Proteomic analysis and lethality of the venom of Aegaeobuthus nigrocinctus, a scorpion of medical significance in the Middle East.

The scorpion Aegaeobuthus nigrocinctus inhabits areas in Turkey and the Levant region of the Middle East where severe/lethal envenomings have been reported. Previous research indicated its extreme venom lethality to vertebrates and distinct envenomation syndrome. We report on the composition of A. nigrocinctus venom from Lebanese specimens using nESI-MS/MS, MALDI-TOF MS, SDS-PAGE and RP-HPLC. Venom lethality in mice was also assessed (LD50 = 1.05 (0.19-1.91) mg/kg, i.p), confirming A. nigrocinctus venom toxicity from Levantine populations. Forty-seven peaks were resolved using RP-HPLC, 25 of which eluted between 20 and 40 % acetonitrile. In reducing SDS-PAGE, most predominant components were <10 kDa, with minor components at higher molecular masses of 19.6, 26.1, 46.3 and 57.7 kDa. MALDI-TOF venom fingerprinting detected 20 components within the 1,000-12,000 m/z range. Whole venom 'shotgun' bottom-up nLC-MS/MS approach, combined with in-gel tryptic digestion of SDS-PAGE bands, identified at least 67 different components belonging to 15 venom families, with ion channel-active components (K+ toxins (23); Na+ toxins (20); Cl- toxins (2)) being predominant. The sequence of a peptide (named α-KTx9.13) ortholog to Leiurus hebraeus putative α-KTx9.3 toxin was fully determined, which exhibited 81-96 % identity to other members of the α-KTx9 subfamily targeting Kv1.x and Ca2+-activated K+ channels. Chlorotoxin-like peptides were also identified. Our study underscores the medical significance of A. nigrocinctus in the region and reveals the potential value of its venom components as lead templates for biomedical applications. Future work should address whether available antivenoms in the Middle East are effective against A. nigrocinctus envenoming in the Levant area.

Antimicrobial, toxicological, and antigenic characteristics of three scorpion venoms from Colombia: Centruroides margaritatus, Tityus pachyurus and Tityus n. sp. aff. metuendus.

The venom fractions of three buthid scorpion species from Colombia, C. margaritatus, T. pachyurus and T. n. sp. aff. metuendus, were examined for antimicrobial and toxicity toward mice and insects. The three venoms were separated into individual fractions using RP-HPLC, resulting in 85 fractions from C. margaritatus, 106 from T. pachyurus, and 70 from T. n. sp. aff. metuendus. The major fractions from the three scorpion venoms, which were eluted between 35 and 50 min, were tested for antimicrobial activity and toxicity. It was confirmed that the venom of the three species contains fractions with antimicrobial peptides that were evaluated against two bacterial strains of public health importance, Pseudomonas aeruginosa and Staphylococcus aureus. The venom of C. margaritatus had two antimicrobial fractions that showed activity against the named tested strains. The venom of T. pachyurus had three fractions that showed activity against S. aureus and two against both bacterial strains. Finally, the venom of T. n. sp. aff. metuendus had one fraction that showed activity against S. aureus, and five fractions showed activity against both bacterial strains. Also, some peptide fractions from the three venoms were toxic to mice. Last, the venoms of C. margaritatus and T. pachyurus were used as immunogens to obtain neutralizing antibodies against its respective venoms and to observe antibody recognition to related and unrelated scorpion venoms. A total of 15 mg of lyophilized antibodies were able to neutralize 1.5⋅LD50 of the venoms from T. n. sp. aff. metuendus, T. pachyurus and C. margaritatus, respectively. This information provides valuable insights into the diversity of each species' venom and their potential role in antimicrobial and venom toxicity.

An overview of some enzymes from buthid scorpion venoms from Colombia: Centruroides margaritatus, Tityus pachyurus, and Tityus n. sp. aff. metuendus

Background: In Colombia, several species of Buthidae scorpions belonging to the genera Centruroides and Tityus coexist, and their stings are considered life-threatening to humans because of their venom neurotoxins. Despite previous studies focusing on neurotoxins from these scorpion genera, little is known about the enzymes present in their venoms and their relationship with whole venom toxicity. Methods: Here, using proteomic and biochemical protocols the enzymatic activities of the venoms of three Colombian scorpion species, C. margaritatus, T. pachyurus, and T. n. sp. aff. metuendus, were compared to establish the presence and absence of enzymes such as phospholipases, hyaluronidases, and proteases that could be related to venom toxicity. Results: C. margaritatus was positive for hyaluronidases, T. n. sp. aff. metuendus for proteases, and T. pachyurus exhibited activity for all three mentioned enzymes. Conclusion: This information provides valuable insights into the specific enzyme diversity of each species' venom and their potential role in venom toxicity, which could contribute to the development of better treatments and prevention strategies for scorpion envenomation.

