Neuromuscular activity of the venoms of the Colombian coral snakes Micrurus dissoleucus and Micrurus mipartitus: An evolutionary perspective

Toxicon 59 (2012) 132–142

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Neuromuscular activity of the venoms of the Colombian coral snakes Micrurus dissoleucus and Micrurus mipartitus: An evolutionary perspective Camila Renjifo a, *, Eric N. Smith b, Wayne C. Hodgson c, Juan M. Renjifo d, Armando Sanchez a, Rodrigo Acosta e, Jairo H. Maldonado f, Alain Riveros a, g a

Departamento de Ciencias Fisiológicas, Facultad de Medicina, Pontificia Universidad Javeriana, Bogotá, Colombia Department of Biology, The University of Texas at Arlington, Arlington, TX 76019, USA Monash Venom Group, Department of Pharmacology, Monash University, Clayton, Australia d Departmento de Biología, Universidad del Magdalena, Santa Marta, Colombia e Departamento de Patologia, Clinica Marly, Bogota, Colombia f Serpentario, Parque Recreativo y Zoológico de Piscilago, Nilo, Tolima, Colombia g Laboratorio de Fisiología, Facultad de Medicina, Universidad Militar Nueva Granada, Colombia b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 June 2011 Received in revised form 23 October 2011 Accepted 28 October 2011 Available online 15 November 2011

The venoms of coral snakes (genus Micrurus) are known to induce a broad spectrum of pharmacological activities. While some studies have investigated their potential human effects, little is known about their mechanism of action in terms of the ecological diversity and evolutionary relationships among the group. In the current study we investigated the neuromuscular blockade of the venom of two sister species Micrurus mipartitus and Micrurus dissoleucus, which exhibit divergent ecological characteristics in Colombia, by using the chick biventer cervicis nerve-muscle preparation. We also undertook a phylogenetic analysis of these species and their congeners, in order to provide an evolutionary framework for the American coral snakes. The venom of M. mipartitus caused a concentration-dependant inhibition (3–10 mg/ml) of nerve-mediated twitches and significantly inhibited contractile responses to exogenous ACh (1 mM), but not KCl (40 mM), indicating a postsynaptic mechanism of action. The inhibition of indirect twitches at the lower venom dose (3 mg/ml) showed to be triphasic and the effect was further attenuated when PLA2 was inhibited. M. dissoleucus venom (10–50 mg/ml) failed to produce a complete blockade of nerve-mediated twitches within a 3 h time period and significantly inhibited contractile responses to exogenous ACh (1 mM) and KCl (40 mM), indicating both postsynaptic and myotoxic mechanisms of action. Myotoxic activity was confirmed by morphological studies of the envenomed tissues. Our results demonstrate a hitherto unsuspected diversity of pharmacological actions in closely related species which exhibit divergent ecological characteristics; these results have important implications for both the clinical management of Coral snake envenomings and the design of Micrurus antivenom. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Micrurus Neurotoxic Myotoxic Chick biventer cervicis Venom

1. Introduction * Corresponding author. Present address: Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Pembroke Place L3 5QA, Liverpool, UK. Tel.: þ44 01517053231x3162. E-mail address: [email protected] (C. Renjifo). 0041-0101/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2011.10.017

Terrestrial New World genera of the Family Elapidae, also known as coral snakes (Micrurus, Leptomicrurus and Micruroides), comprise a group of more than 120 species and subspecies distributed from the Southern United States

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to the pampas of Central Argentina. The genus Micrurus is the most diverse of the three (with around 70 species) and achieves its greatest diversity near the equator (Campbell and Lamar, 2004; Roze, 1967, 1983, 1996), where it is known to inhabit a wide range of environments from lowland rainforests and deserts to highland cloud forests (Campbell and Lamar, 2004). Although little information is available about their specific dietary habits, they are known to be highly diverse and generally dependent on the characteristics of the ecological niche that each species occupies (Campbell and Lamar, 2004; Roze, 1996). Within the genus Micrurus, coral snakes have been traditionally divided into bicolor, monadal and triadal species according to their coloration (and hemipenal shape) and phylogenetic work has supported these distinctions to varying degrees (Campbell and Lamar, 2004; Roze, 1996). Due to the relative rarity and extreme morphological conservatism, coral snake relationships remain poorly understood and their taxonomy unreliable. Recent work carried out by Gutberlet and Harvey (2004), who combined the results of four studies that partially overlap in terms of taxonomic sampling (Roze and Bernal-Carlo, 1987; Slowinski, 1995; Jorge da Silva and Sites, 2001; Sasa and Smith, 2001), represents the most complete, albeit tenuous, hypothesis for coral snake phylogeny available. Coral snake venoms are extremely toxic and their envenoming in humans constitutes a medical emergency (Cecchini et al., 2005; de Roodt et al., 2004; Manock et al., 2008). Although very little is known about the composition and pharmacological effects of Micrurus venoms, it has been reported that the neuromuscular blockade responsible for the fatality of these envenomings (i.e. muscle paralysis and subsequent death by respiratory arrest) is the result of post synaptically acting neurotoxins (also known as three finger toxins (3FTxs) or a-neurotoxins), which bind to the nicotinic acetylcholine (ACh) receptor preventing the binding of the neurotransmitter (i.e. M. frontalis, Micrurus lemniscatus, Micrurus dumerilii and M. spixiii, Micrurus pyrrhocryptus venoms) (Camargo et al., 2011; Cecchini et al., 2005; Serafim et al., 2002; Vital Brazil, 1990; Vital Brazil and Fontana, 1983/84). Furthermore, presynaptic or b-neurotoxins, which are neurotoxic phospholipases A2 (PLA2) (Montecucco and Rossetto, 2000), have also been found to contribute to the mechanism of action of these venoms by inhibiting the release of ACh from the motor nerve endings (i.e. Micrurus corallinus venom) (Silveira de Oliveira et al., 2000; Vital Brazil, 1990; Vital Brazil and Fontana, 1983/84). A variety of other local and systemic effects in patients bitten by different species has also been shown in a number of comparative studies including cardiotoxic, myotoxic, hemolytic, hemorrhagic and, in some cases, edematogenic activities (Barros et al., 1994; Gutierrez et al., 1983; Roze, 1996; Vital Brazil, 1990; Warrell, 2004). Clinical manifestations of snakebite depend strongly on the composition of the venom (Chippaux et al., 1991; Gutierrez et al., 2009; Shashidharamurthy et al., 2002; Warrell, 1989). Likewise, variation in the composition of venom has a significant effect on the success of antivenom therapy. Venom variation has been a subject of much interest and has been frequently associated with strong natural selection towards specific diets, as a result of the

