Androctonus Amoreuxi Research Articles

Virocidal activity of Egyptian scorpion venoms against hepatitis C virus.

BackgroundHepatitis C virus (HCV) is a major global health problem, causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Development of well-tolerated regimens with high cure rates and fewer side effects is still much needed. Recently, natural antimicrobial peptides (AMPs) are attracting more attention as biological compounds and can be a good template to develop therapeutic agents, including antiviral agents against a variety of viruses. Various AMPs have been characterized from the venom of different venomous animals including scorpions.MethodsThe possible antiviral activities of crude venoms obtained from five Egyptian scorpion species (Leiurus quinquestriatus, Androctonus amoreuxi, A. australis, A. bicolor and Scorpio maurus palmatus) were evaluated by a cell culture method using Huh7.5 cells and the J6/JFH1-P47 strain of HCV. Time-of-addition experiments and inactivation of enzymatic activities of the venoms were carried out to determine the characteristics of the anti-HCV activities.ResultsS. maurus palmatus and A. australis venoms showed anti-HCV activities, with 50% inhibitory concentrations (IC50) being 6.3 ± 1.6 and 88.3 ± 5.8 μg/ml, respectively. S. maurus palmatus venom (30 μg/ml) impaired HCV infectivity in culture medium, but not inside the cells, through virocidal effect. The anti-HCV activity of this venom was not inhibited by a metalloprotease inhibitor or heating at 60°C. The antiviral activity was directed preferentially against HCV.ConclusionsS. maurus palmatus venom is considered as a good natural source for characterization and development of novel anti-HCV agents targeting the entry step. To our knowledge, this is the first report describing antiviral activities of Egyptian scorpion venoms against HCV, and may open a new approach towards discovering antiviral compounds derived from scorpion venoms.

Deep intraspecific divergences in the medically relevant fat-tailed scorpions (Androctonus, Scorpiones)

The genus Androctonus, commonly known as fat-tailed scorpions, contains 22 species distributed from Togo and Mauritania in the west, North Africa, through the Middle East and to as far east as India. With 13 species, a substantial amount of this genus’ diversity occurs in North Africa, which is a major hotspot of scorpion sting incidents. Androctonus are among the most medically relevant animals in North Africa. Since venom composition within species is known to vary regionally, the improvement of therapeutic management depends on a correct assessment of the existing regional specific and sub-specific variation. In this study, we assessed the phylogeographical patterns in six species of Androctonus scorpions from North Africa using mitochondrial DNA markers. We sequenced COX1, 12S, 16S and ND1 genes from 110 individuals. Despite lacking basal resolution in the tree, we found taxonomical and geographically coherent clades. We discovered deep intraspecific variation in the widespread Androctonus amoreuxi and Androctonus australis, which consisted of several well-supported clades. Genetic distances between some of these clades are as high as those found between species. North African A. australis have a deep split in Tunisia around the Chott el-Djerid salt-lake. A novel split between A. amoreuxi scorpions was found in Morocco. We also found deep divergences in Androctonus mauritanicus, corresponding to areas attributed to invalidated subspecies. In addition we uncovered a clade of specimens from coastal south Morocco, which could not be ascribed to any know species using morphological characters. Based on these findings we recommend a reassessment of venom potency and anti-venom efficacy between these deep intraspecific divergent clades.

English

Leuirus quinquestriatus, Androctonus amoreuxi and Androctonus australis are venomous scorpion in Egypt. Their venom is complex mixture of salts, neurotoxins, peptides and proteins which has therapeutic applications, and can rapidly kill a broad range of microbes. Estimations of total proteins of the venom indicated that L. quinquestriatus had the highest value than the others (10.64 ± 0.04). Hemolytic activity of human erythrocytes was detected and showed that all concentrations (20, 8 and 5 mg/ml) of tested scorpion species crude venom have hemolytic activity against human erythrocytes. The present study was conducted to evaluate the antimicrobial activity of the three Egyptian scorpion’s venom against four Gram-positive and negative bacterial strains (Bacillus cereus, Bacillus subtillis, Citrobacter freundi and Klibsella pneumonia) in addition to one fungus species Candida albicans. The results show that L. quinquestriatus venom has a significant antibacterial effect against B. subtillis and C. freundi. In contrast, A. amoreuxi and A. australis venoms do not have a noticeable effect on tested microbes. So, the aim of the present study was to investigate the antimicrobial activity of crude venom against Gram-negative, as well as Gram-positive bacteria, fungi and to evaluate the hemolytic activity of the three investigated species on human erythrocytes. Key words: Crude venom, Leuirus quinquestriatus, Androctonus amoreuxi, Androctonus australis, hemolytic activity, antibacterial activity.