LmNaTx15, a novel scorpion toxin, enhances the activity of Nav channels and induces pain in mice.

Polypeptide toxins are major bioactive components found in venomous animals. Many polypeptide toxins can specifically act on targets, such as ion channels and voltage-gated sodium (Nav) channels, in the nervous, muscle, and cardiovascular systems of the recipient to increase defense and predation efficiency. In this study, a novel polypeptide toxin, LmNaTx15, was isolated from the venom of the scorpion Lychas mucronatus, and its activity was analyzed. LmNaTx15 slowed the fast inactivation of Nav1.2, Nav1.3, Nav1.4, Nav1.5, and Nav1.7 and inhibited the peak current of Nav1.5, but it did not affect Nav1.8. In addition, LmNaTx15 altered the voltage-dependent activation and inactivation of these Nav channel subtypes. Furthermore, like site 3 neurotoxins, LmNaTx15 induced pain in mice. These results show a novel scorpion toxin with a modulatory effect on specific Nav channel subtypes and pain induction in mice. Therefore, LmNaTx15 may be a key bioactive component for scorpion defense and predation. Besides, this study provides a basis for analyzing structure-function relationships of the scorpion toxins affecting Nav channel activity.

Scorpion envenomation in the neotropical savannah: Environmental predictors and years of lost life.

Scorpion stings envenomation (SSE) is a growing medical concern in Brazil, particularly in the state of Minas Gerais, which has recorded a significant number of incidents. This study aimed to investigate the potential predictors of scorpion sting incidence and evaluate the cost-effectiveness of interventions in Minas Gerais. Generalized Estimating Equations (GEE) models were constructed using socioeconomic and environmental variables as predictors and scorpion sting incidence as the response variable. The analysis revealed that mean annual temperature and major land use type were significant predictors of scorpion sting incidence, while precipitation and socioeconomic variables showed no relationship with incidence. The total number of cases and annual incidence of scorpion stings overlapped with regions experiencing higher forest conversion and agricultural land use, as well as higher temperatures. The estimated Disability-Adjusted Life Years (DALY) for scorpion sting incidents in Minas Gerais was substantial, indicating the need for effective prevention and treatment measures. The cost per DALY averted varied among municipalities, with some requiring minimal investment while others needing significant funding to address the scorpion risk. Mean annual temperature emerged as the main risk factor for scorpion stings, contributing to increased costs associated with antivenom treatment. These findings highlight the importance of considering environmental factors and implementing targeted interventions to mitigate scorpion sting incidents and reduce associated morbidity and mortality.

Proteomic profile of the venom of three dark-colored Tityus (Scorpiones: Buthidae) from the tropical rainforests of Costa Rica.

OBJECTIVE: We aimed to elucidate the potential differences in the venom peptide sequences of three Tityus species from Costa Rican rainforests: T. jaimei, T. championi and T. dedoslargos, compared to T. cf. asthenes from Colombia, which could explain the low level of scorpionism in Costa Rica, evidenced by the lack of epidemiological data. METHODOLOGY: We applied venom proteomics of peptides purified by RP-HPLC and compared the obtained sequences from venoms of these Tityus species to the sequences previously identified from Tityus inhabiting other Central and South American regions. RESULTS: Venom proteome analysis evidences that most of the putative peptide toxins identified in Costa Rican dark-colored Tityus are very similar to those present in other T. (Atreus) from the region. CONCLUSIONS: Our study suggests that, in the case of potential envenomation by Tityus in Costa Rica, the same level of toxicity should be observed, compared to other cases caused by members of the subgenus from other geographical localities. On the other hand, compared to countries with more accelerated urban expansion, Costa Rican Tityus still inhabit secondary rainforests and do not commonly share the same spaces with humans, so the lack of epidemiological evidence of medical emergencies caused by envenoming by this scorpion group could be more related to ecological and demographic factors and less attributed to the characteristics of the venom.

Mitochondrial swelling in cardiomyocytes: Insights from a murine model of Tityus serrulatus scorpion envenomation.