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species specialization in particular habitats (Barlow et al., 2009; Casewell et al., 2009; Daltry et al., 1996; Kordis and Gubensek, 2000; Nunez et al., 2009; Sanz et al., 2006). Coral snakes represent a group of high interest in this sense due to the diversity of ecosystems they inhabit and the variety of prey items that they feed on, which translates to the consistent lack of antivenom cross reactivity that has been reported to date (Rey-Suárez et al., 2011; Tanaka et al., 2010). To understand the variations in the mechanism of action of coral snake venoms, it is important to consider their ecological and taxonomic characteristics in order to provide a clinical and evolutionary framework for the group. Here we investigated the neuromuscular mechanism of action of venom from the Colombian sister species Micrurus mipartitus and Micrurus dissoleucus (ENS, Personal communication) based on their established ecological differences. M. mipartitus is more commonly found in wet areas, including rain and cloud forests, whilst M. dissoleucus inhabits mostly xeric areas, such as tropical and subtropical thorn woodland and dry to very dry forests (Campbell and Lamar, 2004; Roze, 1996). In order to provide an evolutionary framework for the American coral snakes, we carried out this study alongside a phylogenetic analysis of the species M. mipartitus and M. dissoleucus and their relatives. 2. Materials and methods 2.1. Venom studies 2.1.1. Snakes, venom preparation and storage The snakes used in this study are known at the subspecies level as M. dissoleucus nigrirostris from the region of Magdalena, Colombia and M. mipartitus decussatus from Tolima, Colombia. Venoms were extracted and preserved immediately in liquid nitrogen to subsequently freeze-dry and store at 20  C. The use of animals in the current study was approved by the ethics research committee of the department of Biology, Faculty of Basic Sciences of the Pontificia Universidad Javeriana in Bogota, Colombia. 2.1.2. Chick biventer cervicis nerve-muscle preparation Tissues were dissected from male chicks (4–10 days old) as described previously by Gingsborg and Warriner (1960). Preparations were mounted under 1 g tension in a 15 ml organ bath containing physiological salt solution of the following composition (mM): NaCl, 118.4; NaHCO3, 25; glucose, 11; KCl, 4.7; MgSO4, 1.2; KH2PO4, 1.2 and CaCl2, 2.5, at 34  C and bubbled with carbogen (95% O2 and 5% CO2). In experiments examining the neurotoxic effects of venom, nerve-mediated twitches (i.e. indirect) were evoked by electrical stimulation (0.2 ms duration, 0.1 Hz and supramaximal voltage) using a low-voltage stimulator incorporated in PowerLabÒ. Tissues were allowed to equilibrate for at least 30 min before the addition of venom. Venom (3, 10 and 50 mg/ml), where indicated, was left in contact with the preparations until complete twitch blockade occurred or for a 3 h period. At the conclusion of the experiments, responses to exogenous acetylcholine (ACh, 1 mM for 30 s)

