Crotalus Viridis Research Articles

Effects of novel chemical cues on predatory responses of rodent-specializing rattlesnakes

We examined predatory responses of prairie (Crotalus viridis) and timber (Crotalus horridus) rattlesnakes to mouse carcasses misted with diluted perfumes or water. In Experiment 1, snakes showed equivalent latencies to strike, grasp, and ingest perfume- and water-misted carcasses. In Experiment 2, snakes that struck carcasses misted with perfume, water, or no liquid subsequently showed similar increases in tongue-flicking. In no-strike trials, snakes showed very low levels of tongue-flicking regardless of the type of chemical cues on the carcass. We conclude that during and after strikes, rattlesnakes behaved similarly towards both normal and chemically-novel rodent prey, implying that such prey are equally acceptable to the snakes.

Post-strike orientation of the prairie rattlesnake facilitates location of envenomated prey

Prairie rattlesnakes (Crotalus viridis) typically release adult rodent prey after envenomation. The post-strike head orientation of the snake may facilitate location of the trail left by the rodent. To examine this possibility, mice were presented using a pair of tongs so that no chemical cues were deposited on any surfaces. Snakes exhibited a change in head orientation after predatory strikes, bringing them closer to the departure bearing of the prey. In addition, when trail searching began snakes contacted the departure bearing of the prey first rather than the entry bearing. When rodent trails are available, we expect this initial contact to bias the snake towards selecting the post-envenomation trail.

Biochemical and physiological studies on a kallikrein-like enzyme from the venom of Crotalus viridis viridis (Prairie rattlesnake)

A kallikrein-like enzyme was isolated from Crotalus viridis viridis (Prairie rattlesnake) venom by Sephadex G-50, DEAE-Sephacel and heparin-Sepharose CL-6B column chromatography. The purified enzyme has a molecular mass of 32 kDa and an isoelectric point of 5.4. The enzyme catalyzed the hydrolysis of arginine esters, kallikrein substrates ProPheArgMCA and ZPheArgMCA. The specificity of the enzyme's substrate requirement is demonstrated by the fact that no proteolytic activity was detected against either dimethyl casein or fibrinogen. The enzyme also cleaves kininogen analogs to release bradykinin. Although the enzyme induced contraction of the isolated rat uterus directly at high concentrations, more forceful contractions resulted when the reaction mixture of the enzyme and bovine plasma was applied to the uterus. The reaction mixture of 5 · 10 −11 M of the enzyme and plasma caused contractions equal to that of 10 −9 M of bradykinin. Additionally the enzyme demonstrated capillary permeability-increasing activity and hypotensive activity on the anaesthetized rat, suggesting that the enzyme releases the dilator of the wall of capillaries from plasma. Uterine contraction, capillary permeability-increasing activity and arginine esterolytic activity were inhibited by diisopropyl fluorophosphate, indicating that the serine hydroxyl group is essential for enzymatic and biological activities. It was demonstrated that the NH 2-terminal region of the enzyme has significant similarities in sequence with kallikrein-like enzymes from other snake venoms and porcine pancreatic kallikrein.

Characterization of nicked myotoxin a and its effect on the sarcoplasmic reticulum calcium pump

Myotoxin a, a muscle-necrotizing polypeptide isolated from Crotalus viridis viridis (prairie rattlesnake) venom, was nicked at Met-28 by cyanogen bromide. Amino acid analysis indicated that the methionine content was reduced to zero from the original 1 mol. Judging from circular dichroism, the nicked myotoxin a had a conformation similar to that of original myotoxin. Raman spectra indicated that the conformations of the three disulfide bonds are not affected in nicked myotoxin a. Like the original toxin, nicked myotoxin a was myotoxic and inhibited calcium ion loading activity, although the inhibitory action was slightly lower than that of the original myotoxin a. Both modified and unmodified myotoxin a showed myonecrotic activity as determined by examining histological slides. The modified toxin also inhibited the formation of decavanadate-induced two-dimensional crystalline arrays of the sarcoplasmic reticulum Ca 2+-ATPase just as the original myotoxin a does.

