World Elapids Research Articles

Snake venomics of Micrurus alleni and Micrurus mosquitensis from the Caribbean region of Costa Rica reveals two divergent compositional patterns in New World elapids

Protein composition, toxicity, and neutralization of the venoms of Micrurus alleni and Micrurus mosquitensis, two sympatric monadal coral snakes found in humid environments of the Caribbean region of Costa Rica, were studied. Proteomic profiling revealed that these venoms display highly divergent compositions: the former dominated by three-finger toxins (3FTx) and the latter by phospholipases A2 (PLA2). Protein family abundances correlated with enzymatic and toxic characteristics of the venoms. Selective inhibition experiments showed that PLA2s play only a marginal role in the lethal effect of M. alleni venom, but have a major role in M. mosquitensis venom. Proteomic data gathered from other Micrurus species evidenced that the two divergent venom phenotypes are recurrent, and may constitute a general trend across New World elapids. Further, M. mosquitensis, but not M. alleni, venom contains PLA2-like/Kunitz-type inhibitor complex(es) that resemble the ASIC1a/2-activating MitTx heterodimeric toxin isolated from Micrurus tener venom. The evolutionary origin and adaptive relevance of the puzzling phenotypic variability of Micrurus venoms remain to be understood. An antivenom against the PLA2-predominant Micrurus nigrocinctus venom strongly cross-recognized and neutralized M. mosquitensis venom, but only weakly M. alleni venom.

The venom-gland transcriptome of the eastern coral snake (Micrurus fulvius) reveals high venom complexity in the intragenomic evolution of venoms.

BackgroundSnake venom is shaped by the ecology and evolution of venomous species, and signals of positive selection in toxins have been consistently documented, reflecting the role of venoms as an ecologically critical phenotype. New World coral snakes (Elapidae) are represented by three genera and over 120 species and subspecies that are capable of causing significant human morbidity and mortality, yet coral-snake venom composition is poorly understood in comparison to that of Old World elapids. High-throughput sequencing is capable of identifying thousands of loci, while providing characterizations of expression patterns and the molecular evolutionary forces acting within the venom gland.ResultsWe describe the de novo assembly and analysis of the venom-gland transcriptome of the eastern coral snake (Micrurus fulvius). We identified 1,950 nontoxin transcripts and 116 toxin transcripts. These transcripts accounted for 57.1% of the total reads, with toxins accounting for 45.8% of the total reads. Phospholipases A2 and three-finger toxins dominated expression, accounting for 86.0% of the toxin reads. A total of 15 toxin families were identified, revealing venom complexity previously unknown from New World coral snakes. Toxins exhibited high levels of heterozygosity relative to nontoxins, and overdominance may favor gene duplication leading to the fixation of advantageous alleles. Phospholipase A2 expression was uniformly distributed throughout the class while three-finger toxin expression was dominated by a handful of transcripts, and phylogenetic analyses indicate that toxin divergence may have occurred following speciation. Positive selection was detected in three of the four most diverse toxin classes, suggesting that venom diversification is driven by recurrent directional selection.ConclusionsWe describe the most complete characterization of an elapid venom gland to date. Toxin gene duplication may be driven by heterozygote advantage, as the frequency of polymorphic toxin loci was significantly higher than that of nontoxins. Diversification among toxins appeared to follow speciation reflecting species-specific adaptation, and this divergence may be directly related to dietary shifts and is suggestive of a coevolutionary arms race.

Transcriptomic basis for an antiserum against Micrurus corallinus (coral snake) venom

BackgroundMicrurus corallinus (coral snake) is a tropical forest snake belonging to the family Elapidae. Its venom shows a high neurotoxicity associated with pre- and post-synaptic toxins, causing diaphragm paralysis, which may result in death. In spite of a relatively small incidence of accidents, serum therapy is crucial for those bitten. However, the adequate production of antiserum is hampered by the difficulty in obtaining sufficient amounts of venom from a small snake with demanding breeding conditions. In order to elucidate the molecular basis of this venom and to uncover possible immunogens for an antiserum, we generated expressed sequences tags (ESTs) from its venom glands and analyzed the transcriptomic profile. In addition, their immunogenicity was tested using DNA immunization.ResultsA total of 1438 ESTs were generated and grouped into 611 clusters. Toxin transcripts represented 46% of the total ESTs. The two main toxin classes consisted of three-finger toxins (3FTx) (24%) and phospholipases A2 (PLA2s) (15%). However, 8 other classes of toxins were present, including C-type lectins, natriuretic peptide precursors and even high-molecular mass components such as metalloproteases and L-amino acid oxidases. Each class included an assortment of isoforms, some showing evidence of alternative splicing and domain deletions. Five antigenic candidates were selected (four 3FTx and one PLA2) and used for a preliminary study of DNA immunization. The immunological response showed that the sera from the immunized animals were able to recognize the recombinant antigens.ConclusionBesides an improvement in our knowledge of the composition of coral snake venoms, which are very poorly known when compared to Old World elapids, the expression profile suggests abundant and diversified components that may be used in future antiserum formulation. As recombinant production of venom antigens frequently fails due to complex disulfide arrangements, DNA immunization may be a viable alternative. In fact, the selected candidates provided an initial evidence of the feasibility of this approach, which is less costly and not dependent on the availability of the venom.

