Phenotypic integration in the feeding system of the eastern diamondback rattlesnake (Crotalus adamanteus)

Selection can vary geographically across environments and temporally over the lifetime of an individual. Unlike geographic contexts, where different selective regimes can act on different alleles, age‐specific selection is constrained to act on the same genome by altering age‐specific expression. Snake venoms are exceptional traits for studying ontogeny because toxin expression variation directly changes the phenotype; relative amounts of venom components determine, in part, venom efficacy. Phenotypic integration is the dependent relationship between different traits that collectively produce a complex phenotype and, in venomous snakes, may include traits as diverse as venom, head shape and fang length. We examined the feeding system of the eastern diamondback rattlesnake (Crotalus adamanteus) across environments and over the lifetime of individuals and used a genotype–phenotype map approach, protein expression data and morphological data to demonstrate that: (i) ontogenetic effects explained more of the variation in toxin expression variation than geographic effects, (ii) both juveniles and adults varied geographically, (iii) toxin expression variation was a result of directional selection and (iv) different venom phenotypes covaried with morphological traits also associated with feeding in temporal (ontogenetic) and geographic (functional) contexts. These data are the first to demonstrate, to our knowledge, phenotypic integration between multiple morphological characters and a biochemical phenotype across populations and age classes. We identified copy number variation as the mechanism driving the difference in the venom phenotype associated with these morphological differences, and the parallel mitochondrial, venom and morphological divergence between northern and southern clades suggests that each clade may warrant classification as a separate evolutionarily significant unit.     

Envenomations by Bothrops and Crotalus snakes induce the release of mitochondrial alarmins

Skeletal muscle necrosis is a common manifestation of viperid snakebite envenomations. Venoms from snakes of the genus Bothrops, such as that of B. asper, induce muscle tissue damage at the site of venom injection, provoking severe local pathology which often results in permanent sequelae. In contrast, the venom of the South American rattlesnake Crotalus durissus terrificus, induces a clinical picture of systemic myotoxicity, i.e., rhabdomyolysis, together with neurotoxicity. It is known that molecules released from damaged muscle might act as 'danger' signals. These are known as 'alarmins', and contribute to the inflammatory reaction by activating the innate immune system. Here we show that the venoms of B. asper and C. d. terrificus release the mitochondrial markers mtDNA (from the matrix) and cytochrome c (Cyt c) from the intermembrane space, from ex vivo mouse tibialis anterior muscles. Cyt c was released to a similar extent by the two venoms whereas B. asper venom induced the release of higher amounts of mtDNA, thus reflecting hitherto some differences in their pathological action on muscle mitochondria. At variance, injection of these venoms in mice resulted in a different time-course of mtDNA release, with B. asper venom inducing an early onset increment in plasma levels and C. d. terrificus venom provoking a delayed release. We suggest that the release of mitochondrial 'alarmins' might contribute to the local and systemic inflammatory events characteristic of snakebite envenomations.     

Chemical and functional homology of myotoxin a from prairie rattlesnake venom and crotamine from South American rattlesnake venom

Myonecrosis is a serious result of rattlesnake bite and constitutes a persistent clinical problem. In the current study we have isolated crotamine from the venom of Crotalus durissus terrificus to test its ability to cause structural damage to skeletal muscle, and to make direct chemical comparisons with Myotoxin a, a myotoxic polypeptide we recently isolated from prairie rattlesnake (Crotalus viridis viridis) venom. Disc gel electrophoresis, isoelectric focusing, circular dichroic spectroscopy, and amino acid analysis, all indicated a high degree of chemical similarity. Light microscope histology revealed that crotamine caused vacuolizationof skeletal muscle fibers, qualitatively the same as the vacuolization caused by Myotoxin a. The ability of these two basic snake venom polypeptides to cause structural damage to skeletal muscle fibers has significant implications toward more complete understanding of the cause of snake venom-induced myonecrosis.     

