Crotalus Viridis Research Articles

Ability of wedelolactone, heparin, and para-bromophenacyl bromide to antagonize the myotoxic effects of two crotaline venoms and their PLA 2 myotoxins

We examined the ability of wedelolactone, heparin and para-bromophenacyl bromide to antagonize the myotoxic activity in mice of venoms from Crotalus viridis viridis and Agkistrodon contortrix laticinctus and two phospholipase A 2 myotoxins, CVV myotoxin and ACL myotoxin, isolated from them. Myotoxicity was measured by the increase in plasma creatine kinase (CK) activity at two hours and histological changes in extensor digitorum longus muscle (EDL) at three hours after injection of the test solution. Both heparin and wedelolactone independently reduced the myotoxic effect of both crude venoms and both myotoxins, but wedelolactone was more effective. Wedelolactone plus heparin reduced the myotoxic effect of CVV myotoxin more than either antagonist alone. The PLA 2 inhibitor, para-bromophenacyl bromide ( pBPB), reduced the myotoxic effect of both myotoxins more than either wedelolactone or heparin. On the other hand, the myotoxic effect of polylysine was not reduced by either wedelolactone or para-bromophenacyl bromide, but it was reduced by heparin. These results indicate that wedelolactone, para-bromophenacyl bromide and heparin are antagonists of these two phospholipase A 2 myotoxins, and that antagonism by the first two compounds may be due to a more specific interaction with these proteins than that by the latter.

Allometry and sexual dimorphism in the lung morphology of prairie rattlesnakes, Crotalus viridis viridis

AbstractWe quantified sexual dimorphism and allometric changes in the lung morphology of 160 juvenile and adult specimens of prairie rattlesnake, Crotalus viridis viridis, from a single population. In virtually all lung components, those of males are located more posteriorly than are those of females of the same body size. Males display a longer vascular component than females but there is no sexual dimorphism in size of the avascular component. Thus, males generally have longer lungs than do females at all body sizes. With increasing body size, the lung components are found more anteriorly, relative length of the vascular lung decreases, and relative length of the avascular lung increases in both sexes. However, total lung length increases isometrically with body size. These sexual and allometric differences suggest that intraspecific variation should be taken into account when lung size characters are used in snake systematic studies.

The detection of hemorrhagic proteins in snake venoms using monoclonal antibodies against virginia opossum ( Didelphis virginiana) serum

Most snakes and a few warm-blooded animals have a resistance to snake venoms because of naturally occuring antihemorrhagins found in their sera. The antihemorrhagins in serum of Virginia opossum ( Didelphis virginiana) neutralize hemorrhagic activity by binding to hemorrhagins in snake venoms. The binding characteristic of antihemorrhagins in D. virginiana serum was used to develop a five-step western blot. The detection of hemorrhagic proteins were measured indirectly with antihemorrhagins in Virginia opossum serum and with DV-2LD#2, a monoclonal antibody specific for Virginia opossum antihemorrhagins. Snake venoms were separated by native-PAGE, transferred to a Millipore Immobilon™-P membrane and then incubated with crude Virginia opossum serum. The hemorrhagins in snake venom bind to antihemorrhagins in Virginia opossum serum which react with DV-2LD#2 a monoclonal antibody that is specific for Virginia opossum antihemorrhagins. DV-2LD#2 monoclonal antibody inhibits antihemorrhagic activity in Virginia opossum serum when mixed in equal amounts. The inhibition of antihemorrhagins by DV-2LD#2 monoclonal antibody suggests specificity. DV-2LD#2 monoclonal antibody does not recognize antihemorrhagins in gray woodrat ( Neotoma micropus) serum. The five-step western blot reveals two well-defined bands which represent hemorrhagins found in Western diamondback rattlesnake ( Crotalus atrox) venom. Venoms from 15 different snake species were examined to determine the usefulness of the five-step western blot. Other hemorrhagic venoms (Great Basin rattlesnake ( C. viridis lutosus), Prairie rattlesnake ( C. viridis viridis), Tancitaran dusky rattlesnake ( C. pusillus), Northern Mojave rattlesnake ( C. scutulatus scutulatus type B) and Northern Pacific rattlesnake ( C. v. oreganus)) had one single band in the five-step western blot. DV-2LD#2 did not bind to the non-hemorrhagic venoms and reacted with 50% of the hemorrhagic venoms used in this study. The monoclonal antibody, CAH, reacted with all the hemorrhagic venoms except for the venom of the King cobra ( Ophiophagus hannah) and did not react with the non-hemorrhagic venoms. The hemorrhagic binding site of CAH monoclonal antibody and the antihemorrhagin in Virginia opossum are different binding sites. The five-step western blot will be a very useful assay for determining hemorrhagic activity without using live animals.