Efficacy of a Pressure-Immobilization bandage in delaying the onset of systemic toxicity in a porcine model of eastern coral snake (Micrurus fulvius) envenomation

Study objectives: Pressure-immobilization bandages (PIBs) are first-aid devices that are used in foreign regions where neurotoxic elapid envenomations are common. These devices are theorized to impede lymphatic return to the central circulation, thereby sequestering venom at the bite site and delaying systemic toxicity, which allows additional time to provide the victim with definitive care in the form of antivenom and advanced airway management. Coral snakes are New World elapids with primarily neurotoxic venoms. PIB use has never been formally examined in coral snake envenomations. We evaluate the efficacy of these devices in delaying the onset of systemic toxicity in a porcine model of Micrurus fulvius envenomation. Methods: The study design was a randomized, controlled trial of PIBs in a porcine model of M fulvius envenomation. The setting was a university animal laboratory. Subjects were 12 pigs, ranging from 9.1 to 11.4 kg. The animals were sedated, intubated, and anesthetized. Volatile anesthesia was maintained by animal care technicians at a level to ensure animal comfort and that all pigs were breathing spontaneously. Lyophilized venom was obtained from Natural Toxins Research Center (Kingsville, TX) and resuspended in pure water at a concentration of 10 mg/mL. The lethal dose of M fulvius venom in a human has been estimated at 5 mg, and the venom yield of a large snake can be 20 mg. We chose a 10-mg dose to ensure that all animals would develop severe toxicity during the study. The subjects were injected with 10 mg of M fulvius venom in the subcutaneous tissue of the left distal foreleg. Immediately after injection, the subjects were randomized to either receive a PIB 1 minute after envenomation or to have no treatment. The PIB consisted of an elastic bandage applied to the entire extremity at a pressure such that a finger could be easily passed under. The same operator applied all bandages. Aluminum and foam splints were then loosely taped on either side to immobilize the extremity. Pulse rate, respiratory rate, pulse oximetry, and quality of respirations were recorded at 10-minute intervals. The primary endpoint was survival to 8 hours, and groups were compared using χ 2 analysis. Groups were also compared with respect to time to onset of systemic toxicity in the form of respiratory compromise, and total time of survival. A veterinary pathologist performed necropsies on all animals, and the histology of the envenomation site was examined. One animal from each group was removed because of the discovery of preexisting respiratory pathology. Results: Four of 5 pigs in the treatment group survived to 8 hours, but none in the control group did ( P =.0036). Time to onset of signs of respiratory compromise was 170±33.3 minutes in the control group. Two pigs in the treatment group showed signs of respiratory compromise before 8 hours, 1 at 310 minutes and 1 at 460 minutes. One of these subjects did survive to 8 hours. None of the pigs had histologic changes at the envenomation site consistent with ischemic or pressure-related injury. Conclusion: Pressure immobilization bandages were effective in delaying the onset of systemic venom toxicity in this model of M fulvius envenomation. According to this study, the use of PIBs can be recommended as initial treatment in human envenomations by coral snakes.

Food Habits and Reproductive Biology of the Endemic Melanesian Elapids: Are Tropical Snakes Really Different?

Do the ecological traits of tropical snakes differ from those of temperate-zone species? We examined preserved specimens in museums to quantify body sizes, sexual dimorphism, dietary habits, and reproductive cycles of the proteroglyphous snake lineages endemic to New Guinea and the Solomon Islands for comparison with previously-studied Australian and Asian species. The Melanesian snakes comprise a diverse group in terms of body sizes (adult sizes from 1 meter), body shapes (slender to heavy- bodied), and behavior (nocturnal to diurnal). Males attain larger sizes than do females in Aspidomorphus and Micropechis, but the reverse is true in Loveridgelaps, Salomonelaps, and Toxicocalamus. Aspido- morphus and Toxicocalamus show significant sex differences in relative head length, but species in the other genera do not. All taxa for which we have data on reproductive mode are oviparous, and produce an average of 3 to 9 large eggs per clutch. Relative to maternal body sizes, Melanesian elapids have significantly smaller clutches than do Australian elapids. The two smaller-bodied genera showed dietary specialisation (on earthworms by Toxicocalamus, and on skinks by Aspidomorphus). Toxicocalamus and the Fijian Ogmodon are thus the only Old World elapids to specialise on invertebrate prey. The larger- bodied taxa (Loveridgelaps, Micropechis, Salomonelaps) have more generalised diets, consisting primarily of lizards and frogs. The distinctive features of the Melanesian elapid fauna (low fecundity in all taxa, and earthworm-eating by Toxicocalamus) may reflect adaptations to tropical habitats, but generalizations about the ecological adaptations of tropical snakes remain elusive.

