Crotalus Atrox Research Articles

Chronic Multi-Electrode Electromyography in Snakes.

Knowledge about body motion kinematics and underlying muscle contraction dynamics usually derives from electromyographic (EMG) recordings. However, acquisition of such signals in snakes is challenging because electrodes either attached to or implanted beneath the skin may unintentionally be removed by force or friction caused from undulatory motion, thus severely impeding chronic EMG recordings. Here, we present a reliable method for stable subdermal implantation of up to eight bipolar electrodes above the target muscles. The mechanical stability of the inserted electrodes and the overnight coverage of the snake body with a “sleeping bag” ensured the recording of reliable and robust chronic EMG activity. The utility of the technique was verified by daily acquisition of high signal-to-noise activity from all target sites over four consecutive days during stimulus-evoked postural reactions in Amazon tree boas and Western diamondback rattlesnakes. The successful demonstration of the chronic recording suggests that this technique can improve acute experiments by enabling the collection of larger data sets from single individuals.

Crotalus atrox venom-induced cellular toxicity: Early wound progression involves reactive oxygen species.

Understanding the mechanisms that produce cellular cytotoxicity is fundamental in the field of toxicology. Cytotoxic stimuli can include organic toxins such as hemorrhagic snake venom, which can lead to secondary complications such as the development of necrotic tissue and profuse scarring. These clinical manifestations mimic cytotoxic responses induce by other organic compounds such as organic acids. We used hemorrhagic snake venom and human embryonic kidney cells (HEK 293T) as a model system to better understand the cellular responses involved in venom induced cytotoxicity. Cells stimulated with Crotalus atrox (CA) (western diamondback) venom for 4 or 10h demonstrated significant cytotoxicity. Results from 2',7'-Dichlorodihydrofluorescein diacetate (H2 DCF-DA) assays determine CA venom stimulation induces a robust production of reactive oxygen species (ROS) over a 3-h time course. In contrast, pretreatment with polyethylene glycol (PEG)-catalase or N-acetyl cysteine (NAC) prior to CA venom stimulation significantly blunted H2 DCFDA fluorescence fold changes and showed greater cytoprotective effects than cells stimulated with CA venom alone. Pre- incubating HEK293T cells with the NADPH oxidase (NOX) pan-inhibitor VAS2870 prior venom stimulation significantly minimized the venom-induced oxidative burst at early timepoints (≤2h). Collectively, our experiments show that pre-application of antioxidants reduces CA venom induce cellular toxicity. This result highlights the importance of ROS in the early stages of cytotoxicity and suggests muting ROS production in noxious injuries may increase positive clinical outcomes.

Proteomic Identification and Quantification of Snake Venom Biomarkers in Venom and Plasma Extracellular Vesicles.

The global exploration of snakebites requires the use of quantitative omics approaches to characterize snake venom as it enters into the systemic circulation. These omics approaches give insights into the venom proteome, but a further exploration is warranted to analyze the venom-reactome for the identification of snake venom biomarkers. The recent discovery of extracellular vesicles (EVs), and their critical cellular functions, has presented them as intriguing sources for biomarker discovery and disease diagnosis. Herein, we purified EV’s from the snake venom (svEVs) of Crotalus atrox and C. oreganus helleri, and from plasma of BALB/c mice injected with venom from each snake using EVtrap in conjunction with quantitative mass spectrometry for the proteomic identification and quantification of svEVs and plasma biomarkers. Snake venom EVs from C. atrox and C. o. helleri were highly enriched in 5′ nucleosidase, L-amino acid oxidase, and metalloproteinases. In mouse plasma EVs, a bioinformatic analysis for revealed upregulated responses involved with cytochrome P450, lipid metabolism, acute phase inflammation immune, and heat shock responses, while downregulated proteins were associated with mitochondrial electron transport, NADH, TCA, cortical cytoskeleton, reticulum stress, and oxidative reduction. Altogether, this analysis will provide direct evidence for svEVs composition and observation of the physiological changes of an envenomated organism.

Exceptional long-term sperm storage by a female vertebrate.

