Genus Centruroides Research Articles

Taking the sting out of scorpions: Electrophysiological investigation of the relative efficacy of three antivenoms against medically significant Centruroides species

In this study, we report the innovative application of whole-cell patch-clamp electrophysiology in assessing broad-spectrum neutralisation by three different antivenoms, of venoms from the medically significant scorpion genus Centruroides. Envenomations by as many as 21 species from the Centruroides genus result in up to 300,000 envenomations per year in Mexico, which poses significant and potentially life-threatening pathophysiology. We first evaluated the in vitro manifestation of envenomation against two human voltage-gated sodium (hNaV) channel subtypes: hNaV1.4 and hNaV1.5, which are primarily expressed in skeletal muscles and cardiomyocytes, respectively. The neutralisation of venom activity was then characterised for three different antivenoms using a direct competition model against the more potent target, hNaV1.4. While broad-spectrum neutralisation was identified, variation in neutralisation arose for Centruroides elegans, C. limpidus, C. noxius and C. suffusus venoms, despite the presence of a number of these venoms within the immunising mixture. This raises questions regarding the truly “broad” neutralisation capacity of the antivenoms. This study not only extends previous validation of the in vitro investigation of antivenom efficacy utilising the whole-cell patch-clamp technique but also underscores the potential of this animal-free model in exploring cross-reactivity, experimental scalability, and most importantly, informing clinical management practices regarding the administration of antivenom in Mexico.

Genetic diversity of medically important scorpions of the genus Centruroides (Buthidae) from Panama including two endemic species

With more than 33,000 sting cases and 47 deaths recorded between 2000 and 2016, Panama is the country with the highest incidence of envenomations by scorpions in Central America. Species in the genus Centruroides are responsible for most scorpion sting reports, however, identification at the species level is complicated because the genus has considerable intraspecific morphological variation. To date no molecular data have been reported from Panama that would help to estimate their genetic diversity and validate morphometric identification methods. We provide here the first genetic diversity data of the two endemic species (C. granosus and C. panamensis) and other two species reported in Panama (C. bicolor and C. limbatus). A total of 41 specimens were sequenced for COI and 16S rDNA mitochondrial genes. The phylogenetic concatenated analysis separates the Panamanian samples into four well-supported clades represented by C. bicolor, C. granosus and (C. panamensis + C. limbatus). The two endemic species are not the closest relatives in the tree. Low diversity in combination with its very narrow distribution suggest that C. panamensis is susceptible to environmental degradation. A single specimen of Coiba island is intermediate in the tree structure between C. bicolor and C. panamensis and may represent an early stage of speciation. The haplotype network is also consistent with the phylogenetic trees.

Una nueva especie de alacrán del género Centruroides Marx (1890) (Scorpiones, Buthidae) de la depresión del Balsas, México

Se describe una nueva especie del género Centruroides Marx de la Depresión del Balsas en los Estados de Guerrero, Michoacán y México, en elevaciones que van de los 400 a los 1500 msnm. Esta especies se caracteriza por tener ocho hileras de dentículos en los dedos de la quela del pedipalpo y corresponde, dentro del género, a la sección de alacranes rayados, en la cual se le ha confundido con Centruroides limpidus limpidus Karsch y con Centruroides limpidus tecomanus Hoffmann. Las diferencias se observan en morfometría y patrón de coloración. Esta nueva especie es simpátrica con C. limpidus limpidus en la parte Este de su rango de distribución en el Estado de Michoacán y con C. infamatus infamatus Pocock en la Sierra de Coalcomán y regiones adyacentes en la parte Norte del Estado. Se registra también en una localidad en el Estado de Guerrero y en una localidad en el Estado de México.

The three-dimensional structure of the toxic peptide Cl13 from the scorpion Centruroides limpidus

Cl13 is a toxin purified previously from the venom of the Mexican scorpion Centruroides limpidus. This toxin affects the function of voltage gated Na+-channels, human subtypes Nav1.4, Nav1.5 and Nav1.6 in a similar manner as other known β-toxins from scorpion venoms. Here, we report a correction of the primary structure of Cl13, previously published. The peptide does contain 66 amino acids, but residue 58 is a tryptophan and the last C-terminal amino acid is an amidated lysine, instead of arginine. The main contribution of this communication is the determination of the 3D-structure of Cl13, by solution NMR, showing that Cl13 has the classical cysteine-stabilized α/β (CSα/β) folding. It has a triple stranded antiparallel beta sheet commonly present in scorpion sodium channel β-toxins. In addition, we report and discuss a comparison of Cl13 structure with two other toxins (Cn2 and Css2) from scorpions of the same genus Centruroides, which shows important surface similarities with the structure reported here. Finally, the lack of neutralization of Cl13 toxin by two single-chain antibody fragments (scFvs), named LR and 10FG2, which are capable of neutralizing various toxins from Mexican scorpions, is revised. In particular, 10FG2 is capable of neutralizing toxins Cll1 and Cll2 of the same scorpion C. limpidus. The reasons why LR and 10FG2 are unable of neutralizing Cl13 toxin are discussed.

