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Toxicon 141 (2018) 79e87

Contents lists available at ScienceDirect

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

Venoms of Centruroides and Tityus species from Panama and their

main toxic fractions

n Arenas c, Ligia L. Corrales-García c, d, Roberto Miranda e,
Marcos H. Salazar a, b, Iva

lez a, Jairo Sa
Sara Ve
nchez a, b, Karla Mendoza a, John Cleghorn a, b, Fernando Z. Zamudio c,
Adolfo Castillo , Lourival D. Possani c, Gerardo Corzo c, *, Hildaura Acosta a, **
a

a
n e Informacio
Centro de Investigacio
n de Medicamentos y To
xicos (CIIMET), Facultad de Medicina, Universidad de Panama
, Ciudad de Panama
, Panama
b
Facultad de Ciencias Naturales, Exactas y Tecnología, Universidad de Panama
, Ciudad de Panama

, Panama
c
noma de M

Instituto de Biotecnología, Universidad Nacional Auto exico, Avenida Universidad 2001, Cuernavaca, Morelos, 62210, Mexico
d
Departamento de Alimentos, Facultad de Ciencias Farmac
euticas y Alimentarias, Universidad de Antioquia, AA 1226, Medellín, 050010, Colombia
e
Departamento de Investigacio
n en Entomología M
edica, Instituto Conmemorativo Gorgas de Estudios de la Salud, Ciudad de Panama
, Panama

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

Article history: The scorpionism in Panama is notorious for the confluence and coexistence of buthid scorpions from the
Received 18 August 2017 genera Centruroides and Tityus. This communication describes an overview of the larger representative
Received in revised form toxic venom fractions from eight dangerous buthid scorpion species of Panama: Centruroides (C. granosus,
9 November 2017
C. bicolor, C. limbatus and C. panamensis) and Tityus (T. (A.) asthenes, T. (A.) festae, T. (T.) cerroazul and T. (A.)
Accepted 27 November 2017
Available online 2 December 2017
pachyurus). Their venoms were separated by HPLC and the corresponding sub-fractions were tested for
lethality effects on mice and insects. Many fractions toxic to either mice or insects, or both, were found
and have had their molecular masses determined by mass spectrometry analysis. The great majority of
Keywords:
Amino acid sequence
the lethal components had a molecular mass close to 7000 Da, assumed to be peptides that recognize
Centruroides Naþ-channels, responsible for the toxicity symptoms observed in other buthids scorpion venoms. A toxic
Chromatographic separation peptide isolated from the venom of T. pachyurus was sequenced by Edman degradation, allowing the
Gene cloning synthesis of nucleotide probe for cloning the correspondent gene. The mature toxin based on the cDNA
Lethality test sequencing has the C-terminal residue amidated, contains 62 amino acid packed by 4 disulfide linkages,
Scorpion toxin with molecular mass of 7099.1 Da. This same toxic peptide seems to be present in scorpions of the
Tityus species T. pachyurus collected in 5 different regions of Panama, although the overall HPLC profile is quite
different. The most diverse neurotoxic venom components from the genus Centruroides were found in
the species C. panamensis, whereas T. cerroazul was the one from the genus Tityus. The most common
neurotoxins were observed in the venoms of T. festae, T. asthenes and T. pachyurus with closely related
molecular masses of 7099.1 and 7332 Da.
The information reported here is considered very important for future generation of a neutralizing
antivenom against scorpions from Panama. Furthermore, it will contribute to the growing interest in
using bioactive toxins from scorpions for drug discovery purposes.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction mountains do not form part of the mountain chains of North

America but there are highlands related to the Andean system of
Panama is situated between North and South America, and it is South America near to Colombia. Thus, one of the interesting fea-
considered the central spine of mountains and hills that forms the tures of the geography of Panama seems to be the confluence of
continental separation of both geographical areas. The Panamanian fauna from North and South America. For example, there is the
coexistence of buthid scorpions from the genus Centruroides
(mostly restricted to North America) and from the genus Tityus
* Corresponding author. (commonly situated in South America). Indeed, in Panama, the
** Corresponding author. scorpion stings are considered a serious problem of public health.
E-mail addresses: corzo@ibt.unam.mx (G. Corzo), hildaura6@gmail.com
The Department of Epidemiology of the Ministry of Health
(H. Acosta).

https://doi.org/10.1016/j.toxicon.2017.11.013
0041-0101/© 2017 Elsevier Ltd. All rights reserved.
 Author's Personal Copy