Properties of specific binding site of myotoxin a, a powerful convulsant, in brain microsomes

Myotoxin a, a small basic polypeptide from prairie rattlesnakes (Crotalus viridis viridis), induces myonecrosis and binds to a single class of binding sites in skeletal muscle sarcoplasmic reticulum. In the present study, [125I]myotoxin a with a high specific activity was prepared and it was shown to bind mainly to microsomes in rat whole brain. [125I]Myotoxin a was further shown to bind to microsomes prepared from all regions tested in brain. Its specific binding to whole brain microsomes was of approximately 1.9 times lower affinity (KD = 0.76 microM; Bmax = 13.1 nmol/mg) than that to skeletal muscle sarcoplasmic reticulum. [125I]Myotoxin a binding to brain microsomes was displaced by unlabeled myotoxin a with an IC50 value of 4.5 microM. [125I]Myotoxin a binding was markedly reduced by treatment of microsomes with trypsin, suggesting that the binding site of [125I]myotoxin a is partially proteins. The binding was significantly inhibited by Mg2+ at concentrations above 1 mM. Having looked at several drugs, we noted that [125I]myotoxin a binding was noncompetitively inhibited by spermine, whereas it was enhanced by heparin. On the other hand, the i.c.v. injection of myotoxin a in mice induced potent convulsive effects at 0.05 nmol/mouse or more. This paper is the first to show that the specific binding site of myotoxin a is present in mouse brain and that myotoxin a is a novel peptidic convulsant in mice.

The Sharon Springs Roundup and Prairie Rattlesnake Demography

In the present century rattlesnake roundups have become popular in various parts of the United States as a form of recreation and as a means of generating income for the sponsoring agencies. Various species of rattlesnakes including Crotalus atrox, C. adamanteus, C. horridus, and C. viridis are hunted. The only Kansas roundup was initiated in 1992 in the town of Sharon Springs and C. viridis is the species hunted. Attributes of the Kansas prairie rattlesnake as a game animal are its fertility (mean litter 10.7) and early maturity (third year). About 80% of females produce young each year, unlike more northern populations of the species that reproduce biennially or triennially. In the Kansas prairie, rattlesnakes natural attrition amounts to from one-fourth to one-third annually. Relatively few of the snakes survive for as long as ten years. In order to assure maintenance of a supply of snakes sufficient to provide the recreation and income expected from the roundup, conservation measures must be adopted. Size limits and bag limits should be enforced. About one-fourth of the snakes captured are gravid females, and these should be released unharmed to ensure a continuing supply.

Rattling Behavior of Prairie Rattlesnakes (Crotalus viridis viridis, Viperidae) in Relation to Sex, Reproductive Status, Body Size, and Body Temperature

AbstractWe examined whether sex, reproductive status, body size, or body temperature of prairie rattlesnakes (Crotalus viridis viridis) was related to when snakes rattled in response to an approaching observer. We found that gravid females allowed significantly closer approaches than males, suggesting that females relied on crypsis to avoid predation, possibly because pregnancy constrained their locomotive ability. Smaller snakes allowed significantly closer approaches than did larger snakes. Smaller snakes may be more cryptic or slower, which may influence their waiting to rattle until the observer was close. Overall, we found no consistent relationship between the distance from the observer that a snake rattled and its body temperature. However, cooler gravid females allowed closer approaches by the observer than did warmer gravid females. In summary, reproductive status, body size, and body temperature appear to influence the costs and benefits of crypsis vs. active defense of rattlesnakes.