Females of many vertebrate species have the capacity to store sperm within their reproductive tracts for prolonged periods of time. Termed long-term sperm storage, this phenomenon has many important physiological, ecological, and evolutionary implications, particularly to the study of mating systems, including male reproductive success and post-copulatory sexual selection. Reptiles appear particularly predisposed to long-term sperm storage, with records in most major lineages, with a strong emphasis on turtles and squamates (lizards, snakes, but not the amphisbaenians). Because facultative parthenogenesis is a competing hypothesis to explain the production of offspring after prolonged separation from males, the identification of paternal alleles through genetic analysis is essential. However, few studies in snakes have undertaken this. Here, we report on a wild-collected female Western Diamond-backed Rattlesnake, Crotalus atrox, maintained in isolation from the time of capture in September 1999, that produced two healthy litters approximately one and six years post capture. Genetic analysis of the 2005 litter, identified paternal contribution in all offspring, thus rejecting facultative parthenogenesis. We conclude that the duration of long-term sperm storage was approximately 6 years (71 months), making this the longest period over which a female vertebrate has been shown to store sperm that resulted in the production of healthy offspring.

Unwanted residential wildlife: Evaluating social-ecological patterns for snake removals

Snakes are globally threatened due to anthropogenic pressures. Conflicts between snakes and people occur when residents encounter snakes in their home environments. In collaboration with a local business that provides snake removal services, we examined records from over 2000 snake removals in Phoenix metropolitan area, Arizona, United States between 2018 and 2019. We examined removal locations in relation to neighborhood-level socioeconomic attributes from the American Community Survey and individual demographics from a social survey of 494 respondents. Over 68% of removals were of the venomous Western Diamond-backed Rattlesnake (Crotalus atrox), which is the most common species in the area observed in community-sourced data and publications. Removals occurred throughout the year except winter and peaked during summer rainy season. Snakes were frequently removed from neighborhoods with wealthier and more highly educated residents, greater proportion of Latinx residents, and recently constructed homes. Individual perceptions of snakes as problematic were not related to the number of snakes removed. This research is the first to analyze snake removals in a social-ecological context and underscores the conservation benefits of researchers partnering with a local business to gain spatial and temporal information on an elusive taxon. Similar collaborations could lead to direct conservation action for snakes by researchers learning from community members in cities and by groups willing to use results from research partnerships to inform their practices. Understanding how to maintain biodiversity in urbanizing arid regions could protect snakes if relocating snakes away from areas of high human density translates into fewer snakes killed by people annually.

Examination of the Efficacy and Cross-Reactivity of a Novel Polyclonal Antibody Targeting the Disintegrin Domain in SVMPs to Neutralize Snake Venom.

Snake envenomation can result in hemorrhage, local necrosis, swelling, and if not treated properly can lead to adverse systemic effects such as coagulopathy, nephrotoxicity, neurotoxicity, and cardiotoxicity, which can result in death. As such, snake venom metalloproteinases (SVMPs) and disintegrins are two toxic components that contribute to hemorrhage and interfere with the hemostatic system. Administration of a commercial antivenom is the common antidote to treat snake envenomation, but the high-cost, lack of efficacy, side effects, and limited availability, necessitates the development of new strategies and approaches for therapeutic treatments. Herein, we describe the neutralization ability of anti-disintegrin polyclonal antibody on the activities of isolated disintegrins, P-II/P-III SVMPs, and crude venoms. Our results show disintegrin activity on platelet aggregation in whole blood and the migration of the SK-Mel-28 cells that can be neutralized with anti-disintegrin polyclonal antibody. We characterized a SVMP and found that anti-disintegrin was also able to inhibit its activity in an in vitro proteolytic assay. Moreover, we found that anti-disintegrin could neutralize the proteolytic and hemorrhagic activities from crude Crotalus atrox venom. Our results suggest that anti-disintegrin polyclonal antibodies have the potential for a targeted approach to neutralize SVMPs in the treatment of snakebite envenomations.

Bacterial expression of a snake venom metalloproteinase inhibitory protein from the North American opossum (D. virginiana)