80 M.H. Salazar et al. / Toxicon 141 (2018) 79e87

registered 33 623 cases from 2000 to 2016 with a total of 47 deaths
in a similar period of time (Ministerio de Salud, 2017). Most of these
cases have happened in rural communities, which have difficult
accessing to health centers. Although in Panama, there are fifteen
scorpion species belonging to five genera and to three families
(Hormuridae, Chactidae, and Buthidae) (Borges et al., 2011;
Ministerio de Salud, 2016), the stings of the species Opistacanthus
elatus, Hormuridae (Pocock, 1893), Chactas exsul, Chactidae
(Werner, 1939) and Ananteris platnicki, Buthidae (Lourenço, 1993)
do not appear to have a major medical importance (Ministerio de
Salud, 2016). However, the sting of the species of the buthid scor-
pions from the genus Centruroides and Tityus causes most the en-
venomation accidents.
Regarding the genus Centruroides in Panama, the species Cen-
truroides granosus (Thorell, 1876) is the most common and densely
populated species within urban areas of Panama, and it is the
scorpion that causes the most accidents in Panamanians (Fig. 1).
Other species of Centruroides include Centruroides panamensis
(Quintero and Esposito, 2014), Centruroides limbatus (Pocock, 1898),
Centruroides margaritatus (Gervais, 1841) and Centruroides bicolor
(Pocock, 1898); the latter also associated with severe cases of
poisoning (Miranda et al., 2014; Ministerio de Salud, 2016).
Concerning the genus Tityus in Panama, there are three sub-
genera proposed by Lourenço (2006). The species belonging to the
subgenus Archaeotityus are Tityus ocelote (Francke and Stockwell,
1987) and Tityus tayrona (Lourenço, 1991); to the subgenus Atreus
are Tityus asthenes (Pocock, 1893), Tityus championi (Pocock, 1898),
Tityus festae (Borelli, 1899) and Tityus pachyurus (Pocock, 1897); and
finally, to the subgenus Tityus the specie Tityus cerroazul (Lourenço,
1986). All Tityus species are considered dangerous scorpions in
Panama, and four of them are considered important in the context
of public health; that is: Tityus (Atreus) pachyurus, Tityus (Tityus)
cerroazul, Tityus (Atreus) asthenes and Tityus (Atreus) festae. All of
them have been associated with deathly cases. Between paren-
theses are the newly suggested nomenclatures (Lourenço, 2006).
The species Tityus (Atreus) pachyurus is considered the cause of the
majority of the fatal cases mainly because of the potency of its
venom and because of its wide geographical distribution in Panama
(Fig. 1).
Unfortunately, Panama does not produce its own anti-scorpion
venom. The anti-scorpion serum (SAE) used in Panama comes
from the Institute of Biotechnology of the Central University of
Venezuela (Ministerio de Salud, 2016). It is prepared from plasma of
hyper immunized horses. Their antibodies have been digested with
pepsin and purified. The final product is in the F(ab')2 format. SAE
comes from horses immunized with the venom of Tityus (Atreus)
discrepans (Brito and Borges, 2015), but it has a limited efficacy
(Borges et al., 2012). At present, SAE does not have a Sanitary

Fig. 1. Location of Centruroides and Tityus species from Panama. Scorpions were collected during field trips. Centruroides limbatus, Centruroides panamensis, Centruroides bicolor,

n and CIIMET.
Centruroides granosus, Tityus (Tityus) cerroazul, Tityus (Atreus) festae, Tityus (Atreus) asthenes y Tityus (Atreus) pachyurus. The map was created by Carlos Gordo
 Author's Personal Copy
M.H. Salazar et al. / Toxicon 141 (2018) 79e87
Registry in Panama, and it is acquired through a special import
permit to meet the needs for the country (Ministerio de Salud,
2016).
Given the current limitations for producing anti-scorpion serum
to supply Panama, it is important to carry out relevant studies to
know the most toxic components of the venoms of the genera
Centruroides and Tityus with the aim of producing in a short term a
local anti-scorpion serum by means of scorpion venoms present in
Panama. Therefore, in this work, the scorpion venoms of eight
scorpion species of the genera Centruroides and Tityus were frac-
tionated by HPLC, and their components analyzed to determine
their toxicity (lethality). Initially the venom fractions were assayed
in mice with the aim of observing which venom contains the most
toxic peptide to mammals. In second place the fractions were
assayed into insects with the objective of identifying which com-
ponents have dual activity or just a single activity for mammals or
insects.
The molecular masses of the active fractions were determined
by mass spectrometry. One of the most toxic fractions from Tityus
pachyurus was sequenced by Edman degradation. This information
allowed the preparation of cDNA clone from which the entire
amino acid of the corresponding toxin was deduced.
2. Material and methods
2.1. Biological materials
Species of the genera Centruroides (C. granosus, C. bicolor, C.
limbatus, and C. panamensis) and Tityus (T. asthenes, T. festae, T.
cerroazul and T, pachyurus) were collected from different locations
in Panama (Fig. 1). All scorpion venoms were obtained by manual
stimulation, recovered in dH2O at room temperature, lyophilized
and stored at 40  C until use. Protease inhibitors were not added
to the venom.
2.2. Fractionation of scorpion venoms
Lyophilized crude venoms (2 mg of dry-weight venom) were
resuspended in 500 mL of 0.1% aqueous triflouroacetic acid (TFA),
and the insoluble material was removed by centrifugation at
14,000 g for 5 min. The soluble venom was used directly for HPLC
fractionation using a reverse-phase analytical C18 column (C18,
10  250 mm Vydac, Grace, Germany) equilibrated with water in
0.1% trifluoroacetic acid (TFA), and eluted with a linear gradient of
acetonitrile from 0 to 60% in 0.1%TFA, run for 70 min duration at a
flow rate of 1 mL/min. Effluent absorbance was monitored at
230 nm. Fractions were collected in 1.5 mL Eppendorf tubes and dry
out under high vacuum. The HPLC fractions of interest were further
used for toxicological assays according to Corzo et al. (2009). For
determining the protein content of fractions, after vaccum-drying,
they were resuspended in dH20 and the concentration measured
assuming that one unit of absorbance at 280 nm, in 1 cm cuvette
width, is equal to 1 mg/mL of protein (Torres-Larios et al., 2000;
Garcia-Gomez et al., 2011).
2.3. Mass spectrometry and N-terminal sequencing
The protein fractions were reconstituted to a final concentration
of 500 pmol/5 mL of 50% acetonitrile with 1% acetic acid and directly
applied into a Thermo Scientific LCQ Fleet ion trap mass spec-
trometer (San Jose, CA) using a Surveyor MS syringe pump delivery
system. The eluate at 10 mL/min was split out in order to introduce
only 5% of the sample into the nanospray source (0.5 mL/min). The
spray voltage was set from 1.5 kV and the capillary temperature
was set at 150  C. The fragmentation source was operated at
81
25e35 V of collision energy, 35e45% (arbitrary units) of normalized
collision energy and the scan with wide band was activated. All
spectra were obtained in the positive-ion mode. The data acquisi-
tion and the deconvolution of data were performed on Xcalibur
Windows NT PC data system. The average molecular masses values
are ±1 Da due to the limited resolution of this instrument.
Edman degradation was performed on a Shimadzu PPSQ-31A
(Shimadzu, Kyoto, Japan) automated gas phase sequencer. Sample
(10 mg) was dissolved in 10 mL of 37% CH3CN (v/v) solution and
applied to TFA-treated glass fiber membranes, pre-cycled with
Polybrene (Sigma-Aldrich Co. St. Louis, MO, USA).
2.4. RNA extraction from Tityus pachyurus region Cocl