Histopathological Changes in Liver of Mice after Exper Imental Envenomation with Androctonus Amoreuxi Scorpion Venom

A total of 78 adult male Albino mice were divided into thirteen groups (6 mice in each). One served as a control group and the other twelve groups were venom treated groups. The mice of treated groups were injected with 0.1 ml saline solution in which a particular amount of scorpion venom. The first 6 groups were subcutaneously injected with 1/2 LD50 (0.05 microg/g body weight), while the other 6 groups were injected with 1/4 LD 50 (0.025 microg/g body weight) by the same route. The animals from each group were anesthetized with ethyl ether and sacrificed at different time intervals (3, 6, 9, 12 hrs, 4 & 7days post toxin administration). The microscopic examination of liver tissue obtained from envenomed animals showed variable histopathological changes being severely increased with the time interval of envenoming. The most obvious changes in the liver were acute cellular swelling, hydropic degeneration, congestion of central veins and portal blood vessels. Besides, extramedullary hematopoiesis and invaginations in nuclei of hepatic cells, with formation of intranuclear cytoplasmic inclusions were observed.

Clinicopathological Investigations on Mice Envenomed with Scorpion Venom ( Androctonus Amoreuxi )

The present study assessed the toxicity of Androctonus amoreuxi crude venom on blood and biochemical serum parameters of mice. Adult male Albino mice were divided into three groups, in the control group mice were injected S.C. with saline solution. The second group and the third were injected with the venom S.C. in mice in the following doses 1/4 and 1/2 dose of LD50 respectively. Blood and serum samples were taken after 3 hours, 6 hours, 9 hours, 12 hours, 4 days and 7 days. Hematocrit (Ht), red blood cells (RBC) count, hemoglobin, MCV, MCH & MCHC were performed. Serum biochemical parameters, the levels of total proteins, albumin, globulin, glucose, cholesterol, triglycerides, ALT, AST, ALP, creatinine, uric acid and urea were measured. RBCs, Hob, PCV, MCV, MCH & MCHC showed significant increase, and increase in total protein, albumin and globulin within the experiment. Glucose and cholesterol levels were significantly increase from the beginning. Triglycerides showed significant decrease after 6 hours. Liver enzymes and kidney functions revealed significant changes post-injection.

Surveillance study on scorpion species in Egypt and comparison of their crude venom protein profiles

The present study aimed to focus on all the famous species of scorpions from different regions in Egypt and the attempts to use protein profiles of their venom as a simple tool for taxonomy rather than traditional morphological methods. For this purpose, total protein concentration and protein profiles using SDS–PAGE were measured and the similarity coefficients of the protein bands of different species were calculated. The present results showed that there is one species (Scorpio maruas palmatus) which belongs to the family Scorpionedae, and seven species which belong to the family Buthidae. Four of them fall into the genus Androctonus namely: Androctonus crassicauda, which is very rare in Egypt, Androctonus australis, Androctonus bicolor, and Androctonus amoreuxi. In the electrophoretic analysis the protein bands ranged between14 and 200KDa. A notable find was that all scorpion venom samples examined contained two protein bands with MW of 200 and 95KDa, except in one species. One protein band (125KDa) is common in six species only. Based on this electrophoretic pattern the similarity indices indicate that there is inter-family, inter-genus, and inter-species variation between different scorpion samples. The closest species were A. amoreuxi and A. australis. This study proposes variation in venom protein composition which was measured qualitatively and quantitatively among different scorpion species collected from different regions in Egypt, which throws light on its importance and enables the researchers to consider it a guideline in taxonomy.