Niche partitioning in congeneric scorpions

AbstractSpecies in the scorpion genus Centruroides (Scorpiones: Buthidae) are good candidates for the study of ecological niche partitioning because of their habitat plasticity, widespread geographic distribution, and presence of cryptic species. Currently, three species belonging to three subgroups of Centruroides are distributed along the Isthmus of Tehuantepec in southern Mexico, presenting a rare opportunity to study niche partitioning within a single genus. We examined the environmental, substrate, and habitat conditions of Centruroides flavopictus, Centruroides gracilis, and Centruroides rileyi within Estación de Biología Los Tuxtlas, west of the Isthmus of Tehuantepec. Our results demonstrate habitat partitioning occurs along humidity, temperature, and elevational gradients: individuals of C. gracilis were found in habitats of low humidity and high temperature, and were predominantly found on the substrate in human‐mediated habitats; individuals of C. flavopictus were found in a larger range of humidity and temperature conditions within secondary and primary forest; and individuals of C. rileyi had the greatest habitat specificity, and were found in conditions of low temperature and high humidity on trees within primary forest. Furthermore, individuals of C. rileyi were found significantly higher in trees than those of C. flavopictus. This study represents the first example of niche partitioning within a genus of scorpion, and the first description of the ecological niche in an arboreal‐specialist scorpion.

A new species of the genus Centruroides Marx (Scorpiones, Buthidae) from western Michoacán State, México using molecular and morphological evidence.

A new species of scorpion belonging to the genus Centruroides Marx, 1890 is described from the Coalcomán mountain range, western Michoacán State, Mexico. Its general aspect resembles Centruroidesruana Quijano-Ravell & Ponce-Saavedra, 2016, and C.infamatus (C. L. Koch, 1844), but it is a smaller species having lower pectinal tooth counts; also, males of C.ruana have the pedipalp chelae slightly thicker, whereas C.infamatus has a subaculear tubercle nearer to the base of the aculeus. Another species with similar aspect is Centruroidesornatus Pocock, 1902; however, a preliminary molecular analysis of the mitochondrial gene mRNA 16S showed genetic divergence (measured as p-distance) near to 10% between these species, and lower differences between the new species with respect to C.infamatus (4.63%) and C.ruana (5.07%). The molecular evidence together with the morphological characters (integrative taxonomy) are sufficient for recognizing the Coalcomán population as a separate and valid species.

Dissecting Toxicity: The Venom Gland Transcriptome and the Venom Proteome of the Highly Venomous Scorpion Centruroides limpidus (Karsch, 1879).

Venom glands and soluble venom from the Mexican scorpion Centruroides limpidus (Karsch, 1879) were used for transcriptomic and proteomic analyses, respectively. An RNA-seq was performed by high-throughput sequencing with the Illumina platform. Approximately 80 million reads were obtained and assembled into 198,662 putative transcripts, of which 11,058 were annotated by similarity to sequences from available databases. A total of 192 venom-related sequences were identified, including Na+ and K+ channel-acting toxins, enzymes, host defense peptides, and other venom components. The most diverse transcripts were those potentially coding for ion channel-acting toxins, mainly those active on Na+ channels (NaScTx). Sequences corresponding to β- scorpion toxins active of K+ channels (KScTx) and λ-KScTx are here reported for the first time for a scorpion of the genus Centruroides. Mass fingerprint corroborated that NaScTx are the most abundant components in this venom. Liquid chromatography coupled to mass spectometry (LC-MS/MS) allowed the identification of 46 peptides matching sequences encoded in the transcriptome, confirming their expression in the venom. This study corroborates that, in the venom of toxic buthid scorpions, the more abundant and diverse components are ion channel-acting toxins, mainly NaScTx, while they lack the HDP diversity previously demonstrated for the non-buthid scorpions. The highly abundant and diverse antareases explain the pancreatitis observed after envenomation by this species.

