Paradoxical Exception to Island Tameness: Increased Defensiveness in an Insular Population of Rattlesnakes.

Island tameness results largely from a lack of natural predators. Because some insular rattlesnake populations lack functional rattles, presumably the consequence of relaxed selection from reduced predation, we hypothesized that the Santa Catalina Island, California, USA, population of the southern Pacific rattlesnake (Crotalus helleri, which possesses a functional rattle), would exhibit a decrement in defensive behavior relative to their mainland counterparts. Contrary to our prediction, rattlesnakes from the island not only lacked tameness compared to mainland snakes, but instead exhibited measurably greater levels of defensiveness. Island snakes attempted to bite 4.7 times more frequently as we endeavored to secure them by hand, and required 2.1-fold more time to be pinned and captured. When induced to bite a beaker after being grasped, the island snakes also delivered 2.1-fold greater quantities of venom when controlling for body size. The additional venom resulted from 2.1-fold larger pulses of venom ejected from the fangs. We found no effects of duration in captivity (2-36 months), which suggests an absence of long-term habituation of antipredator behaviors. Breeding bird surveys and Christmas bird counts indicated reduced population densities of avian predators on Catalina compared to the mainland. However, historical estimates confirmed that populations of foxes and introduced mammalian predators (cats and pigs) and antagonists (herbivorous ungulates) substantially exceeded those on the mainland in recent centuries, and therefore best explain the paradoxically exaggerated defensive behaviors exhibited by Catalina's rattlesnakes. These findings augment our understanding of anthropogenic effects on the behaviors of island animals and underscore how these effects can negatively affect human safety.

Crotalus atrox disintegrin reduces hemorrhagic transformation by attenuating matrix metalloproteinase-9 activity after middle cerebral artery occlusion in hyperglycemic male rats.

Hemorrhagic transformation after ischemic stroke is an independent predictor for poor outcome and is characterized by blood vessel rupture leading to brain edema. To date, no therapies for preventing hemorrhagic transformation exist. Disintegrins from the venom of Crotalus atrox have targets within the coagulation cascade, including receptors on platelets. We hypothesized that disintegrins from C. atrox venom can attenuate hemorrhagic transformation by preventing activation of matrix metalloproteinase after middle cerebral artery occlusion (MCAO) in hyperglycemic rats. We subjected 48 male Sprague-Dawley rats weighing 240-260 g to MCAO and hyperglycemia to induce hemorrhagic transformation of the infarction. At reperfusion, we administered either saline (vehicle), whole C. atrox venom (two doses were used), or fractionated C. atrox venom (HPLC Fraction 2). Rats were euthanized 24 hr post-ictus for measurement of infarction and hemoglobin volume. Reversed-phase HPLC was performed to fractionate the whole venom and peaks were combined to form Fraction 2, which contained the disintegrin Crotatroxin. Fraction 2 protected against hemorrhagic transformation after MCAO, and attenuated activation of matrix metalloproteinase-9. Administering matrix metalloproteinase antagonists prevented the protection by Fraction 2. The results of this study indicate that disintegrins found in C. atrox venom may have therapeutic potential for reducing hemorrhagic transformation after ischemic stroke. Moreover, the RP-HPLC fractions retained sufficient protein activity to suggest that gentler and less efficient orthogonal chromatographic methods may be unnecessary to isolate proteins and explore their function.

Comparative analysis of the high molecular mass subproteomes of eight Bothrops snake venoms.

Snake venoms are extremely active biological secretions composed primarily of various classes of enzymes. The genus Bothrops comprises various pit viper species that represent the most medically significant taxa in Central and South America, accounting for more human envenomations and fatalities than any other snakes in the region. Venom proteomes of many Bothrops species have been well-characterized but investigations have focused almost exclusively on proteins smaller than 100 kDa despite expression of larger components being documented in several Bothrops venoms. This study sought to achieve detailed identification of major components in the high molecular mass subproteome of venoms from eight Bothrops species (B. brazili, B. cotiara, B. insularis, B. jararaca, B. jararacussu, B. leucurus, B. moojeni and B. neuwiedi). Enzymes such as metalloproteinases and L-amino acid oxidases were the most prominent components identified in the first size-exclusion chromatography fractions of these venoms. Minor components also identified in the first peaks included 5'-nucleotidase, aminopeptidase, phosphodiesterase, and phospholipases A2 and B. Most of these components disappeared in electrophoretic profiles under reducing conditions, suggesting that they may be composed of more than one polypeptide chain. A significant shift in the molecular masses of these protein bands was observed following enzymatic N-deglycosylation, indicating that they may contain N-glycans. Furthermore, none of the identified high molecular mass proteins were shared by all eight species, revealing a high level of interspecific variability among these venom components.