Response of Neonatal Prairie Rattlesnakes (Crotalus viridis) to Conspecific and Heterospecific Chemical Cues

Socially naive neonate prairie rattlesnakes were observed in three experiments that measured chemosensory behaviors and substrate preference when snakes were given a two-choice presentation of a clean substrate and a substrate marked with ophidian exudates (either from conspecific neonates, conspecific adults, or a bullsnake). Neonates spent more time exploring or resting on substrates marked by conspecifics and avoided areas that contained heterospecific chemical cues. Neonates appear to be able to discriminate conspecific chemical cues from other sympatric species, and they may rely upon this ability to locate communal hibemacula for overwintering.

Evidence of an Alarm Pheromone from the Cloacal Sacs of Prairie Rattlesnakes

Evidence of an Alarm Pheromone from the Cloacal Sacs of Prairie Rattlesnakes

A tecto-rotundo-telencephalic pathway in the rattlesnake: evidence for a forebrain representation of the infrared sense

Rattlesnakes possess a sensory system specialized for the detection of infrared (IR) radiation. IR signals ascend as far as the optic tectum, where they generate a spatiotopic map. It is unknown if such signals reach the forebrain, but the existence of prominent tectothalamic pathways in other vertebrates makes this a distinct possibility. In nonmammalian forms, the major target of ascending tectal visual signals is nucleus rotundus, a thalamic nucleus that projects in turn to the subpallial telencephalon. We sought to determine whether a tecto-rotundo-telencephalic system exists in rattlesnakes and, if so, whether it carries IR as well as visual information. We have identified a thalamic nucleus in the rattlesnake Crotalus viridis that matches the n. rotundus of other reptiles in its topographic location, cytoarchitecture, and connections. Using anterograde and retrograde transport of HRP, we have demonstrated a strong ipsilateral and weaker contralateral tectorotundal projection. Tectorotundal cells lay primarily in the deeper tectal layers, which receive input from the IR system, but also in the superficial, visual layers. In n. rotundus, single units recorded extracellularly invariably responded to visual stimuli, but many were also sensitive to unimodal IR stimuli. IR and visual receptive fields were very large and often bilateral. Some rotundal units appeared sensitive to substrate vibration. Most habituated rapidly. Nucleus rotundus was found to project to a sector of the ipsilateral anterior dorsal ventricular ridge (ADVR) of the telencephalon. Single units in this region of the ADVR resembled those in rotundus, responding to visual, IR, and/or vibrational stimuli and possessing large, often bilateral receptive fields. These findings demonstrate the existence of a tecto-rotundo-telencephalic pathway in rattlesnakes and suggest that this system conveys IR as well as visual information to the forebrain. Ascending tectofugal pathways have been implicated in the discrimination of form. Thus, pattern recognition may have to be added to orientation as a proper function of the IR system of pit vipers.

Classification of myonecrosis induced by snake venoms: Venoms from the prairie rattlesnake ( Crotalus viridis viridis), Western diamondback rattlesnake ( Crotalus atrox) and the Indian cobra ( Naja naja naja)

C. L. Ownby and T. R. Colberg. Classification of myonecrosis induced by snake venoms: venoms from the prairie rattlesnake ( Crotalus viridis viridis), western diamondback rattlesnake ( Crotalus atrox) and the Indian cobra ( Naja naja naja). Toxicon 26, 459 – 474, 1988. — The pathogenesis of myonecrosis induced by three different snake venoms was studied by light microscopic examination of skeletal muscle tissue taken at time periods ranging from 0.25 hr to 4 weeks after an intramuscular injection of the venom into mice. It was possible to identify different types of myonecrosis based on the abnormal morphologic states of the damaged cells. The types of myonecrosis observed correlated with the types of components present in the venom injected. Venoms containing direct acting toxins such as myotoxin a or phospholipase A 2 induced specific types of myonecrosis. Also, venoms containing hemorrhagic toxins produced a type of myonecrosis similar to that induced by pure hemorrhagic toxins. The pathogenesis of each type of myonecrosis could be divided into the same four phases based on the pathologic states of the affected cells and the time after injection. During the ‘early phase’ (0.25 – 3 hr) affected muscle cells were in several different pathologic states reflecting the types of components present in the venom injected. During the ‘intermediate phase’ (6 – 24 hr) the pathologic state of the damaged cells had changed and depending on the venom new states might be present. By the ‘late phase’ (48 – 96 hr) all damaged cells have reached a common pathologic state of necrosis. The ‘final phase’ (1 – 4 weeks) is characterized by regeneration (partial or complete) of muscle cells. Although the number of different types of myonecrosis depended on the type of venom injected, i.e. Naja naja naja venom produced only two different types whereas Crotalus atrox venom produced at least four different types, cells of each type of myonecrosis progressed through the same four phases. In studies of the myotoxicity of snake venoms it is important to examine tissues taken during the early and intermediate phases to obtain accurate and useful information on the types of myonecrosis caused by the venom.