Molecular models of the Mojave rattlesnake (Crotalus scutulatus scutulatus) venom metalloproteinases reveal a structural basis for differences in hemorrhagic activities

Rattlesnake venom can differ in composition and in metalloproteinase-associated activities. The molecular basis for this intra-species variation in Crotalus scutulatus scutulatus (Mojave rattlesnake) remains an enigma. To understand the molecular basis for intra-species variation of metalloproteinase-associated activities, we modeled the three-dimensional structures of four metalloproteinases based on the amino acid sequence of four variations of the proteinase domain of the C. s. scutulatus metalloproteinase gene (GP1, GP2, GP3, and GP4). For comparative purposes, we modeled the atrolysin metalloproteinases of C. atrox as well. All molecular models shared the same topology. While the atrolysin metalloproteinase molecular models contained highly conserved substrate binding sites, the Mojave rattlesnake metalloproteinases showed higher structural divergence when superimposed onto each other. The highest structural divergence among the four C. s. scutulatus molecular models was located at the northern cleft wall and the S’₁-pocket of the substrate binding site, molecular regions that modulate substrate selectivity. Molecular dynamics and field potential maps for each C. s. scutulatus metalloproteinase model demonstrated that the non-hemorrhagic metalloproteinases (GP2 and GP3) contain highly basic molecular and field potential surfaces while the hemorrhagic metalloproteinases GP1 and atrolysin C showed extensive acidic field potential maps and shallow but less dynamic active site pockets. Hence, differences in the spatial arrangement of the northern cleft wall, the S’₁-pocket, and the physico-chemical environment surrounding the catalytic site contribute to differences in metalloproteinase activities in the Mojave rattlesnake. Our results provide a structural basis for variation of metalloproteinase-associated activities in the rattlesnake venom of the Mojave rattlesnake.     

Effect of heparin and antivenom on skeletal muscle damage produced by Bothrops jararacussu venom

We examined the effect of treatment with heparin and polyvalent antivenom on mice muscle Extensor digitorum longus (EDL) regeneration, after damage induced by injection of Bothrops jararacussu crude venom over the muscle of the right posterior limb. The mice were separated into groups and each group received treatment, by intravenous route with either high molecular weight heparin (H), low molecular weight heparin (LMWH), polyvalent antivenom (PAV) or with the combination of PAV plus H or PAV plus LMWH at 15 minutes and 4 hours after the injection of the venom. Myotoxicity was measured by the increase in plasma creatine kinase (CK) activity at two hours after the injection of the venom. The histological changes in EDL at 1, 3, 7 and 21 days after the injection of the venom were analyzed by light microscopy. In each group the normal and regenerated muscle fibers were quantified using Scion Image computer program. We also evaluated in vitro, the influence of these substances in the proteolytic and phospholipase activities of the venom. Heparins decreased the proteolytic activity of the venom but did not affect its phospholipase activity. However the PAV antagonized both activities. PAV and its combinations showed antimyotoxic activity, according to the magnitude of CK plasma levels. At 21 days the regeneration was observed in all animals, also in those that received only the venom. All treatments, except LMWH, promote a significant increase in the number of muscle fibers.     

Energetic and physiological correlates of prey handling and ingestion in lizards and snakes

In this review, we summarize the energetic and physiological correlates of prey handling and ingestion in lizards and snakes. There were marked differences in the magnitude of aerobic metabolism during prey handling and ingestion between these two groups, although they show a similar pattern of variation as a function of relative prey mass. For lizards, the magnitude of aerobic metabolism during prey handling and ingestion also varied as a function of morphological specializations for a particular habitat, prey type, and behavior. For snakes, interspecific differences in aerobic metabolism during prey handling seem to be correlated with adaptations for prey capture (venom injection vs. constriction). During ingestion by snakes, differences in aerobic metabolism might be due to differences in cranial morphology, although allometric effects might be a potentially confounded effect. Anaerobic metabolism is used for prey handling and ingestion, but its relative contribution to total ATP production seems to be more pronounced in snakes than in lizards. The energetic costs of prey handling and ingestion are trivial for both groups and cannot be used to predict patterns of prey-size selection. For lizards, it seems that morphological and ecological factors set the constraints on prey handling and ingestion. For snakes, besides these two factors, the capacity of the cardio-respiratory system may also be an important factor constraining the capacity for prey handling and ingestion.     