Crotalus helleri venom preconditioning reduces postoperative cerebral edema and improves neurological outcomes after surgical brain injury.

INTRODUCTION: Postoperative cerebral edema is a devastating complication in neurosurgical patients. Loss of blood-brain barrier integrity has been shown to lead to the development of brain edema following neurosurgical procedures. The aim of this study was to evaluate preconditioning with Crotalus helleri venom (Cv-PC) as a potential preventive therapy for reducing postoperative brain edema in the rodent SBI model. C. helleri venom is known to contain phospholipase A2 (PLA2), an enzyme upstream to cyclooxygenase-2 (COX-2) in the inflammatory cascade, acts to increase the production of inflammatory mediators, such as prostaglandins. We hypothesize that Cv-PC will downregulate the response of the COX-2 pathway to injury, thereby reducing the inflammatory response and the development of brain edema after SBI. MATERIALS AND METHODS: 75 male Sprague Dawley rats (280-330g) were divided to the following groups-naïve+vehicle, naïve+Cv-PC, sham, vehicle, Cv-PC, Cv-PC+NS398 (COX-2 inhibitor). Vehicle preconditioned and Cv-PC animals received either three daily subcutaneous doses of saline or C. helleri venom at 72h, 48h, and 24h prior to surgery. In Cv-PC+NS398 animals, NS398 was administered intraperitoneally 1h prior to each Cv-PC injection. Sham-operated animals received craniotomy only, whereas SBI animals received a partial right frontal lobectomy. Neurological testing and brain water content were assessed at 24h and 72h after SBI; COX-2 and PGE2 expression was assessed at 24h postoperatively by Western blot and immunohistochemistry, respectively. RESULTS: At 24h after SBI, the vehicle-treated animals were observed to have increased brain water content (83.1±0.2%) compared to that of sham animals (80.2±0.1%). The brain water content of vehicle-treated animals at 72h post-SBI was elevated at 83.3±0.2%. Cv-PC-treated animals with doses of 10% LD50 had significantly reduced brain water content of 81.92±0.7% and 81.82±0.3% at 24h and 72h, respectively, after SBI compared to that of vehicle-treated animals, while Cv-PC with 5% LD50 doses showed brain water content that trended lower but did not reach statistical significance. At 24h and 72h post-SBI, Cv-PC-treated animals had significantly higher neurological score than vehicle-treated animals. The COX-2 over-expression characterized in SBI was attenuated in Cv-PC-treated animals; NS398 reversed the protective effect of Cv-PC on COX-2 expression. Cv-PC tempered the over-expression of the inflammatory marker PGE2. CONCLUSION: Our findings indicate that Cv-PC may provide a promising therapy for reducing postoperative edema and improving neurological function after neurosurgical procedures.

Crotalus atrox venom preconditioning increases plasma fibrinogen and reduces perioperative hemorrhage in a rat model of surgical brain injury.

Perioperative bleeding is a potentially devastating complication in neurosurgical patients, and plasma fibrinogen concentration has been identified as a potential modifiable risk factor for perioperative bleeding. The aim of this study was to evaluate preconditioning with Crotalus atrox venom (Cv-PC) as potential preventive therapy for reducing perioperative hemorrhage in the rodent model of surgical brain injury (SBI). C. atrox venom contains snake venom metalloproteinases that cleave fibrinogen into fibrin split products without inducing clotting. Separately, fibrinogen split products induce fibrinogen production, thereby elevating plasma fibrinogen levels. Thus, the hypothesis was that preconditioning with C. atrox venom will produce fibrinogen spilt products, thereby upregulating fibrinogen levels, ultimately improving perioperative hemostasis during SBI. We observed that Cv-PC SBI animals had significantly reduced intraoperative hemorrhage and postoperative hematoma volumes compared to those of vehicle preconditioned SBI animals. Cv-PC animals were also found to have higher levels of plasma fibrinogen at the time of surgery, with unchanged prothrombin time. Cv-PC studies with fractions of C. atrox venom suggest that snake venom metalloproteinases are largely responsible for the improved hemostasis by Cv-PC. Our findings indicate that Cv-PC increases plasma fibrinogen levels and may provide a promising therapy for reducing perioperative hemorrhage in elective surgeries.