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and potassium chloride (KCl, 40 mM for 30 s) were obtained in the absence of electrical stimulation. In cases where the venom caused complete blockade, the t50 (i.e. time to cause 50% of inhibition of twitches) was calculated in order to provide a quantitative measure of neurotoxicity. Where indicated, neostigmine (50 mM) was added at the t50 time point to examine reversibility of the effects of the venom. In experiments examining the myotoxic effects of venom, muscle-mediated twitches (i.e. direct) were evoked by electrical stimulation (2 ms duration, 0.1 Hz and supramaximal voltage). Prior to the addition of the venom, neuromuscular transmission was abolished by the addition of tubocurarine (10 mM) to the physiological salt solution, ensuring that the twitches were due only to direct muscle stimulation. Venom (10 mg/ml) was left in contact with the preparation until twitch blockade occurred, or for a 3 h period (as above). Histological examination of the tissues was used to confirm myotoxicity. 2.1.3. Inhibition of PLA2 Several studies have shown that presynaptic PLA2 activity can be inhibited in chick biventer cervicis nervemuscle preparations when the safety factor of transmission is lowered by replacing Ca2þ with Sr2þ in the physiological salt solution (Chang et al., 1977; Dodge et al., 1969; Kuruppu et al., 2005; Yu et al., 1993). In order to examine the effect of inhibiting PLA2 activity of the venoms, nerve-mediated twitches (i.e. indirect) were evoked by electrical stimulation (as described above) and tissues were allowed to stabilize in normal physiological salt solution. After 30 min, tissues were washed with Ca2þfree physiological salt solution until twitches were abolished and then allowed to equilibrate for 30 min in physiological salt solution with Sr2þ (10 mM) replacing Ca2þ before the addition of venom (3 mg/ml). 2.1.4. Histology At the conclusion of the experiment, tissues were removed from the organ bath and immediately placed into 10% formaldehyde. Transverse sections of the tissues (14 mm) were cut using a Microtome Sacura (Accu-cut SRM 200) and further placed onto gelatin-coated slides. Tissue sections were fixed for 15 min in 4% formaldehyde, stained with hematoxylin and eosin and examined under a light microscope (Motic Digital Microscope DMB1-223). Areas exhibiting pathological changes were photographed using the program Motic Images Plus 2.0 ML and package Motic MC2000 1.0. 2.1.5. Drugs The following drugs were used: acetylcholine chloride (Sigma); neostigmine (Rotexmedica) and d-tubocurarine (Sigma). All stock solutions were made up in distilled water with further dilutions in physiological salt solution for in vitro experiments. 2.1.6. Analyses of results and statistics Responses were measured via a Grass force displacement transducer (FT03) and recorded on a PowerLab System (ADInstruments) using CHART 5 for Windows. Data

was expressed as mean  SEM. For both neurotoxicity and myotoxicity studies, twitch tension was expressed as a percentage of the pre-treated twitch tension and statistical difference determined by a one-way analysis of variance (ANOVA) at the 180 min (3 h) time point. For myotoxicity studies, baseline tension was expressed as a percentage of the pre-treated baseline tension and statistical difference was determined by a one-way ANOVA at the 180 min time point. Contractures to ACh and KCl were expressed as a percentage of the control and were compared via one-way analysis of variance (ANOVA). All one-way ANOVA were followed by a Dunnett’s post hoc test. Unpaired Student’s t-test was used to analyze the data examining the effect of PLA2 inhibition and the effect of neostigmine versus their respective controls. Statistical significance was indicated when P < 0.05. 2.2. Phylogenetic analyses This study included 36 specimen samples, 22 sequences available through GenBank-18 from Nelson Jorge da Silva and Sites (2001) and four from Castoe et al. (2007), and 14 new sequences. The reconstruction had representatives of all American coral snake genera. Micruroides euryxanthus, sister to other American coral snakes (Castoe et al., 2007), was used as outgroup taxon. The ingroup consisted of 27 taxa, one member of the genus Leptomicrurus, and seven monadal, 16 triadal and two bicolor coral snakes of the genus Micrurus. Voucher information and GenBank accession numbers are listed in Appendix A. All the new samples are supported by a corresponding voucher specimen, tissue, or photograph accessioned in a public research collection. We used the taxonomic nomenclature presented by Campbell and Lamar (2004) for ease of comparison, but followed Passos and Fernandes (2005) in recognizing the subspecies of Micrurus surinamensis as full species. Nomenclatural re-arrangements for specimens analyzed by Jorge da Silva and Sites (2001) are presented and explained in Appendix A. Phylogenetic inference was based on a 850 base pair (bp) segment of the mtDNA protein gene NADH subunit 4 (ND4, 666 bp) and adjacent tRNA coding sequences (Histidine, Serine-AGY, and partial Leucine-UUR, 184 bp). Laboratory methods for DNA isolation and PCR amplification followed those described by Castoe et al. (2007), including the use of primers. ExoSap It (USB Corporation, Cleveland, Ohio, USA) was used to clean amplified fragments and post PCR-cleanup sequencing protocols were performed by the UTA genomics core facility (Arlington, Texas, USA; http://gcf.uta.edu). Sequences were edited using Sequencher 4.1 (GeneCodes, Ann Arbor, Michigan, USA) and aligned manually using GeneDoc (Nicholas and Nicholas, 1997). Homology of DNA characters was inferred from the nucleotide sequences, the inferred amino-acid sequences, and transfer RNA (tRNA) secondary structure models (Kumazawa and Nishida, 1993). Parsimony-based analyses were conducted using PAUP* 4.0b10 (Swofford, 2002). Maximum Parsimony (MP) analysis was conducted under heuristic search criteria using TBR branch swapping and 10 random addition sequence replicates with all characters weighted equally and gap sites