Fine structure of the stinger, histology and histochemistry of the venom gland in the scorpion Androctonus amoreuxi (Buthidae)

The venom apparatus of the scorpion Androctonus amoreuxi has been identified histologically and histochemically in the present study. The results showed that this apparatus composed of a pair of venom glands and a stinger located in the terminal segment called telson. The stinger of the venom apparatus has been studied by the light microscope and SEM. The stinger, located at the end section of the telson, is sickle-shaped. The venom is ejected through a pair of venom pores on its subterminal portion. Both venom ducts extend along the stinger without contact with each other since they are separated by connective tissue cells. The stinger is covered by cuticle and spines. Each venom gland is covered by a sheath of striated muscle and is lined with extensively folded secretory epithelium that consists of non-secretory and secretory venom-producing cells. The venom-producing cells reacted positively to histochemical tests for carbohydrates and proteins. The outcomes also revealed that the venom-producing cells of both glands produce neutral mucosubstances. The structure and secretion of scorpion venom glands are discussed within the context of the present results.

Effects of atropine and propranolol on lung inflammation in experimental envenomation: comparison of two buthidae venoms

BackgroundPrevious works had shown that scorpion venom induced neurotransmitter elevation and an inflammatory response associated with various anatomo-pathological modifications. The most dangerous scorpions species in Algeria responsible for these effects are Androctonus australis hector (Aah) and Androctonus amoreuxi (Aam).ResultsComparison of the physiopathological effects induced by the two venoms showed differences in the kinetic of cytokine release and in lung injury.The lung edema was only observed in response to Aah venom and it was correlated with cell infiltration. In order to better understand the involved mechanism in inflammatory response, we used two antagonists, atropine (non-selective muscarinic antagonist) and propranolol (β adrenergic antagonist), which lead to a decrease of cell infiltration but has no effect on edema forming.ConclusionThese results suggest another pathway in the development of lung injury following envenomation with Aam or Aah venom.

Antimicrobial/cytolytic peptides from the venom of the North African scorpion, Androctonus amoreuxi: Biochemical and functional characterization of natural peptides and a single site-substituted analog

The venoms of scorpions are complex cocktails of polypeptide toxins that fall into two structural categories: those that contain cysteinyl residues with associated disulfide bridges and those that do not. As the majority of lethal toxins acting upon ion channels fall into the first category, most research has been focused there. Here we report the identification and structural characterization of two novel 18-mer antimicrobial peptides from the venom of the North African scorpion, Androctonus amoreuxi. Named AamAP1 and AamAP2, both peptides are C-terminally amidated and differ in primary structure at just two sites: Leu⇒Pro at position 2 and Phe⇒Ile at position 17. Synthetic replicates of both peptides exhibited a broad-spectrum of antimicrobial activity against a Gram-positive bacterium (Staphylococcus aureus), a Gram-negative bacterium (Escherichia coli) and a yeast (Candida albicans), at concentrations ranging between 20μM and 150μM. In this concentration range, both peptides produced significant degrees of hemolysis. A synthetic replicate of AamAP1 containing a single substitution (His⇒Lys) at position 8, generated a peptide (AamAP-S1) with enhanced antimicrobial potency (3–5μM) against the three test organisms and within this concentration range, hemolytic effects were negligible. In addition, this His⇒Lys variant exhibited potent growth inhibitory activity (ID50 25–40μm) against several human cancer cell lines and endothelial cells that was absent in both natural peptides. Natural bioactive peptide libraries, such as those that occur in scorpion venoms, thus constitute a unique source of novel lead compounds with drug development potential whose biological properties can be readily manipulated by simple synthetic chemical means.