e
The RNA from a single postabdominal segment (telson), con-
taining a pair of venom glands of Tityus (A) pachyurus from the

was obtained and stored in FineFix (Milestone Medical
region Cocle
Technologies, Inc., USA). The mRNA was extracted using an
RNAeasy mini kit (Qiagen Inc., Germantown, MD), and also using
the SV Total RNA Isolation System (Promega Co., Madison, WI).
2.5. cDNA library construction and gene cloning
The mRNA was used to synthesize the cDNA by means of an
adapter primer (AP) (30 RACE System for Rapid Amplification of
cDNA Ends, Invitrogen, Carlsbad, CA, USA). The reaction was carried
at 42  C for one hour in presence of 200 enzymatic units of Reverse
transcriptase (SuperScript II RT, Invitrogen, Carlsbad, CA, USA).
Since the N-terminal amino acid sequence of a toxic peptide (Tp32-
Cocle
from the scorpion Tityus pachyurus) was obtained, an oligo-
nucleotide corresponding to its amino acid sequence was designed
for gene cloning. The transcript corresponding to N-terminal pep-
tide from fraction 32 (TpF32-Cocle
) was amplified from the cDNA
using Taq DNA polymerase (2U) (Thermo Fisher Scientific, Whal-
tham, MA, USA) in combination with a degenerated primer based
on N-terminal sequencing of TpF32-Cocle
fraction (AAA-
GATGGNTAYCTRGTBGG) and an equimolar concentration of primer
AUAP (GGCCACGCGTCGACTAGTAC) (30 RACE System for Rapid
Amplification of cDNA Ends, Invitrogen, Carlsbad, CA, USA). A
Mastercycler Gradient thermocycler (Eppendorf, Hamburg, Ger-
many) was used for PCR and the conditions for amplifications were:
3 min denaturation at 94  C, followed by 30 cycles at 94  C for 40 s,
55  C for 40 s and 72  C for 40 s, with a final extension cycle at 72  C
for 10 min. PCR product was gel-purified from the agarose using the
High Pure PCR Product Purification Kit (Roche LifeScience, Penz-
berg, Germany). The resulting DNA fragment was ligated into PCR®
2.1-TOPO TA cloning vector (Invitrogen, Carlsbad, CA, USA). The
ligation reaction was used to transform E. coli XL1 Blue quimio-
competent cells. Ten individual clones were analyzed by colony
PCR using oligonucleotides M13-Forward (50 -GTAAAACGACGGC-
CAG-30 ) and M13-Reverse (50 -CAGGAAACAGCTATGAC-30 ). Four
colonies with the expected DNA size were sequenced at the facil-
ities of the Institute of Biotechnology, UNAM, Mexico. A verified
plasmid containing the correct sequence was used as template to
amplify the gene flanked by different elements; that is, a direct
oligonucleotide introduced the restrictions site BamHI and recog-
nition site to factor Xa (50 -GAGGGATCCATCGAGGGACGCAAA-
GATGGTTAC-30 ); a reverse oligonucleotide introduced a stop codon
and restriction site PstI (50 -CTGCAGCTATTAGCGGCATTTATTTG-
TAGC-30 ). The conditions for amplifications were: 3 min denatur-
ation at 94  C, followed by 30 cycles at 94  C for 30 s, 56  C for 30 s
and 72  C for 30 s, with a final extension cycle at 72  C for 10 min.
PCR product was gel-purified, ligated into pCR®2.1-TOPO TA cloning
vector, transformed in E. coli cells and then sequenced, as described
previously. Both the amplified gene and the expression vector
 Author's Personal Copy