Venoms of Centruroides and Tityus species from Panama and their main toxic fractions

The scorpionism in Panama is notorious for the confluence and coexistence of buthid scorpions from the genera Centruroides and Tityus. This communication describes an overview of the larger representative toxic venom fractions from eight dangerous buthid scorpion species of Panama: Centruroides (C. granosus, C. bicolor, C. limbatus and C. panamensis) and Tityus (T. (A.) asthenes, T. (A.) festae, T. (T.) cerroazul and T. (A.) 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 the lethal components had a molecular mass close to 7000 Da, assumed to be peptides that recognize Na+-channels, responsible for the toxicity symptoms observed in other buthids scorpion venoms. A toxic peptide isolated from the venom of T. pachyurus was sequenced by Edman degradation, allowing the synthesis of nucleotide probe for cloning the correspondent gene. The mature toxin based on the cDNA sequencing has the C-terminal residue amidated, contains 62 amino acid packed by 4 disulfide linkages, with molecular mass of 7099.1 Da. This same toxic peptide seems to be present in scorpions of the 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.

Design and expression of recombinant toxins from Mexican scorpions of the genus Centruroides for production of antivenoms

This manuscript describes the design of plasmids containing the genes coding for four main mammalian toxins of scorpions from the genus Centruroides (C.) of Mexico. The genes that code for toxin 2 of C. noxius (Cn2), toxin 2 from C. suffusus (Css2) and toxins 1 and 2 from C. limpidus (Cll1 and Cll2) were included into individual plasmids carrying the genetic construction for expression of fusion proteins containing a leader peptide (pelB) that directs the expressed protein to the bacterial periplasm, a carrier protein (thioredoxin), the cleavage site for enterokinase, the chosen toxin and a poly-histidine tag (6xHis-tag) for purification of the hybrid protein by immobilized metal ion affinity chromatography after expression in Escherichia coli strain BL21 (DE3). The purified hybrid proteins containing the recombinant toxins (abbreviated Thio-EK-Toxin) were used for immunization of three independent groups of ten mice and four rabbits. Challenging the first group of mice, immunized with recombinant Thio-EK-Css2, with three median lethal doses (LD50) of C. suffusus soluble venom resulted in the survival of all the test animals without showing intoxication symptoms. All control mice (none immunized) died. Similar results were obtained with mice previously immunized with Thio-EK-Cn2 and challenged with C. noxius venom. The third group of mice immunized with both Thio-EK-Cll1 and Thio-EK-Cll2 showed an 80% survival ratio when challenged with only one LD50 of C. limpidus venom, all showing symptoms of intoxication. The sera from rabbits immunized with a combination of the four recombinant toxins were collected separately and used to assess their neutralization capacity in vitro (pre-incubating the serum with the respective scorpion venom and injecting the mixture into mice), using six mice for each serum/venom combination tested. The venoms from the six most dangerous scorpion species of Mexico were assayed: C. noxius, C. suffusus, C. limpidus, C. elegans, C. tecomanus and C. sculpturatus. Two hundred and 50 μL of serum from any of the immunized rabbits were enough to neutralize three LD50 of any of the tested venoms, with mice showing no symptoms of intoxication. These results confirm that the recombinant forms of the main toxins from the most dangerous scorpions of Mexico are excellent immunogens for the production of antivenoms to treat scorpion intoxications.

A new island species of Centruroides Marx, 1890 (Scorpiones: Buthidae) from the southwestern Caribbean

Herein we describe a new species of the Buthidae scorpion genus Centruroides Marx, 1890. It occurs at least in two small offshore islands of the southwestern Caribbean: Cozumel in Mexico and Guanaja in Honduras, based upon type specimens from the former and photographic evidence from the latter. It belongs in the "gracilis" species-group and is most closely related to both Centruroides gracilis (Latreille, 1805) and Centruroides nigrescens (Pocock, 1898).

A new species of scorpion of the genus Centruroides (Scorpiones: Buthidae) from the state of Michoacán, Mexico

Centruroides ruana sp. nov. is described from Felipe Carrillo Puerto, from the Buenavista Municipality in the state of Michoacán, Mexico. The new species was compared with C. balsasensisPonce-Saavedra & Francke, 2004, C. infamatus Koch, 1844, and C. limpidus (Karsch, 1879), which are both morphologically its closest relatives and geographically proximate. The description includes the male holotype, with 7 male and 8 female paratypes. A supplementary morphometric analysis is also presented allowing to distinguish the 4 species.