Dangerous scorpion fauna of Mali

Although the main Malian scorpion species of medical interest, Androctonus amoreuxi, is responsible for severe envenomings and perhaps some deaths, it has hitherto been considered not dangerous for humans. This population is located in the Saharian North-Eastern regions of Mali where it is accompanied by Leiurus quinquestriatus, a well known dangerous species of the Sahara. In the Gao district, divided by the Niger River, less desolate than the Tessalit and Kidal regions, one specimen of the dangerous species Androctonus australis was found. To summarize, Mali harbors at least three dangerous scorpion species: Leiurus quinquestriatus, Androctonus amoreuxi and A. australis, the latter recently having been identified in Mali for the first time. The absence of Androctonus aeneas is surprising in this context because it is found in neighboring countries (Algeria, Niger) and should be detected by new surveys. The possibility of preparing a single scorpion antivenom intended for Saharian and sub-Saharian populations is discussed.

Scorpion envenoming in the North of Mali (West Africa): Epidemiological, clinical and therapeutic aspects

Scorpion envenomation remains a poorly known problem in sub-Saharan Africa, particularly in Mali, where the incidence is high in Northern area of the country (Sahara desert). We conducted a prospective study in two district health centers, Kidal and Tessalit (North-east of Mali), to describe the epidemiological, clinical and therapeutic features of scorpion stings. This study consisted of an exhaustive follow-up from admission to discharge of all patients stung by scorpions. Of a total of 282 cases recorded during one year, 207 (73.4%) occurred in Kidal, and the remaining 75 (26.6%) took place in Tessalit. The annual incidence was significantly higher in Tessalit (437 cases/100 000 population/year) than in Kidal (243 cases/100,000 population/year) (p < 10−6). Two hundred two (71.6%) stings occurred inside human dwellings, 142 (50.4%) during sleeping/resting, especially in August. One hundred ninety-one (67.7%) were on the lower extremities. Nocturnal stings, 168 (59.6%), occurred more often than diurnal stings, 114 (40.4%). Most patients, 163 (57.8%), were admitted less than 1 h after being stung. Local pain at the sting site was the common primary complaint. However, moderate and severe clinical signs were significantly higher in children than in adults (p < 0.05). The death rate (3.9%) was higher in children (3.5%) than in adults (0.3%) (p = 8.10−6; RR = 0.90 [IC: 0.84–0.06]). Of the 22 scorpion species identified, 13 (59.1%) were Leiurus quinquestriiatus, 8 (36.4%) were Androctonus amoreuxi, and 1 (4.5%) specimen was Buthiscus bicalcaratus. From these species, L. quinquestriiatus and A. amoreuxi were responsible of stings. The medical treatment was only symptomatic, and one hundred twenty-eight (45.3%) patients received traditional remedies before seeking medical attention. Our findings suggest that scorpion stings are common in the north of Mali and are a significant threat to human health.

Characterization of three “Birtoxin-like” toxins from the Androctonus amoreuxi scorpion venom

The venom of the North African scorpion Androctonus amoreuxi (Aam) was analyzed using a combination of gel filtration, C18 reverse phase HPLC together with mass spectrometry analysis and bioassays. Three novel Birtoxin-like (BTX-L) peptides of 58 amino acid residues comprising three disulfide bridges were isolated and chemically characterized. One peptide, AamBTX-L3, induced serious toxic symptoms in mice and was lethal at nanogram quantities using intracerebroventricular injection. The three BTX-L peptides were tested in competition experiments on rat brain synaptosomes against the 125I-labeled “classical” α- and β-toxins of reference, as well as with the 125I-KTX, a voltage-gated potassium channel blocker. Only AamBTX-L3 was able to prevent the equilibrium binding of the β-toxin 125I-Css IV to its receptor site 4 with a IC 50 value of 189 nM. Even if previous electrophysiological data allowed the classification of other BTX-L peptides among the β-type toxins, this report clearly shows that AamBTX-L3 is pharmacologically a β-toxin, which recognizes the voltage-gated Na + (Na v) channels from central mammalian neurons. In order to uncover the residues functionally essential for interaction between the AamBTX-L3 with the putative receptor site of 125I-Css IV on Na v1.2, molecular models of the three novel Aam BTX-L molecules were made and their surfaces were compared to the already described Css IV biologically interactive surfaces. A hypothesis is given that in BTX-L3, three residues found in the α-helix play a key role during target binding.