A variety of opossum species are resistant to snake venoms due to the presence of antihemorrhagic and antimyotoxic acidic serum glycoproteins that inhibit several toxic venom components. Two virtually identical antihemorrhagic proteins isolated from either the North American opossum (D. virginiana) or the South American big-eared opossum (D. aurita), termed oprin or DM43 respectively, inhibit specific snake venom metalloproteinases (SVMPs). A better understanding of the structure of these proteins may provide useful insight to determine their mechanism of action and for the development of therapeutics against the global health concern of snake-bite envenomation. The aim of this work is to produce a recombinant snake venom metalloproteinase inhibitor (SVMPI) similar to the above opossum proteins in Escherichia coli and determine if this bacterially produced protein inhibits the proteolytic properties of Western Diamondback rattlesnake (C. atrox) venom. The resulting heterologous SVMPI was produced with either a 6-Histidine or maltose binding protein (MBP) affinity tag on either the C-terminus or N-terminus of the protein, respectively. The presence of the solubility enhancing MBP affinity tag resulted in significantly more soluble protein expression. The inhibitory activity was measured using two complementary assays and the MBP labeled SVMPI showed 7-fold less activity as compared to the 6-Histidine labeled SVMPI. Thus, the bacterially derived SVMPI with an unlabeled N-terminus showed high inhibitory activity (IC50 = 4.5 μM). The use of a solubility enhancing MBP fusion protein construct appears to be a productive way to express sufficient quantities of this mammalian protein in E. coli for further study.

Sonographic extremity assessment of crotalinae envenomation

The evaluation of a patient after Crotalinae envenomation requires assessment of local and systemic signs and symptoms in conjunction with laboratory data. Point-of-care ultrasound (POCUS) is an emerging modality used to evaluate soft tissue changes associated with snake envenomations around the world. However, more data are needed to characterize sonographic findings of snake envenomation. Our study used POCUS to evaluate the depth of soft tissue injury, involvement of the muscles/tendons, and the proximal edge of envenomation. This was a prospective observational study evaluating the sonographic characteristics of Crotalinae envenomation. The eight patients enrolled in the study included one envenomated by a Western diamondback (Crotalus atrox), and the others were envenomated by copperheads (Agkistrodon contortrix). All the patients demonstrated initial subcutaneous cobblestoning on POCUS at the bite site. All digit envenomations revealed edema in the tendon or tendon sheath. None of these cases required acute surgical intervention. There were no sonographic signs of envenomation found below fascia or muscle. Four of the eight patients had POCUS findings of cobblestoning proximal to the edge of envenomation palpated on the extremity. The clinical significance of this is unknown.

Effects of Sodium Silicate Complex against Hemorrhagic Activities Induced by Protobothrops mucrosquamatus Venom.

Protobothrops mucrosquamatus poses a serious medical threat to humans in Southern and Southeastern Asia. Hemorrhage is one of the conspicuous toxicities related to the pathology of P. mucrosquamatus envenoming. Previous in vitro and in vivo studies showed that a silica-derived reagent, sodium silicate complex (SSC), was able to neutralize hemorrhagic and proteolytic activities induced by pit viper venoms, including Crotalus atrox, Agkistrodon contortrix contortrix and Agkistrodon piscivorus leucostoma. In this study, we validated that SSC could neutralize enzymatic and toxic effects caused by the venom of P. mucrosquamatus. We found that SSC inhibited the hemolytic and proteolytic activities induced by P. mucrosquamatus venom in vitro. In addition, we demonstrated that SSC could block intradermal hemorrhage caused by P. mucrosquamatus venom in a mouse model. Finally, SSC could neutralize lethal effects of P. mucrosquamatus venom in the mice. Therefore, SSC is a candidate for further development as a potential onsite first-aid treatment for P. mucrosquamatus envenoming.

Using yeast two-hybrid system and molecular dynamics simulation to detect venom protein-protein interactions

Proteins and peptides are major components of snake venom. Venom protein transcriptomes and proteomes of many snake species have been reported; however, snake venom complexity (i.e., the venom protein-protein interactions, PPIs) remains largely unknown. To detect the venom protein interactions, we used the most common snake venom component, phospholipase A2s (PLA2s) as a “bait” to identify the interactions between PLA2s and 14 of the most common proteins in Western diamondback rattlesnake (Crotalus atrox) venom by using yeast two-hybrid (Y2H) analysis, a technique used to detect PPIs. As a result, we identified PLA2s interacting with themselves, and lysing-49 PLA2 (Lys49 PLA2) interacting with venom cysteine-rich secretory protein (CRISP). To reveal the complex structure of Lys49 PLA2-CRISP interaction at the structural level, we first built the three-dimensional (3D) structures of Lys49 PLA2 and CRISP by a widely used computational program-MODELLER. The binding modes of Lys49 PLA2-CRISP interaction were then predicted through three different docking programs including ClusPro, ZDOCK and HADDOCK. Furthermore, the most likely complex structure of Lys49 PLA2-CRISP was inferred by molecular dynamic (MD) simulations with GROMACS software. The techniques used and results obtained from this study strengthen the understanding of snake venom protein interactions and pave the way for the study of animal venom complexity.