Sheath fluid control to permit stable flow in rapid mix flow cytometry.

BACKGROUND: Flow cytometry is a potentially powerful tool to analyze the kinetics of ligand binding, cell response and molecular assembly. The difficulty in adding reactant to cells, achieving adequate mixing, delivering those cells to the laser focal point and establishing stable flow, has historically limited flow cytometry to systems with reactions times longer than 5 s. With the advent of automated syringes and flow injection methods, sample injection times shorter than 1 s have become routine. However, an inherent problem in acquiring time courses starting under 1 s is that rapid sample introduction through the flow tip to the detection point perturbs laminar flow. The purpose of this work was to determine if stable flow could be reestablished more quickly if the sheath flow was reduced during sample introduction, returning to normal sheath and sample rates afterward. METHODS: We used programmable syringes and valves to control sample mixing as well as sheath and sample delivery through the flow tip to the detection point for stream-in-air detection. Stable flow was monitored by mean particle fluorescence during sample introduction. RESULTS: With no sheath reduction, stable flow recovered after more than 1 s. By reducing sheath flow during the short period (300 msec) of sample mixing and delivery, stable laminar flow recovered within 200 msec. CONCLUSIONS: This use of automated syringes to control both sheath and sample flow provides a potential for robust sample handling applicable to kinetic as well as high throughput flow cytometric analysis.     

Influence of the venom delivery system on intraoral prey transport in snakes

We compared intraoral prey transport in venomous snake species from four families (two atractaspidids, nine elapids, three colubrids, 44 viperids) with that in eight non-venomous colubrid species, most feeding on similar mammalian prey. The morphology of the venom delivery system suggests that intraoral prey transport performance should be slightly decreased in atractaspidids, unmodified in most elapids and venomous colubrids, and increased or unmodified in vipers, as compared to that in non-venomous colubrid snakes. Our measurements of relative intraoral prey transport performance show that differences among families do not match expectations based on morphology or past studies. Decreased performance in Atractaspis results from reduction and loss of teeth on the medial palatal elements and dentaries, but affects only early phases of ingestion. Although joint and bone features of elapids and colubrids are similar, intraoral prey transport performance is significantly lower in elapids than in colubrids. Predicted enhancement of intraoral prey transport performance in vipers as compared to colubrids was not borne out by measurements, presumably because palatopterygoid movement during intraoral prey transport is reduced in many viper species to limit fang erection. Absence of significant performance differences between colubrids and viperids might suggest that evolution of the viperid venom delivery system was subject to little selection pressure from intraoral prey transport. Another possibility is that there are trade-offs between intraoral prey transport and strike performance in vipers related to relative skull mass and jaw fragility. Immobilizing prey prior to intraoral transport places less demand on transport performance in vipers. In this model, the conservative kinesis and greater robustness of the colubrid palate has greater potential for transporting live prey with less risk of injury.     

How tubular venom-conducting fangs are formed

Elapids, viperids, and some other groups of colubroid snakes have tubular fangs for the conduction of venom into their prey. The literature describing the development of venom-conducting fangs provides two contradictory accounts of fang development. Some studies claim that the venom canal forms by the infolding of a deep groove along the surface of the tooth to produce an enclosed canal. In other works the tubular fang is said to form by the deposition of material from tip to base, so that the canal develops without any folding. This study was undertaken to examine fang development and to account for the disagreement in the literature by determining whether fang formation varies among groups of venomous snakes and whether it differs between embryos and adults. Adult and embryonic representatives of elapids and viperids were examined. All fangs examined, elapid and viperid, embryos and adults, were found to develop into their tubular shape by the addition of material to the basal end of the tooth rather than by the folding inward of an ungrooved tooth to form a tubular fang. In some cases, the first fang that develops in embryonic snakes differs morphologically from all those formed subsequently.     