Crystallization of the Ca 2+-ATPase of skeletal muscle sarcoplasmic reticulum Inhibition by myotoxin a

Decavanadate produces extensive ordered arrays of Ca 2+-ATPase molecules on sarcoplasmic reticulum (SR) vesicle surfaces [(1984) J. Bioenerg. Biomembranes 16, 491–505] and the basic unit of these crystalline structures seems to be a dimer of Ca 2+-ATPase [(]983) J. Ultrastruct. Res. 24, 454–464; (1984) J. Mol. Biol. 174, 193–204]. Myotoxin a, isolated from the venom of the prairie rattlesnake Crotalus viridis viridis, is a muscle-degenerating polypeptide and its primary site of interaction is the SR membrane, where it uncouples Ca 2+-translocation from Ca 2+-dependent ATP hydrolysis [(1986) Arch. Biochem. Biophys. 246, 90–97]. The effect of myotoxin a on decavanadate-induced two-dimensional Ca 2+-ATPase crystals of SR membranes has been investigated. The toxin inhibits the formation of two-dimensional SR-membrane crystals and disrupts previously formed crystals in a time- and concentration-dependent manner, which parallels the uncoupling of ATP hydrolysis from Ca 2+ translocation. Two-dimensional crystalline arrays of the SR membrane have a typical diffraction pattern which, after myotoxin a treatment, displays a progressive loss of order. Decavanadate is an uncompetitive inhibitor of the Ca 2+-ATPase enzyme-myotoxin a complex. The present results suggest that a Ca 2+-ATPase dimer is required for coupling Ca 2+ translocation to Ca 2+-dependent ATP hydrolysis.

Myotoxina-Phospholipid Interactions, an Attempt by Intrinsic Fluorescence to Define a Possible Mode of Action for the Toxin on Membranes

Intrinsic fluorescence of myotoxin a from Crotalus viridis viridis has been proved to be due to tryptophan residues, and this knowledge allows one to detect, for the first time, an interaction of the toxin with phospholipids. The formation of lipid-protein species is followed by a blue shift of about 8 nm and a quenching of up to 50% of the emission of tryptophans. These changes could result from the positioning of Trp 32,34 residues at the lipid interface and/or a local conformational change of the toxin. In the μM range, complexes are formed only with charged lipids; charge complementarity of two partners is needed regardless of the physical state of the lipids. The binding is electrostatic, and the affinity of myotoxin a decreases in parallel with the net charge of the phospholipid interface and/or that of the toxin. Similarly, calcium-ion addition, pH changes, and an increase in ionic strength can dissociate the complexes. Their stability increases in the following sequence: phosphatidylinositol < phosphatidylserine < phosphatidylglycerol < phosphatidic acid < cardiolipin. It is proposed that the toxin lies at the interface, without penetrating the membrane. On binding, the toxin does not severely change the structure of lipid vesicles nor their permeability; however, significant changes in the lipid chain order are detected with synthetic lipids, both in their gel and fluid phases. The possible consequences of the existence of myotoxin a-charged phospholipid complexes are discussed and are compared to the behavior of other related basic toxins of snake venom acting on membrane.