Supracategorical fear information revealed by aversively conditioning multiple categories

ABSTRACT Fear-generalization is a critical function for survival, in which an organism extracts information from a specific instantiation of a threat (e.g., the western diamondback rattlesnake in my front yard on Sunday) and learns to fear – and accordingly respond to – pertinent higher-order information (e.g., snakes live in my yard). Previous work investigating fear-conditioning in humans has used functional magnetic resonance imaging (fMRI) to demonstrate that activity patterns representing stimuli from an aversively-conditioned category (CS+) are more similar to each other than those of a neutral category (CS-). Here we used fMRI and multiple aversively-conditioned categories to ask whether we would find only similarity increases within the CS+ categories or also similarity increases between the CS+ categories. Using representational similarity analysis, we correlated several models to activity patterns underlying different brain regions and found that, following fear-conditioning, between-category and within-category similarity increased for the CS+ categories in the insula, superior frontal gyrus (SFG), and the right temporal pole. When specifically investigating fear-generalization, these between- and within-category effects were detected in the SFG. These results advance prior pattern-based neuroimaging work by exploring the effect of aversively-conditioning multiple categories and indicate an extended role for such regions in potentially representing supracategorical information during fear-learning.

Soft Matter Mechanics and the Mechanisms Underpinning the Infrared Vision of Snakes

Summary Pit-bearing snakes (vipers, pythons, and boas) have the extraordinary ability to “see” and accurately locate their prey and predators in total darkness. These animals use the infrared radiation emanating from objects that are warmer relative to the background environment to form a thermal image. Although enormous progress has been made to identify the key physiological features that enable the infrared vision of these snakes and a few other animals, the precise thermoelectric transduction mechanism that mediates the conversion of infrared heat to processable electrical signals has remained elusive. In this work, we quantitatively outline how cells in the snake's pit membrane organ act as apparent pyroelectric materials and convert infrared radiation into electrical signals. Despite the exceptional simplicity of our proposed mechanism and model, we are able to explain many central experimental results pertaining to the transduction process.

101 Thromboelastography versus Conventional Coagulation Tests in Pit Viper Envenomation and Antivenom Response

Pit viper envenomations represent a significant burden of disease worldwide, leading to severe hematologic derangements such as venom-induced consumptive coagulopathy (VICC). Recognizing this problem, the WHO recently launched a campaign to cut snake bite deaths in half by 2030. Immediacy in detection and treatment of coagulopathy with antivenom is paramount to reducing morbidity and mortality. Traditionally, administration of the pit viper antivenom, CroFab®, has been based on clinical signs and symptoms and conventional coagulation tests (CCT) such as fibrinogen, PT, PTT and INR. The purpose of this study was to assess the efficacy of TEG in detecting response to antivenom administration. Blood samples from 25 healthy adult volunteers were mixed with different concentrations of western diamondback rattlesnake (Crotalus atrox) venom (50% and 100% LD50). Each group of envenomated blood samples was treated with 4, 6, and 10 vial equivalents (vialeq) of CroFab®. All samples were assessed with CCTs including: PT, PTT, INR and Fibrinogen, as well as with TEG measures of reaction time (R), amplification (k), rate of clot formation (alpha angle), and clot strength (MA). Data was analyzed to determine the rate of return to normal range CCT and TEG values as a surrogate for return to normal coagulation. For the 50% LD50 group, CCT parameters of PT, PTT and INR returned to normal in 24%-32% of samples across all CroFab® doses. For the same group, 72% of Fibrinogen samples returned to normal at 4 vialeq with a max of 88% at 10 vialeq. TEG parameters R, k, alpha angle and MA returned to normal at a rate of 80%-96% across all CroFab doses. For the 100% LD50 group, CCT parameters of PT, PTT, and INR had a maximum of 24% return to normal for all CroFab® doses. For the same group, 16% of Fibrinogen samples returned to normal at 4 vialeq with a max of 88% at 10 vialeq. TEG parameters showed incremental increases towards normal range with 92-96% of samples in range at 10 vialeq. Pit viper envenomations are a global health threat with significant morbidity and mortality and suboptimal methods to guide the utilization of the expensive and often limited CroFab® antivenom. In this in vitro model, TEG was shown to be more sensitive than CCTs in tracking antivenom-induced recovery from VICC. Additionally, our findings represent a step towards developing a dose-response curve for antivenom administration with the potential to reduce both the amount of antivenom used and the side effects associated with its use.