A Phylogenetic Analysis of the New World Coral Snakes (Elapidae: Leptomicrurus, Micruroides, and Micrurus) Based on Allozymic and Morphological Characters

The phylogenetic relationships of 18 species of New World snakes (Elapidae: Leptomi- crurus, Micruroides, and Micrurus) were examined with morphological and allozymic characters analyzed by parsimony. Two species of kraits (Elapidae: Bungarus) were included as outgroups. Coral snake mor- phology was found to be very conservative and few informative morphological characters were found. The general relationships of all snakes are discussed in light of the morphological synapomorphies identified in this study. The results indicate that Micrurus is paraphyletic because of the exclusion of Leptomicrurus. This is rectified by placing Leptomicrurus into the synonymy of Micrurus. The New World snakes are a large group of small to moderate-sized, dangerously ven- omous snakes in the family Elapidae. The 61 species currently recognized are divided among three genera: Micrurus (57 species), Leptomicru- rus (three species), and Micruroides (one species). Micrurus is found in a wide range of terrestrial habitats throughout its range from the south- eastern United States to southern South Amer- ica. Leptomicrurus is found in tropical lowland moist and wet forests in Amazonia. The mono- typic Micruroides is found in the Sonoran Desert of the southwestern United States and adjacent northwestern Mexico. Although various Old World elapids (e.g., Calliophis, Maticora, Aspi- delaps) are often termed coral snakes, the term is restricted herein to New World elapids.

Karyotypes of Five Species of Coral Snakes (Micrurus)

There are more than 50 species of coral snakes classified in the genera Micrurus and Micruroides (Roze, 1982). The evolutionary relationships within this group, as well as between coral snakes and Old World elapids, have been studied only to a partial extent. Most studies have been based on morphological analyses (e.g., Savage and Vial, 1973; Roze, 1982), although immunochemical comparisons of serum albumins have also been used (Cadle and Sarich, 1981; Cadle and Gorman, 1981). Chromosome studies have shown extensive karyotype variation between elapid species (Singh, 1972a; Singh, 1974; Graham, 1977; Gutierrez and Bolafios, 1979; Gorman, 1981). Thus, karyology may become a useful tool in understanding the phylogenetic relationships among elapid snakes in general, and within coral snakes in particular. Karyotypes of five species of Micrurus have been reported: M. lemniscatus (Becak and Becak, 1969), M. fulvius (Graham, 1977), M. nigrocinctus, M. alleni, and M. mipartitus (Gutierrez and Bolafios, 1979, 1981). In this work we describe the karyotypes of five additional species of Micrurus: M. elegans, M. diastema, M. hippocrepis, M. browni, and M. surinamensis. To obtain the karyotypes, the method of Gutierrez and Bolafios (1979), based on the simultaneous injection of phytohemagglutinin and colchicine, was used. From each species, at least 30 mitotic figures were studied to determine the diploid number; from these, four were selected and photographed to prepare idiograms. Chromosomes were classified using the nomenclature proposed by Levan et al. (1964). The Nombre Fondamentale (NF) was defined as the number of arms of the macrochromosomes. The specimens used in this study were collected in the following locations: M. browni, two males from Volcan de Agua, Guatemala; M. diastema sapperi, one male from Izabal, Guatemala; M. diastema apiatus, one female from Purulha, Baja Verapaz, Guatemala; M. elegans, three males from Purulha, Baja Verapaz, Guatemala; and M. surinamensis, one male from Guyanas. All of the specimens studied are kept at the Museum of Zoology of the Universidad de Costa Rica. In M. elegans the diploid number is 30, with 16 macro and 14 microchromosomes and with an NF of

An immunological assessment of the phylogenetic position of New World coral snakes

The New World coral snakes (micrurines), genera Micrurus and Micruroides have recently been seen as derived from a lineage of South American colubrids, rather than from a common lineage with Old World elapids and sea snakes as traditionally accepted. We compared serum albumins of representative coral snakes, Old World elapids, sea snakes, and neotropical colubrids immunologically. Phylogenetic analysis of the biochemical data unambiguously allies the micrurines with the family Elapidae as it is currently understood. Using the albumin molecular clock calibration derived from other terrestrial vertebrates. we suggest a late Oligocene‐early Miocene separation between the New and Old World elapid lineages. This requires a movement of elapid stocks from Asia into North America, and supporting evidence for this model is derived from several paleontological sources. We suggest that a number of extant micrurine lineages have had long independent histories.