Fission-fusion dynamics in the social networks of a North American pitviper.

Many animal species exist in fission-fusion societies, where the size and composition of conspecific groups change spatially and temporally. To help investigate such phenomena, social network analysis (SNA) has emerged as a powerful conceptual and analytical framework for assessing patterns of interconnectedness and quantifying group-level interactions. We leveraged behavioral observations via radiotelemetry and genotypic data from a long-term (>10 years) study on the pitviper Crotalus atrox (western diamondback rattlesnake) and used SNA to quantify the first robust demonstration of social network structures for any free-living snake. Group-level interactions among adults in this population resulted in structurally modular networks (i.e., distinct clusters of interacting individuals) for fidelis use of communal winter dens (denning network), mating behaviors (pairing network), and offspring production (parentage network). Although the structure of each network was similar, the size and composition of groups varied among them. Specifically, adults associated with moderately sized social groups at winter dens but often engaged in reproductive behaviors-both at and away from dens-with different and fewer partners. Additionally, modules formed by individuals in the pairing network were frequently different from those in the parentage network, likely due to multiple mating, long-term sperm storage by females, and resultant multiple paternity. Further evidence for fission-fusion dynamics exhibited by this population-interactions were rare when snakes were dispersing to and traversing their spring-summer home ranges (to which individuals show high fidelity), despite ample opportunities to associate with numerous conspecifics that had highly overlapping ranges. Taken together, we show that long-term datasets incorporating SNA with spatial and genetic information provide robust and unique insights to understanding the social structure of cryptic taxa that are understudied.

Intracellular Zinc Trafficking during Crotalus atrox Venom Wound Development.

In this study, we examined zinc trafficking in human umbilical vein endothelial cells (HUVEC) stimulated with Crotalus atrox (CA venom) snake venom. We utilized MTS cytotoxicity assays to monitor the cytotoxic range of CA venom. HUVEC monolayers stimulated with 10 µg/mL CA venom for 3 h displayed cellular retraction, which coincided with 53.0 ± 6.5 percent viability. In contrast, venom concentrations of 100 µg/mL produced a complete disruption of cellular adherence and viability decreased to 36.6 ± 1.0. The zinc probe Fluozin-3AM was used to detect intracellular zinc in non-stimulated controls, HUVEC stimulated with 10 µg/mL CA venom or HUVEC preincubated with TPEN for 2 h then stimulated with 10 µg/mL CA venom. Fluorescent intensity analysis returned values of 1434.3 ± 197.4 for CA venom demonstrating an increase of about two orders of magnitude in labile zinc compared to non-stimulated controls. Endothelial response to CA venom induced a 96.1 ± 3.0- and 4.4 ± 0.41-fold increase in metallothionein 1X (MT1X) and metallothionein 2A (MT2A) gene expression. Zinc chelation during CA venom stimulation significantly increased cell viability, suggesting that the maintenance of zinc homeostasis during envenomation injury improves cell survival.

Biochemical and computational approaches to understand venom toxin-toxin interaction.

Snake venoms are mainly composed of proteins and peptides (venom toxins). The venom transcriptomes and proteomes have been extensively investigated; however, venom toxin-toxin interactions remain poorly characterized. We detected the interaction of venom Asp49-PLA2 and 3FTx using biochemical and computational approaches. A stable structure of Asp49-PLA2-3FTx was identified, and the interface of Asp49-PLA2 and 3FTx was analyzed. The approaches will shed light on understanding the venom complexity and deciphering the synergistic effects of venom toxins.

Characterization of the venom and external morphology of a natural hybrid between Crotalus atrox and Crotalus mictlantecuhtli.

Here we report, for the first time, a natural hybrid between Crotalus atrox and C. mictlantecuhtli based on intermediate characteristics of the external morphology and venom. Morphologically, the individual had characteristics of both parent species. The hybrid's venom exhibited an intermediate composition including the presence of crotoxin which has never been documented in C. atrox but is well documented in C. mictlantecuhtli. The hybrid's venom was highly toxic and showed an intermediate proteolytic activity between the parental species. The two Mexican antivenoms were able to neutralize the hybrid's venom's lethality.