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excluded. Weighted parsimony (WP) analysis was conducted utilizing a tri-level weighting scheme (Benabib et al., 1997; Flores-Villela et al., 2000) with gaps coded as a fifth base. Tri-level weighting incorporates three different levels of information on the structure and inferred function of nucleotide substitutions. Under this WP scheme, transitions have a weight of 1, transversions are weighted 2, and any nucleotide substitution that is inferred to cause an aminoacid substitution is weighted þ1 more. MP and WP employed ACCTRAN optimization of character state changes. Tree searching was conducted with 500 random addition sequence replicates, and all searches used the tree-bisectionregrafting method of proposing new topologies. MP and WP bootstrap analyses (Felsenstein, 1985) involved 2000 pseudoreplicates with 2–10 random addition sequences each. To search for appropriate models of nucleotide evolution for probabilistic analyses we employed the program Modeltest 3.7 (Posada and Crandall, 1998) with an Akaike selection criterion. We partitioned these analyses by codon position for bases in open reading frames and applied a single model search for the tRNA fragment. This scheme resulted in four partitions. Models selected for these partitions are listed in Appendix B. Bayesian Markov chain Monte Carlo (MCMC) analyses (Yang and Rannala, 1997) were conducted in MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). Two parallel MCMC runs were conducted over 10 million generations, with sampling occurring every 1000 generations. The first 5  103 generations from each run were discarded as burn-in. Other than our partitioning scheme, we used default settings in MrBayes. 3. Results 3.1. Venom studies 3.1.1. Neurotoxicity M. mipartitus venom caused a concentration-dependent (3–10 mg/ml) inhibition of indirect twitches in the chick biventer cervicis nerve-muscle preparation (t50 ¼ 88.3  6.1 min for 3 mg/ml; 48.2  5.7 min for 10 mg/ml) (Fig. 1a; n ¼ 5–6; one-way ANOVA, P < 0.05). The inhibition of indirect twitches at 3 mg/ml displayed a triphasic effect consisting of a small initial decrease, followed by a transient increase and a final inhibition of twitches. Venom (3 and 10 mg/ml) significantly inhibited the contracture to exogenous ACh (1 mM; n ¼ 5–6; P < 0.05, one-way ANOVA) but not KCl (40 mM; n ¼ 5–6; P > 0.05, one-way ANOVA) Fig. 1b. M. dissoleucus venom (10–50 mg/ml) significantly inhibited indirect twitches (Fig. 1c; n ¼ 5–6; one-way ANOVA, P < 0.05), but failed to produce a complete blockade within a 3 h time period in the chick biventer cervicis nerve-muscle preparation. Therefore, it was not possible to calculate the t50 for this venom. In contrast to M. mipartitus, M. dissoleucus venom (3, 10 and 50 mg/ml) significantly inhibited contracture on both exogenous (1 mM; n ¼ 5–6; P < 0.05, one-way ANOVA) and KCl (40 mM; n ¼ 5–6; P < 0.05, one-way ANOVA, Fig. 1d). 3.1.2. PLA2 inhibition Complete inhibition of indirect twitches in the chick biventer cervicis nerve-muscle preparation was still

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obtained within a similar time frame when PLA2 activity of M. mipartitus venom was inhibited (t50 ¼ 37.7  5.3 min). Furthermore, the triphasic inhibitory response produced in normal physiological conditions (3 mg/ml) was not observed when Ca2þ (2.5 mM) was replaced with Sr2þ (10 mM); instead, a single, inhibitory effect was obtained. Twitch tension at the t50 time point for 3 mg/ml of M. mipartitus venom when indirectly stimulated under PLA2 inhibition conditions was significantly different in comparison to twitch tension obtained under normal conditions (Fig. 2a; n ¼ 5; P < 0.05, Student unpaired ttest). Interestingly, in the presence of Sr2þ, M. dissoleucus venom (3 mg/ml) showed a stronger and quicker inhibitory response in comparison with the one obtained under normal conditions (Fig. 2b; n ¼ 5; P < 0.05, Student unpaired t-test). 3.1.3. Reversal of the neurotoxic effect The addition of neostigmine (5 mM) to the chick biventer cervicis nerve-muscle preparation at the t50 time point, following the earlier addition of M. mipartitus venom (10 mg/ml, n ¼ 5), produced a transient reversal of twitch inhibition, but failed to prevent blockade which occurred in a similar time frame to inhibition in the absence of neostigmine (Fig. 3; n ¼ 5; P > 0.05, Student unpaired ttest). 3.1.4. Myotoxicity M. dissoleucus venom (10 mg/ml) caused a significant inhibition of direct twitches compared to the control (Fig. 4; n ¼ 5; P < 0.05; one-way ANOVA), indicating myotoxic activity in the venom. These results were further supported by a significant increase in the baseline tension of the muscle (10 mg/ml) under normal physiological conditions (Fig. 5; n ¼ 5; P < 0.05; one-way ANOVA). On the contrary, M. mipartitus venom (10 mg/ml), failed to produce a significant inhibition of direct twitches compared to the control (Fig. 4; n ¼ 5; P > 0.05; one-way ANOVA) but caused an increase in baseline tension, which was significant in comparison with the control (Fig. 5; n ¼ 5; P < 0.05; oneway ANOVA). 3.1.5. Histology Light microscopy studies of tissues exposed to M. dissoleucus venom (10 mg/ml and 50 mg/ml) produced concentration-dependent morphological changes when compared to control tissues (Fig. 6a–c). Changes included shrinkage of myofibers, vacuolation, edema, and appearance of cellular infiltrate and necrotic cells. 3.2. Phylogenetic reconstruction The parsimony and Bayesian reconstructions were congruent with each other in the majority of wellsupported clades, through posterior probabilities and bootstrap support. The preferred phylogenetic hypothesis with a tree-length of 119 steps from 296 parsimony informative characters is presented in Fig. 7 with parsimony bootstrap support and Bayesian posterior probabilities. Discrepancies of taxa labels between GenBank sequences and published results by Jorge da Silva and Sites (2001)