Isolation of a crotalase-like protease with alpha-fibrinogenase activity from the western diamondback rattlesnake, Crotalus atrox.

Venom toxins were isolated from rattlesnake (Crotalus atrox) venom by cation-exchange chromatography. Seven major fractions could be obtained by single-step ion-exchange chromatography with two fractions showing essentially apparent homogeneity by SDS-gel electrophoresis. All fractions showed various extents of specific proteolytic activity against alpha- or beta-chains of fibrinogen molecules. Further characterization of one of the purified fractions with alpha-fribrinogenase activity indicated that it is a single-chain thrombin-like protease with a molecular mass of about 30 kDa. It is relatively heat stable, inhibited by phenylmethanesulfonyl fluoride, N alpha-p-tosyl-L-phenylalanine chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone but not by soybean trypsin inhibitor and beta-mercaptoethanol. Amino acid analysis showed that the enzyme possesses an amino acid composition very similar to thrombin and crotalase characterized before from the closely related snake venoms. N-Terminal sequence analysis of the enzyme corroborated the close similarity between this enzyme and those sequences of crotalase and kallikrein-like enzymes characterized from the same Crotalidae snake family. This study is in contrast to the previous reports which indicated a lack of thrombin- and crotalase-like enzyme in the venom of Western diamondback rattlesnake.     

Venom apparatus of the Brazilian tarantula <Emphasis Type="Italic">Vitalius dubius</Emphasis> Mello-Leitão 1923 (Theraphosidae)

Tarantula venoms are a cocktail of proteins and peptides that have been increasingly studied in recent years. In contrast, less attention has been given to analyzing the structure of the paired cephalic glands that produce the venom. We have used light, electron, and confocal microscopy to study the organization and structure of the venom gland of the Brazilian tarantula Vitalius dubius. The chelicerae are hairy chitinous structures, each with a single curved hollow fang that opens via an orifice on the anterior surface. Internally, each chelicera contains striated muscle fiber bundles that control fang extension and retraction, and a cylindrical conical venom gland surrounded by a thick well-developed layer of obliquely arranged muscle fibers. Light microscopy of longitudinal and transverse sections showed that the gland secretory epithelium consists of a sponge-like network of slender epithelial cell processes with numerous bridges and interconnections that form lacunae containing secretion. This secretory epithelium is supported by a basement membrane containing elastic fibers. The entire epithelial structure of the venom-secreting cells is reinforced by a dense network of F-actin intermediate filaments, as shown by staining with phalloidin. Neural elements (axons and acetylcholinesterase activity) are also associated with the venom gland. Transmission electron microscopy of the epithelium revealed an ultrastructure typical of secretory cells, including abundant rough and smooth endoplasmic reticulum, an extensive Golgi apparatus, and numerous mitochondria.     

Venom variation in hemostasis of the southern Pacific rattlesnake (Crotalus oreganus helleri): isolation of hellerase

Envenomations by the southern Pacific rattlesnake (Crotalus oreganus helleri) are the most common snakebite accidents in southern California. Intraspecies venom variation may lead to unresponsiveness to antivenom therapy. Even in a known species, venom toxins are recognized as diverse in conformity with interpopulational, seasonal, ontogenetic and individual factors. Five venoms of individual C. oreganus helleri located in Riverside and San Bernardino counties of southern California were studied for their variation in their hemostatic activity. The results demonstrated that Riverside 2 and San Bernardino 1 venoms presented the highest lethal activity without hemorrhagic activity. In contrast, San Bernardino 2 and 3 venoms had the highest hemorrhagic and fibrinolytic activities with low lethal and coagulant activities. Riverside 1, Riverside 2 and San Bernardino 1 venoms presented a significant thrombin-like activity. San Bernardino 2 and 3 venoms presented an insignificant thrombin-like activity. In relation to the fibrinolytic activity, San Bernardino 3 venom was the most active on fibrin plates, which was in turn neutralized by metal chelating inhibitors. These results demonstrate the differences amongst C. oreganus helleri venoms from close localities. A metalloproteinase, hellerase, was purified by anionic and cationic exchange chromatographies from San Bernardino 3 venom. Hellerase exhibited the ability to break fibrin clots in vitro, which can be of biomedically importance in the treatment of heart attacks and strokes.     

