Scorpion venoms from the Buthidae family: A dual study of proteomic composition and anticancer potentials.

Armenia is home to a rich diversity of unique species. Scorpion venom is a complex mixture of biologically active compounds that has been used in traditional medicine for centuries. Recent studies have confirmed that scorpion venom contains antimicrobial peptides effective against various bacteria. Additionally, it exhibits immunosuppressive and anticancer properties. The aim of this study is to investigate the composition and selected biological activities of venom from two scorpion species native to Armenia. The proteolytic and hemolytic activities of venom and hemolymph from Mesobuthus caucasicus and Androctonus crassicauda scorpions were analyzed using 12% SDS-PAGE. Protein fractions were separated based on their molecular weight under denaturing conditions. Antibacterial activity of the crude venom, hemolymph, and isolated protein fractions was assessed using the agar diffusion method. The venom and hemolymph of Mesobuthus caucasicus and Androctonus crassicauda exhibit narrow-spectrum antibacterial activity. Notably, the protein composition of venom and hemolymph differs between the two species. Antibacterial effects are attributed to only two specific protein and/or glycoprotein fractions present in the venom. Furthermore, nutritional status appears to influence the protein profile of both venom and hemolymph. In addition to their antimicrobial properties, the venom and hemolymph also demonstrate proteolytic and hemolytic activities. The venom and hemolymph of Mesobuthus caucasicus and Androctonus crassicauda demonstrate distinct protein compositions and exhibit significant antibacterial, proteolytic, and hemolytic activities, highlighting their potential for biotechnological and therapeutic applications.

ABSTRACTScorpion stings are a global health problem, potentially impacting thousands, especially across northern Africa and the Middle East. However, scorpions are comparatively understudied compared to other venomous animals, and little is known about their distributions and associated spatial patterns of sting risk. Timely access to medical treatment after scorpion stings is crucial to positive medical outcomes, but it remains poorly understood how accessible health care is for populations at risk. Thus, mapping hotspots of sting risk and identifying vulnerable populations can greatly enhance mitigation strategies. In this study, we used ecological niche models to map the distribution of eight of the most dangerous scorpion species to quantify sting risk across Iran. We identified the areas where people are most vulnerable to scorpion stings based on exposure to scorpions and limited accessibility to healthcare centers. We identified sting risk areas for each species across their distribution ranges. Androctonus crassicauda has the widest distribution range. On the contrary, Orthochirus iranus has the smallest distribution range. Regions of greatest vulnerability are located in the central parts of Iran, as well as the south and southwest of the country. Our results provide valuable information for decision makers by supporting targeted awareness‐raising programs and antivenom distribution among the most vulnerable populations.

Background:Envenomation by poisonous creatures is a major global cause of mortality. Its concomitant impact on the postmortem corpse decomposition and associated insect succession pattern is still poorly understood. Purpose of the study: This study comparatively investigates the impact of envenomization with the venoms of the snake Walterinnesia aegyptia L. versus the scorpion, Androctonus crassicauda L., on rabbit corpse decomposition and beetle succession. Methods: Three groups of rabbits (five animals each) were injected with the snake venom, the scorpion venom, or 0.9% saline (control) prior to euthanasia with CO2. The corpse decomposition stages and beetle succession were monitored over 11 days. Results: Four stages of decomposition with venom-dependent duration variation were observed. The scorpion-envenomized corpses showed a longer decay stage and a delayed dry stage. A total of 1094 beetles belonging to 27 species of 14 families were reported. Histeridae, Dermestidae, Scarabaeidae, and Tenebrionidae were the most diversified and prevalent families. Chrysomelidae, Elateridae, Hybosoridae, and Ptinidae were incidentally attracted to control corpses, while Nitidulidae and Zopheridae were only found on control and snake-envenomized ones. Four species belonging to the families Anthicidae, Histeridae, Scarabaeidae, and Tenebrionidae were predominant on all corpses. Four species belonging to the families Chrysomelidae, Curculionidae, Elateridae, and Hybosoridae were distinctively associated with the control corpses. Conclusions: These findings provided evidence that envenomation impacted the decomposition process and beetle succession in a venom-dependent manner, which could be significant for forensic investigations.