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Fig. 1. The effect of (a) M. mipartitus (3 mg/ml, n ¼ 5 and 10 mg/ml, n ¼ 6) or (c) M. dissoleucus (3 mg/ml, n ¼ 5; 10 mg/ml, n ¼ 6 and 50 mg/ml, n ¼ 5) venom and control (n ¼ 6) on the twitch tension of indirectly-evoked (0.2 ms, 0.1 Hz and supramaximal voltage) twitches of the isolated chick biventer cervicis nerve-muscle preparation. The effect of (b) M. mipartitus (3 mg/ml, n ¼ 5 and 10 mg/ml, n ¼ 6) and (d) M. dissoleucus (3 mg/ml, n ¼ 5; 10 mg/ml, n ¼ 6 and 50 mg/ml, n ¼ 5) venom and control (n ¼ 6) on ACh and KCl responses on the isolated chick biventer cervicis nerve-muscle preparation. *P < 0.05 significantly different from control (oneway ANOVA followed by a Dunnett post hoc test).

were solved by replicating their analyses (see Appendix A). The tree clearly presents a cohesive monadal group, Leptomicrurus set aside from other South American taxa, and two sister groups of South American triadal coral snakes. One group contains Amazonian and southern South American coral snakes (M. decoratus–M. cf. lemniscatus) alongside the subjects of the present study, M. dissoleucus and M. mipartitus, and the coral snakes allied to M. lemniscatus, which include M. surinamensis and M. hemprichii. M. dissoleucus and M. mipartitus form a monophyletic clade with strong support, even though the first is triadal and the second bicolor in body pattern. Most relationships inferred by Jorge da Silva and Sites (2001) for the South American triadal coral snakes were corroborated by our results, with the exception of the basal placement of M. surinamensis,

which is strongly nested within one of the two clades of triadal species. 4. Discussion Venoms evolve as a complex assemblage of substances that interact synergistically to immobilize, kill and digest prey items (Daltry et al., 1996; Kordis and Gubensek, 2000). Considerable interspecific, intersubspecific and interpopulational variation in the composition of Micrurus venoms has been previously demonstrated and has been attributed mainly to their wide differences in habitat composition and hence, prey items (Jorge da Silva and Aird, 2001; Tanaka et al., 2010). Our phylogenetic results corroborated a strong monophyletic relationship between M. dissoleucus

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Fig. 2. The effect of (a) M. mipartitus and (b) M. dissoleucus venom (3 mg/ml) in normal physiological salt solution conditions (n ¼ 5) or in physiological salt solution containing Sr2þ (n ¼ 3) instead of Ca2þ on the twitch tension of indirectly-evoked (0.2 ms, 0.1 Hz and supramaximal voltage) twitches of the isolated chick biventer cervicis nerve-muscle preparation. *P < 0.05 significantly different (Student unpaired t-test).

and M. mipartitus and although little is known about their dietary habits (Campbell and Lamar, 2004, Ayerbe et al., 1990; Roze, 1996; Ruthven, 1922) the ecosystems they inhabit are known to have very distinct reptile and amphibian faunas. In the current study, we investigated the neuromuscular properties of venoms from the two Colombian sister taxa, M. mipartitus and M. dissoleucus. For venoms that cause mainly neurotoxic symptoms in envenomed patients, it is desirable to have knowledge of their lethality based on parameters such as ‘quantity’ (i.e. LD50: concentration of venom that kills 50% of mice, usually over a 24–48 h period) and on ‘how quick’ (i.e. t50/90: time taken to cause 50% or 90% inhibition of nerve-mediated twitches) the effects occur (Reviewed in: Hodgson and Wickramaratna, 2002). The LD50 for M. dissoleucus venom has not been reported yet, which may be due to a number of factors including the difficulty of obtaining this species, together with its small size and therefore the small amount of venom obtained in each extraction. In the present study,

we were unable to determine the t50 value for this venom due to the lack of complete inhibition of indirect twitches, despite the use of relatively high concentrations (i.e. 50 mg/ ml). Is widely known that M. mipartitus is one of the most medically important species in Central and South America (Tanaka et al., 2010) and has been reported to have a murine LD50 of 0.47 mg/g i.p. (Otero et al., 1992). Additionally, isolated three finger toxins (3FTxs) Mm-8 and Mm-14 from M. mipartitus venom have been shown to have a high lethal potency with a murine LD50 of 0.06 mg/g and 0.07 mg/g respectively (Rey-Suárez et al., 2011). The t50 value determined in the present study for the crude venom of M. mipartitus (i.e. 88.3  6.1 min for 3 mg/ml; 48,2  5.7 min for 10 mg/ml) is the first reported for coral snake venom and is relatively low when compared to other Elapids such as death adders and sea snakes. It has been previously found that in some cases, very ‘lethal’ venoms