Detection of isoforms and isomers of rattlesnake myotoxins by capillary electrophoresis and matrix-assisted laser desorption time-of-flight mass spectrometry

Capillary electrophoresis (CE) was used to study myotoxins, members of a highly homologous family of small, basic, non-enzymatic proteins found in rattlesnake venoms. Several rattlesnake species were investigated and conditions were set to optimize the one-step separation of the myotoxins from the rest of the venom components. Myotoxin containing venoms, when subjected to CE analysis, yielded multiple myotoxin peaks. The myotoxin were collected and further analysed by matrix-assisted laser desorption time-of-flight mass spectrometry. In some instances, multiple mass peaks were observed from the mass spectra, suggesting the existence of myotoxin isoforms. Furthermore, each venom that contained myotoxin yielded fractions with indistinguishable masses, indicating that myotoxin isomers were present. The isomers were readily detected in the electropherograms since they exist in a ca. 1:4 ratio, data already established for myotoxin a from the prairie rattlesnake. An in-column incubation method was used to address the re-equilibration of the separated isomers.

Straight-line movement and competitive mate searching in prairie rattlesnakes,Crotalus viridis viridis

Males compete in various ways for mating and reproductive success. Phylogenetic factors and local ecology affect female spatial and temporal distributions, which in turn influence the form of male competition, sexual selection and mating systems. In prairie rattlesnake,Crotalus viridis viridispopulations where (1) males seek females during a brief reproductive period, (2) females are relatively few, and (3) females are widely and unpredictably distributed spatially into small discrete clusters, males should show efficient mate searching more so than time-consuming ‘handling’ (e.g. fighting, mate persuasion). Natural history studies and computer and mathematical modelling generate this expectation. This long-term field study of prairie rattlesnakes in Wyoming indicated that straight-line (i.e. fixed-bearing) movement by males is critical for mate location and, thus, for mating success. Males that searched along straight-line paths located and mated with more females than those having less straight movement paths. Fighting was observed rarely, and no relationship was found between males’ success at mate location and body mass and/or snout–vent length. Thus a range of traits may mediate competition and mating success among male snakes.

Reassessment of Phylogenetic Relationships among Pitviper Genera Based on Mitochondrial Cytochrome b Gene Sequences

Crotalines are biomedically important, venomous snakes that are widely distributed across Asia and the Western Hemisphere. Phylogenetic relationships and contingent taxonomy of these pitvipers, however, remain problematic. This has been particularly true for snakes of the moccasin complex of the tribe Agkistrodontini (Gloyd and Conant, 1990). Species of this group were assigned to the genus Agkistrodon (senso lato), first described by de Beauvois (1799) but more recently have been divided into three Asian genera (Calloselasma, Deinagkistrodon, and Hypnale) and a more narrowly redefined Agkistrodon (Gloyd, 1979). This general division is supported by immunological evidence (Cadle, 1992; Minton, 1990). The redefined genus Agkistrodon remains controversial. Morphological evidence led Hoge and Romano-Hoge (1981) to group Old World Agkistrodon species in a separate genus, which they named Gloydius. Cranial musculoskeletal data were interpreted by Goombridge (1986) as favoring recognition of Gloydius, whereas Kardong (1990) viewed the same data as compatible with retention of Agkistrodon for Old World species. Using osteological characters, Brattstrom (1964) supported a monophyletic Agkistrodon and suggested that the bushmaster, Lachesis muta, and the rattlesnakes (genera Crotalus and Sistrurus) formed a monophyletic group. Finally, molecular data (Knight et al., 1992) indicate that the genus Agkistrodon, as defined by Gloyd (1979), is paraphyletic; restriction fragment length polymorphism (RFLP) of mitochondrial DNA and 16S ribosomal RNA sequences group New World species of Agkistrodon with the New World rattlesnake, Crotalus molossus, and the tropical New World pit viper, Cerrophidion godmani, rather than with Old World species of Agkistrodon. In this study, we used mitochondrial cytochrome b gene sequences to test two phylogenetic hypotheses: (1) the genus Agkistrodon as presently defined is monophyletic; and (2) the bushmaster, Lachesis muta, and the rattlesnakes, represented by Crotalus viridis, constitute a monophyletic clade.

