Coral Snake Venom Research Articles

The challenge of protecting against all venomous coral snakes

The challenge of protecting against all venomous coral snakes

In vivo neutralization of coral snake venoms with an oligoclonal nanobody mixture in a murine challenge model

Oligoclonal mixtures of broadly-neutralizing antibodies can neutralize complex compositions of similar and dissimilar antigens, making them versatile tools for the treatment of e.g., infectious diseases and animal envenomations. However, these biotherapeutics are complicated to develop due to their complex nature. In this work, we describe the application of various strategies for the discovery of cross-neutralizing nanobodies against key toxins in coral snake venoms using phage display technology. We prepare two oligoclonal mixtures of nanobodies and demonstrate their ability to neutralize the lethality induced by two North American coral snake venoms in mice, while individual nanobodies fail to do so. We thus show that an oligoclonal mixture of nanobodies can neutralize the lethality of venoms where the clinical syndrome is caused by more than one toxin family in a murine challenge model. The approaches described may find utility for the development of advanced biotherapeutics against snakebite envenomation and other pathologies where multi-epitope targeting is beneficial.

Quantifying venom production: A study on Micrurus snakes in Mexico

Our study quantifies venom production in nine Mexican coral snake species (Micrurus), encompassing 76 specimens and 253 extractions. Noteworthy variations were observed, with M. diastema and M. laticollaris displaying diverse yields, ranging from 0.3 mg to 59 mg. For animals for which we have length data, there is a relationship between size and venom quantity. Twenty-eight percent of the observed variability in venom production can be explained by snake size, suggesting that other factors influence the amount of obtained venom. These findings are pivotal for predicting venom effects and guiding antivenom interventions. Our data offer insights into Micrurus venom yields, laying the groundwork for future research and aiding in medical response strategies. This study advances understanding coral snake venom production, facilitating informed medical responses to coral snake bites.

First Insights into the Venom Composition of Two Ecuadorian Coral Snakes.

Micrurus is a medically relevant genus of venomous snakes composed of 85 species. Bites caused by coral snakes are rare, but they are usually associated with very severe and life-threatening clinical manifestations. Ecuador is a highly biodiverse country with a complex natural environment, which is home to approximately 20% of identified Micrurus species. Additionally, it is on the list of Latin American countries with the highest number of snakebites. However, there is no local antivenom available against the Ecuadorian snake venoms, and the biochemistry of these venoms has been poorly explored. Only a limited number of samples collected in the country from the Viperidae family were recently characterised. Therefore, this study addressed the compositional patterns of two coral snake venoms from Ecuador, M. helleri and M. mipartitus, using venomics strategies, integrating sample fractionation, gel electrophoresis, and mass spectrometry. Chromatographic and electrophoretic profiles of these snake venoms revealed interspecific variability, which was ascertained by mass spectrometry. The two venoms followed the recently recognised dichotomic toxin expression trends displayed by Micrurus species: M. helleri venom contains a high proportion (72%) of phospholipase A2, whereas M. mipartitus venom is dominated by three-finger toxins (63%). A few additional protein families were also detected in these venoms. Overall, these results provide the first comprehensive views on the composition of two Ecuadorian coral snake venoms and expand the knowledge of Micrurus venom phenotypes. These findings open novel perspectives to further research the functional aspects of these biological cocktails of PLA2s and 3FTxs and stress the need for the preclinical evaluation of the currently used antivenoms for therapeutic purposes in Ecuador.

Pharmacological Screening of Venoms from Five Brazilian Micrurus Species on Different Ion Channels.