Immune system hyperactivation is involved with clinical severity and pathological alterations during scorpion envenomation. In a murine model, mice inoculated with a lethal dose of Tityus serrulatus scorpion venom presented mitochondrial swelling in cardiomyocytes, with other structures such as sarcomeres and intercalated disks preserved. Treatment with dexamethasone or knockout animals to the interleukin-1ß receptor do not undergo mitochondrial changes in cardiomyocytes during envenomation.

Pioneering in vitro characterization of macrophage response induced by scorpion venoms from the Brazilian Amazon.

There are several scorpion species of medical relevance around the world. Some of them are well characterized by their toxins and clinical outcomes. Brazilian Amazon has a great amount of these arthropods that have an impact in the scorpionism events specifically in this region of Brazil. Recently, several studies pointed out the immune system activation during scorpion envenouming as an important facet of scorpionism, inducing a sepsis-like state that culminates in clinical severity and death. In this work, we characterized the macrophage response of three species of clinical relevance in Brazilian Amazon: Tityus silvestris, T. metuendus and T. obscurus and one specie with no toxic effects to humans, Brotheas amazonicus. All the four species analyzed were able to induce pro- and anti-inflammatory cytokine production in a J774.1 murine macrophage model. This activation was dependent on TLR2/TLR4/MyD88 activation and abolished by TLRs antagonists. These results suggest that the venoms of the four species analyzed were able to induce macrophage response in agreement to the well-established immune activation by T. serrulatus venom. Our findings provide new insights into the clinical repercussions of scorpionism of uncharacterized species and point to new biotechnological applications of these venoms and possible supportive therapies in scorpionism.

On the noxious black Amazonian scorpion, Tityus obscurus (Scorpiones, Buthidae): Taxonomic notes, biology, medical importance and envenoming treatment.

Tityus obscurus has caused mild, moderate and severe accidents of medical relevance in the eastern Brazilian Amazon and French Guiana. Tityus obscurus has sexual dimorphism although males and females have uniform black coloration. In the Amazon, one of the habitats of this scorpion is seasonally flooded forests (igapós and várzeas). However, most stings occur in terra firme forest areas (non-flooded region), where most rural communities are located. Adults and children stung by T. obscurus may experience an "electric shock" sensation for more than 30 h after the sting. Our data shows that people inhabiting remote forest areas, including rubber tappers, fishermen and indigenous people, with no access to anti-scorpion serum, use parts of native plants, such as seeds and leaves, against pain and vomiting caused by scorpion stings. Although there is a technical effort to produce and distribute antivenoms in the Amazon, many cases of scorpion stings are geographically unpredictable in this region, due to the lack of detailed knowledge of the natural distribution of these animals. In this manuscript, we compile information on the natural history of T. obscurus and the impact of its envenoming on human health. We identify the natural sites that host this scorpion in the Amazon, in order to warn about the risk of human envenoming. The use of specific antivenom serum is the recommended treatment for accidents involving venomous animals. However, atypical symptoms not neutralized by the available commercial antivenom are reported in the Amazon region. Facing this scenario, we present some challenges to the study of venomous animals in the Amazon rainforest and possible experimental bottlenecks and perspectives for establishing a method aimed at producing an efficient antivenom.

The remarkably enzyme-rich venom of the Big Bend Scorpion (Diplocentrus whitei).

Scorpion venoms have long been studied for their peptide discovery potential, with modern high-throughput venom-characterization techniques paving the way for the discovery of thousands of novel putative toxins. Research into these toxins has provided insight into the pathology and treatment of human diseases, even resulting in the development of one compound with Food and Drug Administration (FDA) approval. Although most of this research has focused on the toxins of scorpion species considered medically significant to humans, the venom of harmless scorpion species possess toxins that are homologous to those from medically significant species, indicating that harmless scorpion venoms may also serve as valuable sources of novel peptide variants. Furthermore, as harmless scorpions represent a vast majority of scorpion species diversity, and therefore venom toxin diversity, venoms from these species likely contain entirely new toxin classes. We sequenced the venom-gland transcriptome and venom proteome of two male Big Bend scorpions (Diplocentrus whitei), providing the first high-throughput venom characterization for a member of this genus. We identified a total of 82 toxins in the venom of D. whitei, 25 of which were identified in both the transcriptome and proteome, and 57 of which were only identified in the transcriptome. Furthermore, we identified a unique, enzyme-rich venom dominated by serine proteases and the first arylsulfatase B toxins identified in scorpions.