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 10 h 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 (≤2 h). 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.

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.

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 (ß SS = 0.588, p = 0.199) or females (ß SS = 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.

Toxin transcripts in Crotalus atrox venom and in silico structures of toxins.

The western diamondback rattlesnake (Crotalus atrox) is a common and widespread North American pit viper species, and its venom possesses medical applications. In this research, we identified 14 of the most common transcripts encoding 11 major venom toxins including transcripts for a three-finger toxin (3FTx) from the crude venom of C. atrox. In silico three-dimensional (3D) structures of 9 venom toxins were predicted by using deduced toxin amino acid sequences and a computer programme-MODELLER. The accuracy of all predicted toxin structures was evaluated by five stereochemical structure parameters including discrete optimised protein energy (DOPE) score, root mean square deviation (RMSD), Z-score, overall quality factor (ERRAT), and φ/ψ dihedral angle distribution of toxin backbone Cα residues, resulting that the overall predicted models are satisfied quality evaluation checks. Our present toxin transcripts and simulated individual toxin structures are important not only for revealing species-specific venom gene expression profiles, but also for predicting the toxin-toxin interactions and designing the structure-based toxin inhibitors for the treatment of snakebites.

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 neutralization efficacy of expired polyvalent antivenoms: An alternative option.

The expense of production and distribution of snakebite antivenom, as well as its relatively infrequent use, has caused antivenom to be increasingly difficult to obtain and ultimately producing an alarming global shortage. Unused, expired antivenom may represent a significant, untapped resource to ameliorate this crisis. This study examines the efficacy of expired antivenom over time using in vitro, whole blood clotting, and platelet function statistics. Representatives from three years for four different global brands of polyvalent antivenom were chosen and tested against their corresponding venoms as well as other venoms that could display cross-reactivity. These antivenoms include Wyeth Polyvalent (U.S.; exp. 1997, 2001, 2003), Antivipmyn® (Mexico; exp. 2005, 2013, 2017), Biotecfars Polyvalent (Venezuela; exp. 2010, 2014, 2016), and SAIMR (South Africa; exp. 1997, 2005, 2017). Venoms of species tested were Crotalus atrox against Wyeth; C. atrox and Crotalus vegrandis against Antivipmyn®; C. atrox, C. vegrandis and Bothrops colombiensis against Biotecfar; and Bitis gabonica and Echis carinatus against South African Institute for Medical Research (SAIMR). Parameters recorded were activated clotting time (ACT), clotting rate (CR), and platelet function (PF). Preliminary results are encouraging as the antivenoms maintained significant efficacy even 20 y after their expiration date. We anticipate these results will motivate further studies and provide hope in the cases of snakebite emergencies when preferable treatments are unavailable.

In vitro effects of Crotalus atrox snake venom on chick and mouse neuromuscular preparations.

The neuromuscular effect of venoms is not a major clinical manifestation shared between rattlesnakes native to the Americas, which showed two different venom phenotypes. Taking into account this dichotomy, nerve muscle preparations from mice and chicks were used to investigate the ability of Crotalus atrox venom to induce in vitro neurotoxicity and myotoxicity. Unlike crotalic venoms of South America, low concentrations of C. atrox venom did not result in significant effects on mouse neuromuscular preparations. The venom was more active on avian nerve-muscle, showing reduction of twitch heights after 120 min of incubation with 10, 30 and 100 µg/mL of venom with diminished responses to agonists and KCl. Histological analysis highlighted that C. atrox was myotoxic in both species of experimental animals; as evidenced by degenerative events, including edematous cells, delta lesions, hypercontracted fibers and muscle necrosis, which can lead to neurotoxic action. These results provide key insights into the myotoxicity and low neurotoxicity of C. atrox in two animal models, corroborating with previous genomic and proteomic findings and would be useful for a deeper understanding of venom evolution in snakes belonging to the genus Crotalus.