Coral snake venoms from the Micrurus genus are a natural library of components with multiple targets, yet are poorly explored. In Brazil, 34 Micrurus species are currently described, and just a few have been investigated for their venom activities. Micrurus venoms are composed mainly of phospholipases A2 and three-finger toxins, which are responsible for neuromuscular blockade—the main envenomation outcome in humans. Beyond these two major toxin families, minor components are also important for the global venom activity, including Kunitz-peptides, serine proteases, 5′ nucleotidases, among others. In the present study, we used the two-microelectrode voltage clamp technique to explore the crude venom activities of five different Micrurus species from the south and southeast of Brazil: M. altirostris, M. corallinus, M. frontalis, M. carvalhoi and M. decoratus. All five venoms induced full inhibition of the muscle-type α1β1δε nAChR with different levels of reversibility. We found M. altirostris and M. frontalis venoms acting as partial inhibitors of the neuronal-type α7 nAChR with an interesting subsequent potentiation after one washout. We discovered that M. altirostris and M. corallinus venoms modulate the α1β2 GABAAR. Interestingly, the screening on KV1.3 showed that all five Micrurus venoms act as inhibitors, being totally reversible after the washout. Since this activity seems to be conserved among different species, we hypothesized that the Micrurus venoms may rely on potassium channel inhibitory activity as an important feature of their envenomation strategy. Finally, tests on NaV1.2 and NaV1.4 showed that these channels do not seem to be targeted by Micrurus venoms. In summary, the venoms tested are multifunctional, each of them acting on at least two different types of targets.

Co-occurrence patterns between false coral snake Atractus latifrons (Günther, 1868) (Serpentes: Dipsadidae) and venomous coral snakes from the Amazon.

Batesian mimicry may result in remarkable cases of phenotypic convergence that represent classic examples of evolution through natural selection. The existence of mimicry systems among coral snakes, however, remains controversial because of contradictions between the predictions of mimetic theory and the empirical patterns of co-occurrence and species abundance. Here, we analyze the geographic distribution of coral snake species of the genus Micrurus and populations of the false coral snake Atractus latifrons in Amazonia, and perform ecological niche modeling (ENM) analyzes to generate potential geographic distributions of species of Micrurus and A. latifrons, identify patterns of co-occurrence and assess whether the distribution of A. latifrons coincides with the distribution of Micrurus species, which could suggest the existence of a possible mimetic relationship between the species. We identified six Micrurus species that may represent mimetic models for A. latifrons. The results of the co-occurrence analysis corroborates the results from ENM, indicating that chromatic patterns of A. latifrons and their respective model species are aggregated. Our study suggests that all color patterns of A. latifrons - including the tricolor monads, and the more common tricolor dyads and tricolor tetrads - may benefit from the resemblance with other Micrurus species as perfect and imperfect mimics.

In vivo treatment with varespladib, a phospholipase A2 inhibitor, prevents the peripheral neurotoxicity and systemic disorders induced by Micrurus corallinus (coral snake) venom in rats

In this study, we investigated the action of varespladib (VPL) alone or in combination with a coral snake antivenom (CAV) on the local and systemic effects induced by Micrurus corallinus venom in rats. Adult male Wistar rats were exposed to venom (1.5 mg/kg – i.m.) and immediately treated with CAV (antivenom:venom ratio 1:1.5 ‘v/w’ – i.p.), VPL (0.5 mg/kg – i.p.), or both of these treatments. The animals were monitored for 120 min and then anesthetized to collect blood samples used for haematological and serum biochemical analysis; after euthanasia, skeletal muscle, renal and hepatic tissue samples were collected for histopathological analysis. M. corallinus venom caused local oedema without subcutaneous haemorrhage or apparent necrosis formation, although there was accentuated muscle morphological damage; none of the treatments prevented oedema formation but the combination of CAV and VPL reduced venom-induced myonecrosis. Venom caused neuromuscular paralysis and respiratory impairment in approximately 60 min following envenomation; CAV alone did not prevent the neurotoxic action, whereas VPL alone prevented neurotoxic symptoms developing as did the combination of CAV and VPL. Venom induced significant increase of serum CK and AST release, mostly due to local and systemic myotoxicity, which was partially prevented by the combination of CAV and VPL. The release of hepatotoxic serum biomarkers (LDH and ALP) induced by M. corallinus venom was not prevented by CAV and VPL when individually administered; their combination effectively prevented ALP release. The venom-induced nephrotoxicity (increase in serum creatinine concentration) was prevented by all the treatments. VPL alone or in combination with CAV significantly prevented the venom-induced lymphocytosis. In conclusion, VPL shows to be effective at preventing the neurotoxic, nephrotoxic, and inflammatory activities of M. corallinus venom. In addition, VPL acts synergistically with antivenom to prevent a number of systemic effects caused by M. corallinus venom.