Lipidomic profiling of the Brazilian yellow scorpion venom: new insights into inflammatory responses following Tityus serrulatus envenomation.

Due to the high prevalence and clinical relevance, scorpionism is a critical public health issue in several Brazilian regions. Tityus serrulatus, commonly known as the Brazilian yellow scorpion, is the most venomous genus found in Brazilian fauna and associated with severe clinical manifestations such as localized pain, hypertension, sweating, tachycardia and complex hyperinflammatory responses. In general, T. serrulatus venom contains a complex mixture of active compounds, including proteins, peptides, and amino acids. Although knowledge of the protein fractions of scorpion venom is available, venom lipid components are not yet comprehensively known. The aim of the present study was to determine and characterize the lipid constituents/profile of the T. serratus venom utilizing liquid chromatography coupled with high-resolution mass spectrometry. Lipid species (164 in total) belonging to 3 different lipid categories, glycerophospholipids, sphingolipids, and glycerolipids, were identified. A further search on MetaCore/MetaDrug platform, which is based upon a manually curated database of molecular interactions, molecular pathways, gene-disease associations, chemical metabolism, and toxicity information, exhibited several metabolic pathways for 24 of previously identified lipid species, including activation of nuclear factor kappa B and oxidative stress pathways. Further several bioactive compounds, such as plasmalogens, lyso-platelet-activating factors, and sphingomyelins, associated with systemic responses triggered by T. serrulatus envenomation were detected. Finally, lipidomic data presented provide advanced and valuable information to better comprehend the mechanisms underlying the complex pathophysiology induced by T. serrulatus envenomation.

Epidemiological, toxicological and physiopathological characteristics of scorpion stings and their management in Morocco: A literature review.

All around the world, scorpion envenomation represents a public health issue. In Morocco, it represents the first cause of poisoning. The aim of this review is to highlight the epidemiological, toxicological and physiopathological characteristics of scorpion stings and envenomation in Morocco as well as their management. According to the bibliography, the most areas affected by scorpion stings and envenomation are those in the center and south of Morocco. Lethality rate is more important in children than adult. Age, admission class, season, serum venom concentration, and the time elapsed between the scorpion sting and medical intervention are risk factors for a poor prognosis. Until now, we have only known the LD50 of 14 scorpion species from three genera: Androctonus, Buthus and Hottentota, and only 6 species, out of 61 moroccan scorpions, have been assessed for their venom toxicity on mice organs. The venom of harmful moroccan scorpions induces several histopathological changes in the myocardium, brain, liver, pulmonary alveoli, and kidneys in rats, as well as severe consequences in the lungs with intra-alveolar hemorrhage. There was also a significant increase in serum enzyme levels of aspartate transaminase (AST), alanine transaminase (ALT), creatine phosphokinase (CPK), and lactate dehydrogenase (LDH), as well as high levels of plasma albumin, creatine and glucose. In the absence of a specific treatment, the management of scorpion envenomation is based only on symptomatic therapy. We noted a widely use of traditional remedies. Despite the magnitude of the scorpion problem in Morocco, it remains understudied and continues to claim lives, particularly given the lack of an efficient etiological cure.

The nociceptive response induced by different classes of Tityus serrulatus neurotoxins: The important role of Ts5 in venom-induced nociception.

Scorpion sting envenomations (SSE) are feared by the intense pain that they produce in victims. Pain from SSE is triggered mainly by the presence of neurotoxins in the scorpion venom that modulates voltage-gated ion channels. In Brazil, SSE is mostly caused by Tityus serrulatus, popularly known as yellow scorpion. Here, we evaluated experimental spontaneous nociception induced by T. serrulatus venom as well as its isolated neurotoxins Ts1, Ts5, Ts6, Ts8, and Ts19 frag II, evidencing different degrees of pain behavior in mice. In addition, we developed a mice-derived polyclonal antibody targeting Ts5 able to neutralize the effect of this neurotoxin, showing that Ts5 presents epitopes capable of activating the immune response, which decreased considerably the nociception produced by the whole venom. This is the pioneer study to explore nociception using different classes of T. serrulatus neurotoxins on nociception (α-NaTx, ß-NaTx, α-KTx, and ß-KTx), targeting potassium and sodium voltage-gated channels, besides demonstrating that Ts5 plays an important role in the scorpion sting induced-pain.