82 M.H. Salazar et al. / Toxicon 141 (2018) 79e87

pQE30 were digested with BamHI and PstI (New England Biolabs,
Ipswich, MA, USA). After subsequent purifications, a ligation reac-
tion (10 mL) was carried out with T4 DNA ligase (Fermentas, Car-
isbad, CA, USA) with a 5-fold gene excess over plasmid during
16 h at 16  C; ten microliters of the ligation reaction were used to
transfect competent E. coli XL1 Blue cells. Ten individual clones
were analyzed by colony PCR using oligonucleotides pQE30-
Forward (50 -GAGCGGATAACAATTATAA-30 ) and pQE30-Reverse (50 -
GGTCATTACTGGATCTAT-30 ). Four colonies with the expected DNA
size were sequenced at the facilities of the Institute of Biotech-
nology, UNAM, Mexico.
25e35 min are principally voltage-dependent potassium channel
(Kv) peptide blockers containing 3 or 4 disulfide bridges. Likewise,
venom components that elute over 35e45 min period are generally
the most toxic components that affect mammals and insects. Those
35e45 min venom components affect the voltage-dependent so-
dium channel (Nav) (Batista et al., 2004). Usually these components
contain 4 disulfide bridges and consist approximately of 60e70
amino acid residues. Enzymes like hyaluronidases and proteases

2.6. Biological activities

2.6.1. Mice
The protocol used for assaying the activity of peptides in vivo
was followed according to the guidelines of our Institute Commit-
tee of Animal Welfare, keeping the number of animals to a mini-
mum. Male mice (CD-1, 20 g body weight) were tested by
intracranial (i.c.) and intraperitoneal injection (i.p.). Peptide frac-
tions were diluted up to 50e100 mL, to achieve the desired con-
centration, with deionized water (dH20). The injection, with a 10 mL
micro-syringe fitted with a glass capillary, was performed mid-way
between the left eye and the left ear (intracranial). Negative con-
trols were done with dH20 only and positive controls with the
neurotoxic scorpion peptide CssII isolated in our laboratory
(Hernandez-Salgado et al., 2009). Male mice (CD-1, 20 g body
weight) were tested also by intra-peritoneal injection according to
a standard protocol (Organization, W.H., 1981, WHO, Geneva). Mice
were observed for toxicity symptoms up to 24 h. Toxicity assess-
ments in mice were classified as follows; mild toxicity was
considered when the mice showed alterations such as restless and
piloerection in its behavior after venom fraction injection; moder-
ate toxicity was when the mice showed additional symptoms of
intoxication such as sialorrhea and/or hyperactivity, but recovered
before 24 h; severe toxicity was considered when the post-24-hour
injection the mice still showed signs of intoxication (hypoactivity,
forced abdominal breathing, partial paralysis of limbs, exoph-
thalmus) but it finally was recovered and lethal when the mice
died. Most of these symptoms are dose-dependent. The amounts of
protein used are indicated in the headings of the table results.

2.6.2. Insects
Crickets (Gryllus bimaculatus) were injected intrathoracically
(i.t.) between the second and third pair of legs, with 1 mg/mL dose
(5 mL) of each peptide fraction previously dissolved in dH20. Con-
trols were done with distillated water only and the crickets were
closely observed up to 2 h. Toxicity assessments in crickets were
classified as follows: it was considered mild toxicity when, after
injection, the cricket lost the capacity to standing up when placed
backwards, but recovered after a few seconds; moderate toxicity
when the cricket recovered after 30 min and severe toxicity when
the animal did not recover after one hour of the injection. The word
“paralysis” was used when the cricket lost all mobility for 2 h, but
finally recovered. The lethal effect was said, when the crickets died
after injection.

3. Results and discussion

According to the literature, most of the buthid scorpion venom

components, which elute over the first 10 min period of elution
(please check elution gradient conditions) are mainly heterocyclic
compounds associated with pain as shown in other species of
scorpions previously studied (Basu et al., 1990; Quintero-
Hernandez et al., 2013). Venom components that elute over
Fig. 2. Reverse-phase HPLC profiles of venoms from Centruroides species collected
in Panama. C. bicolor (A); C. limbatus (B); C. granosus (C); and C. panamensis (D). Each
one of the chromatograms were obtained by application of 2 mg of dry-weight venom
for the four species. Numbers on top of some sub-fractions were used to identify the
components for further analysis (lethality tests, molecular mass and sequencing). A
picture of each scorpion is included in the corresponding HPLC profile.
 Author's Personal Copy

M.H. Salazar et al. / Toxicon 141 (2018) 79e87 83

Table 1
Biological activities in mice and insects of venom fractions from Centruroides species from Panama.

C. bicolor DL50 ¼ 3.4 mg/kg Fraction Mice i.c. (1 mg) Mice i.p. (50 mg) Insect i.t. (5 mg) Mm (±1 Da)
11 Mild NI NT 2611.6
25 Moderate NI NT 4008.9
27 Lethal NI NT 4220.7, 4332.1
28 Moderate NI Paralysis 4048.4
30 Moderate NI Mild 6904.7, 7002.0
36 Lethal Moderate Paralysis 7306.0, 7623.0
38 Lethal Severe Paralysis 7177.3, 6943.8
41 Lethal Lethal Moderate 7487.1
43 Lethal NI Moderate 7048.6, 6979.8
48 Moderate NI NT ND
C. limbatus DL50 ¼ 5.70 mg/kg Fraction Mice i.c. (1 mg) Mice i.p. (50 mg) Insect i.t. (5 mg) Mm (±1 Da)
38 Lethal Severe Paralysis 7516.3
39 Lethal NI Paralysis 7049.2
C. granosus DL50 ¼ 2.9 mg/kg Fraction Mice i.c. (1 mg) Mice i.p. (50 mg) Insect i.t. (5 mg) Mm (±1 Da)
40 Lethal Moderate Paralysis 6940.3
42 Moderate NI Paralysis 7149.1, 7513.4
43 Lethal Moderate Moderate 7148.3,7514.2
C. panamensis Fraction Mice i.c. (1 mg) Mice i.p. (50 mg) Insect i.t. (5 mg) Mm (±1 Da)
12 Mild NI Mild 3051.8, 3022.1
32 Mild NI Paralysis 6995.5
35 Mild NI NT 7188.1, 7609.1
36 Mild NI Moderate 6944.1, 7608.5
38 Mild NI NT 7195.31
41 Lethal Moderate Paralysis 7487.4
44 Lethal Moderate Moderate 7195.5

