Discovery of a New Analgesic Peptide, Leptucin, from the Iranian Scorpion, Hemiscorpius lepturus

Hemiscorpius lepturus scorpion stings do not induce considerable pain based on epidemiological surveys conducted in the southwest part of Iran. Accordingly, this study was aimed to identify the analgesic molecule in H. lepturus venom by analyzing a cDNA library of the scorpion venom gland looking for sequences having homology with known animal venom analgesic peptides. The analgesic molecule is a cysteine rich peptide of 55 amino acids. the synthetic peptide was deprotected and refolded. RP-HPLC, Ellman’s, and DLS assays confirmed the refolding accuracy. Circular dichroism (CD) showed helix and beta sheet contents. This peptide, called leptucin, demonstrated 95% analgesic activity at the dose of 0.48 mg/kg in hot plate assay. Leptucin at the doses of 0.32, 0.48, and 0.64 mg/kg showed 100% activity in thermal tail flick test. No hemolysis or cytotoxicity was observed at 8 and 16 µg. Histopathology evaluations indicated no hepatotoxicity, nephrotoxicity, and cardiotoxicity. We thus report that leptucin is the analgesic agent of H. lepturus venom. Regarding the high in vivo efficacy of leptucin and the fact it shows no observable toxicity, it could be suggested as a drug lead in a preclinical study of acute pain as well as the study of its mechanism of action.

Species diversity and abundance of scorpions in Ahvaz city, Southwest Iran

Mansouri NJS, Akbarzadeh K, Jahanifard E, Vazirianzadeh B, Rafinejad J. 2021. Species diversity and abundance of scorpions in Ahvaz city, Southwest Iran. Biodiversitas 22: 763-768. Arthropods are one of the most important and diverse phyla of the animal kingdom. This phylum includes several classes of insects and arachnids, which are of medical and veterinary importance. Scorpions are at the top of the group of venomous arthropods in terms of having venomous bites and causing death. Scorpions live mainly in tropical and subtropical regions of the world and therefore have higher diversity and richness in these regions. This study aimed to investigate the biodiversity and abundance of scorpions in Ahvaz city, Khuzestan province, Iran. It has the highest species diversity and species richness in Iranian scorpions. Scorpions were collected from five regions in Ahvaz using various methods. In total 237 scorpions were collected and identified. They belonged to two families, seven genera and seven species: Mesobuthus eupeus (65%), Hemiscorpius lepturus (23.2%), Androctonus crassicauda (3.8%), Compsobuthus rugosulus (3.4%), Orthochirus zagrosensis (2.5%), Apistobuthus susanae (1.7%), Buthacus macrocentrus (0.4%). Except for H. lepturus (Hemiscorpiidae), other species belonged to the Buthidae family. That all of them were non-burrowing. M. eupeus was the dominant species diversity analysis indicated higher species richness and species diversity for summer (Margalef = 1.07; Shannon = 1.55). Logistic regression analysis showed that the variables of geographical location, humidity, and temperature had a significant relationship with the abundance of scorpion species, while height had no statistically significant.

Cardiotoxic Effects of Hemiscorpius Lepturus Scorpion Venom Fractions in Rats

Background: Scorpion stings are responsible for many deaths in humans; however, the toxicity mechanisms of the venoms from many species are not well studied. We investigated the cardiotoxicity of the crude venom from H. lepturus scorpion and its isolated fractions, F-I to F-VI. Methods: The scorpion’s venom was extracted into six fractions by chromotagraphy. Healthy male Wistar rats (N=72) were equally divided into eight groups of nine: G1: Controls (0.5ml. normal saline), G2: Crude venom (1000µg/kg), G3: F-I (120µg/kg), G4: F-II (430µg/kg), G5: F-III (80 µg/kg), G6: F-IV (180µg/kg), G7: F-V (60µg/kg), and G8: F-VI (130µg/kg). Blood samples were obtained by cardiac puncture at 1, 3 and 24 hours after the venom injection. The serum levels of AST, LDH, CPK, CK-MB and troponin-I were determined. Upon euthanasia, the hearts were removed from the rats and examined microscopically for histopathology. Results: In groups receiving crude venom and F-VI, we observed multifocal fragmentation of myocardial fibers, hemorrhage, degeneration and disappearance of striations in cardiac muscles as compared to the controls. The findings showed that AST and LDH activity in groups 2, 4 and 8, CPK activity in groups 2, 4, 6 and 8 and CK-MB activity and troponin-I levels in groups 2 and 8 increased significantly compared to those in the control group. Conclusion: There was evidence of significant cardiotoxicity in the group receiving crude venom and F-VI. Although alterations in the enzymatic and troponin-I levels were observed in other groups, the greatest cardiotoxicity of H. lepturus venom was caused by fraction VI.

