Millions of years of evolution have fine-tuned the ability of venom peptides to rapidly incapacitate both prey and potential predators. Toxicofera reptiles are characterized by serous-secreting mandibular or maxillary glands with heightened levels of protein expression. These glands are the core anatomical components of the toxicoferan venom system, which exists in myriad points along an evolutionary continuum. Neofunctionalisation of toxins is facilitated by positive selection at functional hotspots on the ancestral protein and venom proteins have undergone dynamic diversification in helodermatid and varanid lizards as well as advanced snakes. A spectacular point on the venom system continuum is the long-glanded blue coral snake (Calliophis bivirgatus), a specialist feeder that preys on fast moving, venomous snakes which have both a high likelihood of prey escape but also represent significant danger to the predator itself. The maxillary venom glands of C. bivirgatus extend one quarter of the snake’s body length and nestle within the rib cavity. Despite the snake’s notoriety its venom has remained largely unstudied. Here we show that the venom uniquely produces spastic paralysis, in contrast to the flaccid paralysis typically produced by neurotoxic snake venoms. The toxin responsible, which we have called calliotoxin (δ-elapitoxin-Cb1a), is a three-finger toxin (3FTx). Calliotoxin shifts the voltage-dependence of NaV1.4 activation to more hyperpolarised potentials, inhibits inactivation, and produces large ramp currents, consistent with its profound effects on contractile force in an isolated skeletal muscle preparation. Voltage-gated sodium channels (NaV) are a particularly attractive pharmacological target as they are involved in almost all physiological processes including action potential generation and conduction. Accordingly, venom peptides that interfere with NaV function provide a key defensive and predatory advantage to a range of invertebrate venomous species including cone snails, scorpions, spiders, and anemones. Enhanced activation or delayed inactivation of sodium channels by toxins is associated with the extremely rapid onset of tetanic/excitatory paralysis in envenomed prey animals. A strong selection pressure exists for the evolution of such toxins where there is a high chance of prey escape. However, despite their prevalence in other venomous species, toxins causing delay of sodium channel inhibition have never previously been described in vertebrate venoms. Here we show that NaV modulators, convergent with those of invertebrates, have evolved in the venom of the long-glanded coral snake. Calliotoxin represents a functionally novel class of 3FTx and a structurally novel class of NaV toxins that will provide significant insights into the pharmacology and physiology of NaV. The toxin represents a remarkable case of functional convergence between invertebrate and vertebrate venom systems in response to similar selection pressures. These results underscore the dynamic evolution of the Toxicofera reptile system and reinforces the value of using evolution as a roadmap for biodiscovery.
Over the past 20 years, exposure to mycotoxin producing mold has been recognized as a significant health risk. Scientific literature has demonstrated mycotoxins as possible causes of human disease in water-damaged buildings (WDB). This study was conducted to determine if selected mycotoxins could be identified in human urine from patients suffering from chronic fatigue syndrome (CFS). Patients (n = 112) with a prior diagnosis of CFS were evaluated for mold exposure and the presence of mycotoxins in their urine. Urine was tested for aflatoxins (AT), ochratoxin A (OTA) and macrocyclic trichothecenes (MT) using Enzyme Linked Immunosorbent Assays (ELISA). Urine specimens from 104 of 112 patients (93%) were positive for at least one mycotoxin (one in the equivocal range). Almost 30% of the cases had more than one mycotoxin present. OTA was the most prevalent mycotoxin detected (83%) with MT as the next most common (44%). Exposure histories indicated current and/or past exposure to WDB in over 90% of cases. Environmental testing was performed in the WDB from a subset of these patients. This testing revealed the presence of potentially mycotoxin producing mold species and mycotoxins in the environment of the WDB. Prior testing in a healthy control population with no history of exposure to a WDB or moldy environment (n = 55) by the same laboratory, utilizing the same methods, revealed no positive cases at the limits of detection.
The most common technique used to detect ochratoxin A (OTA) in food matrices is based on extraction, clean-up, and chromatography detection. Different clean-up cartridges, such as immunoaffinity columns (IAC), molecular imprinting polymers (MIP), Mycosep™ 229, Mycospin™, and Oasis® HLB (Hydrophilic Lipophilic balance) as solid phase extraction were tested to optimize the purification for red wine, beer, roasted coffee and chili. Recovery, reproducibility, reproducibility, limit of detection (LOD) and limit of quantification (LOQ) were calculated for each clean-up method. IAC demonstrated to be suitable for OTA analysis in wine and beer with recovery rate >90%, as well as Mycosep™ for wine and chili. On the contrary, MIP columns were the most appropriate to clean up coffee. A total of 120 samples (30 wines, 30 beers, 30 roasted coffee, 30 chili) marketed in Italy were analyzed, by applying the developed clean-up methods. Twenty-seven out of 120 samples analyzed (22.7%: two wines, five beers, eight coffees, and 12 chili) resulted positive to OTA. A higher incidence of OTA was found in chili (40.0%) more than wine (6.6%), beers (16.6%) and coffee (26.6%). Moreover, OTA concentration in chili was the highest detected, reaching 47.8 µg/kg. Furthermore, three samples (2.5%), two wines and one chili, exceeded the European threshold.
