Toxicon 59 (2012) 132–142

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Toxicon
journal homepage: www.elsevier.com/locate/toxicon

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
b
Department of Biology, The University of Texas at Arlington, Arlington, TX 76019, USA
c
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

a r t i c l e i n f o a b s t r a c t

Article history: The venoms of coral snakes (genus Micrurus) are known to induce a broad spectrum of
Received 16 June 2011 pharmacological activities. While some studies have investigated their potential human
Received in revised form 23 October 2011 effects, little is known about their mechanism of action in terms of the ecological diversity
Accepted 28 October 2011
and evolutionary relationships among the group. In the current study we investigated the
Available online 15 November 2011
neuromuscular blockade of the venom of two sister species Micrurus mipartitus and
Micrurus dissoleucus, which exhibit divergent ecological characteristics in Colombia, by
Keywords:
using the chick biventer cervicis nerve-muscle preparation. We also undertook a phylo-
Micrurus
Neurotoxic genetic analysis of these species and their congeners, in order to provide an evolutionary
Myotoxic framework for the American coral snakes. The venom of M. mipartitus caused a concen-
Chick biventer cervicis tration-dependant inhibition (3–10 mg/ml) of nerve-mediated twitches and significantly
Venom 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.

1. Introduction

Terrestrial New World genera of the Family Elapidae,

* 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: renjifo@liv.ac.uk (C. Renjifo).
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

0041-0101/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.toxicon.2011.10.017
 C. Renjifo et al. / Toxicon 59 (2012) 132–142
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 char-
acteristics 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 composi-
tion and pharmacological effects of Micrurus venoms, it has
been reported that the neuromuscular blockade respon-
sible 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 car-
diotoxic, 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
133
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 mecha-
nism 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 evolu-
tionary 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 decussa-
tus 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 supra-
maximal voltage) using a low-voltage stimulator incorpo-
rated 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)
134 C. Renjifo et al. / Toxicon 59 (2012) 132–142
and potassium chloride (KCl, 40 mM for 30 s) were ob-
tained 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 nerve-
muscle preparations when the safety factor of trans-
mission 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 Ca2þ-
free physiological salt solution until twitches were abol-
ished and then allowed to equilibrate for 30 min in physi-
ological 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 displace-
ment 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 statis-
tical difference determined by a one-way analysis of vari-
ance (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 euryx-
anthus, 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 spec-
imen, 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 amplifica-
tion 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 frag-
ments 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 repli-
cates with all characters weighted equally and gap sites
 C. Renjifo et al. / Toxicon 59 (2012) 132–142
excluded. Weighted parsimony (WP) analysis was conduct-
ed 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 amino-
acid 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-bisection-
regrafting method of proposing new topologies. MP and WP
bootstrap analyses (Felsenstein, 1985) involved 2000 pseu-
doreplicates with 2–10 random addition sequences each.
To search for appropriate models of nucleotide evolu-
tion 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 con-
ducted over 10 million generations, with sampling occur-
ring 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 exoge-
nous 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
135
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 t-
test). 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 t-
test).
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; one-
way ANOVA).
3.1.5. Histology
Light microscopy studies of tissues exposed to M. dis-
soleucus 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 appear-
ance of cellular infiltrate and necrotic cells.
3.2. Phylogenetic reconstruction
The parsimony and Bayesian reconstructions were
congruent with each other in the majority of well-
supported clades, through posterior probabilities and
bootstrap support. The preferred phylogenetic hypothesis
with a tree-length of 119 steps from 296 parsimony infor-
mative 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)
136 C. Renjifo et al. / Toxicon 59 (2012) 132–142

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 (one-
way 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, Lep-
tomicrurus 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. lem-
niscatus, 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 interpopu-
lational 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
 C. Renjifo et al. / Toxicon 59 (2012) 132–142 137

a b
100 100
Twitch tension (% of the initial)

Twitch tension (% of the initial)