The Effect of Various Fractions of Hemiscorpius Lepturus Scorpion (Scorpionida: Hemiscorpiidae) Venom on Hemostatic System in Peripheral Blood of Rats in Comparison to Whole Venom

Background: Hemiscorpius lepturus is the most critical scorpion in Khuzestan, Iran, responsible for the majority of deaths caused by scorpion stings. Objectives: This prospective randomized experimental study was performed from June to August 2019 to investigate the effects of various venom fractions on the hemostatic system. Methods: Lyophilized venom of H. lepturus was separated into six fractions by Sephadex G-50 gel chromatography. A total of 96 Albino male rats were treated in eight equal groups; Control: 0.5 ml normal saline (IP); Whole venom group: H. lepturus Lyophilized venom, one mg/kg, IP; F1-F6 groups: Isolated venom fraction 1-6, 0.12 mg/kg, 0.43 mg/kg, 0.08 mg/kg, 0.18 mg/kg, 0.06 mg/kg and 0.13 mg/kg, IP, respectively. Blood sampling was performed at 1, 3, and 24 hours after injection. Results: Plasma fibrinogen was significantly elevated in the whole venom group, while it was significantly decreased in F3, F5, and F6 groups compared to the control group. Prothrombin time (PT) and activated partial thromboplastin time (APTT) assessment revealed that there was a significant prolongation of both tests in whole venom and fraction 5 and 6 receiving groups while fraction 2 only caused a significant PT extension, compared to the control group. Conversely, fraction 3 and 4 resulted in PT and APTT in comparison to the control group. The concentration of FDP and D-dimer was significantly increased in whole venom and F2 group compared to the control group at all sampling times. Conclusions: It seems that H. lepturus venom has both procoagulant and anticoagulant properties which are distinct characteristics of it. Further purification and sequencing of the amino acids in the fraction of peptides can lead to a more precise identification of the mechanisms of venom-induced coagulopathies.

Antigenic and Substrate Preference Differences between Scorpion and Spider Dermonecrotic Toxins, a Comparative Investigation

The Hemiscorpius lepturus scorpion and brown spider Loxosceles intermedia represent a public health problem in Asia and America, respectively. Although distinct, these organisms contain similar toxins responsible for the principal clinical signs of envenomation. To better understand the properties of these toxins, we designed a study to compare recombinant Heminecrolysin (rHNC) and rLiD1, the major phospholipase D toxins of scorpion and spider venom, respectively. Using a competitive ELISA and a hemolytic inhibition test, we come to spot a cross reaction between scorpion and spider venoms along with an epitopic similarity between rHNC and rLiD1 associated with neutralizing antibodies. Results show that the ability of the rHNC to hydrolyze lysophosphatidylcholine (LPC) is equivalent to that of rLiD1 to hydrolyze sphingomyelin and vice-versa. rHNC exclusively catalyze transphosphatidylation of LPC producing cyclic phosphatidic acid (cPA). The in-silico analysis of hydrogen bonds between LPC and toxins provides a possible explanation for the higher transphosphatidylase activity of rHNC. Interestingly, for the first time, we reveal that lysophosphatidic acid (LPA) can be a substrate for both enzymes using cellular and enzymatic assays. The finding of the usage of LPA as a substrate as well as the formation of cPA as an end product could shed more light on the molecular basis of Hemiscorpius lepturus envenomation as well as on loxoscelism.