Effectiveness of Centruroides scorpion antivenom compared to historical controls

BackgroundEnvenomation by North American scorpions of genus Centruroides is associated with a syndrome of neurotoxicity and respiratory compromise that disproportionately affects rural children. Severe scorpion envenomation is rare, which makes treatment difficult to study using conventional controlled clinical trials; and small-scale placebo-controlled trials conducted in tertiary centers are of limited generalizability to the community setting. Open label studies, although safer and easier to conduct, are of limited value unless a suitable comparator group is used. Historical controls may be appropriate when concurrent controls are not feasible or ethical. MethodsA successful placebo-controlled, double-blind clinical trial design was adapted for community use in Arizona and Mexico. A comparator population was established by replacement of the placebo group with a retrospective cohort and preservation of criteria for inclusion, exclusion, dosing and endpoint assessment. Study endpoints were selected to demonstrate the clearest possible difference between treatment groups, while minimizing confounders. Results were summarized and endpoints were directly compared between groups and with the prior double-blind study. ResultsThe clinical syndrome remained evident in 95.9% of the historical cohort (93/97) 4 h after admission, and their cumulative dose of midazolam given between baseline and discharge was 5.29 ± 8.68 mg/kg (range 0–62.8). Among 78 prospectively treated cases, none received midazolam and only 2 (2.8%) remained symptomatic at 4 h. Venom was detectable in the plasma of all antivenom recipients tested, and it dropped by 90% of baseline in 95% of cases studied. ConclusionsThe results of this pragmatic study strongly support the findings of the double-blind, placebo controlled clinical trial of the same antivenom. Recipients of antivenom at rural sites improved at a rate similar to that in the intensive care (ICU) setting, and historical cases resolved at a rate similar to that for untreated ICU controls. Use of antivenom in the primary care setting appeared to be safe and effective and to eliminate the need for intensive care or for transport to a tertiary care center, in all subjects prospectively studied.

Species Delimitation and Morphological Divergence in the Scorpion Centruroides vittatus (Say, 1821): Insights from Phylogeography

Scorpion systematics and taxonomy have recently shown a need for revision, partially due to insights from molecular techniques. Scorpion taxonomy has been difficult with morphological characters as disagreement exists among researchers with character choice for adequate species delimitation in taxonomic studies. Within the family Buthidae, species identification and delimitation is particularly difficult due to the morphological similarity among species and extensive intraspecific morphological diversity. The genus Centruroides in the western hemisphere is a prime example of the difficulty in untangling the taxonomic complexity within buthid scorpions. In this paper, we present phylogeographic, Ecological Niche Modeling, and morphometric analyses to further understand how population diversification may have produced morphological diversity in Centruroides vittatus (Say, 1821). We show that C. vittatus populations in the Big Bend and Trans-Pecos region of Texas, USA are phylogeographically distinct and may predate the Last Glacial Maximum (LGM). In addition, we suggest the extended isolation of Big Bend region populations may have created the C. vittatus variant once known as C. pantheriensis.

A new species of the genus Centruroides Marx, 1890 (Scorpiones: Buthidae) from Guatemala

A new species of the genus Centruroides Marx, 1890 is described from northeastern Guatemala on basis to an adult male. By its general pattern and slight sexual dimorphism, the new species looks like C. flavopictus (Pocock, 1898), a larger species from Veracruz, Mexico, with higher pectinal tooth count (males: 21 to 24 teeth) and stronger subaculear tubercle. It also resembles Centruroides chamulaensis Hoffmann, 1932, from Chiapas, Mexico, a smaller species with small to obsolete subaculear tubercle, stronger metasomal carinae, pedipalp chelae narrower than patella, and anterior margin of carapace almost straight (V-shaped in the new species).

Scorpionism in Central America, with special reference to the case of Panama

Scorpionism in the Americas occurs mainly in Mexico, northern South America and southeast Brazil. This article reviews the local scorpion fauna, available health statistics, and the literature to assess scorpionism in Central America. Notwithstanding its high toxicity in Mexico, most scorpion sting cases in Guatemala, Belize, El Salvador, Nicaragua, and Costa Rica are produced by species in the genus Centruroides that are only mildly toxic to humans despite the existence of ion channel-active toxins in their venoms. Regional morbidity is low with the exception of Panama, where an incidence of 52 cases per 100,000 inhabitants was recorded for 2007, with 28 deaths from 1998 to 2006. Taxa belonging to the genus Tityus (also present in the Atlantic coast of Costa Rica) are responsible for fatalities in Panama, with Tityus pachyurus being the most important species medically. Most Tityus species inhabiting Panama are also found in northern South America from which they probably migrated upon closure of the Panamanian isthmus in the Miocene era. Incorporation of Panama as part of the northern South American endemic area of scorpionism is thereby suggested based on the incidence of these accidents and the geographical distribution of Panamanian Tityus species.