Characterization of the two myotoxin a isomers from the prairie rattlesnake ( Crotalus viridis viridis) by capillary zone electrophoresis and fluorescence quenching studies

The two myotoxin α isomers from the venom of the prairie rattlesnake Crotalus viridis viridis have different isoelectric points, as determined by capillary zone electrophoresis. The p I values are 10.50 and 10.57, respectively, and both are higher than the previously reported p I value for myotoxin α. The difference in the isoelectric points between the two isomers is attributed to altered surface charge as a result of the conformational change in myotoxin α. Both isomers exist in crude venom, discounting the possibility that they are artifacts formed during the purification process. Fluorescence quenching of myotoxin α reveals heterogeneity of the tryptophans, possibly due to different environments. The fraction of the total tryptophan fluorescence quenched by iodide is 81% and is attributed to solvent-accessible tryptophan residues at the protein surface. The 19% non-quenchable tryptophans probably represent residues that are shielded from the solvent exposure. The ratio of buried to exposed tryptophans is similar to the ratio of isomers seen by capillary zone electrophoresis and reverse-phase high-performance liquid chromatography ( C. 1:4).

Coelomic and Muscular Cross-Sectional Areas in Three Families of Snakes

Coelomic and Muscular Cross-Sectional Areas in Three Families of Snakes

Response to Rodent Saliva by Two Species of Rodentiophagous Snakes

Brown tree snakes (Boiga irregularis) and prairie rattlesnakes (Crotalus viridis) responded with higher rates of tongue flicking to rodent saliva than to water. Both materials were presented on cotton-tipped applicators touched gently to the snakes' lips. Rattlesnakes also struck more frequently at applicators bearing saliva than at control applicators. Since rodents frequently lick themselves during bouts of grooming behavior, saliva is certainly a component of the chemicals associated with rodent integuments. It is concluded that this association has given rise in rodentiophagous predators to a sensitivity to saliva or to salivary components.

Inhibition of RPE cell-mediated matrix adhesion and collagen gel contraction by crovidisin, a collagen-binding snake venom protein

PURPOSE. Cell-mediated collagen gel contraction plays an important role in the pathogenesis of proliferative vitreoretinopathy (PVR). Anti-adhesion therapy has been suggested as a promising strategy in the treatment of PVR. Crovidisin, a snake venom protein isolated from Crotalus viridis, has been shown to bind selectively to collagen and to inhibit collagen-induced platelet aggregation. In the present study, the effectiveness of crovidisin in inhibiting the attachment of retinal pigment epithelial (RPE) cells to collagen, and RPE cell-mediated collagen gel contraction, was evaluated. METHODS. Fluorescein isothiocyanate (FITC)-conjugated crovidisin was prepared and used to evaluate its binding affinity for collagen type I, fibronectin, vitronectin, and laminin. The inhibitory effect of crovidisin on RPE cell-mediated extracellular matrix attachment and collagen gel contraction was evaluated by cell adhesion and type I collagen gel contraction assays. The cytotoxic effect of crovidisin was examined with a cell proliferation assay, using the Alamar blue method. Flavoridin, an Arg-Gly-Asp-containing peptide from viper venom, was used for comparison. RESULTS. FITC-conjugated crovidisin bound selectively to collagen type I with high affinity. It did not bind to other matrix proteins, including fibronectin, vitronectin and laminin, nor to RPE cells. Crovidisin inhibited RPE cell attachment to type I collagen in a dose-dependent manner. This inhibitory effect was enhanced by the presence of flavoridin. Crovidisin also dose-dependently inhibited RPE cell-mediated type I collagen gel contraction. Crovidisin was non-toxic to RPE cells. CONCLUSIONS. Crovidisin, a snake venom-derived collagen-binding protein, possessing an inhibitory activity on RPE cell-collagen interaction and RPE cell-mediated collagen gel contraction, may be a useful tool for studying cell-collagen interaction, and a potential anti-adhesion therapeutic agent for ocular disorders in which cell-collagen interaction is involved, such as PVR.