100 Neostigmine 50µ M

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Fig. 3. The effect of the addition of neostigmine (5 mM) at the t50 on the twitch tension of indirectly-evoked (0.2 ms, 0.1 Hz and supramaximal voltage) twitches of the isolated chick biventer cervicis nerve-muscle preparation treated previously with M. mipartitus venom (10 mg/ml; n ¼ 5) and control. *P > 0.05 no significant difference (Student unpaired ttest).

0

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Time (min) Fig. 4. The effect of M. mipartitus and M. dissoleucus venom (10 mg/ml) on the twitch tension of directly-evoked (2 ms duration, 0.1 Hz and supramaximal voltage) twitches of the isolated chick biventer cervicis nervemuscle preparation. *P < 0.05 (one-way ANOVA compared to control; n ¼ 5).

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1 25 1.25 1

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Time (min) Fig. 5. The effect of M. mipartitus (10 mg/ml, n ¼ 6) and M. dissoleucus (10 mg/ ml, n ¼ 6) venom and control (n ¼ 6) on the baseline tension of the directlyevoked (2 ms duration, 0.1 Hz and supramaximal voltage) twitches of the isolated chick biventer cervicis nerve-muscle preparation. *P < 0.05, significantly different from control (one-way ANOVA followed by Dunnett post hoc test).

(based on LD50 values) can take a significant amount of time to produce its effects, therefore ranking lower in the in vitro technique (Hodgson and Wickramaratna, 2002). We suggest that this might be the case for M. mipartitus venom and that determination of both parameters would be desirable for both Mm-8 and Mm-14, which represent a total of 27.9% and 15.3% respectively in the crude venom (Rey-Suarez et al., 2011). Neostigmine has been previously used in the treatment of patients following Elapid envenoming (Vieira and Bucaretchi, 2007). However, in the present study, the anticholinesterasic agent was only capable of producing a transient reversal of the neurotoxic effects of M. mipartitus venom – possibly indicating that the neurotoxins are unlikely to be readily reversible. According to our results, we suggest that anticholinesterase therapy is likely to have limited clinical effectiveness in envenoming by this species and further investigation is warranted to elucidate its potential therapeutic success. Although a complete inhibitory effect of indirect twitches failed to occur with M. dissoleucus venom, it did significantly inhibit the contracture to exogenous ACh suggesting the presence of post synaptically acting neurotoxins. Interestingly, inhibition of PLA2 activity of M.

Fig. 6. Longitudinal sections of the chick biventer cervicis nerve-muscle preparations exposed to (a) control; M. dissoleucus venom (b) 10 mg/ml and (c) 50 mg/ml. All the photographs were taken at 400. Arrows indicate vacuolation (A), cellular infiltrate (B) and edema (C).

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Fig. 7. The phylogenetic relationship of American coral snakes with emphasis on the triadal and bicolor clades of South America. This preferred phylogeny is one of six equally parsimonious trees differing only in the placement of M. d. diastema and M. spixii obscurus. These taxa are marked with solid dots and their alternative placements with open dots. Parsimony bootstrap support is noted above nodes and Bayesian posterior probabilities below. Taxa discussed in this paper are marked with an asterisk. Some sequences had incorrect taxon labels in Genbank (AF228435 as M. lemniscatus lemniscatus; AF228438 as M. lemniscatus Clone1; AF228439 as M. lemniscatus Clone2; AF228437 as M. lemniscatus carvalhoi 2; AF228436 as M. lemniscatus carvalhoi Cl).

dissoleucus venom showed to be quicker and possibly more potent than the one obtained under normal conditions. Whilst this could represent an important participation of PLA2 in the neuromuscular activity of the venom, further studies need to be carried out in order to confirm it. Furthermore, in agreement with previous studies on the bioactivity of Micrurus venoms (Cecchini et al., 2005; Serafim et al., 2002), M. mipartitus also displayed postsynaptic activity, as evidenced by the concentrationdependent inhibition of indirect twitches and significant abolishment of the contracture to exogenous ACh. Our results correlate with studies carried out by Rey-Suarez et al. (2011) who have suggested that the lethal action of M. mipartitus is exerted predominantly by 3FTxs with postsynaptic activity. Accordingly, inhibition of PLA2

activity of M. mipartitus venom demonstrated that the final neuromuscular blockade is not affected in time or potency, which is perhaps not surprising since 3FTxs are a have been reported to be a major component of this venom, representing the 60% in the proteome (Rey-Suárez et al., 2011). The triphasic effect observed during the inhibition of indirect twitches by M. mipartitus venom (3 mg/ml) was characterized by a small decrease, followed by a brief transient increase, and a final inhibition of twitches. This triphasic effect has previously been shown to happen with known presynaptic neurotoxins such as taipoxin, notexin and b-bungarotoxin in chick biventer cervicis preparations in vitro (Harris, 1991; Montecucco and Rossetto, 2000; Pungercar and Krizaj, 2007) and has been suggested to involve PLA2 activity (Harvey, 1990; Montecucco and