Serum production of Androctonus crassicauda (scorpion) venom: Evaluating the impact of high-quality protein diets

This study investigates the coxosternal or ventral sternal region, in scorpions and spiders. We col-lected 10 specimens of Scorpions from provinces Baghdad in region Abo Kreab and Salahuddin provinces in Tikrit then 7 specimens of Spiders were collected from variant regions in provinces Baghdad and various genera and families were collected and processed for examination. The aim was to compare the morphology of the coxosternal between these two arachnid groups and analyze the underlying functional and evolutionary implications of any structural variations. So the result of this study on coxosternal region of scorpions in the Family: Buthidae like Androctonus crassicau-da, is not wide the majority of members of this family have a triangular cut and the female genital cover is divided, coxosternal region in Orthochirus scrobiculosus do not extend forward and do not form lobes, edges of the scales grainy and it is finely grained the majority of individuals have a tri-angular cut, Mesobuthus eupeus the ventral side is shiny and wide with strong hairs and the ster-num is triangular, coxosternal region in Family: Scorpionidae like Scorpio Maurus in the ventral side do not have lobes extending forward, the sternum is pentagonal. But in Family: Salticidae of spiders the coxosternal region in Hasarius adansoni, are oval shape sternum or variable in Thyene imperialis, and Evarcha seyun, are circle shape sternum or variable, coxosternal region in Ne-oscona subfusca, family Araniedae are heart-shaped or triangular. The expected results include identifying distinct coxosternal shapes in scorpions compared to spiders. These variations might be linked to specific Roles like locomotion and internal organ protection. The determined geomor-phology gets to render Understandings into the evolutionary relationships betwixt scorpions and spiders. This comparative analysis is expected to Add to a better understanding of how the cox-osternal plays a role in the biology of scorpions and spiders. The known variations get bid im-portant information along the development and practical adjustations inside these arachnids

Background:Venom of scorpions are complex bioactive polypeptides. To gain greater insights into the structural and functional impacts of toxins from Androctonus crassicauda (Buthidae), a dangerously venomous scorpion species, its venom was isolated, purified, and characterized.Methods:Long chain toxin with four disulfide bonds purified by size exclusion chromatography and reversed-phase HPLC and characterized by amino acid sequencing and molecular weight determination.Results:The primary structure analysis exhibits a neurotoxin named AnCra2 with 7302.24 Da molecular weight and 64 amino acid residues that cause paralysis and lead to death in NIH mice. The LD50 of AnCra2 was determined to be 0.61±0.04 μg/mice. Phylogenetic analysis displays the toxin has 97% sequence similarity with alpha toxins reported from north African scorpions that affect voltage-gated sodium channels (VGSC), also proposed that differentiation among the scorpions of family Buthidae is affected by the geographical conditions and efficiency in evolutionary variations. AnCra2 exposed binding residues have a high affinity for receptor residues in site-3 (segment-3) of VGSC that are approved by three-dimensional structure and homology modeling.Conclusion:Purified AnCra2 seems to be a new putative Alpha neurotoxin in homology with the structure of neurotoxins that act on VGSC as a pharmaceutical tool.

This is the first preliminary study on the scorpions of Kuwait. Three species of scorpions were identified, described and illustrated from the State of Kuwait. Identified species belong to family Buthidae: Apistobuthus susanae, Androctonus crassicauda and Compsobuthus pallidus. Measurements were given for Apistobuthus susanae. Morphometric measurements were given for the three species, along with comparison of Apistobuthus susanae and Apistobuthus pterygocercus.

Phospholipase A2 is the most abundant venom gland enzyme, whose activity leads to the activation of the inflammatory response by accumulating lipid mediators. This study aimed to identify, classify, and investigate the properties of venom PLA2 isoforms. Then, the present findings were confirmed by chemically measuring the activity of PLA2. The sequences representing PLA2 annotation were extracted from the Androctonus crassicauda transcriptome dataset using BLAS searches against the local PLA2 database. We found several cDNA sequences of PLA2 classified and named by conducting multiple searches as platelet-activating factor acetylhydrolases, calcium-dependent PLA2s, calcium-independent PLA2s, and secreted PLA2s. The largest and smallest isoforms of these proteins range between approximately 70.34 kDa (iPLA2) and 17.75 kDa (cPLA2). Among sPLA2 isoforms, sPLA2GXIIA and sPLA2G3 with ORF encoding 169 and 299 amino acids are the smallest and largest secreted PLA2, respectively. These results collectively suggested that A. crassicauda venom has PLA2 activity, and the members of this protein family may have important biological roles in lipid metabolism. This study also revealed the interaction between members of PLA2s in the PPI network. The results of this study would greatly help with the classification, evolutionary relationships, and interactions between PLA2 family proteins in the gene network.