Scorpion Envenomation and Antivenom Therapy

Scorpion envenomation is a dangerous and common global event that can result in a variety of toxic clinical effects. These are typically managed with supportive care or antivenom. Antivenom use is controversial because of conflicting evidence of effectiveness for adrenergic toxicity. However, both controlled and uncontrolled studies have shown that antivenom is effective in resolving neuromotor toxicity associated with envenomations by the scorpions of genus Centruroides.

Biochemical, genetic and physiological characterization of venom components from two species of scorpions: Centruroides exilicauda Wood and Centruroides sculpturatus Ewing

Current literature concerning the taxonomic names of two possibly distinct species of scorpions from the genus Centruroides ( sculpturatus and/or exilicauda) is controversial. This communication reports the results of biochemical, genetic and electrophysiological experiments conducted with C. exilicauda Wood of Baja California (Mexico) and C. sculpturatus Ewing of Arizona (USA). The chromatographic profile fractionation of the soluble venom from both species of scorpions is different. The N-terminal amino acid sequence for nine toxins of C. exilicauda was determined and compared with those from C. sculpturatus. Lethality tests conducted in mice support the idea that C. exilicauda venom should be expected to be medically less important than C. sculpturatus. Thirteen genes from the venomous glands of the scorpion C. exilicauda were obtained and compared with previously published sequences from genes of the species C. sculpturatus. Genes coding for cytochrome oxidase I and II of both species were also sequenced. A phylogenetic tree was generated with this information showing important differences between them. Additionally, the results of electrophysiological assays conducted with the venom from both species on the Ca 2+-dependent K +-channels, showed significant differences. These results strongly support the conclusion that C. exilicauda and C. sculpturatus are in fact two distinct species of scorpions.

A large number of novel Ergtoxin-like genes and ERG K +-channels blocking peptides from scorpions of the genus Centruroides

Twenty-three novel sequences similar to Ergtoxin (ErgTx) were obtained by direct sequencing of peptides or deduced from gene cloned using cDNAs of venomous glands of Centruroides ( C.) elegans, C. exilicauda, C. gracilis, C. limpidus limpidus, C. noxius and C. sculpturatus. These peptides have from 42 to 47 amino acid residues cross-linked by four disulfide bridges. They share sequence similarities (60–98% compared with ErgTx1) and were shown to block ERG K +-channels of F-11 clone (N18TG-2×rat DRG) cultured cells. An unrooted phylogenetic tree analysis of these peptides showed that they conform at least five different subfamilies, of which three are novel subfamilies.

Cloning and characterization of the cDNAs encoding Na + channel-specific toxins 1 and 2 of the scorpion Centruroides noxius Hoffmann

Using a cDNA library prepared from venomous glands of the Mexican scorpion Centruroides noxius Hoffmann the genes that encode toxins 1 and 2 were identified, cloned and sequenced. In view of the proposed mechanism for processing the mature peptides coded by these two genes, the corresponding peptide-toxins were sequenced de novo. Mass spectrometric and 1H-NMR analyses of the C-terminal peptide produced by enzymatic digestion of both toxins indicated that the last residue is serine-amide. Sequence comparison revealed that these two genes have a similarity of 56% and 80% at the amino acid and nucleotide levels, respectively. Small corrections to the published primary structures were introduced: Cn toxin 1 has an extra serine residue at position 65 and the residue in position 60 is a proline, while the amino acids at positions 34 and 35 of Cn 2 are, respectively, tyrosine and glycine. Sequence comparison of toxins from the genus Centruroides suggests the presence of at least three classes of distinct peptides in these venoms.

Cloning of Genes Encoding Scorpion Toxins: An Interpretative Review

AbstractScorpion toxins (Stox) are polypeptides intensively investigated because they are excellent models to study protein-structure-function relationships and exquisite tools to access ion channel functions. Recently, techniques of molecular biology have been used to clone the genes encoding toxins specific for Na+-channels. Genes from scorpions of the genus Androctonus, Centruroides, Leiurus, Tityus and Bothus have been cloned and their nucleotide (nt) sequence determined. Data from complementary DNA (cDNA) cloning of Stox from the genus before mentioned and genomic cloning of Stox genes from scorpions of the genus Tityus and Androctonus have provided information for the deduction of the structure of the transcriptional units encoding toxin genes of these scorpions. It seems that transcription of these genomic regions generates pre messenger RNAs (pre-mRNAs) of approximately 800nt containing an intron of approximately 470nt, located within the region encoding the signal peptide. The processing of these...