Preparation and Biological Evaluation of 67Gallium- Labeled Iranian Hemiscorpius Lepturus Scorpion Venom

Background: The Hemiscorpius lepturus (H. lepturus) is a deadly scorpion species living in the southern Iran. Objective: H. lepturus induces delayed toxicity symptoms and understanding the long term biodistribution/ biokinetic of the venom is of great interest in toxicology. Methods: A Ga-67 labeled venom was prepared using a DOTA -conjugated venom followed by radiolabeling using 67GaCl3 at 40°C for 90 min. The purification of the radiolabeled venom was performed using size exclusion-chromatography (radiochemical purity 71%). The radiolabeled venom was stable in the final solution in the presence of human serum at 37°C for 72 hours. The tissue distribution was studied in blood, heart, liver, spleen, muscle, brain, kidney, intestine and skin tissues at the intervals of 1, 4, 24, 48 and 72 hours using tissue counting and SPECT imaging. Results: The radiolabeled venom mixture obtained with an estimated molar activity of 0.52 MBq/μg. The main accumulation tissues during the first 72 hours were kidneys, blood, liver, intestines, stomach and skin, respectively. Therefore, it is likely that H. lepturus’ clinical effects and renal toxicity are primary and caused by direct effects of the H. lepturus venom. Conclusion: The results have largely shown the direct clinical effects on the studied tissues during the 72-hour period and antivenom administration can strongly alleviate the toxicity effects as early as 72 hours in the management of the patients.

Novel Mutant Phospholipase D from Hemiscorpius lepturus Acts as A Highly Immunogen in BALB/c Mice Against the Lethality of Scorpion Venom

Hemiscorpius lepturus (H. lepturus) which belongs to the Scorpionidae family, is the deadliest scorpion in Iran. It causes pathological manifestations like dermonecrosis, hemolysis, renal failure, necrotic ulcers, and in some cases, even death. The venom of this scorpion is well-known for its cytotoxic effects in comparison with the other venomous scorpions which show significant neurotoxic effects. Due to the painless nature of the sting of this scorpion, the clinical symptoms occur in victims 24 to 72 h post-sting. In our previous studies during the last decade, we demonstrated that the medical complications are attributable to the presence of phospholipase D (PLD) as a major toxin in the venom. With the purpose of designing and constructing a vaccine against H. lepturus for humans, animal model experiments were performed. To achieve this goal, non-toxic PLD was developed by mutation of two critical catalytic residues—His12 and His48—into alanines and the product was then denominated mut-rPLD1. The in-vivo tests showed that the mice immunized with interval doses of 10 µg of mut-rPLD1, were completely protected against 10× the LD100 of the venom. In conclusion, this mutant may be an effective vaccine candidate against scorpion envenomation by H. lepturus in future clinical studies.

Presence probability of Hemiscorpius lepturus Peters, 1861 using maximum entropy approach in the western areas of Zagros Mountains, Iran

Aim: The purpose of this research was to use environmental variables for predicting the probability of Hemiscorpius lepturus existence in the provinces where situated in the west of the Zagros Mountains. Materials and Methods: In this study, 64 occurrence records of the H. lepturus were extracted from the published documents available in electronic databases. MaxEnt model was used for predicting the ecological niches of this species. Normalized difference vegetation index (NDVI) and 19 climatic variables were used as the environmental variables affecting the distribution of this scorpion. The Jackknife test in the model was used to indicate the importance of variables to predict the probability of the presence of the studied species. The logistic threshold that was evaluated using a logistic regression algorithm showed the converting of the probability model into a binary model. The model was evaluated by area under the curve (AUC). The probability presence map of this scorpion was then prepared in ArcGIS 10.5 Software. Results: The results of the analysis showed that the most important environmental factor on the distribution of H. lepturus was the maximum temperature of the warmest month (Bio5) with a contribution rate of 43% and permutation importance of 8%. The Jackknife test revealed that NDVI did not gain any value when it used independently in the model. The logistic threshold was reported 0.255 for the maximum test sensitivity plus specificity. The AUC of the model was 0.7698, shows an acceptable value for model validity. Overall the hot spots for this toxic scorpion seem to be in Khuzestan, Lorestan, and Ilam Provinces of the studied area. Conclusion: Regarding our findings, MaxEnt algorithm, in combination with geographic information system contributed to revealing the effects of environmental variables on the probability of H. lepturus presence in the west of Zagros Mountains. These visualized maps as a warning alarm can be helpful to policymakers for managing, controlling, and monitoring the scorpionism in high-risk areas.