Assignment of the aromatic 1H-NMR resonance of myotoxin a isolated from the venom of Crotalus virdis viridis

The 400 MHz 1H-NMR spectrum of myotoxin a from the venom of Crotalus viridis viridis is described. The identification of spin systems in the aromatic region corresponding to the six aromatic residues of myotoxin a was completed using both one- and two-dimensional NMR spectroscopy and the pH dependence of chemical shifts. Assignments of these spin systems to specific residues was possible for the singly occuring amino acids Tyr-1 and Phe-12. Resonances from Tyr-1, His-5 and His-10 were shifted significantly from their random coil values in a pH-dependent manner. These shift perturbations were deemed evidence of a helical arrangement of the amino terminal region which placed these residues in close proximity to each other.

An Active Prairie Rattlesnake Den Taken Over by Foxes

An Active Prairie Rattlesnake Den Taken Over by Foxes

Strike-induced chemosensory searching in rattlesnakes: A rodent specialist (Crotalus viridis) differs from a lizard specialist (Crotalus pricei)

Prairie rattlesnakes (Crotalus viridis) exhibited a sustained high rate of tongue flicking after a predatory strike whether or not rodent odors were present in the poststrike environment. For the lizard specialist, C. pricei, strike-induced chemosensory searching was maintained at a high level only when chemical cues were available following the strike.

Characterization of the biological and immunological properties of fractions of prairie rattlesnake ( Crotalus viridis viridis) venom

C. L. Ownby and T. R. Colberg. Characterization of the biological and immunological properties of fractions of prairie rattlesnake ( Crotalus viridis viridis) venom. Toxicon 25, 1329 – 1342, 1987. — Prairie rattlesnake ( Crotalus viridis viridis) venom was separated using liquid column chromatography. The fractions were tested for biological activity in mice and for immunological reactivity against polyvalent (Crotalidae) antivenom and a monovalent antivenom to the crude venom. Several of the basic fractions and most of the non-basic fractions had hemorrhagic activity. Six of eight basic fractions had direct myotoxic activity, two of the basic fractions produced edema 30 min after injection, and two were lethal. Polyvalent antivenom contained few antibodies to the fractions of this venom, reacting with only two of the basic fractions. Monovalent antivenom formed mulitiple precipitin bands with almost all of the fractions. These results clearly demonstrate that most of the venom components are antigenic and immunogenic. Immunodiffusion using the monovalent antivenom demonstrated that C. v. viridis venom contains many antigens common to 10 other crotaline venoms. One of the hemorrhagic components was present in five of the other venoms tested, one of the direct myotoxic components was present in three other venoms, and one of the lethal components was common to two other venoms. Another highly active hemorrhagic component was common to all of the venoms tested except that of Trimeresurus flavoviridis.

Trigeminal responses to thermal stimulation of the oral cavity in rattlesnakes ( Crotalus viridis) before and after bilateral anesthetization of the facial pit organs

Multiunit neural responses from the superficial maxillary branch of the trigeminal nerve in prairie rattlesnakes were elicited by intraoral thermal stimulation. The responses to oral stimulation were shown to be independent of responses obtained by thermal stimulation of the loreal pits. Histological examination of the dorsal lip, palate, and fang sheath regions revealed dense ramifying neurons in the epidermal layers of the fang sheaths that were morphologically similar to suspected infrared sensitive neurons in the pit membranes.

Isolation and partial characterization of two lethal toxins from prairie rattlesnake (Crotalus viridis viridis) venom: Terry R. Colberg and Charlotte L. Ownby (Department of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma 74078, U.S.A.)

Isolation and partial characterization of two lethal toxins from prairie rattlesnake (Crotalus viridis viridis) venom: Terry R. Colberg and Charlotte L. Ownby (Department of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma 74078, U.S.A.)

Amino acid sequences of myotoxins from Crotalus viridis concolor venom

A. L. Bieber, R. H. McParland and R. R. Becker. Amino acid sequences of myotoxins from Crotalus viridis concolor venom. Toxicon 25, 677 – 680, 1987. — Myotoxins I and II were isolated from the venom of Crotalus viridis concolor. Complete sequences were derived for each reduced, alkylated toxin with data obtained by a single run on a gas phase sequencer and from fragments derived by cyanogen bromide cleavage. The results demonstrate that microheterogeneity is present in myotoxin II. The newly established sequences were compared with 3447 protein sequences in the Protein Information Resource database. The only homologous proteins found were other known myotoxins from rattlesnake venoms, namely myotoxin a, crotamine and peptide C.