Abbreviations used: i.c., intracranial injection; i.p., intraperitoneal injection; i.t., intrathoracic injection; Mm, average molecular masses, the values are ±1 Da due to the limited
resolution of our instrument; NT, non-toxic after 2 h; NI, no injection; ND, not detected, n ¼ 2. The last column contains the molecular masses obtained from the given fraction.
Some fractions still contain more than one component. Molecular masses in bold means the most representative toxin peptides from the venom of Centruroides species in
Panama.

elute after 50 min period. Knowing this information, we selected

the major fractions of each scorpion venoms, within the 35e45 min
period, for assaying toxicity in mice and insects.

3.1. Neurotoxins from the venom of Centruroides species

The venoms of Centruroides (here after abbreviated C.):

C. granosus, C. bicolor, C. limbatus, and C. panamensis were frac-
tionated using RP-HPLC (Fig. 2). The most abundant fractions
(labeled with numbers) were tested on CD-1 mice and on insects to
evaluate its toxicity (Table 1). The fractions 27, 36, 38, 41 and 43
from C. bicolor were neurotoxic when injected intracraneally;
however, only the fraction 41 was lethal when injected i.p. to mice.
The fractions 28, 36 and 38 were paralytic to crickets. The fractions
38 and 39 from C. limbatus were neurotoxic when injected intra-
craneally into mice, and also provoked paralysis to crickets. How-
ever, none was lethal when injected i.p. to mice. Interestingly, only
the fraction 38 via i.c. provoked a nasal hemorrhage. The fractions
40 and 43 from C. granosus were neurotoxic when injected intra-
craneally (i.c.); however, none was lethal when injected intraperi-
toneally (i.p.) to mice. Both fractions (40 and 42) were paralytic
when injected into crickets. The fractions 41 and 44 from
C. panamensis were neurotoxic when injected intracraneally, but
none was lethal when injected i.p. to mice. The fractions 32 and 41
were paralytic to crickets. Now taking into account the elution
times and the molecular masses found (most of which were around
7000 kDa), it is very likely, based on the literature data (Possani
et al., 2000) that most of the biological active fractions are venom
peptides that affect the Nav of mammals and insects (Table 1).
Moreover, the fractions obtained from all four species of Centrur-
oides seem to be different according to their elution times and
molecular masses (see Fig. 2). Fractions 41 and 43 from C. bicolor Fig. 3. Reverse-phase HPLC profiles of venoms from Tityus species collected in
have elution times and molecular masses similar to that of fractions Panama. Tityus (A) asthenes (A); Tityus (A) festae (B); and Tityus (T) cerroazul (C). Each
one of the chromatograms were obtained by application of 2 mg of dry-weight venom
39 from C. limbatus and 41 from C. panamensis. The fact that they for the three species studied. Numbers on top of some sub-fractions were used to
have closely related molecular masses (7048.6 vs 7049.2 and 7487.4 identify the components for further analysis (lethality tests, molecular mass and
vs 7487.4 Da, respectively) do not necessary means the primary sequencing). A picture of each scorpion is included in the corresponding HPLC profile.
 Author's Personal Copy

84 M.H. Salazar et al. / Toxicon 141 (2018) 79e87

Table 2
Biological activities in mice and insects of venom fractions from Tityus (A) asthenes, T. (A) festae and T. (T) cerroazul species from Panama.

T. (A) asthenes DL50 ¼ 4.2 mg/kg Fraction Mice i.c. (1 mg) Mice i.p. (50 mg) Insect i.t. (5 mg) Mm (±1 Da)
27 Mild NI NT 7332.9
28 Lethal Lethal Paralysis 6522.2, 7101.5
29 Mild NI NT 7399.3
30 Lethal Moderate Lethal 7400.0, 7760.0
31 Mild NI NT 7098.0
T. (A) festae DL50 ¼ 4.9 mg/kg Fraction Mice i.c. (1 mg) Mice i.p. (50 mg) Insect i.t. (5 mg) Mm (±1 Da)
36 Lethal Moderate Mild 7332.0
37 Lethal Severe Paralysis 7398.8, 7073.6, 7100.0
39 Lethal Moderate NT 6969.6, 7399.0
44 Moderate NI NT 7394.5
T. (T) cerroazul DL50 ¼ 0.9 mg/kg Fraction Mice i.c. (1 mg) Mice i.p. (50 mg) Insect i.t. (5 mg) Mm (±1 Da)
15 Lethal Mild NT 4393.3, 3955.6
26 Moderate NI NT 7364.7
30 Lethal Mild NT 7046.0
31 Lethal Severe NT 6995.9, 7046.0
35 Moderate NI Lethal 6969.2, 10780.6
36 Lethal Severe NT 6968.8
40 Moderate NI NT 7356.0