Intraspecific venom variation in the medically significant Southern Pacific Rattlesnake (Crotalus oreganus helleri): biodiscovery, clinical and evolutionary implications.

Due to the extreme variation of venom, which consequently results in drastically variable degrees of neutralization by CroFab antivenom, the management and treatment of envenoming by Crotalus oreganus helleri (the Southern Pacific Rattlesnake), one of the most medically significant snake species in all of North America, has been a clinician's nightmare. This snake has also been the subject of sensational news stories regarding supposed rapid (within the last few decades) evolution of its venom. This research demonstrates for the first time that variable evolutionary selection pressures sculpt the intraspecific molecular diversity of venom components in C. o. helleri. We show that myotoxic ß-defensin peptides (aka: crotamines/small basic myotoxic peptides) are secreted in large amounts by all populations. However, the mature toxin-encoding nucleotide regions evolve under the constraints of negative selection, likely as a result of their non-specific mode of action which doesn't enforce them to follow the regime of the classic predator-prey chemical arms race. The hemorrhagic and tissue destroying snake venom metalloproteinases (SVMPs) were secreted in larger amounts by the Catalina Island and Phelan rattlesnake populations, in moderate amounts in the Loma Linda population and in only trace levels by the Idyllwild population. Only the Idyllwild population in the San Jacinto Mountains contained potent presynaptic neurotoxic phospholipase A2 complex characteristic of Mohave Rattlesnake (Crotalus scutulatus) and Neotropical Rattlesnake (Crotalus durissus terrificus). The derived heterodimeric lectin toxins characteristic of viper venoms, which exhibit a diversity of biological activities, including anticoagulation, agonism/antagonism of platelet activation, or procoagulation, appear to have evolved under extremely variable selection pressures. While most lectin α- and ß-chains evolved rapidly under the influence of positive Darwinian selection, the ß-chain lectin of the Catalina Island population appears to have evolved under the constraint of negative selection. Both lectin chains were conspicuously absent in both the proteomics and transcriptomics of the Idyllwild population. Thus, we not only highlight the tremendous biochemical diversity in C. o. helleri's venom-arsenal, but we also show that they experience remarkably variable strengths of evolutionary selection pressures, within each toxin class among populations and among toxin classes within each population. The mapping of geographical venom variation not only provides additional information regarding venom evolution, but also has direct medical implications by allowing prediction of the clinical effects of rattlesnake bites from different regions. Such information, however, also points to these highly variable venoms as being a rich source of novel toxins which may ultimately prove to be useful in drug design and development. BIOLOGICAL SIGNIFICANCE: These results have direct implications for the treatment of envenomed patients. The variable venom profile of Crotalus oreganus helleri underscores the biodiscovery potential of novel snake venoms.

Poisons, toxungens, and venoms: redefining and classifying toxic biological secretions and the organisms that employ them.

Despite extensive study of poisonous and venomous organisms and the toxins they produce, a review of the literature reveals inconsistency and ambiguity in the definitions of 'poison' and 'venom'. These two terms are frequently conflated with one another, and with the more general term, 'toxin.' We therefore clarify distinctions among three major classes of toxins (biological, environmental, and anthropogenic or man-made), evaluate prior definitions of venom which differentiate it from poison, and propose more rigorous definitions for poison and venom based on differences in mechanism of delivery. We also introduce a new term, 'toxungen', thereby partitioning toxic biological secretions into three categories: poisons lacking a delivery mechanism, i.e. ingested, inhaled, or absorbed across the body surface; toxungens delivered to the body surface without an accompanying wound; and venoms, delivered to internal tissues via creation of a wound. We further propose a system to classify toxic organisms with respect to delivery mechanism (absent versus present), source (autogenous versus heterogenous), and storage of toxins (aglandular versus glandular). As examples, a frog that acquires toxins from its diet, stores the secretion within cutaneous glands, and transfers the secretion upon contact or ingestion would be heteroglandular-poisonous; an ant that produces its own toxins, stores the secretion in a gland, and sprays it for defence would be autoglandular-toxungenous; and an anemone that produces its own toxins within specialized cells that deliver the secretion via a penetrating wound would be autoaglandular-venomous. Adoption of our scheme should benefit our understanding of both proximate and ultimate causes in the evolution of these toxins.