Oreganus Helleri Research Articles

A genome assembly for the southern Pacific rattlesnake, Crotalus oreganus helleri, in the western rattlesnake species complex.

Rattlesnakes play important roles in their ecosystems by regulating prey populations, are involved in complex coevolutionary dynamics with their prey, and exhibit a variety of unusual adaptations, including maternal care, heat-sensing pit organs, hinged fangs, and medically-significant venoms. The western rattlesnake (Crotalus oreganus) is one of the widest ranging rattlesnake species, with a distribution from British Columbia, where it is listed as threatened, to Baja California and east across the Great Basin to western Wyoming, Colorado and New Mexico. Here, we report a new reference genome assembly for one of six currently recognized subspecies, C. oreganus helleri, as part of the California Conservation Genomics Project (CCGP). Consistent with the reference genomic sequencing strategy of the CCGP, we used Pacific Biosciences HiFi long reads and Hi-C chromatin-proximity sequencing technology to produce a de novo assembled genome. The assembly comprises a total of 698 scaffolds spanning 1,564,812,557 base pairs, has a contig N50 of 64.7 Mb, a scaffold N50 of 110.8 Mb, and BUSCO complete score of 90.5%. This reference genome will be valuable for studies on the genomic basis of venom evolution and variation within Crotalus, in resolving the taxonomy of C. oreganus and its relatives, and for the conservation and management of rattlesnakes in general.

Proteomic Identification and Quantification of Snake Venom Biomarkers in Venom and Plasma Extracellular Vesicles.

The global exploration of snakebites requires the use of quantitative omics approaches to characterize snake venom as it enters into the systemic circulation. These omics approaches give insights into the venom proteome, but a further exploration is warranted to analyze the venom-reactome for the identification of snake venom biomarkers. The recent discovery of extracellular vesicles (EVs), and their critical cellular functions, has presented them as intriguing sources for biomarker discovery and disease diagnosis. Herein, we purified EV’s from the snake venom (svEVs) of Crotalus atrox and C. oreganus helleri, and from plasma of BALB/c mice injected with venom from each snake using EVtrap in conjunction with quantitative mass spectrometry for the proteomic identification and quantification of svEVs and plasma biomarkers. Snake venom EVs from C. atrox and C. o. helleri were highly enriched in 5′ nucleosidase, L-amino acid oxidase, and metalloproteinases. In mouse plasma EVs, a bioinformatic analysis for revealed upregulated responses involved with cytochrome P450, lipid metabolism, acute phase inflammation immune, and heat shock responses, while downregulated proteins were associated with mitochondrial electron transport, NADH, TCA, cortical cytoskeleton, reticulum stress, and oxidative reduction. Altogether, this analysis will provide direct evidence for svEVs composition and observation of the physiological changes of an envenomated organism.

Neutralization of crotamine by polyclonal antibodies generated against two whole rattlesnake venoms and a novel recombinant fusion protein

Crotamine is a paralyzing toxin (MW: ~5 kDa) found in different proportions in some rattlesnake venoms (up to 62%). Mexican pit viper antivenoms have shown low immunoreactivity against crotamine, which is an urgent quality to be improved. The objective of this work was to evaluate the ability of a novel recombinant fusion protein composed of sphingomyelinase D and crotamine, and two whole venoms from Crotalus molossus nigrescens and C. oreganus helleri to produce neutralizing antibodies against crotamine. These immunogens were separately used for immunization procedures in rabbits. Then, we generated three experimental antivenoms to test their cross-reactivity via western-blot against crotamine from 7 species (C. m. nigrescens, C. o. helleri, C. durissus terrificus, C. scutulatus salvini, C. basiliscus, C. culminatus and C. tzabcan). We also performed pre-incubation neutralization experiments in mice to measure the neutralizing potency of each antivenom against crotamine induced hind limb paralysis. Our antivenoms showed broad recognition across crotamine from most of the tested species. Also, neutralization against crotamine paralysis symptom was successfully achieved by our three antivenoms, albeit with different efficiencies. Our results highlight the use of crotamine enriched venoms and our novel recombinant fusion protein as promising immunogens to improve the neutralizing potency against crotamine for the improvement of Mexican antivenoms.

Biological activities of a new crotamine-like peptide from Crotalus oreganus helleri on C2C12 and CHO cell lines, and ultrastructural changes on motor endplate and striated muscle

Crotamine and crotamine-like peptides are non-enzymatic polypeptides, belonging to the family of myotoxins, which are found in high concentration in the venom of the Crotalus genus. Helleramine was isolated and purified from the venom of the Southern Pacific rattlesnake, Crotalus oreganus helleri. This peptide had a similar, but unique, identity to crotamine and crotamine-like proteins isolated from other rattlesnakes species. The variability of crotamine-like protein amino acid sequences may allow different toxic effects on biological targets or optimize the action against the same target of different prey. Helleramine was capable of increasing intracellular Ca2+ in Chinese Hamster Ovary (CHO) cell line. It inhibited cell migration as well as cell viability (IC50 = 11.44 μM) of C2C12, immortalized skeletal myoblasts, in a concentration dependent manner, and promoted early apoptosis and cell death under our experimental conditions. Skeletal muscle harvested from mice 24 h after helleramine injection showed contracted myofibrils and profound vacuolization that enlarged the subsarcolemmal space, along with loss of plasmatic and basal membrane integrity. The effects of helleramine provide further insights and evidence of myotoxic activities of crotamine-like peptides and their possible role in crotalid envenomings.

