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.

When one phenotype is not enough: divergent evolutionary trajectories govern venom variation in a widespread rattlesnake species.

Understanding the origin and maintenance of phenotypic variation, particularly across a continuous spatial distribution, represents a key challenge in evolutionary biology. For this, animal venoms represent ideal study systems: they are complex, variable, yet easily quantifiable molecular phenotypes with a clear function. Rattlesnakes display tremendous variation in their venom composition, mostly through strongly dichotomous venom strategies, which may even coexist within a single species. Here, through dense, widespread population-level sampling of the Mojave rattlesnake, Crotalus scutulatus, we show that genomic structural variation at multiple loci underlies extreme geographical variation in venom composition, which is maintained despite extensive gene flow. Unexpectedly, neither diet composition nor neutral population structure explain venom variation. Instead, venom divergence is strongly correlated with environmental conditions. Individual toxin genes correlate with distinct environmental factors, suggesting that different selective pressures can act on individual loci independently of their co-expression patterns or genomic proximity. Our results challenge common assumptions about diet composition as the key selective driver of snake venom evolution and emphasize how the interplay between genomic architecture and local-scale spatial heterogeneity in selective pressures may facilitate the retention of adaptive functional polymorphisms across a continuous space.

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.

Interfang Distances of Rattlesnakes: Sexual, Interspecific, and Body Size-related Variation, and Implications for Snakebite Research and Management.

OBJECTIVES: Snakebite severity corresponds to size of snake because the amount of venom a snake injects is positively associated with snake size. Because fang marks are often present on snakebite patients, we tested whether the relationship between snake length and distance between fang puncture wounds can be generalized for rattlesnakes of genus Crotalus. METHODS: We measured 2 interfang distances from 79 rattlesnakes of both sexes, 5 species, and varying body length: 1) distance between fang bases in anesthetized snakes, and 2) distance between fang punctures in a membrane-covered beaker bitten defensively. RESULTS: Statistical analyses supported our 2 hypotheses, that 1) body size-related fang divergence during fang protraction (ie, anterolateral movement during fang erection), and 2) the relationship between snake length and interfang distance are similar between the sexes and among different rattlesnake species. We therefore derived a general equation to estimate snake length based on distance between fang marks, and recommended 5 snake size categories: very small (<10 mm), small (10-15 mm), medium (15-20 mm), large (20-25 mm), and very large (>25 mm). CONCLUSIONS: The distance between fang marks on a snakebite patient may be used to estimate the size or size category of the offending snake, which in some cases may have predictive value for overall clinical severity of a given envenomation. Assessing interfang distance from puncture wounds can improve snakebite research and anticipation of snakebite severity.

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.

Sensationalistic journalism and tales of snakebite: are rattlesnakes rapidly evolving more toxic venom?

Recent reports in the lay press have suggested that bites by rattlesnakes in the last several years have been more severe than those in the past. The explanation, often citing physicians, is that rattlesnakes are evolving more toxic venom, perhaps in response to anthropogenic causes. We suggest that other explanations are more parsimonious, including factors dependent on the snake and factors associated with the bite victim's response to envenomation. Although bites could become more severe from an increased proportion of bites from larger or more provoked snakes (ie, more venom injected), the venom itself evolves much too slowly to explain the severe symptoms occasionally seen. Increased snakebite severity could also result from a number of demographic changes in the victim profile, including age and body size, behavior toward the snake (provocation), anatomical site of bite, clothing, and general health including asthma prevalence and sensitivity to foreign antigens. Clinical management of bites also changes perpetually, rendering comparisons of snakebite severity over time tenuous. Clearly, careful study taking into consideration many factors will be essential to document temporal changes in snakebite severity or venom toxicity. Presently, no published evidence for these changes exists. The sensationalistic coverage of these atypical bites and accompanying speculation is highly misleading and can produce many detrimental results, such as inappropriate fear of the outdoors and snakes, and distraction from proven snakebite management needs, including a consistent supply of antivenom, adequate health care, and training. We urge healthcare providers to avoid propagating misinformation about snakes and snakebites.

Venom Collection from Spiders and Snakes: Voluntary and Involuntary Extractions ("Milking") and Venom Gland Extractions.