No evidence of male-biased sexual selection in a snake with conventional Darwinian sex roles.

Decades of research on sexual selection have demonstrated that ‘conventional’ Darwinian sex roles are common in species with anisogamous gametes, with those species often exhibiting male-biased sexual selection. Yet, mating system characteristics such as long-term sperm storage and polyandry have the capacity to disrupt this pattern. Here, these ideas were explored by quantifying sexual selection metrics for the western diamond-backed rattlesnake (Crotalus atrox). A significant standardized sexual selection gradient was not found for males (βSS = 0.588, p = 0.199) or females (βSS = 0.151, p = 0.664), and opportunities for sexual selection (Is) and selection (I) did not differ between males (Is = 0.069, I = 0.360) and females (Is = 0.284, I = 0.424; both p > 0.05). Furthermore, the sexes did not differ in the maximum intensity of precopulatory sexual selection (males: s′max = 0.155, females: s′max = 0.080; p > 0.05). Finally, there was no evidence that male snout–vent length, a trait associated with mating advantage, is a target of sexual selection (p > 0.05). These results suggest a lack of male-biased sexual selection in this population. Mating system characteristics that could erode male-biased sexual selection, despite the presence of conventional Darwinian sex roles, are discussed.

Hyperimmune horse serum and immunopurified antibodies impact on human fibroblast cytokine response to Crotalus atrox venom

Hyperimmune horse serum and immunopurified antibodies impact on human fibroblast cytokine response to Crotalus atrox venom

Human fibroblast response to Crotalus atrox venom and model wound healing

Human fibroblast response to Crotalus atrox venom and model wound healing

Integrative Framework for Long-Term Activity Monitoring of Small and Secretive Animals: Validation With a Cryptic Pitviper

The use of miniature accelerometer (ACT) data-loggers for remote and continuous recording of animal movement behavior is becoming increasingly common. Until recently, size constraints limited most animal-borne ACT applications to large-bodied species. We capitalized on the ongoing miniaturization and advancement of these technologies and associated computational techniques to develop a framework for long-term, low-frequency ACT monitoring of activity in small and secretive terrestrial species. We achieved this by internally implanting coupled radio transmitters and tri-axial ACTs in rattlesnakes (Crotalus atrox). Periodic field-validation observations of behavior were used to train and test supervised learning models (Random Forest, RF; Generalized Linear Elastic Net, GLMNET) for activity classification. The best performing RF model classified periods of movement and immobility in rattlesnakes with high accuracy (movement = 96%, immobile = 99%), and was applied to extensive ACT field datasets (median = 35 days, range = 6–289 days; N = 12) to produce activity budgets at multiple temporal scales. In general, these cryptic ambush predators were found to be highly sedentary, with activity budgets characterized by long periods of immobility interrupted by punctuated bouts of movement. The same temporal daily activity pattern was conserved across all active seasons (spring, summer non-mating, summer mating, fall), as most movement always occurred during the evening or nocturnal diel periods. Contrary to movement timing, daily movement duration was seasonally variable, as movement increased during the summer-mating season – possibly reflecting a combination of more favorable weather conditions (onset of rainy season) and mate-searching efforts by male rattlesnakes. This radio telemetry-accelerometry (RT-ACT) framework provides an objective and flexible set of data collection and processing procedures for long-term ACT field datasets. Expanding our coarse-scale behavioral classification scheme could provide a foundation for future investigations using the RT-ACT framework to explore relationships between individual behavioral decisions and performance in snakes and other small and secretive terrestrial vertebrates.

Repurposing Cancer Drugs Batimastat and Marimastat to Inhibit the Activity of a Group I Metalloprotease from the Venom of the Western Diamondback Rattlesnake, Crotalus atrox.