Evaluation of in vivo Lethality and in vitro Cytotoxic Effect of Odontobuthus bidentatus Scorpion Venom.

The results of numerous studies have revealed that some deadly scorpion venoms are composed of various bioactive molecules that have significant cytotoxic effects on cancer cells. In this study, the in vivo lethality and cytotoxic effect of Odontobuthus bidentatus venom were evaluated in different cancer cell lines. Through MTT assay, the cytotoxic effects of O. bidentatus scorpion venom were analyzed on the MCF-7, A549, AGS, HepG2, and Ht-29 cancer cell lines and Hu02 normal cells. To this end, six venom fractions were obtained through a Sephadex G-50 column, and the cytotoxic effects of isolated fractions were evaluated on A549 lung cancer cells. The median lethal dose of O. bidentatus scorpion venom was determined at 0.73 mg/kg by intravenous administration of different venom doses in male BALB/c mice according to the Spearman-Karber method. The O. bidentatus scorpion whole venom had a significant cytotoxic effect on MCF-7, A549, and AGS cells. The treatment of A549 cells with various concentrations of fraction F1 showed that this fraction significantly induced growth inhibitory effect on the cells in a dose-dependent manner, compared to untreated cells.

Characterization of Four Medically Important Toxins from Centruroides huichol Scorpion Venom and Its Neutralization by a Single Recombinant Antibody Fragment.

Centruroides huichol scorpion venom is lethal to mammals. Analysis of the venom allowed the characterization of four lethal toxins named Chui2, Chui3, Chui4, and Chui5. scFv 10FG2 recognized well all toxins except Chui5 toxin, therefore a partial neutralization of the venom was observed. Thus, scFv 10FG2 was subjected to three processes of directed evolution and phage display against Chui5 toxin until obtaining scFv HV. Interaction kinetic constants of these scFvs with the toxins were determined by surface plasmon resonance (SPR) as well as thermodynamic parameters of scFv variants bound to Chui5. In silico models allowed to analyze the molecular interactions that favor the increase in affinity. In a rescue trial, scFv HV protected 100% of the mice injected with three lethal doses 50 (LD50) of venom. Moreover, in mix-type neutralization assays, a combination of scFvs HV and 10FG2 protected 100% of mice injected with 5 LD50 of venom with moderate signs of intoxication. The ability of scFv HV to neutralize different toxins is a significant achievement, considering the diversity of the species of Mexican venomous scorpions, so this scFv is a candidate to be part of a recombinant anti-venom against scorpion stings in Mexico.

The Enzymatic Core of Scorpion Venoms.

Enzymes are an integral part of animal venoms. Unlike snakes, in which enzymes play a primary role in envenomation, in scorpions, their function appears to be ancillary in most species. Due to this, studies on the diversity of scorpion venom components have focused primarily on the peptides responsible for envenomation (toxins) and a few others (e.g., antimicrobials), while enzymes have been overlooked. In this work, a comprehensive study on enzyme diversity in scorpion venoms was performed by transcriptomic and proteomic techniques. Enzymes of 63 different EC types were found, belonging to 330 orthogroups. Of them, 24 ECs conform the scorpion venom enzymatic core, since they were determined to be present in all the studied scorpion species. Transferases and lyases are reported for the first time. Novel enzymes, which can play different roles in the venom, including direct toxicity, as venom spreading factors, activators of venom components, venom preservatives, or in prey pre-digestion, were described and annotated. The expression profile for transcripts coding for venom enzymes was analyzed, and shown to be similar among the studied species, while being significantly different from their expression pattern outside the telson.

Scorpion Species with Smaller Body Sizes and Narrower Chelae Have the Highest Venom Potency.

Scorpionism is a global health concern, with an estimation of over one million annual envenomation cases. Despite this, little is known regarding the drivers of scorpion venom potency. One widely held view is that smaller scorpions with less-developed chelae possess the most potent venoms. While this perception is often used as a guide for medical intervention, it has yet to be tested in a formal comparative framework. Here, we use a phylogenetic comparative analysis of 36 scorpion species to test whether scorpion venom potency, as measured using LD50, is related to scorpion body size and morphology. We found a positive relationship between LD50 and scorpion total length, supporting the perception that smaller scorpions possess more potent venoms. We also found that, independent of body size, scorpion species with long narrow chelae have higher venom potencies compared to species with more robust chelae. These results not only support the general perception of scorpion morphology and potency, but also the presence of an ecology trade-off with scorpions either selected for well-developed chelae or more potent venoms. Testing the patterns of venom variations in scorpions aids both our ecological understanding and our ability to address the global health burden of scorpionism.