Signaling Pathways in Proton and Non-proton ASIC1a Activation.

Acid-sensing ion channels (ASICs) regulate synaptic activities and play important roles in neurodegenerative diseases as well as pain conditions. Classically, ASICs are described as transiently activated by a reduced pH, followed by desensitization; the activation allows sodium influx, and in the case of ASIC1a-composed channels, also calcium to some degree. Several factors are emerging and extensively analyzed as modulators, activating, inhibiting, and potentiating specific channel subunits. However, the signaling pathways triggered by channel activation are only starting to be revealed.The channel has been recently shown to be activated through a mechanism other than proton-mediated. Indeed, the large extracellular loop of these channels opens the possibility that other non-proton ligands might exist. One such molecule discovered was a toxin present in the Texas coral snake venom. The finding was associated with the activation of the channel at neutral pH via the toxin and causing intense and unremitting pain.By using different pharmacological tools, we analyzed the downstream signaling pathway triggered either by the proton and non-proton activation for human, mouse, and rat ASIC1a-composed channels in in vitro models. We show that for all species analyzed, the non-protonic mode of activation determines the activation of the ERK signaling cascade at a higher level and duration compared to the proton mode.This study adds to the growing evidence of the important role ASIC1a channels play in different physiological and pathological conditions and also hints at a possible pathological mechanism for a sustained effect.

Cross-reactivity of some Micrurus venoms against experimental and therapeutic anti-Micrurus antivenoms

We developed experimental equine polyvalent and monovalent antivenoms against the venoms of Micrurus (M.) fulvius, M. nigrocinctus and M. surinamensis and studied their immunochemical reactivity on the venoms used as immunogens and on M. pyrrhocryptus, M altirostris and M. balyocoriphus venoms. Assessment of the neutralizing capacity of the polyvalent experimental antivenom was based on inhibition of lethality (preincubation and rescue assay experiments in mice) and indirect hemolytic and phospholipase activities. The immunochemical reactivity and neutralizing capacity were compared with those of two therapeutic antivenoms used for the treatment of coral snake envenomation in North America and in Argentina. In general, the experimental antivenom conferred a comparable level of neutralization against the venoms used as immunogens when compared to the therapeutic antivenoms and a certain level of cross-neutralization against the other venoms. The results suggest the need for additional venoms in the immunogenic mixture used, in order to obtain a broad spectrum anti-Micrurus antivenom with a good neutralizing potency. Paraspecific neutralization of South American coral snake venoms, although present at a higher level than the neutralization conferred by available nonspecific Micrurus therapeutic antivenoms, was rather low in relation to the specific neutralizing capacity.

FIRST CONFIRMED RECORD OF THE VENOMOUS CORAL SNAKE MICRURUS IBIBOBOCA (SERPENTES, ELAPIDAE) IN MINAS GERAIS, BRAZIL

We report for the first time the occurrence of the venomous snake species Micrurus ibiboboca in the state of Minas Gerais, Brazil, based on a specimen collected in an Atlantic Forest area. This is the sixth record of a venomous coral snake in Minas Gerais and increases to 160 the number of known snake species in the state.

Being a bright snake: Testing aposematism and mimicry in a neotropical forest

AbstractBased on color patterns and behavioral similarities, venomous coral snake Micrurus corallinus (Elapidae) may act as a model for two polymorphic species, Erythrolamprus aesculapii (Dipsadidae) and Micrurus decoratus (Elapidae). Plasticine replicas were used to investigate the aposematism of these coloration patterns and whether these species may be part of mimetic complexes in two Atlantic Forest localities in Southeast Brazil. Coral replicas were more avoided when set upon a white background, evincing that the pattern may act aposematically in contrast with light substrates. Birds attacked all four patterns equally during the mimicry experiments. Birds of prey, known to be effective in predating snakes, are quite abundant in the study areas, which may have led to this lack of avoidance. Accordingly, they predated more adult‐sized replicas, which could be more dangerous. Interestingly, opossum avoided the Micrurus corallinus and Erythrolamprus aesculapii replicas that resembled the model. This suggests that opportunistic predators, as the opossum may be important selective agents in mimicry complexes.Abstract in Portuguese is available with online material.