Abstract The diet of the Pharaoh Eagle-Owl, Bubo ascalaphus, was investigated based on 338 pellets collected from caves and underground caves in Ara’r region, northern Saudi Arabia. Small mammals constituted the highest number of consumed prey (75.75%), followed by arthropods (20%), birds (2.9%) and reptiles (1.26%). The Libyan Jird, Meriones libycus, was the most consumed rodent (26.46%) followed by Sundevall’s Jird, Meriones crassus (20.47%), while the least were Cheesman Gerbil, Gerbillus cheesmani, and Wagner’s Gerbil, Gerbillus dasyurus. At least three species of scorpions, Androctonus crassicauda, Compsbuthus sp. and Scorpio sp., and two species of reptiles (Ptyodactylus hasselquistii and Trapellus agnetae) were recovered. Study of owl pellet contents proved to be a valuable tool to study species composition in unexplored regions. Also, our findings substantiate the fact that the Pharaoh Eagle-Owl is an opportunistic species that adapts to available preys in its habitat.

Antihelimentic effect of Androctonus crassicauda scorpion venom against Trichuris arvicolae isolated from Psammomys obesus in Egypt

Androctonus crassicauda is the most medically relevant scorpion and understanding its genetic forms is essential for improvement of anti-venom sera, and risk management of scorpionism. Present study was designed to identify the variations of mitochondrial genes in different populations of A. crassicauda. Adults of A. crassicauda were collected from Zanjan Province during 2016-2017. Genomic DNA of samples was extracted and fragments of mitochondrial 16S, COI and ND1 genes were amplified and some of the amplicons were sequenced. Haplotype of samples were identified by multiple alignment of sequences, then phylogenetic trees of haplotypes were constructed. Fragments of 352bp, 618bp and 680bp were amplified from 16S, COI and ND1 genes respectively. Nucleotide sequence in COI fragments was conserved, however, five haplotypes with some specific polymorphic sites were detected in 16S and ND1 fragments. Haplotype I was dominant and found in all areas. Other haplotypes were rare and limited to specific regions. Analysis of the phylogenetic trees inferred from 16S and COI genes, confirmed a strong positive correlation between geographic and genetic distance. Mitochondrial COI, 16S and ND1 genes were detected suitable for identifying the population structure. Five genotypes were found using 16S and ND1 genes. To prepare and improve the anti-venoms quality, additional studies are necessary to identify the toxin electrophoretic profile and geographical/ecological niche models of these genotypes in future.

Background:In this study aimed to show the role of autophagy acting as a seesaw between apoptosis and necroptosis in certain vital organs under the effects of the Aegaeobuthus nigricinctus venom and different dosages of the Androctonus crassicauda antivenom administration in mice.Methods:In the venom group (VG), mice (n= 6) were inoculated with 2LD50A. nigrocinctus venom. In the antivenom administered groups (AVG), the effects of the potency of the A. crassicauda antivenom were evaluated to have a neutralization effect against 20LD50 of the A. nigrocinctus venom. After histopathological examination, expressions of mammalian target of rapamycin (mTOR) as an autophagy activator, receptor-interacting serine/threonine-protein kinase 3 (RIPK3) as a necroptosis activator, and caspase-3, caspase-9 as the markers of apoptotic cell death signals were evaluated by the immunoperoxidase method in addition to DNA in-situ fragmentations by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method.Results:Only in VG, caspases and TUNEL expressions were found to be higher after the envenomation process in contrast to the elevated RIPK3 expressions. mTOR expressions remained almost stable in the organs. In AG, mTOR expressions were further increased in the 30LD50 and 40LD50 groups.Conclusion:There were an increased mTOR expression and stabilized caspases and TUNEL expression in these subgroups, the RIPK3 expressions were found to be low when compared with all of the antivenom administration groups. Increasing doses of the antivenom drifts more the cells to autophagy while cell fate in organs under envenomation getting rid of apoptosis and necroptosis pathways.

Androctonus crassicauda scorpion species lives in nature both as a prey and a predator. So it has venom that can be effective for hunting and protection. It can cause venoming and death by stinging people and animals. It is of great importance to know the ecological characteristics and density of this scorpion species, especially in regions where venoming cases are high. In the present study, Şanlıurfa province, where the scorpion species Androctonus crassicauda is prevalent, was chosen as the research area. 289 samples were collected after field studies in all districts of Şanlıurfa province. Owing to desolated and stony structure, Androctonus crassicauda was observed to be more intensive in Harran district. It was generally found from April to October. The most abundant period of the species is June, July, and August. Considering the seasonal expectation, test results of correlation for a series with non-normal distribution were listed in two options, Kendall'stau_b correlation coefficient and Spearman'srho correlation coefficient. There was a positive correlation of 0,168 (16%) at 1% significance between month and population for the former (Kendall'stau_b) coefficient. A positive correlation of 0,231 (23%) at 1% significance level between month and population for the latter (Spearman'srho) coefficient. In the view of regional expectation, results of correlation test for a non-normal distribution were presented in two options. A negative correlation of 0,099 (9%) was found at 5% significance level between region and population for the former coefficient. There was a negative correlation of 0,128 (12%) at 5% significance level between region and population for the second coefficient. The study revealed a variation between seasons and districts.