Venom collection (often called "milking") provides the toxic secretions essential for studying animal venoms and/or generating venom products. Methods of venom collection vary widely, falling into three broad categories: voluntary venom extraction (inducing the animal to willingly release its venom), involuntary venom extraction (glandular massage, electrical stimulation, or administration of induction chemicals to promote venom expulsion), and venom gland extraction (surgical aspiration or trituration of homogenized gland tissue). Choice of method requires consideration of animal species, animal welfare, human safety (avoiding envenomation), venom yield and composition desired, and level of toxin purity required. Here, we summarize the materials and methods used to obtain venom by each of these approaches from spiders and snakes.

Denim clothing reduces venom expenditure by rattlesnakes striking defensively at model human limbs.

STUDY OBJECTIVE: Venomous snakebites can be painful, costly, and potentially life threatening. We seek to learn whether ordinary clothing (denim material from blue jeans) interferes with the kinematics of venom delivery, thereby reducing the amount of venom injected by a representative viper into a human limb. METHODS: In a laboratory study, we used model human limbs (warm, saline solution-filled gloves) to elicit defensive strikes from small and large southern Pacific rattlesnakes (Crotalus oreganus helleri). Each snake was videotaped biting a bare glove and a denim-covered glove. RESULTS: The snakes injected significantly less venom into denim-covered gloves than bare gloves during defensive strikes, with a 60% reduction for small snakes and 66% for large snakes. Latency to bite, number of bites, and duration of fang contact during the bite were similar for the 2 glove types, suggesting that the 2 targets elicited similar defensive behaviors and strikes. Several findings suggested that denim interfered with venom delivery, including the high proportion of dry bites for denim-covered gloves and the large quantity of venom spilled harmlessly on the denim cover. Large rattlesnakes struck more readily, maintained longer fang contact during the bite, and delivered 26 to 41 times more venom into gloves than small snakes. CONCLUSION: In our model, denim clothing proved effective at reducing venom injection by both small and large rattlesnakes. Wearing long denim pants as an alternative to shorts may provide a simple, low-cost means of reducing the severity of snakebites.

Case reports of envenomation and venom composition differences between two Arizona populations of the Southwestern Speckled Rattlesnake, Crotalus pyrrhus (Cope, 1867).

Envenomations by the Southwestern Speckled Rattlesnake (Crotalus pyrrhus) are fairly rare. Previous descriptions in the literature do not include locality, an important factor in the clinical symptoms or syndromes of snakebites resulting from geographic variation in venom composition. Here, we describe two cases of envenoming by C. pyrrhus from two Arizona localities (Tinajas Altas Mountains, Yuma County, and Phoenix Mountains, Maricopa County). Both patients experienced swelling, but neither demonstrated coagulopathy, thrombocytopenia, or hypofibrinogenemia. The Phoenix Mountains patient developed hemorrhagic bullae and tissue damage in his bitten extremity, necessitating the amputation of the distal portion of his middle finger. Treatment for both consisted of medication for pain, isotonic crystalloid, and antivenom therapy with recovery in each case. Based on visual inspection of 1D-gels and RP-HPLC chromatograms, venom samples were largely similar but appeared to differ quantitatively for several toxin families between and within populations.

Surgically-induced brain injury: where are we now?

Neurosurgical procedures cause inevitable brain damage from the multitude of surgical manipulations utilized. Incisions, retraction, thermal damage from electrocautery, and intraoperative hemorrhage cause immediate and long-term brain injuries that are directly linked to neurosurgical operations, and these types of injuries, collectively, have been termed surgical brain injury (SBI). For the past decade, a model developed to study the underlying brain pathologies resulting from SBI has provided insight on cellular mechanisms and potential therapeutic targets. This model, as seen in a rat, mouse, and rabbit, mimics a neurosurgical operation and causes commonly encountered post-operative complications such as brain edema, neuroinflammation, and hemorrhage. In this review, we elaborate on SBI and its clinical impact, the SBI animal models and their clinical relevance, the importance of applying therapeutics before neurosurgical procedures (i.e., preconditioning), and the new direction of applying venom-derived proteins to attenuate SBI.

The effects of rearing method on social behaviors of mentored, captive-reared juvenile California condors.