Resistance of California ground squirrels ( Spermophilus Beecheyi) to the venom of the northern Pacific rattlesnake ( Crotalus Viridis Oreganus): A study of adaptive variation

N. S. Poran, R. G. Coss and E. Benjamini. Resistance of California ground squirrels ( Spermophilus beecheyi) to the venom of the northern Pacific rattlesnake ( Crotalus viridis oreganus): a study of adaptive variation. Toxicon 25, 767 – 777, 1987.—Recent studies have documented natural resistance to snake venom in a number of diverse mammalian species. The present paper documents for the first time variation in such resistance within one single species, the California ground squirrel ( Spermophilus beecheyi). This species is a frequent prey of the northern Pacific rattlesnake ( Crotalus viridis oreganus) in certain habitats. Venom resistance was tested directly in two populations of ground squirrels by injection of 1 – 40 mg/kg venom doses. One population was obtained from a habitat with a high rattlesnake density; the other population came from a rattlesnake-free habitat. Dramatic differences in the response to venom between these populations were manifested, based on a variety of criteria, such as mortality, necrosis and healing time. Resistance to venom was also examined by ld 50 tests in groups of mice pre-injected with ground squirrel sera from three rattlesnake-adapted California populations and a non-adapted Arctic population ( S. parryii) from snake-free central Alaska. The California ground squirrel sera were 3.3 – 5.3 times more effective in the in vivo neutralization of venom than the sera from Arctic ground squirrels. Moreover, the level of protection by the sera as reflected by the ld 50 values was highly correlated ( P < 0.005) with the level of in vitro squirrel serum - venom binding as quantified by radioimmunoassay (RIA). A subsequent RIA revealed that binding levels of sera from 14 California ground squirrel populations correlated significantly ( P < 0.025) with local rattlesnake densities; i.e. sera pools from populations sympatric with rattlesnakes exhibited the highest binding, whereas populations living in habitats where rattlesnakes are rare or absent typically exhibited the lowest binding levels, several of which approximated the Arctic control. Taken together, these results demonstrate intraspecific variation that is probably the result of differential natural selection due to northern Pacific rattlesnakes. This intraspecific variation should be taken into consideration when testing for natural resistance in wild-caught species.

Fractionation and immunological characterization of prairie rattlesnake (Crotalus viridis viridis) venom: Charlotte L. Ownby and Terry R. Colberg (Department of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma 74078, U.S.A.)

Fractionation and immunological characterization of prairie rattlesnake (Crotalus viridis viridis) venom: Charlotte L. Ownby and Terry R. Colberg (Department of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma 74078, U.S.A.)

Evolution of isozyme characters in the tuatara,Sphenodon punctatus

Abstract Isozyme characters of the tuatara, Sphenodon punctatus, were examined by starch gel electrophoresis. The characters investigated included: (1) the number of presumptive structural gene loci encoding for each of 30 enzyme systems; (2) the formation of interlocus heteropolymers in polymeric, multilocus enzyme systems; and (3) the tissue-specific distribution of 51 presumptive structural gene loci among 15 tissues. The number of loci controlling the various enzyme systems in the tuatara was similar to that reported in other vertebrates. No unexpected duplicated loci were observed although some silencing may have occurred. All anticipated interlocus heteropolymers were observed, except for dihydrolipoamide dehydrogenase; most loci were widely distributed among the tissues examined. With respect to the first two characters, Sphenodon appears to exhibit the primitive condition. However, analysis of the tissue-specific distribution of loci suggests that, when compared to other members of the class Reptilia, the tuatara is not necessarily the most primitive taxon. For this character, the tuatara appeared more primitive than the prairie rattlesnake yet more derived than the American alligator. The results suggest that isozyme characters may be of value in examining both the intra- and interpopulation genic variability of the tuatara as well as the phylogenetic relationships of Sphenodon and other reptilian taxa.