Abbreviations used: i.c., intracranial injection; i.p., intraperitoneal injection; i.t., intrathoracic injection; Mm, average molecular masses, the values are ±1 Da due to the limited
resolution of our instrument; NT, non-toxic after 2 h; NI, no injection; ND, not detected, n ¼ 2. The last column contains the molecular masses obtained from the given fraction.
Some fractions still contain more than one component. Molecular masses in bold means the most representative toxin peptides from the venom of Tityus species in Panama.

sequence is identical. This must be verified experimentally.
3.2. Neurotoxins from the venom of Tityus species
The venoms from T. (A) asthenes, T. (A) festae and T. (T) cerroazul
were fractionated using reverse phase chromatography (Fig. 3). The
abundant fractions were also tested on CD-1 mice and insects to
evaluate their toxicities (Table 2). The fractions 28 and 30 from T. (A)
asthenes were lethal when injected intracraneally in mice. Fraction
28 causes specific symptomatology behavior. It provoked nasal
hemorrhage; it was lethal when injected i.p. to mice, and it was
paralytic to crickets. The fraction 30 was lethal to crickets. The
fractions 36, 37 and 39 from T. (A) festae were lethal when injected
intracraneally into mice; however, none was lethal when injected

Table 3
Biological activities in mice and insects of venom fractions from Tityus (T) pachyurus collected at different geographical areas of Panama.

Bocas del Toro DL50 ¼ 3.3 mg/kg Fraction Mice i.c. (1 mg) Mice i.p. (50 mg) Insect i.t. (5 mg) Mm (±1 Da)
13 Lethal Severe Moderate 7332.0
14 Lethal Severe Paralysis 7101.8, 7434.0

DL50 ¼ 2.6 mg/kg
Cocle Fraction Mice i.c. (1 mg) Mice i.p. (50 mg) Insect i.t. (5 mg) Mm (±1 Da)
14 Tremors NI Paralysis 973.0
27 Mild NI NT 969.0, 7345.0
31 Mild NI NT 7313.7, 6036.7
32 Lethal Severe Paralysis 7332.4
33 Lethal Severe Paralysis 7100.7, 6521.6
34 Lethal NI NT 7075.0
37 Lethal NI NT 10680.1, 7400.6
40 Mild NI NT 7338.5
41 Mild NI NT 7354.9
44 Mild NI NT 7628.6
46 Mild NI NT ND

Oeste DL50 ¼ 3.3 mg/kg
Panama Fraction Mice i.c. (1 mg) Mice i.p. (50 mg) Insect i.t. (5 mg) Mm (±1 Da)
22 Mild NI NT 4487.5
29 Mild NI NT 1001.3, 733.5
31 Lethal NI NT 2676.8
32 Severe Severe NT 6037.5, 7295.0, 7332.0
33 Lethal Severe Moderate 7432.7, 7100.2
35 Lethal NI Paralysis 6971.0
36 Lethal NI NT 7398.6, 10680.0
37 Lethal NI NT ND
41 Mild NI NT 7627.4, 7173.1

n DL50 ¼ 2.6 mg/kg
Colo Fraction Mice i.c. (1 mg) Mice i.p. (50 mg) Insect i.t. (5 mg) Mm (±1 Da)
27 Moderate NI NT ND
29 Lethal Moderate NT 7332.6
30 Lethal Severe Moderate 7100.0, 7332.5
34 Lethal Severe NT 7399.3
Herrera DL50 ¼ 2.0 mg/kg Fraction Mice i.c. (1 mg) Mice i.p. (50 mg) Insect i.t. (5 mg) Mm (±1 Da)
24 Moderate NI Paralysis 7863.0, 2676.5
28 Lethal Severe NT 7332.6, 10800.0
29 Lethal Severe NT 7099.7, 7331.3
32 Severe Severe NT 7399.7, 10563.0

Abbreviations used: i.c., intracranial injection; i.p., intraperitoneal injection; i.t., intrathoracic injection; Mm, average molecular masses, the values are ±1 Da due to the limited
resolution of our instrument; NT, non-toxic after 2 h; NI, no injection; ND, no detected, n ¼ 2. The last column shows the molecular masses determined from these fractions.
Molecular masses in bold means the most representative toxin peptides from the venom of Tityus species in Panama.
 Author's Personal Copy

M.H. Salazar et al. / Toxicon 141 (2018) 79e87 85

i.p. to mice. The fraction 37 was paralytic to crickets. The fractions

15, 30, 31 and 36 from T. (T) cerroazul were lethal to mice when
injected intracraneally. Fraction 15 provokes an instantaneous dead
of mice via i.c. None of the fractions were lethal when injected i.p.
to mice, but fractions 31 and 36 showed severe intoxication. In
crickets, fraction 35 was lethal at the doses assayed.
Furthermore, the venoms of T. (A) pachyurus collected at
different geographical areas (La Amistad National Park-Bocas del
Toro, El Montuoso Forest Reserve -Herrera, Tulú-Cocle
, San Lorenzo
National Park-Colo
n and Altos de Campana National Park - Panama