Functional, proteomic and transcriptomic characterization of the venom from Micrurus browni browni: Identification of the first lethal multimeric neurotoxin in coral snake venom

Proteomic characterization of Micrurus browni browni venom showed approximately 41 components belonging to 9 protein families, mainly phospholipases A2 (PLA2s) and three-finger toxins (3FTxs). Venom gland transcriptome yielded 39 venom transcripts belonging to 10 protein families. Functional characterization identified a multimeric toxin, here designated Brownitoxin-1, which comprises at least one PLA2 and one 3FTx. Its components have no or very low lethality individually but become extremely lethal when combined; both were partially characterized. Other two lethal components were identified: A neurotoxic PLA2, and a postsynaptic α-neurotoxin. LD50s as well as PLA2 and nAChR-blocking activities were determined for whole venom and isolated components. Application of venom to murine neuromuscular preparations caused a progressive decrease of twitch force that was irreversible after washing. Inhibition of PLA2 activity with p-bromophenacyl bromide (pBPB) showed that approximately 90% of toxicity is dependent on this activity. Non-lethal components include diverse 3FTxs, at least three enzymatically active PLA2s and the nociceptive toxin MitTx. No evidence of specificity towards prey was observed. This work is one of the most complete characterizations of a coral snake venom so far and its findings highlight the relevance of protein complexes in venom function. SignificanceThis study represents a profound analysis of the venom of the coral snake Micrurus browni browni, including a venom proteome, venom gland transcriptomic data and functional studies of whole venom and isolated toxins. It significantly contributes to the understanding of North American coral snake venoms, which are currently largely unknown. It includes characterization of relevant venom components, one of which represents the first description of a lethal multimeric neurotoxin in coral snake venom. This work highlights the importance of protein complexes in coral snake venom and could serve as a basis for the finding of several other multimeric toxins. Finally, we report the absence of taxon specificity, which has been previously reported in the venoms of other snakes of the same genus.

Renal effects of venoms of Mexican coral snakes Micrurus browni and Micrurus laticollaris

The Micrurus snake venoms mainly cause systemic complications, essentially neurotoxicity. Previous studies, however, have described that they are involved in the occurrence of acute kidney injury (AKI) in animal models. AKI pathogenesis in snakebites is multifactorial and involves immunological reactions, hemodynamic disturbances, and direct nephrotoxicity. The aim of this study was to compare the nephrotoxic effects of coral snake venoms from M. browni (MbV) and M. laticollaris (MlV) on the proximal tubular epithelial cell line (LLC-MK2) and isolated perfused kidney. Using an MTT assay, both venoms significantly reduced cell viability at higher concentrations (25–100 μg/mL). MlV (10 μg/mL) increased the perfusion pressure (PP) at 60, 90 and 120 min, while the MbV did it only at 90 and 120 min. Renal vascular resistance (RVR) decreased at 60 min and increased at 120 min with MbV, but decreased at 60, 90 and 120 min with MlV. Urinary flow (UF) alterations were not observed with MlV, but MbV elevated them at 90 and 120 min. Both venoms significantly decreased the glomerular filtration rate (GFR), %TNa+, %TK+ and %TCl− levels as of 60 min of perfusion. Oxidative stress analysis revealed that both venoms behaved similarly, reducing glutathione and increasing malondialdehyde levels. Kidney injury is not usually described in clinical cases of Micrurus snakebites. However, the potential for nephrotoxicity should be considered in the overall picture of envenomation.

Postmortem histopathology and detection of venom by ELISA following suicide by cobra (Naja kaouthia) envenomation

Forensic proof of venomous snakebite is challenging, particularly when the snake species involved is not endemic to the region in which the event occurred. This case report describes postmortem histopathological findings and venom detection following suicide by monocled cobra (Naja kaouthia) envenomation, at a location (Texas, USA) where that animal is not normally encountered. External, internal and histopathological examination was performed according to the standard of the local office of the medical examiner. Tissue homogenate and blood were assayed with a Naja-specific enzyme-linked immunosorbent assay (ELISA), using locally obtained coral snake (Micrurus) venom as negative control. External evidence of puncture marks, edema and erythema were associated with local evidence of microscopic necrosis, acute inflammation, vascular congestion and lymphadenitis. Cobra venom was present in cardiac serum and at higher concentrations in serum, soft tissue and lymph nodes near the puncture sites. In the absence of commercial kits for venom detection, confirmation of snake envenomation postmortem can be achieved by correlation of specific ELISA with local findings. Exclusion of related local snake venom is necessary to confirm the specificity of the assay.