A novel enzyme-linked immunosorbent assay based on magnetic nanoparticles and biotin/streptavidin-HRP (MNP-bsELISA) was developed for rapid and sensitive detection of zearalenone (ZEN). The detection signal was enhanced and the sensitivity of the assay was improved by combined use of antibody-conjugated magnetic nanoparticles and biotin-streptavidin system. Under the optimized conditions, the regression equation for quantification of ZEN was y = -0.4287x + 0.3132 (R² = 0.9904). The working range was 0.07-2.41 ng/mL. The detection limit was 0.04 ng/mL and IC50 was 0.37 ng/mL. The recovery rates of intra-assay and inter-assay ranged from 92.8%-111.9% and 91.7%-114.5%, respectively, in spiked corn samples. Coefficients of variation were less than 10% in both cases. Parallel analysis of cereal and feed samples showed good correlation between MNP-bsELISA and liquid chromatograph-tandem mass spectrometry (R² = 0.9283). We conclude that this method is suitable for rapid detection of zearalenone in cereal and feed samples in relevant laboratories.
Australia is the stronghold of the front-fanged venomous snake family Elapidae. The Australasian elapid snake radiation, which includes approximately 100 terrestrial species in Australia, as well as Melanesian species and all the world’s sea snakes, is less than 12 million years old. The incredible phenotypic and ecological diversity of the clade is matched by considerable diversity in venom composition. The clade’s evolutionary youth and dynamic evolution should make it of particular interest to toxinologists, however, the majority of species, which are small, typically inoffensive, and seldom encountered by non-herpetologists, have been almost completely neglected by researchers. The present study investigates the venom composition of 28 species proteomically, revealing several interesting trends in venom composition, and reports, for the first time in elapid snakes, the existence of an ontogenetic shift in the venom composition and activity of brown snakes (Pseudonaja sp.). Trends in venom composition are compared to the snakes' feeding ecology and the paper concludes with an extended discussion of the selection pressures shaping the evolution of snake venom.
Cnidarian envenomations are the leading cause of severe and lethal human sting injuries from marine life. The total amount of venom discharged into sting-site tissues, sometimes referred to as “venom load”, has been previously shown to correlate with tentacle contact length and sequelae severity. Since <1% of cnidae discharge upon initial tentacle contact, effective and safe removal of adherent tentacles is of paramount importance in the management of life-threatening cubozoan stings. We evaluated whether common rinse solutions or scraping increased venom load as measured in a direct functional assay of venom activity (hemolysis). Scraping significantly increased hemolysis by increasing cnidae discharge. For Alatina alata, increases did not occur if the tentacles were first doused with vinegar or if heat was applied. However, in Chironex fleckeri, vinegar dousing and heat treatment were less effective, and the best outcomes occurred with the use of venom-inhibiting technologies (Sting No More(®) products). Seawater rinsing, considered a "no-harm" alternative, significantly increased venom load. The application of ice severely exacerbated A. alata stings, but had a less pronounced effect on C. fleckeri stings, while heat application markedly reduced hemolysis for both species. Our results do not support scraping or seawater rinsing to remove adherent tentacles.