Oeste, Fig. 1) were also fractionated and tested on CD-1 mice and
insects to evaluate their toxicities (Table 3, Fig. 4). The fractions 13
and 14 from Bocas del Toro were lethal to mice when injected
intracraneally, but were not lethal when injected i.p. to mice. In
crickets, fraction 14 caused paralysis. The fractions 32, 33, 34 and 37
from Cocle
were lethal to mice when injected intracraneally. Frac-
tion 14 provokes just tremors in mice. When injected to mice i.p.,
fractions 32 y 33 provoked acute intoxications. Also, fractions 14, 32
and 33 were paralytic to crickets. The fractions 31, 33, 35, 36 and 37
from Panama
Oeste were lethal to mice when injected intra-
craneally. Fraction 36 via i.p. provoked sialorrhea. The fractions 32
and 33 provoked acute intoxications. Only fraction 35 was paralytic
to crickets. Fractions 29, 30 and 34 from Colo
n were lethal to mice
when injected intracraneally. Fraction 30 provoked nasal hemor-
rhages and fraction 34 provoked tremors in mice. When injected to
mice i.p., fractions 30 and 34 provoked severe intoxications. Only
fraction 30 was moderately toxic to crickets. Finally, fractions 28
and 29 from Herrera were lethal to mice when injected intra-
craneally. Fraction 29 provoked nasal hemorrhage via i.c. When
injected to mice i.p., fractions 28 and 29 provoked severe in-
toxications. Only fraction 24 was paralytic to crickets. Fig. 4 shows
that the HPLC profile of the venom from the same species, collected
in different geographical areas, showed clear differences profiles.
However, there are common peptides present in all runs
(Fig. 4AeE) eluting around 39 min, whose molecular masses found
are probably identical (close to 7332 and 7100 Da, see discussion
below).
According to the elution times and molecular masses, we can
suggest that fractions 32 and 33 from Cocle
having molecular
masses of 7332.4 and 7100.7 Da, respectively, are similar to frac-
tions 13 and 14 from Bocas del Toro, 32 and 33 from Panama
Oeste,
29 and 30 from Colo
n, and 28 and 29 from Herrera. The molecular
masses of 7332.4 and 7100.7 Da seems to be the most abundants in
T. (A) pachyurus. These proteins also seem to be present in T. (A)
asthenes (fractions 27 and 28) and T. (A) festae (fraction 36 and 37)
(Table 2) being the most abundants in all those venoms. However,
these proteins do not seem to be present in the venom of T. (T)
cerroazul.
In conclusion, the HPLC separations and identification of the
toxic fractions reported here are important information for future
work aimed at determining the structure and function of these
peptides, as well as, for selection of which venoms or fractions
should be taken into consideration for anti-venom production,
important object of this communication, as mentioned in the
Introductory section.

3.2.1. N-terminal sequencing of the largest fraction with toxic

activity from the venom of Tityus (A) pachyurus from Cocl
e
Some of the interesting fractions were the number 32 and 33
from Tityus (A) pachyurus Cocle
, which were toxic to mice and
crickets, and also they have elution times and molecular masses
(7332.2 and 7099.2 Da, respectively) similar to the most abundant
peptides from the venoms of T. (A) asthenes (fractions 27 and 28)
and T. (A) festae (fraction 36 and 37). These molecular masses are
also found in the venom of Tityus (A) pachyurus collected in
Fig. 4. Reverse-phase HPLC profiles of venoms from Tityus (A) pachyurus. Bocas del
Toro (A); El Tulú-Cocl
e (B); PNAC- Panama Oeste (C); San Lorenzo National Park - Colo

n
(D); Reserva Forestal El Montuoso-Herrera (E). Same amount of protein was applied in
each case (2 mg of dry-weight venom) and numbers on top of fractions were used for
identification of the components for further analysis. Since all chromatograms come
from the same scorpion, only the first one contains its picture.
 Author's Personal Copy
86 M.H. Salazar et al. / Toxicon 141 (2018) 79e87
Table 4

and its alignment with transcripts obtained from its own venom glands, from Tityus obscurus
Sequence alignment of TpF32 from the venom of Tityus (A.) pachyurus Cocle
(H1ZZI0.1 and P60213.3) and from Tityus (A.) discrepans (Q1I169.1).
Toxin Amino Acid Sequencea


TpF32-Cocle KDGYLVGNDGCKYSCNTYPK……

-A1
Cocle KDGYLVGNDGCKYSCLTRPGHYCASECSRVKGKDGYCYAWMACYCYNMPNWVKTWSRATNKCGKRK
H1ZZI0.1 KDGYLVGNDGCKYNCLTRPGHYCANECSRVKGKDGYCYAWMACYCYNMPNWVKTWSRATNKCGKRK
P60213.3 KDGYLVGNDGCKYNCLTRPGHYCANECSRVKGKDGYCYAWMACYCYSMPDWVKTWSRSTNRCGRGK
Q1I169.1 KDGYLVGNDGCKYSCSTRPGHYCASECSRVKGKDGYCYAWLACYCYNMPNWAPIWNSATNRCRGRK
a
The amino acid sequences correspond to the expected mature peptide (underlined) and the extra amino acids that codes for a posttranslational modification to produce a