80 80

60 60

40 40

20 Ca2+ 20 Ca2+
Sr2+ Sr2+
0 0
0 30 60 90 120 150 180 0 30 60 90 120 150 180
Time (min.) Time (min.)

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 we were unable to determine the t50 value for this venom
dietary habits (Campbell and Lamar, 2004, Ayerbe et al., due to the lack of complete inhibition of indirect twitches,
1990; Roze, 1996; Ruthven, 1922) the ecosystems they despite the use of relatively high concentrations (i.e. 50 mg/
inhabit are known to have very distinct reptile and ml).
amphibian faunas. In the current study, we investigated the Is widely known that M. mipartitus is one of the most
neuromuscular properties of venoms from the two medically important species in Central and South America
Colombian sister taxa, M. mipartitus and M. dissoleucus. (Tanaka et al., 2010) and has been reported to have
For venoms that cause mainly neurotoxic symptoms in a murine LD50 of 0.47 mg/g i.p. (Otero et al., 1992). Addi-
envenomed patients, it is desirable to have knowledge of tionally, isolated three finger toxins (3FTxs) Mm-8 and
their lethality based on parameters such as ‘quantity’ (i.e. Mm-14 from M. mipartitus venom have been shown to have
LD50: concentration of venom that kills 50% of mice, usually a high lethal potency with a murine LD50 of 0.06 mg/g and
over a 24–48 h period) and on ‘how quick’ (i.e. t50/90: time 0.07 mg/g respectively (Rey-Suárez et al., 2011). The t50
taken to cause 50% or 90% inhibition of nerve-mediated value determined in the present study for the crude venom
twitches) the effects occur (Reviewed in: Hodgson and of M. mipartitus (i.e. 88.3  6.1 min for 3 mg/ml;
Wickramaratna, 2002). The LD50 for M. dissoleucus venom 48,2  5.7 min for 10 mg/ml) is the first reported for coral
has not been reported yet, which may be due to a number snake venom and is relatively low when compared to other
of factors including the difficulty of obtaining this species, Elapids such as death adders and sea snakes. It has been
together with its small size and therefore the small amount previously found that in some cases, very ‘lethal’ venoms
of venom obtained in each extraction. In the present study,

100

120 Neostigmine 50µ M

Twitch-tension (% of the initial)

80
Twitch tension (% of the initial)

100

80 60 *
60

40
40

20
M. mipartitus (10 µg/ml) 20 Controls
0
M. mipartitus
0 30 60 90 120 150 180 M. dissoleucus
Time (min.) 0
0 30 60 90 120 150 180
Fig. 3. The effect of the addition of neostigmine (5 mM) at the t50 on the Time (min)
twitch tension of indirectly-evoked (0.2 ms, 0.1 Hz and supramaximal
voltage) twitches of the isolated chick biventer cervicis nerve-muscle Fig. 4. The effect of M. mipartitus and M. dissoleucus venom (10 mg/ml) on
preparation treated previously with M. mipartitus venom (10 mg/ml; the twitch tension of directly-evoked (2 ms duration, 0.1 Hz and supra-
n ¼ 5) and control. *P > 0.05 no significant difference (Student unpaired t- maximal voltage) twitches of the isolated chick biventer cervicis nerve-
test). muscle preparation. *P < 0.05 (one-way ANOVA compared to control; n ¼ 5).
138 C. Renjifo et al. / Toxicon 59 (2012) 132–142

1 25
1.25 (based on LD50 values) can take a significant amount of
Control *
time to produce its effects, therefore ranking lower in the
M mipartitus
M.
1 in vitro technique (Hodgson and Wickramaratna, 2002). We
M di
M. dissoleucus
l
suggest that this might be the case for M. mipartitus venom
Baseline tension (g)

and that determination of both parameters would be

0.75
desirable for both Mm-8 and Mm-14, which represent
*
a total of 27.9% and 15.3% respectively in the crude venom
0.5
(Rey-Suarez et al., 2011).
Neostigmine has been previously used in the treatment
0 25
0.25 of patients following Elapid envenoming (Vieira and
Bucaretchi, 2007). However, in the present study, the
0 anticholinesterasic agent was only capable of producing
a transient reversal of the neurotoxic effects of M. mipar-
-0.25 titus venom – possibly indicating that the neurotoxins are
0 30 60 90 120 150 180 unlikely to be readily reversible. According to our results,
Time (min) we suggest that anticholinesterase therapy is likely to have
limited clinical effectiveness in envenoming by this species
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 directly- and further investigation is warranted to elucidate its
evoked (2 ms duration, 0.1 Hz and supramaximal voltage) twitches of the potential therapeutic success.
isolated chick biventer cervicis nerve-muscle preparation. *P < 0.05, Although a complete inhibitory effect of indirect
significantly different from control (one-way ANOVA followed by Dunnett twitches failed to occur with M. dissoleucus venom, it did
post hoc test).
significantly inhibit the contracture to exogenous ACh
suggesting the presence of post synaptically acting neuro-
toxins. 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).
 C. Renjifo et al. / Toxicon 59 (2012) 132–142 139

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 post-
synaptic activity, as evidenced by the concentration-
dependent 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, repre-
senting 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
140 C. Renjifo et al. / Toxicon 59 (2012) 132–142
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 electrophysio-
logical 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 inhi-
bition 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 neuro-
muscular 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; Rey-
Suárez 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 anti-
venom 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 evolu-
tionary 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 vari-
ability 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 spon-
sored research, including Laurie Vitt (University of Okla-
homa, 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 DEB-
9705277 and DEB-0102383), Cesar Jaramillo (Círculo Her-
petológico 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 Flores-
Villela), Ministerio de Ambiente y Energia (Costa Rica, to
 C. Renjifo et al. / Toxicon 59 (2012) 132–142
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 Desar-
rollo 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 Desar-
rollo 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 Cien-
cias Fisiologicas, Pontificia Universidad Javeriana. Phylo-
genetic 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 (DEB-
0416000, 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.
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