Puppet-reared and parent-reared captive-bred California condor (Gymnogyps californianus) juveniles were studied before their release into the wild. Behavioral data were collected during social interactions within two cohorts of juveniles (N = 11) and their adult mentors (N = 5). The purposes of this study were to (1) document the social behaviors of mentored juvenile California condors, and (2) compare social behaviors for two different rearing methods (puppet-reared versus parent-reared) during two phases of the mentoring process (San Diego Wild Animal Park versus release sites). Of the 17 behaviors examined by 2 x 2 analyses of variance (ANOVAs), two significant interactions between the rearing method and mentoring phase were found: pulls feathers and feeds alone. For both behaviors, parent-reared condors engaged in these activities more often at the zoo and less often at the release pens than did the puppet-reared condors. The main effect of rearing was also significant for two behaviors: near others, and receives contact aggression from other. Parent-reared birds were more likely to be near another bird and receive contact aggression, regardless of mentoring phase, than puppet-reared birds. The effect size for 16 of the 17 behaviors was greater for the rearing method than for mentoring phase. Rearing method differences may persist long-term, as parent-reared adult mentors were significantly more aggressive than puppet-reared adult mentors. Dominance relations were examined for both cohorts, with the first cohort exhibiting a strong linear relationship (h' = 0.86, P = 0.018), whereas the second cohort exhibited a moderate but non-significant linear hierarchy (h' = 0.63, P = 0.21). The rearing method had no effect on dominance among the juveniles, but adults were probably dominant to juveniles (P = 0.052; the difference was nearly significant). Although social behaviors between the two rearing groups were similar in most respects, this study is the first to document measurable differences between puppet- and parent-reared captive-bred California condor juveniles. Zoo Biol 27:1-18, 2008. (c) 2007 Wiley-Liss, Inc.

Complement inactivating proteins and intraspecies venom variation in Crotalus oreganus helleri.

Complement inactivating properties were detected in venom from the southern California distribution of Crotalus oreganus helleri (Southern Pacific Rattlesnake). This activity showed strong geographic bias to the San Bernardino Mountain range, and venom from this area reacted strongly with Fraction 5 antiserum (AF5). However, venoms from the San Jacinto Mountain range, which have been previously shown to contain Mojave toxin, did not inhibit complement and did not react with AF5. AF5 has been previously shown to recognize a protease in C. scutulatus venom that inactivates complement, but the identity of this protein has remained unknown. Using a functional venomic approach, utilizing two-dimensional gel electrophoresis coupled with liquid chromatography and tandem mass spectrometry (LC/MS/MS), we have identified catrocollastatin and hemorrhagic toxin II (HT-2) as the primary proteins recognized by AF5. The information we present within this manuscript further illustrates the now well-known reality of intraspecies venom variation and the challenges faced in providing comprehensive polyvalent antivenoms.

Diverse habitat use during two life stages of the critically endangered Bahama Oriole (Icterus northropi): community structure, foraging, and social interactions.

Our ability to prevent extinction in declining populations often depends on effective management of habitats that are disturbed through wildfire, logging, agriculture, or development. In these disturbed landscapes, the juxtaposition of multiple habitat types can be especially important to fledglings and young birds, which may leave breeding grounds in human-altered habitat for different habitats nearby that provide increased foraging opportunities, reduced competition, and higher protection from predators. In this study, we evaluated the importance of three habitat types to two life stages of the critically endangered Bahama Oriole (Icterus northropi), a synanthropic songbird endemic to Andros, The Bahamas. First, we determined the avian species composition and relative abundance of I. northropi among three major vegetation types on Andros: Caribbean pine (Pinus caribaea) forest, coppice (broadleaf dry forest), and anthropogenic areas, dominated by nonnative vegetation (farmland and developed land). We then compared the foraging strategies and social interactions of two age classes of adult Bahama Orioles in relation to differential habitat use. Bird surveys late in the Bahama Oriole's breeding season indicated the number of avian species and Bahama Oriole density were highest in coppice. Some bird species occurring in the coppice and pine forest were never observed in agricultural or residential areas, and may be at risk if human disturbance of pine forest and coppice increases, as is occurring at a rapid pace on Andros. During the breeding season, second-year (SY) adult Bahama Orioles foraged in all vegetation types, whereas after-second-year (ASY) adults were observed foraging only in anthropogenic areas, where the species nested largely in introduced coconut palms (Cocos nucifera). Additionally, SY adults foraging in anthropogenic areas were often observed with an ASY adult, suggesting divergent habitat use for younger, unpaired birds. Other aspects of foraging (vegetation features, food-gleaning behavior, and food items) were similar for the two age classes. Older Bahama Orioles exhibited relatively higher rates of social interactions (intraspecific and interspecific pooled) in anthropogenic areas, and won more interaction outcomes compared to younger adults. Our findings concur with those of other studies indicating dry broadleaf forest is vitally important to migrating, wintering, and resident birds, including the critically endangered Bahama Oriole, which appears to depend heavily on this vegetation type during certain life stages.