Isolation, characterization and crystallization of a phospholipase A 2 myotoxin from the venom of the prairie rattlesnake ( Crotalus viridis viridis)

A myotoxin with phospholipase A 2 (PLA 2) activity was isolated from the venom of the prairie rattlesnake ( Crotalus viridis viridis, CVV) by cation-exchange chromatography. The toxin contains 123 amino acids and has an estimated mol. wt of 14,000. It is basic, with a p I above 9. Comparison of the N-terminal 33 residues of this myotoxin with other PLA 2 proteins from snake venoms showed that CVV myotoxin has highest homology (91%) to one isoform of the B component of crotoxin from Crotalus durissus terrificus venom, and less homology (73–75%) to mojave toxin from Crotalus scutulatus scutulatus venom and agkistrotoxin from Agkistrodon halys Pallas venom. It has the least homology (40–43%) to PLA 2s from venom of two other snakes in the Crotalus genus which are neither neurotoxic nor myotoxic. CVV myotoxin induces the type of myonecrosis typical of snake venom myotoxins with the PLA 2 structure, i.e. rapid disruption of the plasma membrane as indicated by the presence of delta lesions, hypercontraction and clumping of the myofilaments, and necrosis of affected skeletal muscle cells. Inhibition of the phospholipase activity of the toxin with p-bromophenacyl bromide inhibits the myotoxic activity, indicating that for some myotoxins with the PLA 2 structure, the catalytic activity is important for myotoxic activity. This is the first report of the isolation of a non-neurotoxic, single-chain PLA 2 myotoxin from the venom of a snake from the Crotalus genus.

The Nucleotide Sequence of the Translated and Untranslated Regions of a cDNA for Myotoxinafrom the Venom of Prairie Rattlesnake (Crotalus viridis viridis)

Myotoxina, isolated fromC. viridis viridisvenom, is a myonecrotizing agent present in many snake venoms. A cDNA library was prepared from mRNA obtained from the venom glands of aC. viridis viridis.The complete base sequence of a cDNA corresponding to an mRNA encoding myotoxinawas determined. The 5′ untranslated region has 15 nucleotides, while the 3′ untranslated region has 109 nucleotides. The translated portion of the myotoxinacDNA encodes a start methionine, a signal peptide, the myotoxinapeptide sequence, and an additional lysine residue. It is likely that myotoxinais secreted as the cDNA encodes a signal peptide immediately 5′ to the myotoxinapeptide code.

Presence of Cover Influences Response to Prey by Prairie Rattlesnakes (Crotalus Viridis)

Predatory behavior of captive prairie rattlesnakes (Crotalus viridis) was observed when artificial rock burrows were and were not present in terraria. Euthanized mice were suspended from tongs in front of the snakes, each of which immediately struck and released these prey. The rodent was placed 30–35 cm from the snake. We measured latency to retrieve the envenomated carcass and to swallow the carcass once it was retrieved. Retrieval was faster in snakes living in terraria containing cover than in snakes without cover. Swallowing time did not differ between the conditions. Burrows were used not only as refugia during inactive periods, but also as centers of activity during predatory episodes.

The Effect of Moonlight on Activity Patterns of Adult and Juvenile Prairie Rattlesnakes (Crotalus viridis viridis)

This study investigated the effect of moonlight on the nocturnal activity patterns of prairie rattlesnakes (Crotalus viridis viridis). The effect of simulated moonlight on six adults and eight juvenile prairie rattlesnakes was tested under laboratory conditions in which temperature, feeding frequency, and photoperiod were controlled. The snakes were maintained and tested under a summer photoperiod of 14L: 10D h cycle. The activity of each snake was measured using an index of tracking in the sand floor of a test chamber under simulated new, half, three-quarters, and full moon light (0.06, 0.35, 1.00, and 2.10 lux, respectively). Adult snake activity was significantly greater in new moonlight (starlight only) when com- pared to activity in three-quarter and full moonlight. The adults also significantly increased activity in open areas in dim moonlight. Variations in simulated moonlight had no effect on the activity of the juvenile rattlesnakes or in their use of edge and open areas. Bright moonlight avoidance by adult snakes may be a strategy that reduces detection by visually hunting predators and may also be influenced by the activity patterns of their nocturnal rodent prey. The lack of moonlight avoidance by juvenile rattlesnakes may be related to utilization of nocturnally inactive prey, such as lizards and neonatal rodents.