Complete primary structure of a galactose-specific lectin from the venom of the rattlesnake Crotalus atrox. Homologies with Ca2(+)-dependent-type lectins

The complete primary structure of a galactose-specific lectin contained in the venom of the rattlesnake, Crotalus atrox, was determined. The lectin is composed of two covalently linked, identical subunits, each consisting of 135 amino acid residues. Under physiological conditions the lectin proved to be highly aggregated. The venom lectin contained 9 half-cystines, 8 of which formed four intrasubunit disulfide bridges (Cys3-Cys14, Cys31-Cys131, Cys38-Cys133, and Cys106-Cys123), while Cys86 was involved in an intersubunit disulfide bridge. Because of the high content of disulfide bridges, the intact lectin was extremely resistant to tryptic digestion. The determined amino acid sequence was found to be homologous with those of the so-called carbohydrate recognition domains of Ca2(+)-dependent-type lectins in animal. Among them, 8 amino acid residues (Cys31, Gly69, Trp92, Pro97, Cys106, Asp120, Cys123, and Cys131) were completely conserved. Leu40, Trp67, and Trp81 were also well conserved. The rattlesnake venom lectin showed high hemagglutinating activity. These results, together with the occurrence of similar lectins in crotalid venoms, suggest that these lectins have evolved in order to make the venom a more effective weapon to capture prey animals.     

Functional subdivision of the venom gland musculature and the regulation of venom expulsion in rattlesnakes

A combination of histology, whole muscle force physiology, glycogen depletion, and venom expulsion analyses using transonic probes to measure venom flow and fluid pressure transducers to measure venom pressure was performed on the m. compressor glandulae and m. pterygoideus glandulae. The m. pterygoideus glandulae has less than one-third the cross-sectional area of the m. compressor glandulae, and produces approximately one-fifth the total twitch and tetanic force; however, in situ surface stimulation of the muscle produces venom flow and pressure levels that are similar to those produced by the m. compressor glandulae. The similarity in venom output following stimulation reflects in part the functional role of the larger m. compressor glandulae in jaw adduction, but also the functional subdivisions within this muscle. The m. compressor glandulae is divided into a series of columnar fascicles that run from the surface of the muscle to the venom gland. The combined results of clearing and staining and glycogen depletion studies suggest that these fascicles may represent functional compartments. Identical stimulations applied to different regions of the m. compressor glandulae result in up to a six-fold difference in venom expulsion. This functional specialization may play a role in the regulation of venom flow during offensive and defensive strikes.     

Activation, aggregation, and product inhibition of cobra venom phospholipase A2 and comparison with other phospholipases

The kinetics of phospholipid hydrolysis by cobra venom phospholipase A2 were examined and compared to those of phospholipase A2 from porcine pancreas, Crotalus adamanteus (rattlesnake) venom, and bee venom. Only the enzyme from Naja naja naja (cobra) venom was found to be activated significantly by phosphorylcholine-containing compounds when hydrolyzing phosphatidylethanolamine. The cobra venom enzyme was also the only one in which these activators induced protein aggregation. The parallel specificity for activators and aggregators suggests that these two phenomena are linked. Product effects were also shown to vary between these four phospholipases. These effects manifest themselves in nonlinear time courses, in changes in steady state velocity, and in the differential effects of serum albumin on reaction rates. Different effects were even seen for the same enzyme when acting on different substrates. A model is presented to account for these observations; its main features are enzyme activation by an activator molecule, whose specificity depends on the enzyme, and an activator-induced aggregation of the enzyme.     