The cytotoxicity of the venom of 25 species of Old World elapid snake was tested and compared with the morphological and behavioural adaptations of hooding and spitting. We determined that, contrary to previous assumptions, the venoms of spitting species are not consistently more cytotoxic than those of closely related non-spitting species. While this correlation between spitting and non-spitting was found among African cobras, it was not present among Asian cobras. On the other hand, a consistent positive correlation was observed between cytotoxicity and utilisation of the defensive hooding display that cobras are famous for. Hooding and spitting are widely regarded as defensive adaptations, but it has hitherto been uncertain whether cytotoxicity serves a defensive purpose or is somehow useful in prey subjugation. The results of this study suggest that cytotoxicity evolved primarily as a defensive innovation and that it has co-evolved twice alongside hooding behavior: once in the Hemachatus + Naja and again independently in the king cobras (Ophiophagus). There was a significant increase of cytotoxicity in the Asian Naja linked to the evolution of bold aposematic hood markings, reinforcing the link between hooding and the evolution of defensive cytotoxic venoms. In parallel, lineages with increased cytotoxicity but lacking bold hood patterns evolved aposematic markers in the form of high contrast body banding. The results also indicate that, secondary to the evolution of venom rich in cytotoxins, spitting has evolved three times independently: once within the African Naja, once within the Asian Naja, and once in the Hemachatus genus. The evolution of cytotoxic venom thus appears to facilitate the evolution of defensive spitting behaviour. In contrast, a secondary loss of cytotoxicity and reduction of the hood occurred in the water cobra Naja annulata, which possesses streamlined neurotoxic venom similar to that of other aquatic elapid snakes (e.g., hydrophiine sea snakes). The results of this study make an important contribution to our growing understanding of the selection pressures shaping the evolution of snake venom and its constituent toxins. The data also aid in elucidating the relationship between these selection pressures and the medical impact of human snakebite in the developing world, as cytotoxic cobras cause considerable morbidity including loss-of-function injuries that result in economic and social burdens in the tropics of Asia and sub-Saharan Africa.
Despite the medical urgency presented by cubozoan envenomations, ineffective and contradictory first-aid management recommendations persist. A critical barrier to progress has been the lack of readily available and reproducible envenomation assays that (1) recapitulate live-tentacle stings; (2) allow quantitation and imaging of cnidae discharge; (3) allow primary quantitation of venom toxicity; and (4) employ rigorous controls. We report the implementation of an integrated array of three experimental approaches designed to meet the above-stated criteria. Mechanistically overlapping, yet distinct, the three approaches comprised (1) direct application of test solutions on live tentacles (termed tentacle solution assay, or TSA) with single image- and video-microscopy; (2) spontaneous stinging assay using freshly excised tentacles overlaid on substrate of live human red blood cells suspended in agarose (tentacle blood agarose assays, or TBAA); and (3) a “skin” covered adaptation of TBAA (tentacle skin blood agarose assay, or TSBAA). We report the use and results of these assays to evaluate the efficacy of topical first-aid approaches to inhibit tentacle firing and venom activity. TSA results included the potent stimulation of massive cnidae discharge by alcohols but only moderate induction by urine, freshwater, and “cola” (carbonated soft drink). Although vinegar, the 40-year field standard of first aid for the removal of adherent tentacles, completely inhibited cnidae firing in TSA and TSBAA ex vivo models, the most striking inhibition of both tentacle firing and subsequent venom-induced hemolysis was observed using newly-developed proprietary formulations (Sting No More™) containing copper gluconate, magnesium sulfate, and urea.
Centipedes are among the oldest extant venomous predators on the planet. Armed with a pair of modified, venom-bearing limbs, they are an important group of predatory arthropods and are infamous for their ability to deliver painful stings. Despite this, very little is known about centipede venom and its composition. Advances in analytical tools, however, have recently provided the first detailed insights into the composition and evolution of centipede venoms. This has revealed that centipede venom proteins are highly diverse, with 61 phylogenetically distinct venom protein and peptide families. A number of these have been convergently recruited into the venoms of other animals, providing valuable information on potential underlying causes of the occasionally serious complications arising from human centipede envenomations. However, the majority of venom protein and peptide families bear no resemblance to any characterised protein or peptide family, highlighting the novelty of centipede venoms. This review highlights recent discoveries and summarises the current state of knowledge on the fascinating venom system of centipedes.
Venomous animals have evolved with sophisticated bio-chemical strategies to arrest prey and defend themselves from natural predators. In recent years, peptide toxins from venomous animals have drawn considerable attention from researchers due to their surprising chemical, biochemical, and pharmacological diversity. Similar to other venomous animals, centipedes are one of the crucial venomous arthropods that have been used in traditional medicine for hundreds of years in China. Despite signifying pharmacological importance, very little is known about the active components of centipede venoms. More than 500 peptide sequences have been reported in centipede venomous glands by transcriptome analysis, but only a small number of peptide toxins from centipede has been functionally described. Like other venomous animals such as snakes, scorpions, and spiders, the venom of centipedes could be an excellent source of peptides for developing drugs for treatments as well as bio-insecticides for agrochemical applications. Although centipede venoms are yet to be adequately studied, the venom of centipedes as well as their components described to date, should be compiled to help further research. Therefore, based on previous reports, this review focusses on findings and possible therapeutic applications of centipede venoms as well as their components.