Comparison of chela size and pincer force in scorpions; getting a first grip

A key feature of the ancient body plan of scorpions is the pincer or chela. These multifunctional structures vary considerably in size and shape between different scorpion species. Here we provide the first comparative data on the pinching performance of the chelae of seven species of scorpions exemplifying the extremes of the shape range from slender to robust; Leiurus quinquestriatus, Androctonus amoreuxi, Androctonus australis, Hadogenes sp., Pandinus imperator, Scorpio maurus and Pandinus cavimanus (in the order of decreasing chela height to width ratio). Size-corrected chela height correlates highly with maximum pinch force. Independent contrasts suggest that the correlation of chela width, height and fixed finger length with maximum pinch force is independent of phylogeny, suggesting an adaptive component to the evolution of chela shape and performance.

Phylogeography of Androctonus species (Scorpiones: Buthidae) in Tunisia: Diagnostic characters for linking species to scorpionism

A fragment of the mitochondrial (mt) 16S ribosomal RNA gene was amplified by PCR and sequenced from individual adult scorpions of the genus Androctonus, which were sampled from central and southern Tunisia and identified using an explicit set of morphological characters. Phylogenetic analyses placed the mtDNA haplotypes in three well-supported monophyletic lineages, corresponding to the morphospecies Androctonus aeneas, Androctonus amoreuxi and Androctonus australis. The latter species was the most abundant and widespread, and it was characterized by two mtDNA sub-lineages each of which predominated only north or south of the Chott el Jerid, a seasonally flooded saline depression that divides non-Mediterranean Tunisia. The divergence of the two mtDNA lineages was dated by mtDNA molecular clocks, indicating that the formation of the Chott el Jerid is unlikely to have been the barrier generating the vicariant evolution of the two lineages of A. australis, although it may have impeded their mixing following secondary contact. Both regional mtDNA lineages were found in A. australis hector and A. australis garzonii, indicating that these two morphological forms are neither monophyletic nor geographically isolated and, therefore, should not be treated as species or subspecies. It is recommended that no subspecies of A. australis should be recognized in North Africa and toxicologists should cease the taxonomic error of referring to a species “ Androctonus australis Hector”. The morphological form “hector” has no proven association with an increased risk of scorpionism compared with “garzonii”. However, it might be prudent to produce anti-venom in Tunisia by using both morphological forms of A. australis collected each side of the Chott el Jerid, because of the evidence for regional variation in toxins. The highest risk for scorpion stings occurs in the central region, where the new diagnostic markers should be used to discover any association between Androctonus species and scorpionism.

Full characterization of three toxins from the Androctonus amoreuxi scorpion venom

In this study, we have characterized the immunological and pharmacological properties of the three major α-type toxins from the scorpion Androctonus amoreuxi, AamH1, AamH2 and AamH3, which were previously described as putative toxins from cDNAs [Chen, T. et al., 2003. Regul. Pept. 115, 115–121]. The immunological tests (ELISA, RIA) have demonstrated that AamH1, AamH2 and AamH3 belong to the immunological groups 3 and 4 of α-type toxins. Analysis of the three toxin effects on currents through rat brain (rNav1.2), rat muscle (rNav1.4) and Drosophila (DmNav1) sodium channels expressed in Xenopus oocytes revealed that AamH1 and AamH2, but not AamH3, have anti-insect and anti-mammal activities and can be classified as α-like toxins. While AamH1 removes fast inactivation only in neuronal rNav1.2 channel and has no effect on muscular rNav1.4 channel, AamH2 affects both neuronal rNav1.2 and muscular rNav1.4 channels. AamH3 was lethal to mice by intracerebroventricular injection despite its lack of activity on the neuronal rNav1.2 channel. Finally, we have shown that the A. amoreuxi venom was better neutralized by the antiserum raised against the venom of Buthus occitanus tunetanus than by the antisera raised against scorpion venoms from the same genus Androctonus.