Snakebite envenomation causes over 140,000 deaths every year, predominantly in developing countries. As a result, it is one of the most lethal neglected tropical diseases. It is associated with incredibly complex pathophysiology due to the vast number of unique toxins/proteins present in the venoms of diverse snake species found worldwide. Here, we report the purification and functional characteristics of a Group I (PI) metalloprotease (CAMP-2) from the venom of the western diamondback rattlesnake, Crotalus atrox. Its sensitivity to matrix metalloprotease inhibitors (batimastat and marimastat) was established using specific in vitro experiments and in silico molecular docking analysis. CAMP-2 shows high sequence homology to atroxase from the venom of Crotalus atrox and exhibits collagenolytic, fibrinogenolytic and mild haemolytic activities. It exerts a mild inhibitory effect on agonist-induced platelet aggregation in the absence of plasma proteins. Its collagenolytic activity is completely inhibited by batimastat and marimastat. Zinc chloride also inhibits the collagenolytic activity of CAMP-2 by around 75% at 50 μM, while it is partially potentiated by calcium chloride. Molecular docking studies have demonstrated that batimastat and marimastat are able to bind strongly to the active site residues of CAMP-2. This study demonstrates the impact of matrix metalloprotease inhibitors in the modulation of a purified, Group I metalloprotease activities in comparison to the whole venom. By improving our understanding of snake venom metalloproteases and their sensitivity to small molecule inhibitors, we can begin to develop novel and improved treatment strategies for snakebites.

The origin and diversification of a novel protein family in venomous snakes

The genetic origins of novelty are a central interest of evolutionary biology. Most new proteins evolve from preexisting proteins but the evolutionary path from ancestral gene to novel protein is challenging to trace, and therefore the requirements for and order of coding sequence changes, expression changes, or gene duplication are not clear. Snake venoms are important novel traits that are comprised of toxins derived from several distinct protein families, but the genomic and evolutionary origins of most venom components are not understood. Here, we have traced the origin and diversification of one prominent family, the snake venom metalloproteinases (SVMPs) that play key roles in subduing prey in many vipers. Genomic analyses of several rattlesnake (Crotalus) species revealed the SVMP family massively expanded from a single, deeply conserved adam28 disintegrin and metalloproteinase gene, to as many as 31 tandem genes in the Western Diamondback rattlesnake (Crotalus atrox) through a number of single gene and multigene duplication events. Furthermore, we identified a series of stepwise intragenic deletions that occurred at different times in the course of gene family expansion and gave rise to the three major classes of secreted SVMP toxins by sequential removal of a membrane-tethering domain, the cysteine-rich domain, and a disintegrin domain, respectively. Finally, we show that gene deletion has further shaped the SVMP complex within rattlesnakes, creating both fusion genes and substantially reduced gene complexes. These results indicate that gene duplication and intragenic deletion played essential roles in the origin and diversification of these novel biochemical weapons.

Toxicological profile of medically relevant Crotalus species from Mexico and their neutralization by a Crotalus basiliscus/Bothrops asper antivenom

Specimens of the Crotalus genus represent a potential snakebite problem in Mexico, and despite the great number of species of Crotalus present in this country, only a few of them are relevant from a medical point of view. Crotalus envenomed patients can present a range of signs and symptoms, depending on the species involved, and their treatment is indistinctly with either of the anti-viperid antivenoms available in the Mexican Public Health System. One of these antivenoms is produced by immunization of horses with a mixture of only two venoms: Crotalus basiliscus and Bothrops asper venoms. In light of the high variability found in Crotalus species venom composition, it is important to demonstrate the cross-neutralization of this antivenom against other Crotalus species. Therefore, in this work the toxic variability of eight medically important Crotalus venoms from Mexico and its neutralization by the Crotalus basiliscus/Bothrops asper antivenom were assessed. The present study evidenced the variability of toxic and enzymatic activities among the following Crotalus venoms: (1) Crotalus atrox, (2) Crotalus basiliscus, (3) Crotalus culminatus, (4) Crotalus simus, (5) Crotalus tzabcan, (6) Crotalus scutulatus salvini, (7) Crotalus scutulatus scutulatus-A, and (8) Crotalus scutulatus scutulatus-B. All venoms studied possess lethal and hemorrhagic activity on a murine model, although there are important variations among the species; in contrast, the PLA2 activity was similar for all venoms. Interestingly, only C. simus venom exhibited coagulant activity on human plasma under 100 μg. The antivenom neutralized the lethality and all the other assessed activities for all venoms tested. However, the dose required varied depending on the venom and the evaluated activity. Our preclinical data support the recommendation of using this antivenom to clinically manage Crotalus snakebites produced by the species assessed in this study. Nonetheless, only clinical trials could categorically validate these results.