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.

Mosquito Capture Rate Using CO2-Baited Traps in Relation to Distance From Water and Height: Implications for Avian Disease Transmission.

Accumulating evidence suggests that enzootic transmission of pathogens such as West Nile virus (WNV) by mosquitoes is governed by host-bird interactions, including mosquito preferences for specific species and developmental stages of host birds, host bird availability, and host defensive behavior. Here, we examined how the attack rate of five mosquito species in southern California was influenced by the position of CO2-baited traps in relation to distance from water and trap height. We identified 44,207 female mosquitoes representing five species: Aedes vexans Meigen, Anopheles franciscanus McCracken, Anopheles hermsi Barr & Guptavanij, and the two most abundant species which are also WNV vectors, Culex erythrothorax Dyar and Culex tarsalis Coquillett. Mosquito captures decreased markedly with trap height, and also decreased with distance from a riparian area but not with distance from an open water source lacking a vegetated border. The results of this study suggest that WNV-competent ornithophilic mosquitoes may amplify the virus especially in reservoir birds that roost or nest close to the ground and near riparian vegetation.

Venom-spraying behavior of the scorpion Parabuthus transvaalicus (Arachnida: Buthidae).

Many animals use chemical squirting or spraying behavior as a defensive response. Some members of the scorpion genus Parabuthus (family Buthidae) can spray their venom. We examined the stimulus control and characteristics of venom spraying by Parabuthus transvaalicus to better understand the behavioral context for its use. Venom spraying occurred mostly, but not always, when the metasoma (tail) was contacted (usually grasped by forceps), and was absent during stinging-like thrusts of the metasoma apart from contact. Scorpions were significantly more likely to spray when contact was also accompanied by airborne stimuli. Sprays happened almost instantaneously following grasping by forceps (median=0.23s) as a brief (0.07-0.30s, mean=0.18s), fine stream (<5° arc) that was not directed toward the stimulus source; however, rapid independent movements of the metasoma and/or telson (stinger) often created a more diffuse spray, increasing the possibility of venom contact with the sensitive eyes of potential scorpion predators. Successive venom sprays varied considerably in duration and velocity. Collectively, these results suggest that venom spraying might be useful as an antipredator function and can be modulated based on threat.

Geographic variation and genetic structure in the Bahama Oriole (Icterus northropi), a critically endangered synanthropic species.

Bird species may exhibit unexpected population structuring over small distances, with gene flow restricted by geographic features such as water or mountains. The Bahama Oriole (Icterus northropi) is a critically endangered, synanthropic island endemic with a declining population of fewer than 300 individuals. It now remains only on Andros Island (The Bahamas), which is riddled with waterways that past studies assumed did not hinder gene flow. We examined 1,858 base pairs of mitochondrial DNA sequenced from four gene regions in 14 birds (roughly 5% of the remaining population) found on the largest land masses of Andros Island (North Andros and Mangrove Cay/South Andros). We sought to discern genetic structuring between the remaining subpopulations and its relationship to current conservation concerns. Four unique haplotypes were identified, with only one shared between the two subpopulations. Nucleotide and haplotype diversity were higher for the North Andros subpopulation than for the Mangrove Cay/South Andros subpopulation. Analysis of molecular variance (AMOVA) yielded a Wright's fixation index (F st) of 0.60 (P Fst = 0.016), with 40.2% of the molecular variation explained by within-population differences and 59.8% by among-population differences. Based on the mitochondrial regions examined in this study, we suggest the extant subpopulations of Bahama Oriole exhibit significant population structuring over short distances, consistent with some other non-migratory tropical songbird species.

The dilemma of choosing a reference character for measuring sexual size dimorphism, sexual body component dimorphism, and character scaling: cryptic dimorphism and allometry in the scorpion Hadrurus arizonensis.