Coral Snake Envenomations in Central and South America

Snakebites are increasingly recognized as a neglected tropical disease. The purposes of this review were to determine the significance of coral snakebites in Central and South America and how best to treat these envenomations. Questions that needed to be answered included: What is the incidence of coral snake envenomation? What are the features of coral snake envenomation? What are the pharmacological and nonpharmacological ways to treat these bites? Multiple case reports and series describe the clinical features of coral snake envenomations, which account for fewer than 5% of all snakebites in Central and South America. Coral snake venom is a complex mixture of neurotoxins and other biologically active substances. There are multiple coral snake antivenoms available, but no single antivenom can be used for all species. Coral snake envenomations are characterized by motor and sensory neurotoxicity. However, muscle damage, renal injury, hematologic toxicity, electrocardiographic abnormalities, and nonspecific signs and symptoms are also possible consequences. Supportive care and antivenom are the mainstays of therapy. Pressure immobilization may prevent systemic absorption, and neostigmine may protect against paralysis. Death is uncommon if treatment is initiated promptly. Nonspecific inhibitors of various venom components may serve as a bridge to definitive therapy.

A Quantitative Analysis of Micrurus Coral Snakes Reveals Unexpected Variation in Stereotyped Anti-Predator Displays Within a Mimicry System.

SynopsisWarning signals in chemically defended organisms are critical components of predator–prey interactions, often requiring multiple coordinated display components for effective communication. When threatened by a predator, venomous coral snakes (genus Micrurus) display a vigorous, non-locomotory thrashing behavior that has previously been qualitatively described. Given the high contrast and colorful banding patterns of these snakes, this thrashing display is hypothesized to be a key component of a complex aposematic signal under strong stabilizing selection across species in a mimicry system. By experimentally testing snake response across simulated predator cues, we analyzed variation in the presence and expression of a thrashing display across five species of South American coral snakes. Although the major features of the thrash display were conserved across species, we found that predator cue type, snake body size, and species identity predict significant inter- and intraspecific variation in the propensity to perform a display, the duration of thrashing, and the curvature of snake bodies. We also found an interaction between curve magnitude and body location that clearly shows which parts of the display vary most across individuals and species. Our results suggest that contrary to the assumption that all Micrurus species and individuals perform the same display, a high degree of variation exists despite presumably strong selection to conserve a common signal. This quantitative behavioral characterization presents a new framework for analyzing the non-locomotory motions displayed by snakes in a broader ecological context, especially for signaling systems with complex interaction across multiple modalities.

Anticoagulant activity of krait, coral snake, and cobra neurotoxic venoms with diverse proteomes are inhibited by carbon monoxide

A phenomena of interest is the in vitro anticoagulant effects of neurotoxins found in elapid venoms that kill by paralysis. These enzymes include phospholipase A2 (PLA2), and it has recently been demonstrated that carbon monoxide inhibits the PLA2-dependent neurotoxin contained in Mojave rattlesnake type A venom. The purpose of this investigation was to assess if the anticoagulant activity of elapid venoms containing PLA2 and/or three finger toxins could be inhibited by carbon monoxide. Venoms collected from Bungarus multicinctus, Micrurus fulvius, and five Naja species were exposed to carbon monoxide via carbon monoxide releasing molecule-2 prior to placement into human plasma. Coagulation kinetics were assessed via thrombelastography. Compared with plasma without venom addition, all venoms had significant anticoagulant effects, with a 160-fold range of concentrations having similar anticoagulant effects in a species-specific manner. Carbon monoxide significantly inhibited the anticoagulant effect of all venoms tested, but inhibition was not complete in all cases. Given that individual neurotoxin activity often depends on intact activity that includes anticoagulant action, it may be possible that carbon monoxide inhibits neurotoxicity. Future investigation is justified to assess such carbon monoxide mediated inhibition with purified neurotoxins in vitro and in vivo.