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Rossetto, 2000; Pungercar and Krizaj, 2007; Su and Chang, 1984). While our study was carried out with crude venom and is difficult to draw conclusions about its presynaptic mechanism of action, earlier studies have reported that the presence of postsynaptic neurotoxins, which have a much quicker onset of action, can often mask the effects of other toxins (Hodgson and Wickramaratna, 2002); and this could perhaps explain the fact that the effect was not observed when a higher concentration of M. mipartitus venom was tested. Studies carried out by Rey-Suarez et al. (2011) found PLA2s from M. mipartitus venom, which are related to enzymes from M. corallinus and Old world elapids, and suggest the likely participation of this components in a presynaptic mechanism of action. In the current study, although the inhibition of PLA2 activity of M. mipartitus venom (by replacing Ca2þ with Sr2þ in the physiological salt solution) showed an attenuation of the triphasic effect, we suggest that further studies involving electrophysiological measurements during the facilitation period on isolated toxin components could help elucidate if PLA2 is involved in a possible presynaptic mechanism of action of this venom. It has previously been reported that PLA2 enzymes are the result of two independent toxin recruitment events and both neurotoxicity and myotoxicity are its major properties (Fry and Wuster, 2004; Harvey et al., 1994; Lynch, 2007). M. mipartitus venom showed little evidence for a myotoxic effect, as evidenced by its lack of response to KCl and absence of a significant inhibition of direct twitches. While a statistically significant increase in baseline tension was obtained with a venom concentration of 10 mg/mL, lack of myotoxicity was further confirmed by no obvious morphological changes in the examined tissues (data not shown). Our results are in agreement with studies carried out by Rey-Suarez et al. (2011), where creatine kinase levels of mice injected with 10 mg of crude venom from M. mipartitus were shown to be within the normal ranges when compared to the highly myotoxic venom of Micrurus nigrocinctus. On the other hand, M. dissoleucus venom produced a significant inhibition to KCl response, a significant inhibition of muscle-mediated twitches and a marked increase in baseline tension suggesting a myotoxic mechanism of action. Additionally, light microscopy studies of tissues exposed to this venom confirmed damage to the muscle, as evidenced by pathological changes such as vacuolation with a prominent damage of the intracellular material and hyper contraction of the muscle fibers. Previous studies carried out on M. nigrocinctus venom have shown that coral snakes are capable inducing local myotoxicity in neuromuscular preparations exposed to the crude venom (Goularte et al., 1995, 1999) as well as after intramuscular injection in mice (Cecchini et al., 2005; Gutierrez et al., 1986, 1980, 1983). We suggest that further in vivo studies, involving M. dissoleucus venom should be carried out in order to confirm this effect. It has been well established that venom variability can affect the success of antivenom therapy, which relies on the venoms chosen for the immunization protocol (Casewell et al., 2010; Galan et al., 2004; Visser et al., 2008). And several studies have reported the consistent lack of cross

reactivity between groups (de Roodt et al., 2004; ReySuárez et al., 2011; Tanaka et al., 2010) Therefore, the significant differences that we report for the mechanism of action of M. mipartitus and M. dissoleucus venom may have important implications for the design of Micrurus antivenom and further investigation is required to clarify any potential therapeutic consequences. In conclusion, this study corroborates the existence of a broad spectrum of pharmacological activities in a single lineage. Although M. mipartitus and M. disoleucus venoms caused neurotoxic effects in vitro, which appear to be postsynaptic in origin, the venom of M. dissoleucus showed a myotoxic mechanism of action that seems to be accent from M. mipartitus venom. Accordingly, we suggest that despite the close phylogenetic relationship and evolutionary history between M. mipartitus and M. dissoleucus, the differences between the mechanisms of action of the venoms could be attributed to the dissimilar characteristics present in the ecosystems they inhabit and as a possible consequence, the prey items available to each species. Further studies need to be conducted in order to determine their specific dietary habits and their effect on the variability of venom composition to confirm this hypothesis and its therapeutic implications. Acknowledgments CR is grateful to acknowledge all the staff members of Departamento de Ciencias Fisiologicas PUJ to those actively involved in the development of this project: Dr. Gabriel Pascual, Dr. Henry Aceros and Dr. Dario Riascos. The Department of Pathology at PUJ, the National Institute of Health in Bogotá and specially to Mario Alejandro Lozano, Luis Ucross, Alvaro Andres Velasquez, Jairo Sanchez, Jaime Ramirez Avila, Dr. Santiago Ayerbe, Claudia Cifuentes, Dr. Silvia Lopez and Dr. Bryan Grieg Fry for their support during the study and to Nicholas R. Casewell for the critical reading and discussion of the manuscript. ENS is grateful to acknowledge several researchers who provided tissues under their care, obtained during sponsored research, including Laurie Vitt (University of Oklahoma, obtained through NSF grants DEB-9200779 and DEB-9505518), Roy W. McDiarmid and Steve W. Gotte, USNM, Carl J. Franklin and Jonathan Campbell (University of Texas at Arlington, obtained through NSF grants DEB9705277 and DEB-0102383), Cesar Jaramillo (Círculo Herpetológico de Panamá), William Duellman (KU), Mahmood Sasa M. (Clodomiro Picado), Fred Sheldon and Donna Dittmann (LSU), Travis LaDuc (TNHC), Jens Vindum (CAS), and Robert Murphy (ROM). Todd A. Castoe and Matthew Ingrasci kindly generated and edited some of the DNA sequences. Jeffrey Streicher kindly assisted with the Bayesian reconstruction. For help in obtaining permits to Aleyda Martinez and Mauricio Rivera (Ministerio de Ambiente, Vivienda y Desarrollo Territorial, Colombia), Juan M. Daza and Vivian P. Páez (Universidad de Antioquia), Andrew J. Crawford and Nelsy R. Pinto (Universidad de Los Andes, Colombia), and Andréa Acevedo. Collecting and exportation permits were issued by the Secretaria de Medio Ambiente y Recursos Naturales (Mexico, to Oscar FloresVillela), Ministerio de Ambiente y Energia (Costa Rica, to