Background:Scorpion neurotoxins such as those that modify the mammalian voltage-gated sodium ion channels (Nav) are the main responsible for scorpion envenomation. Their neutralization is crucial in the production of antivenoms against scorpion stings.Methods:In the present study, two in silico designed genes - one that codes for a native neurotoxin from the venom of the Anatolian scorpion Androctonus crassicauda, named Acra 4 - and another non-native toxin - named consensus scorpion toxin (SccTx) obtained from the alignment of the primary structures of the most toxic neurotoxins from the Middle Eastern and North African scorpions - were recombinantly expressed in E. coli Origami. Results:Following bacterial expression, the two expressed neurotoxins, hereafter named HisrAcra4 and HisrSccTx, were obtained from inclusion bodies. Both recombinant neurotoxins were obtained in multiple Cys-Cys isoforms. After refolding, the active protein fractions were identified with molecular masses of 8,947.6 and 9,989.1 Da for HisrAcra4 and HisrSccTx, respectively, which agreed with their expected theoretical masses. HisrAcra4 and HisrSccTx were used as antigens to immunize two groups of rabbits, to produce either anti-HisrAcra4 or anti-HisrSccTx serum antibodies, which in turn could recognize and neutralize neurotoxins from venoms of scorpion species from the Middle East and North Africa. The antibodies obtained from rabbits neutralized the 3LD50 of Androctonus australis, Leiurus quinquestriatus hebraeus and Buthus occitanus venoms, but they did not neutralize A. crassicauda and A. mauritanicus venoms. In addition, the anti-HisrAcra4 antibodies did not neutralize any of the five scorpion venoms tested. However, an antibody blend of anti-HisrAcra4 and anti-HisrSccTx was able to neutralize A. crassicauda and A. mauritanicus venoms. Conclusions:Two recombinant Nav neurotoxins, from different peptide families, were used as antigens to generate IgGs for neutralizing scorpion venoms of species from the Middle East and North Africa.

Teh venoms of teh scorpions Androctonus crassicauda and Leuris abdullahbayrami, scorpion species each of teh two members of teh Buthidae family, were analyzed by MALDI–TOFMS in a mass range between 1 and 50 kDa. Interspecies differentiation was evaluated over peptide and protein molecules in dis mass range. Teh similarities and differences between two different scorpion species were revealed wif teh TEMPprincipal component analysis study, which was conducted wif spectral patterns including peptide and protein profiles. Teh similarity rate of teh LAB-123 and teh LAB460 scorpion venoms of teh same species was found as 66% while teh similarity rates of venoms of teh ACR species to teh LAB species ranged from zero to 37%. It was demonstrated dat scorpion venoms belonging to two different species from teh Buthadia family can be differentiated wif teh halp of dendrogram and gel profile, CCI color matrix, 3D or 2D-scattering profile, spectral mass loading data formed by peptide and protein spectral patterns of eleven scorpion venoms. It is anticipated dat dis approach, which was used for teh first time wif teh application of MALDI-TOFMS-based PCA analysis for teh differentiation of scorpion venoms, will be useful in differentiating venoms wif different spectral patterns.

Androctonus crassicauda is the most medically relevant animal and understanding its morphological characteristics is essential in the production of antiscorpion sera. Adults of A. crassicauda were collected from different areas of Zanjan Province and the morphometric parameters and the cuticular fluorescence patterns of samples were studied. The crude venom of samples was extracted by electric stimulation, and their biochemical properties were analyzed by the SDS-PAGE method. Values of the morphometric parameters depended on sex and altitude of the area. Except for values of the pectinal organ, these parameters in females were higher than in males. No significant difference was in the number, shape, and intensity of cuticular fluorescence patterns. The body length of males in high and lowlands was 72.53±1.53 and 77.33±2.70mm, respectively. Females' body lengths in that area were 81.66±2.19 and 86.55±2.33mm, respectively. Analysis of toxin proteins showed two isotypes that the 12, 13, 15, 16, 18, and 19kDa proteins were in all areas. However, the 41 and 74kDa proteins, and 46 and 63kDa proteins were detected in low and highlands, respectively. Black fat-tailed scorpion has a considerable dominancy and developing preventive programs and providing treatment facilities in studied areas are necessary. Values of the morphological parameters and venom electrophoresis patterns depended on the geographical location. Therefore, pool crude toxin is suggested for the production of effective antivenoms. Moreover, additional field complementary works in the geographic information system based niche modeling and mass fingerprinting of scorpion venoms are suggested for screening effective isotypes.