Identification of Lys49-PLA2 from crude venom of Crotalus atrox as a human neutrophil-calcium modulating protein.

We fortuitously observed a human neutrophil intracellular free-calcium concentration ([Ca(2+)]i) increasing activity in the commercially available phosphodiesterase I (PDE I), which is actually dried crude venom of Crotalus atrox. As this activity was not observed with another commercially available pure PDE I, we tried to find out the causative molecule(s) present in 'crude' PDE, and identified Lys49-phospholipase A2 (Lys49-PLA2 or K49-PLA2), a catalytically inactive protein which belongs to the phospholipase A2 family, by activity-driven three HPLC (reverse phase, size exclusion, reverse phase) steps followed by SDS-PAGE and LC-MS/MS. K49-PLA2 induced Ca(2+) infl ux in human neutrophils without any cytotoxic eff ect. Two calcium channel inhibitors, 2-aminoetoxydiphenyl borate (2-APB) (30 µM) and SKF-96365 (20 µM) signifi cantly inhibited K49-PLA2-induced [Ca(2+)]i increase. These results suggest that K49-PLA2 modulates [Ca(2+)]i in human neutrophils via 2-APB- and SKF-96365-sensitive calcium channels without causing membrane disruption.

Identification of Lys49-PLA2 from crude venom of Crotalus atrox as a human neutrophil-calcium modulating protein

We fortuitously observed a human neutrophil intracellular free-calcium concentration ([Ca2+]i) increasing activity in the commercially available phosphodiesterase I (PDE I), which is actually dried crude venom of Crotalus atrox. As this activity was not observed with another commercially available pure PDE I, we tried to find out the causative molecule(s) present in 'crude' PDE, and identified Lys49-phospholipase A2 (Lys49-PLA2 or K49-PLA2), a catalytically inactive protein which belongs to the phospholipase A2 family, by activity-driven three HPLC (reverse phase, size exclusion, reverse phase) steps followed by SDS-PAGE and LC-MS/MS. K49-PLA2 induced Ca2+ infl ux in human neutrophils without any cytotoxic eff ect. Two calcium channel inhibitors, 2-aminoetoxydiphenyl borate (2-APB) (30 microM) and SKF-96365 (20 microM) signifi cantly inhibited K49-PLA2-induced [Ca2+]i increase. These results suggest that K49-PLA2 modulates [Ca2+]i in human neutrophils via 2-APB- and SKF-96365-sensitive calcium channels without causing membrane disruption.

Irreversible inactivation of snake venom l-amino acid oxidase by covalent modification during catalysis of l-propargylglycine.

Snake venom l-amino acid oxidase (SV-LAAO, a flavor-enzyme) has attracted considerable attention due to its multifunctional nature, which is manifest in diverse clinical and biological effects such as inhibition of platelet aggregation, induction of cell apoptosis and cytotoxicity against various cells. The majority of these effects are mediated by H2O2 generated during the catalytic conversion of l-amino acids. The substrate analog l-propargylglycine (LPG) irreversibly inhibited the enzyme from Crotalus adamanteus and Crotalus atrox in a dose- and time-dependent manner. Inactivation was irreversible which was significantly protected by the substrate l-phenylalanine. A Kitz-Wilson replot of the inhibition kinetics suggested formation of reversible enzyme-LPG complex, which occurred prior to modification and inactivation of the enzyme. UV-visible and fluorescence spectra of the enzyme and the cofactor strongly suggested formation of covalent adduct between LPG and an active site residue of the enzyme. A molecular modeling study revealed that the FAD-binding, substrate-binding and the helical domains are conserved in SV-LAAOs and both His223 and Arg322 are the important active site residues that are likely to get modified by LPG. Chymotrypsin digest of the LPG inactivated enzyme followed by RP-HPLC and MALDI mass analysis identified His223 as the site of modification. The findings reported here contribute towards complete inactivation of SV-LAAO as a part of snake envenomation management.