.
C-amide; residues L16, R18 and G20 in italics differ from the N-terminal of TpF32-Cocle
b
-A1.
Identity to Cocle
c
Theoretical molecular mass based on four disulfide bridges and C-amidation.
different geographical areas of Panama (see Fig. 1). The fraction
with higher concentration (TpF32-Cocle
) was submitted to amino
acid sequencing by Edman degradation and the first 20 residues
were identified: KDGYLVGNDGXKYSXNTYPK (Table 4). The
cysteine residue (labeled X) was surmised based on similar N-ter-
minal sequence found. Table 4 shows the N-terminal sequence
experimentally determined (TpF32-Cocle
), the amino acid
sequence deduced from the clone gene of Cocle
-A1, and three
additional peptide sequences (H1ZZI01, P60213.3, Q1I169.1) from
toxins obtained from Genebank system of the species T. obscurus
and T (A) discrepans scorpion venom (Borges et al., 2006). They are
all quite similar, with identity better than 87%.
3.2.2. RNA isolation, cDNA library construction and Tityus (A)
pachyurus fraction 32 gene identification and its corresponding
peptide
The mRNA was extracted, and afterward, used to synthesize the
cDNA. The transcript of Tityus (A) pachyurus Cocle
(TpF32-Cocle
)
was amplified by PCR based on degenerated oligonucleotide and
AUAP primer (30 RACE System for Rapid Amplification of cDNA
Ends, Invitrogen). Two bands of 250 and 350 base pairs (bp) were
obtained after PCR amplification. Bands were gel-purified from
agarose and then ligated into pCR®2.1-TOPO TA cloning vector.
After transformation into E. coli XL1 Blue chemically-competent
cells, 30 clones were obtained and 10 of them were analyzed by
colony PCR using oligonucleotides M13-Forward and M13-Reverse
and amplifying a band of 550 bp approximately. Four plasmids from
the evaluated colonies were confirmed by DNA sequencing. A
verified plasmid with correct TpF32-Cocle
gene was amplified by
PCR using oligonucleotides, which introduced restriction sites. This
PCR product as well as the pQE30 vector, were digested with BamHI
and PstI and after respective purification, both were ligated. The
ligation reaction was transformed into competent E. coli XL1 Blue
cells obtained approximately >100 clones and 10 of them were
analyzed by colony PCR using oligonucleotides pQE30-Forward and
pQE30-Reverse, where a band of 380 bp was expected. Plasmids
from 4 colonies were purified, sequenced and finally confirming the
expected amino acidic TpF32-Cocle
N-terminal sequence; however,
it corresponds to the theoretical molecular mass of 7099.1 Da from
fraction TpF33-Cocle
, and correspond to the experimental molec-
ular masses found in the fractions 28 and 30 from T. asthenes and
T. festae, respectively, and in the fractions 14, 33, 33, 30 and 29 from
T. pachyurus from the areas from Bocas del Toro, El Tulú-Cocle
,
Panam
a Oeste, San Lorenzo National Park e Colo
n, and Reserva
Forestal El Montuoso-Herrera, respectively. Based on these findings
we assume that this toxic peptide is the same, regardless the
geographical region of collection. The mature peptide as shown in
Table 4 should contain 62 amino acid residues, with 4 disulfide
linkage and the C-terminal amino acid (Cysteine) amidated. Ac-
cording to Becerril et al. (1993), when the nucleotide sequence ends
with glycine followed by basic amino acids, the precedent residue
Identityb (%) Mmexp (Da) Mmtheoc (Da)
e 7332.4 ND
100 ND 7099.1
97 ND 7153.2
90 ND 7171.2
87 ND 7150.1
used the amine from glycine and becomes amidated, while the
basic(s) residue(s) are eliminated. The molecular masses experi-
mentally obtained from these fractions can be considered identical
(please note that the mass spectrometry equipment has limited
resolution; so the molecular masses are ±1 mass unit difference
depending on the sample). Furthermore, the molecular masses
reported here are average molecular masses. The theoretical mo-
lecular mass expected from the cloned gene (Cocle
-A1) is within
the same value (7,099.1); however, the transcript that codes for
TpF32-Cocle
has not been found yet.
4. Conclusions
This work shows the venom complexity of eight buthid scorpion
species of medical importance from Panama
. The results support
evidence of divergent protein composition in the venom of the
genus Centruroides, and more similar composition in the venoms of
the scorpion species Tityus (Atreus) asthenes, pachyurus and festae,
in contrast with the composition of the venom from T. (T) cerroazul.
The protein peptides from fractions Tp32 and TpF33-Cocle
, which
are similar to other venom components within the same Tityus
(Atreus) subgenus, seems to be an excellent candidate to be used as
immunogen for production of neutralizing antibodies against the
current Tityus antivenoms, for the population of Panama.
An interesting observation was that the HPLC profiles of the
venoms from the Centruroides species were more complex than
that of the venoms from Tityus species. Fractions from the venom of
Centruroides scorpions eluting around 25 to 35 min, assumed to be
peptides that block Kþ-channels, are more diverse and abundant
than those from Tityus species. Fractions that elute from 35 to
45 min on the HPLC system are very likely peptides that modify the
function of Naþ-channels, according to the molecular masses
experimentally determined.
More work is under way in order to elucidate the structure and
physiological actions of the principal components from the species
of Centruoides and Tityus scorpions of Panama, responsible for the
envenomation symptoms, which certainly should help developing
much better antivenoms for the country.
Acknowledgements
This work was financed by grants from Panama funds: Proyecto
VIP-UP (CUFI-2015-CS-P-010), and SENACYT (SUM08-005, COL10-
45, INF10-051), and Mexicans funds: DGAPA-UNAM No. IN203416
to LDP, and DGAPA-UNAM No. IN204415 and SEP-CONACyT No.
240616 to GC.
Transparency document
Transparency document related to this article can be found
online at https://doi.org/10.1016/j.toxicon.2017.11.013.
 Author's Personal Copy

M.H. Salazar et al. / Toxicon 141 (2018) 79e87 87

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