Intraspecific molecular and morphological variations among geographically isolated populations are useful for understanding the evolutionary processes, which is considered early stage of allopatric speciation. Also, the knowledge of the regional variation of scorpion venom composition is needed to improve antivenom therapeutic management. Androctonus crassicauda (Olivier, 1807) is the most common and medically important species in Arabia and the Middle East. Therefore, this study aimed to investigate the geographic morphological variation among A.crassicaudapopulations, regarding its geographical distribution in unexplored arid regions in Saudi Arabia. Samples were collected and examined morphologically under a dissecting microscope from different four eco-geographical regions. The results of ANOVA and multivariate statistical analyses provide strong evidence of geographical variation. The two populations from OTU3 and OUT4 showed the greatest degree of morphological difference from populations of OUT1 and OUT2. Each OTU3 and OTU4 populations showed significant speciation without overlapping in the two groups, while the remaining overlapped groups comprised two other populations. Several body variables influenced male separation, including carapace posterior width, metasoma 3rd length, and metasoma 2nd length. For females, telson length, metasoma 1st width, and sternite 7th width were highly influential variables. Such variation may suggest the existence of cryptic taxa within A. crassicauda populations in Saudi Arabia. Moreover, metasoma ratios can be used as good indicators in intraspecific variation studies of Scorpions.

Background:Annually, thousands of scorpion stings and rare mortalities are recorded in the Kurdistan Region of Iraq. In the present study, we prepared a checklist for the geographical distribution of scorpion fauna composition, new localities data, and scorpion sting profile for the years 2019–2020 in the region.Methods:Random field surveys for scorpion collection were carried out in 22 localities in the Kurdistan Region from June 2020 to October 2021. Clinical data related to scorpion stings in 2019 and 2020 were obtained from the medical records of public health offices in the region.Results:In this survey, 166 specimens were collected. They were composed of seven scorpion species belonging to three families including: Buthidae, Hemiscorpiidae, and Scorpionidae. The collected species were Androctonus crassicauda, Compsobuthus matthiesseni, Hottentotta saulcyi, Mesobuthus mesopotamicus, Orthochirus fomichevi, Hemiscorpius lepturus, and Scorpio kruglovi. Moreover, three scorpion species comprising Buthacus tadmorensis, Hottentotta mesopotamicus, and Calchas anlasi were recorded in the previous study conducted in the Kurdistan region. In 2019–2020, 3726 scorpion stings were reported, with a peak in the summer season. Most of the patients were 15–49 years old. In the current study, medically significant scorpion species have been identified.Conclusion:The outcome of the present work will increase the awareness of the Kurdish community regarding the distribution of dangerous scorpion species and consequently reduce their stings.

Abstract One of the innovative methods in cancer treatment with fewer side effects is the use of active principles produced by animals and microorganisms to generate new drugs with diagnosis and treatment capability for cancer. Androctonus crassicauda scorpion venom has high potential as an anti-tumor agent and has the ability to interfere with human physiology but is still unknown. Therefore, labeling venom with radionuclides is very important to study the biological distribution of toxins for diagnostic and therapeutic purposes of various diseases, especially cancer. In this research, the toxic fraction of this venom was labeled with 99mTc. The radiochemical purity of the labeled toxic fraction was obtained by using chromatographic systems. Animal biodistribution studies were performed after injecting the labeled compound into normal rats and 4T1 breast cancer-bearing mice. The radiochemical purity of 90% was obtained for labeled compound. Biological distribution studies showed relatively rapid clearance of the labeled compound from the blood circulation system. The accumulation of the labeled compound in the liver and kidney was the highest among the organs, which could indicate the possibility of toxin excretion metabolism from liver and the kidneys. The accumulation rate of the labeled compound after 15 min was 7% in the breast cancer mass, and the ratio of the distribution of the labeled compound in the breast cancer mass to the blood was 155% at 15 min post injection. The study results indicated that scorpion venom labeling with 99mTc could be a useful tool for the biodistribution and kinetic studies of the venoms for clinical use. On the other hand, based on the results of the biological distribution of the labeled compound in tumor-bearing mice, the 99mTc-venom complex can be used as an imaging agent to diagnose breast cancer. Moreover at the same time, with some measures, it can be used as a therapeutic agent for breast cancer with more consideration. We hope that the results of this study will be a step in the future for the clinical diagnostic and therapeutic purposes of cancer using natural products.