Sexual differences in morphology, ranging from subtle to extravagant, occur commonly in many animal species. These differences can encompass overall body size (sexual size dimorphism, SSD) or the size and/or shape of specific body parts (sexual body component dimorphism, SBCD). Interacting forces of natural and sexual selection shape much of the expression of dimorphism we see, though non-adaptive processes may be involved. Differential scaling of individual features can result when selection favors either exaggerated (positive allometry) or reduced (negative allometry) size during growth. Studies of sexual dimorphism and character scaling rely on multivariate models that ideally use an unbiased reference character as an overall measure of body size. We explored several candidate reference characters in a cryptically dimorphic taxon, Hadrurus arizonensis. In this scorpion, essentially every body component among the 16 we examined could be interpreted as dimorphic, but identification of SSD and SBCD depended on which character was used as the reference (prosoma length, prosoma area, total length, principal component 1, or metasoma segment 1 width). Of these characters, discriminant function analysis suggested that metasoma segment 1 width was the most appropriate. The pattern of dimorphism in H. arizonensis mirrored that seen in other more obviously dimorphic scorpions, with static allometry trending towards isometry in most characters. Our findings are consistent with the conclusions of others that fecundity selection likely favors a larger prosoma in female scorpions, whereas sexual selection may favor other body parts being larger in males, especially the metasoma, pectines, and possibly the chela. For this scorpion and probably most other organisms, the choice of reference character profoundly affects interpretations of SSD, SBCD, and allometry. Thus, researchers need to broaden their consideration of an appropriate reference and exercise caution in interpreting findings. We highly recommend use of discriminant function analysis to identify the least-biased reference character.

Mojave toxin in venom of Crotalus helleri (Southern Pacific Rattlesnake): molecular and geographic characterization.

Mojave toxin (MT) was detected in five of 25 Crotalus helleri (Southern Pacific rattlesnake) sampled using anti-MT antibodies and nucleotide sequence analysis. All of the venoms that were positive for MT were collected from Mt San Jacinto in Riverside Co., California. Since this population is geographically isolated from C. scutulatus scutulatus (Mojave rattlesnake), it is unlikely that this finding is due to recent hybridization. MT concentration differences between C. helleri and C. s. scutulatus reflected the presence of 'isoforms' of the toxin in the venom. Whereas C. s. scutulatus generally has several isoforms of the toxin (detected by Western blotting), only one 'isoform' that focused at pI 5.1 was detected in C. helleri. Both acidic and basic subunits of MT sequences were obtained from C. helleri DNA with primers specific for MT, but only from snakes that had MT in their venom. The sequence identity of the C. helleri acidic subunit to the C. s. scutulatus subunit was 84.9%, whereas the sequence identity of the C. helleri basic subunit was 97% to the C. s. scutulatus basic subunit. Using casein, fibrin, and hide powder azure as substrates, assays for proteolytic activity suggested that C. helleri possesses several different types of metalloproteinases in their venom. However, proteolytic activity was not detected, or present in reduced amounts, in specimens having MT. Clinical neurotoxicity following envenomation by certain populations of C. helleri may be due to MT.

Pressure immobilization delays mortality and increases intracompartmental pressure after artificial intramuscular rattlesnake envenomation in a porcine model.

STUDY OBJECTIVES: We determine the effect of pressure immobilization on mortality and intracompartmental pressure after artificial intramuscular Crotalus atrox envenomation in a porcine model. METHODS: We prospectively studied 20 pigs using a randomized, controlled design. After anesthesia, C atrox venom (20 mg/kg) was injected with a 22-gauge needle 10 mm deep into the tibialis anterior muscle of the hind leg. Pigs were randomized to receive either pressure immobilization (applied 1 minute after envenomation and maintained throughout the duration of the experiment) or no pressure immobilization. We measured time to death, intracompartmental pressure before venom injection and at 2 hours after injection, and leg circumference at a standardized location before injection and immediately postmortem. Duration of survival was compared using Kaplan-Meier survival analysis. RESULTS: The dose of venom resulted in 100% mortality. The median survival was longer in the pressure immobilization group (191 minutes, range 140 to 240 minutes) than in the control group (median 155 minutes, range 119 to 187 minutes). The difference between the groups was 36 minutes (95% confidence interval [CI] 2 to 64 minutes; P =.0122). The mean intracompartmental pressures were 67+/-13 mm Hg+/-SD with pressure immobilization and 24+/-5 mm Hg without pressure immobilization. The difference between groups was 43 mm Hg (95% CI 32 to 53 mm Hg). The mean circumferences were 14.3 cm in the pressure immobilization group and 19.1 cm in the control group. The difference between groups was -4.8 cm (95% CI -5.7 to -3.9 cm). CONCLUSION: Compared with control animals without treatment, the pressure immobilization group had longer survival, less swelling, and higher intracompartmental pressures after artificial, intramuscular C atrox envenomation in our porcine model.