Novel three-finger toxins from Micrurus dumerilii and Micrurus mipartitus coral snake venoms: Phylogenetic relationships and characterization of Clarkitoxin-I-Mdum

Micrurus mipartitus and M. dumerilii are the most medically important coral snakes in Colombia. Proteomic characterization of their venoms has previously shown that proteins of the three-finger toxin (3FTx) family are abundant components, especially in M. mipartitus (61%) and to a lesser extent in M. dumerilii (28%). In order to increase knowledge on these toxins, in this work a major 3FTx of M. dumerilii venom (8% of the venom proteins), named Clarkitoxin-I-Mdum, was isolated and characterized. Its amino acid sequence comprises 66 residues, with an isotope-averaged molecular mass of 7537 ± 2 Da and a theoretical pI of 9.36, presenting the conserved pattern of eight cysteines that classifies it as a short-chain (type I) 3FTx. Clarkitoxin-I-Mdum was not lethal to mice by intravenous or intracerebroventricular route and was not cytolytic to myogenic cells in vitro. On the other hand, five coding sequences for 3FTxs were obtained from the venom gland of M. mipartitus. These novel toxin sequences were named Mm3FTx-01 to Mm3FTx-05, all of them also presenting the eight conserved cysteines of short-chain 3FTxs. Phylogenetic analysis revealed high variability of 3FTxs from Micrurus, and ELISA using antibodies raised to the major 3FTxs from M. mipartitus and M. dumerilii confirmed their immunochemical divergence. These results highlight the relevance of performing further studies aiming at a deeper understanding of the functional and antigenic relationships among specific Micrurus toxins, with important implications for the production of antivenoms.

Inhibition of Kv2.1 Potassium Channels by MiDCA1, A Pre-Synaptically Active PLA2-Type Toxin from Micrurus dumerilii carinicauda Coral Snake Venom.

MiDCA1, a phospholipase A2 (PLA2) neurotoxin isolated from Micrurus dumerilii carinicauda coral snake venom, inhibited a major component of voltage-activated potassium (Kv) currents (41 ± 3% inhibition with 1 μM toxin) in mouse cultured dorsal root ganglion (DRG) neurons. In addition, the selective Kv2.1 channel blocker guangxitoxin (GxTx-1E) and MiDCA1 competitively inhibited the outward potassium current in DRG neurons. MiDCA1 (1 µM) reversibly inhibited the Kv2.1 current by 55 ± 8.9% in a Xenopus oocyte heterologous system. The toxin showed selectivity for Kv2.1 channels over all the other Kv channels tested in this study. We propose that Kv2.1 channel blockade by MiDCA1 underlies the toxin’s action on acetylcholine release at mammalian neuromuscular junctions.

Comparative venomics of Brazilian coral snakes: Micrurus frontalis, Micrurus spixii spixii, and Micrurus surinamensis

A comparative venom proteomic analysis of the Brazilian southern coral snake, M. frontalis, the Amazon coral snake M. spixii spixii, and the aquatic coral snake M. surinamensis is reported. Venoms from M. frontalis and M. s. spixii were composed mainly (>90% of the total venom proteome) by 3FTxs and PLA2s in different proportions, and minor proteins from 2 to 5 protein families. Conversely, the aquatic coral snake expressed a streamlined (95%) 3FTx venom with low abundance (4.2%) of PLA2 molecules. A compositional-lethal activity for natural prey correlation analysis suggests that M. surinamensis venom may has evolved under strong pressure to quickly immobilize aquatic prey. On the other hand, venoms from M. frontalis and M. s. spixii, whose diet consist mainly of amphisbaenians and colubrid snakes, may have been shaped through balancing selection. Our work provides strong evidence for the occurrence in M. frontalis venom, but not in those from M. s. spixi and M. surinamensis, of a KUN-PLA2 complex homologue to heterodimeric venom toxins from some long-tailed monadal coral snakes that target acid-sensing receptors ASIC1a/2 evoking pain. The M. frontalis protein would represent the first example of a KUN-PLA2 heterodimer in a South American short-tailed triadal coral snake venom.