, whereas inclusion of the extract into the pipette solution strongly increased single-channel activity, suggesting a direct effect on the pore-forming channel subunit. Despite its robust effect on I(Ca,L) C47 enhanced cardiac force of contraction by only a fraction of the maximum increase caused by high extracellular concentrations of Ca(2+) and failed to increase vascular tone. These findings suggest that the effect of C47 is restricted to the Ca(2+) channel.

The I-superfamily of conotoxins represents a new class of peptides in the venom of some Conus species. These toxins are characterized by four disulfide bridges and inhibit or modify ion channels of nerve cells. When testing venoms from 11 Conus species for a functional characterization, blocking activity on potassium channels (like Kv1.1 and Kv1.3 channels, but not Kv1.2 channels) was detected in the venom of Conus capitaneus, Conus miles, Conus vexillum and Conus virgo. Analysis at the cDNA level of these venoms using primers designed according to the amino acid sequence of a potassium channel blocking toxin (ViTx) from C. virgo confirmed the presence of structurally homologous peptides in these venoms. Moreover, peptides belonging to the I-superfamily, but with divergent amino acid sequences, were found in Conus striatus and Conus imperialis. In all cases, the sequences of the precursors' prepro-regions exhibited high conservation, whereas the sequences of the mature peptides ranged from almost identical to highly divergent between species. We then performed phylogenetic analyses of new and published mitochondrial 16S rDNA sequences representing 104 haplotypes from these and numerous other Conus species, using Bayesian, maximum-likelihood, maximum-parsimony and neighbor-joining methods of inference. Cone snails known to possess I-superfamily toxins were assigned to five different major clades in all of the resulting gene trees. Moreover, I-superfamily conopeptides were detected both in vermivorous and piscivorous species of Conus, thus demonstrating the widespread presence of such toxins in this speciose genus beyond evolutionary and ecological groups.

The sequence of a long-chain neurotoxin (71 amino acid residues, 10 half-cystines) from the venom of the African banded water cobra (Boulengerina annulata) was determined by Edman degradation. It exhibits high sequence similarity with long-chain neurotoxins from the venoms of four species of African cobras (genus Naja), which are collectively more similar to the Boulengerina toxin than to those of Asian Naja species. These results are discussed in the light of phylogenetic hypotheses on the relationships of African cobras.

We isolated the most lethal toxins in the venom of the Malayan krait (Bungarus candidus), one of the medically most important snake species in southeast Asia. Three beta-BTx like basic neurotoxins, T1-1, T1-2, and T2, with PLA2 activity were isolated from pooled venom of eight B. candidus from southern Thailand by cation-exchange chromatography, followed by adsorption chromatography on hydroxylapatite and RP-HPLC, with 14-, 16-, and 4-fold increases in toxicity compared to crude venom. The LDs50 determined in mice weighing 18-20 g were 0.26, 0.22, and 0.84 micro g per mouse with i.v. injection. T1-1 and T1-2 possessed comparable lethal toxicities to those of beta1-BTx, the most toxic neurotoxin in B. multicinctus venom, and the major neurotoxin in B. flaviceps venom. The apparent molecular weights of the native toxins were approximately 25-25.5 kDa. They consist of two polypeptide chains with apparent molecular weights of 15.5-16.5 and 8-8.5 kDa, respectively. The amino terminal sequences of the two chains of each of the toxins determined by Edman degradation exhibited considerable similarity with those of the A-chains and B-chains of beta-BTxs in the venom of Bungarus multicinctus.