Opossum peptide that can neutralize rattlesnake venom is expressed in Escherichia coli

An eleven amino acid ribosomal peptide was shown to completely neutralize Western Diamondback Rattlesnake (Crotalus atrox) venom in mice when a lethal dose of the venom was pre‐incubated with the peptide prior to intravenous injection. We have expressed the peptide as a concatenated chain of peptides and cleaved them apart from an immobilized metal affinity column using a protease. After ultrafiltration steps, the mixture was shown to partially neutralize rattlesnake venom in mice. Preliminary experiments are described here that suggest a potential life‐saving therapy could be developed. To date, no recombinant therapies targeting cytotoxic envenomation have been reported. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:81–86, 2017     

Dissociation of maximal O2 uptake from O2 delivery in canine gastrocnemius in situ

To test the hypothesis that maximal O2 uptake (VO2max) can be limited by O2 diffusion in the peripheral tissue, we kept O2 delivery [blood flow X arterial O2 content (CaO2)] to maximally contracting muscle equal between 1) low flow-high CaO2 and 2) high flow-low CaO2 conditions. The hypothesis predicts, because of differences in the capillary PO2 profile, that the former condition will result in both a higher VO2max and muscle effluent venous PO2 (PVO2). We studied the relations among VO2max, PVO2, and O2 delivery during maximal isometric contractions in isolated, in situ dog gastrocnemius muscle (n = 6) during these two conditions. O2 delivery was matched by varying arterial O2 partial pressure and adjusting flow to the muscle accordingly. A total of 18 matched O2 delivery pairs were obtained. As planned, O2 delivery was not significantly different between the two treatments. In contrast, VO2max was significantly higher [10.4 +/- 0.5 (SE) ml.100 g-1.min-1; P = 0.01], as was PVO2 (25 +/- 1 Torr; P less than 0.01) in the low flow-high CaO2 treatment compared with the high flow-low CaO2 treatment (9.1 +/- 0.4 ml.100 g-1.min-1 and 20 +/- 1 Torr, respectively). The rate of fatigue was greater in the high flow-low CaO2 condition, as was lactate output from the muscle and muscle lactate concentration. The results of this study show that VO2max is not uniquely dependent on O2 delivery and support the hypothesis that VO2max can be limited by peripheral tissue O2 diffusion.     

云南孟加拉种眼镜蛇蛇毒因子在灵长类动物内应用的免疫原性研究

眼镜蛇蛇毒因子(CVF)能特异性清除机体循环中的补体C3,从而可能在防治补体介导的损伤或疾病中发挥重要的治疗作用.云南孟加拉种眼镜蛇蛇毒因子(Y-CVF)较文献报道的其他各种CVF具有更高的活性和较少的用药量.为探讨Y-CVF静脉使用是否诱导灵长类动物体内产生特异性中和抗体和异种天然抗体,给2只正常食蟹猴每两周静脉注射一次治疗剂量(0.05mg/kg)的Y-CVF,共4次,检测注射前后不同时间点血清内补体C3水平、总补体活性(CH50)、抗Y-CVF抗体和抗猪内皮细胞异种抗体的变化.结果显示,前2次注射Y-CVF后均有良好的清除补体效果,第3次注射Y-CVF后补体仪被部分灭活,第4次注射Y-CVF后则基本无效.免疫印迹和酶联免疫吸附试验均证实特异性抗Y-CVF抗体产生,且其滴度随着Y-CVF注射次数增加而递增.多次注射Y-CVF后,并没有在血清内榆测到明显的抗猪内皮细胞抗体的变化.因此,多次静脉注射Y-CVF能诱导灵长类动物产生特异性抗体,从而导致Y-CVF失效,但未发现抗α-Gal异种天然抗体明显增加.