A new Kaliotoxin selective towards Kv1.3 and Kv1.2 but not Kv1.1 channels expressed in oocytes

In this paper were described the purification, the sequencing, and the immunological and biological characterization of a new Kaliotoxin analog, Aam-KTX, from the venom of the scorpion Androctonus amoreuxi. The toxin effects on three cloned Kv channels (Kv1.1, Kv1.2, and Kv1.3) were investigated in Xenopus oocytes using electrophysiology experiments. The Aam-KTX preference for Kv1.3 channel versus Kv1.2 was expected (EC50 values, 1.1±0.02 and 10.4±1.5nM, respectively) but its total inefficacity on Kv1.1 was very surprising. 3D molecular modeling of Aam-KTX brought putative answers to this difference in selectivity.

Molecular cloning of a novel putative potassium channel-blocking neurotoxin from the venom of the North African scorpion, Androctonus amoreuxi

Scorpion venoms are a particularly rich source of neurotoxic proteins/peptides that interact in a highly specific fashion with discrete subtypes of ion channels in excitable and non-excitable cells. Here we have employed a recently developed technique to effect molecular cloning and structural characterization of a novel putative potassium channel-blocking toxin from the same sample of venom from the North African scorpion, Androctonus amoreuxi. The deduced precursor open-reading frame is composed of 59 amino acid residues that consists of a signal peptide of approximately 22 amino acid residues followed by a mature toxin of 37 amino acid residues. The mature toxin contains two functionally important residues (Lys 27 and Tyr 36), constituting a functional dyad motif that may be critical for potassium channel-blocking activity that can be affirmed from structural homologs as occurring in the venoms from other species of Androctonus scorpions. Parallel proteomic/transcriptomic studies can thus be performed on the same scorpion venom sample without sacrifice of the donor animal.

Chromosomal Studies on Four Egyptian Scorpion Species of Genus Androctonus (Family: Buthidae)

Chromosomal analysis of 4 Egyptian species of family Buthidae (Androctonus australis, Androctonus bicolor, Androctonus amoreuxi and Androctonus crassicauda) have been studied. The 4 buthid scorpions species have the same diploid chromosome number of 2n=24. These species have a unique combination of cytogenetic features including holocentric chromosomes. Multivalent chromosomes of regular and irregular form were observed during the first meiotic division. To the best of the authors knowledge, these results are reported for the first time in Egypt.

Allozymic differentiation of Tunisian populations of Androctonus species and Buthus occitanus (Scorpiones: Buthidae)

We examined the population structure and phylogeny of Buthus occitanus (Amoreux, 1789), Androctonus amoreuxi (Audouin & Savigny 1812) and Androctonus australis (Linnaeus, 1758) in Tunisia, using horizontal starch gel and polyacrylamide gel electrophoresis to resolve allozymes at 18 loci. Populations of B. occitanus and A. amoreuxi showed little genetic variation, whereas A. australis populations showed moderate genetic variation. Moreover, despite the restricted dispersal abilities of these species, the allozyme analysis indicated very low levels of intraspecific population differentiation. The Island model of population differentiation best fitted the intraspecific variation, both for mainland populations and for the slightly more differentiated populations from two Mediterranean islands. No diagnostic, fixed alleles were observed between the species of Androctonus, which concurs with the results of nuclear ribosomal DNA analysis. Two subspecies of A. australis, A. a. garzonii Goyffon & Lamy, 1973 and A. a. hector Vachon, 1948, were not resolved by a phylogenetic analysis of the allozyme data. The most likely reasons for the slight population differentiation are either low mutation rates or recent range extensions promoted by human activities. The possibility of indirect gene flow between Androctonus species should also be explored. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 81, 255–265.

Isolation of scorpion ( Androctonus amoreuxi) putative alpha neurotoxins and parallel cloning of their respective cDNAs from a single sample of venom

The venoms of buthid scorpions are known to contain basic, single-chain protein toxins (alpha toxins) consisting of 60–70 amino acid residues that are tightly folded by four disulfide bridges. Here we describe isolation and sequencing of three novel putative alpha toxins (AamH1-3) from the venom of the North African scorpion, Androctonus amoreuxi, and subsequent cloning of their precursor cDNAs from the same sample of venom. This experimental approach can expedite functional genomic analyses of the protein toxins from this group of venomous animals and does not require specimen sacrifice for cloning of protein toxin precursor cDNAs.