Rattlesnake venom poisoning in horses: 32 cases (1973-1993)

Objective To determine the clinical manifestations, morbidity, mortality, and treatment methods for rattlesnake venom poisoning in horses. Design Retrospective analysis of medical records. Animals 27 horses with acute venom poisoning attributable to prairie rattlesnakes, and 5 with chronic problems subsequent to a rattlesnake bite. Results Most horses were bitten on or near the muzzle while on pasture, resulting in head swelling, dyspnea, and epistaxis. Additional manifestations of acute poisoning included fever, tachycardia, tachypnea, cardiac arrhythmia, hemolytic anemia, thrombocytopenia, hemorrhage, thrombosis of venipuncture sites, colic, diarrhea, and prehensile and masticatory dysfunction. Chronic problems included cardiac disease, pneumonia, laminitis, pharyngeal paralysis, and wound complications. The most common chronic problem was cardiac disease. The most commonly used treatments were antibiotics, nonsteroidal anti-inflammatory drugs, tetanus prophylaxis, and airway support. Mortality in the 27 acutely affected horses was 18.5%; the overall mortality was 25%. Clinical Implications Horses bitten by prairie rattlesnakes may develop multiple, often severe, acute or chronic manifestations of poisoning involving various organ systems. Thorough clinical evaluation, effective treatment, supportive care, and close observation are indicated in horses with rattlesnake venom poisoning. (J Am Vet Med Assoc 1996;208:1866-1871)

Immunological studies of rabbit antibodies against hemorrhagic fractions of Crotalus viridis viridis venom: Role of crossreacting antibodies in neutralization

1. Crude Prairie rattlesnake ( Crotalus viridis viridis) venom was fractionated by HPLC DEAE anion exchange chromatography. Both acidic and basic protein fractions were assayed for hemorrhagic activity in vivo. 2. The acidic fractions that showed the highest hemorrhagic activities were pooled and divided into two samples. One sample was treated with EDTA and heat to inactivate and denature the hemorrhagic toxins; the other sample was left in native form. Both denatured and native samples were used as immunogens for production of polyvalent antibodies in rabbits. 3. Neutralizing ability for hemorrhage of the antiserum raised against the native sample was about 50% greater than that of antiserum raised against the denatured sample. There was no correlation between ELISA reactivity and neutralizing ability of the antisera. 4. Antiserum raised against native hemorrhagic fractions showed extensive ELISA crossreactivities with the nonhemorrhagic basic proteins. The crossreacting antibodies accounted for 61% of the total ELISA reactivity and 54% of the total neutralizing ability of the antiserum. Treatment of the hemorrhagic fractions with EDTA induced a fundamental conformational change of the molecules, which was reflected in significantly increased ELISA reactivities. Antivenom raised in rabbits had high neutralizing potency compared to Wyeth antivenom (0.38 mg IgG was able to completely neutralize the hemorrhagic lesions induced by 10 μg crude C. v. viridis venom). However, when it was quantitatively compared with commercial Wyeth antivenom, no significant advantages were found.

Evidence for isomerization in myotoxin a from the prairie rattlesnake ( Crotalus viridis viridis)

Myotoxin a, from the venom of the prairie rattlesnake, Crotalus viridis viridis, exists as a temperature-dependent equilibrium of two interconverting forms. Reverse-phase high-performance liquid chromatography (RP-HPLC) shows that the two forms interconvert slowly enough at 25 °C to be seen as two separate peaks with a molar ratio of c. 1:4. Each peak can be isolated and individually injected to give the same two peaks in the same ratio of areas. The two peaks merge during chromatography at elevated temperatures, indicating an increase in the rate of interconversion. At low temperature, c. 5 °C, the individual peaks can be isolated and maintained for several days without reaching equilibrium. Mass analysis by matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry shows that myotoxin a is present in both RP-HPLC peaks, suggesting that the two resolved forms are conformational isomers. Capillary zone electrophoresis (CZE) also shows two resolved, but interconvertible peaks over a range of pH values. Furthermore, RP-HPLC chromatograms of myotoxin a at concentrations from 0.013 mM to 0.41 mM maintain a consistent ratio of peak areas, without evidence of dimerization. Two-dimensional 1H-NMR nuclear Overhauser enhancement spectroscopy indicates the presence of a cis-proline peptide bond, consistent with an equilibrium mixture of cis-trans isomers; however, addition of peptidyl-prolyl cis-trans isomerase (PPI) does not enhance the rate of equilibration of the RP-HPLC peaks isolated at c. 5 °C.