Induced pathophysiological alterations by the venoms of the most dangerous Moroccan scorpions Androctonus mauretanicus and Buthus occitanus: A comparative pathophysiological and toxic-symptoms study.

Scorpion envenomation is a serious public health issue. Androctonus mauretanicus (Am) and Buthus occitanus (Bo) are the most dangerous scorpions in Morocco. Despite their medical relevance, no study has yet related their kinetics of symptom apparition and the consequent tissue disorders at the same interval post-injection. This work achieved the first comparative pathophysiological and toxic-symptoms study between the Am and Bo venoms from a biochemical, toxicological and physiopathological standpoint. The activity of venoms and their subletal dose were determined by administration of increasing concentrations of the venoms. 30, 60 and 120 min following the experimental envenomation in mice, the profile of clinical symptoms was underlined and the main organs: brain, heart, lungs, liver and kidneys were removed for histological examination. The Am venom is a rich source of proteins and three-times more toxic than the Bo. The most observed clinical symptoms are neurological and cardiopulmonary. The Am venom caused histopathological alterations at 30, 60, and 120 min which were more important than the Bo. This study highlighted that both venoms exhibited a strong toxicity with variable intensities. Moreover, we showed the presence of correlation between the level of histopathological disorders observed and the intensity of signs appeared at the same time following venom inoculation.

A Buthus martensii Karsch scorpion sting targets Nav1.7 in mice and mimics a phenotype of human chronic pain.

ABSTRACT: Gain-of-function and loss-of-function mutations in Nav1.7 cause chronic pain and pain insensitivity, respectively. The preferential expression of Nav1.7 in the peripheral nervous system and its role in human pain signaling make Nav1.7 a promising target for next-generation pain therapeutics. However, pharmacological agents have not fully recapitulated these pain phenotypes, and because of the lack of subtype-selective molecular modulators, the role of Nav1.7 in the perception of pain remains poorly understood. Scorpion venom is an excellent source of bioactive peptides that modulate various ion channels, including voltage-gated sodium (Nav) channels. Here, we demonstrate that Buthus martensii Karsch scorpion venom (BV) elicits pain responses in mice through direct enhancement of Nav1.7 activity and have identified Makatoxin-3, an α-like toxin, as a critical component for BV-mediated effects on Nav1.7. Blocking other Nav subtypes did not eliminate BV-evoked pain responses, supporting the pivotal role of Nav1.7 in BV-induced pain. Makatoxin-3 acts on the S3-S4 loop of voltage sensor domain IV (VSD4) of Nav1.7, which causes a hyperpolarizing shift in the steady-state fast inactivation and impairs inactivation kinetics. We also determined the key residues and structure-function relationships for the toxin-channel interactions, which are distinct from those of other well-studied α toxins. This study not only reveals a new mechanism underlying BV-evoked pain but also enriches our knowledge of key structural elements of scorpion toxins that are pivotal for toxin-Nav1.7 interactions, which facilitates the design of novel Nav1.7 selective modulators.

Protein profile of scorpion venom from Hottentotta rugiscutis and its immunogenic potential in inducing long term memory response.

The scorpions of the Buthidae family exhibit diverse toxins with proven pharmacological activities and yet underexplored. The Hottentotta rugiscutis is a commonly found south-Indian buthid scorpion, whose venom proteomic profile is unknown. In this study, the venom was biochemically and immunologically characterized by SDS-PAGE, MALDI-TOF MS, Western blot and ELISA. The regional and seasonal variation in the venom composition from the same species was also assessed at the molecular mass level. The venom was further studied in albino mice to understand its impact on various blood parameters. The venom has varied MW proteins from 6 to 275 kDa, four of them were found to be major immunodominant proteins. The mass spectra have revealed that some proteins are predominantly present in the venom of 3-4.5 kDa or 6.5-8.0 kDa, which could be the K+ or Na+ channel blockers respectively whose ratio varied by season. The obtained venom-mass spectra could also be used as H. rugiscutis specific finger-print in identifying the region-specific species. The venom was found to elicit a stress-induced innate immune response in mice, giving rise to a strong Th2 mediated humoral immune response. Overall, this study has provided a glimpse of the venom composition and its immunogenicity.