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Mahmood Sasa), the US Fish and Wildlife service (USA, to ENS), Texas Parks and Wildlife (USA, to Carl J. Franklin and ENS), and the Ministerio de Ambiente, Vivienda y Desarrollo Territorial (Colombia, Res. No. 0445, 14 March 2008, to ENS). The use of Colombian samples for phylogenetic analyses was possible under the Genetic Resources Access permit to ENS (Ministerio de Ambiente, Vivienda y Desarrollo Territorial de Colombia, Res. No. 0445, 14 March 2008). Funding Bioactivity experiments were part of Camila Renjifo’s undergraduate thesis work at the Departamento de Ciencias Fisiologicas, Pontificia Universidad Javeriana. Phylogenetic analysis of this project was provided by a grant from Instituto Bioclon (to ENS), an NSF Collaborative Research grant to Christopher L. Parkinson and ENS (DEB0416000, 0416160), and startup package to Christopher L. Parkinson (UCF) and ENS (UTA). Conflict of interest The authors declare that there are no conflicts of interest. Appendix. Supplementary material Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.toxicon. 2011.10.017. References Ayerbe, S., Tidwell, M.A., Tidwell, M., 1990. Observación sobre la biología y comportamiento de la serpiente coral "Rabo de Ají" (Micrurus mipartitus). Descripción de una Subespecie Nueva. Novedades Colombianas, 2, pp. 30–41. Barlow, A., Pook, C.E., Harrison, R.A., Wuster, W., 2009. Coevolution of diet and prey-specific venom activity supports the role of selection in snake venom evolution. Proc. Biol. Sci. 276, 2443–2449. Barros, A.C., Fernandes, D.P., Ferreira, L.C., Dos Santos, M.C., 1994. Local effects induced by venoms from five species of genus Micrurus sp. (coral snakes). Toxicon 32, 445–452. Benabib, M., Kjer, K.M., Sites, J.W., 1997. Mitochondrial DNA sequencebased phylogeny and the evolution of viviparity in the Sceloporus scalaris group (Reptilia, Squamata). Evolution 51, 1262–1275. Camargo, T.M., de Roodt, A.R., da Cruz-Hofling, M.A., Rodrigues-Simioni, L. , 2011. The neuromuscular activity of Micrurus pyrrhocryptus venom and its neutralization by commercial and specific coral snake antivenoms. J. Venom Res. 2, 24–31. Campbell, J.A., Lamar, W.W., 2004. The Venomous reptiles of the western hemisphere, 2 ed. Cornell University Press, Ithaca, New York. Casewell, N.R., Harrison, R.A., Wuster, W., Wagstaff, S.C., 2009. Comparative venom gland transcriptome surveys of the saw-scaled vipers (Viperidae: Echis) reveal substantial intra-family gene diversity and novel venom transcripts. BMC Genomics 10, 564. Casewell, N.R., Cook, D.A., Wagstaff, S.C., Nasidi, A., Durfa, N., Wuster, W., Harrison, R.A., 2010. Pre-clinical assays predict pan-African Echis viper efficacy for a species-specific antivenom. PLoS neglected tropical diseases 4, e851. Castoe, T.A., Smith, E.N., Brown, R.M., Parkinson, C.L., 2007. Higher-level phylogeny of Asian and American coralsnakes, their placement within the Elapidae (Squamata), and the systematic affinities of the enigmatic Asian coralsnake Hemibungarus calligaster Wiegmann, 1834. Zool. J. Linn Soc-Lond 151, 809–831. Cecchini, A.L., Marcussi, S., Silveira, L.B., Borja-Oliveira, C.R., RodriguesSimioni, L., Amara, S., Stabeli, R.G., Giglio, J.R., Arantes, E.C., Soares, A.M., 2005. Biological and enzymatic activities of Micrurus sp. (Coral) snake venoms. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 140, 125–134.

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