A myotoxin from the venom of Crotalus oreganus helleri: Its role in snake envenoming

A myotoxin from the venom of Crotalus oreganus helleri: Its role in snake envenoming

The effects of temperature on the defensive strikes of rattlesnakes.

Movements of ectotherms are constrained by their body temperature owing to the effects of temperature on muscle physiology. As physical performance often affects the outcome of predator-prey interactions, environmental temperature can influence the ability of ectotherms to capture prey and/or defend themselves against predators. However, previous research on the kinematics of ectotherms suggests that some species may use elastic storage mechanisms when attacking or defending, thereby mitigating the effects of sub-optimal temperature. Rattlesnakes (Crotalus spp.) are a speciose group of ectothermic viperid snakes that rely on crypsis, rattling and striking to deter predators. We examined the influence of body temperature on the behavior and kinematics of two rattlesnake species (Crotalus oreganus helleri and Crotalus scutulatus) when defensively striking towards a threatening stimulus. We recorded defensive strikes at body temperatures ranging from 15-35°C. We found that strike speed and speed of mouth gaping during the strike were positively correlated with temperature. We also found a marginal effect of temperature on the probability of striking, latency to strike and strike outcome. Overall, warmer snakes are more likely to strike, strike faster, open their mouth faster and reach maximum gape earlier than colder snakes. However, the effects of temperature were less than would be expected for purely muscle-driven movements. Our results suggest that, although rattlesnakes are at a greater risk of predation at colder body temperatures, their decrease in strike performance may be mitigated to some extent by employing mechanisms in addition to skeletal muscle contraction (e.g. elastic energy storage) to power strikes.

Does Rattling Deter? The Case of Domestic Dogs.

Although the rattling of rattlesnakes (Crotalus and Sistrurus) is widely accepted as being aposematic, the hypothesis that rattling deters approach from the snake's potentially dangerous adversaries has not been well tested. In a controlled study using rattling recorded from captive rattlesnakes (C. oreganus helleri) and a variety of comparison sounds or no-sound controls, domestic dogs (Canis familiaris) showed no hesitation to approach camouflaged speakers projecting the recorded rattles. The dogs were equally likely to approach speakers projecting rattling as they were to approach speakers playing control sounds, or speakers that were silent. Furthermore, the dogs spent no less time in front of the speakers projecting the rattles than they did in front of speakers projecting control sounds or no sound. The dogs' reactions may not be representative of other species with whom rattlesnakes come into contact, but the data suggest a need for some circumspection about the role of rattling in the rattlesnake's defensive repertoire. Our results also suggest that dogs may be vulnerable to envenomation because they fail to react to the sound of rattling with avoidance.

Purification and characterization of cysteine rich-secretory proteins (crisps) from the venom of the southern pacific rattlesnake (crotalus oreganus helleri): Their role on blood and lymphatic endothelial cell permeability

Purification and characterization of cysteine rich-secretory proteins (crisps) from the venom of the southern pacific rattlesnake (crotalus oreganus helleri): Their role on blood and lymphatic endothelial cell permeability

Expression, purification and characterization of a new recombinant crotamine isoform from the venom gland of Crotalus oreganus helleri

Expression, purification and characterization of a new recombinant crotamine isoform from the venom gland of Crotalus oreganus helleri

Expression, purification, and characterization of the new recombinant crotamine isoform from the venom gland of Crotalus oreganus helleri on antibacterial activity

Expression, purification, and characterization of the new recombinant crotamine isoform from the venom gland of Crotalus oreganus helleri on antibacterial activity

The effects of hybridization on divergent venom phenotypes: Characterization of venom from Crotalus scutulatus scutulatus × Crotalus oreganus helleri hybrids

Hybridization between divergent species can be analyzed to elucidate expression patterns of distinct parental characteristics, as well as to provide information about the extent of reproductive isolation between species. A known hybrid cross between two rattlesnakes with highly divergent venom phenotypes provided the opportunity to examine occurrence of parental venom characteristics in the F1 hybrids as well as ontogenetic shifts in the expression of these characters as the hybrids aged. Although venom phenotypes of adult rattlesnake venoms are known for many species, the effect of hybridization on phenotype inheritance is not well understood, and effects of hybridization on venom ontogeny have not yet been investigated. The current study investigates both phenomena resulting from the hybridization of a male snake with type I degradative venom, Crotalus oreganus helleri (Southern Pacific Rattlesnake), and a female snake with type II highly toxic venom, Crotalus scutulatus scutulatus (Mojave Rattlesnake). SDS-PAGE, enzymology, Western blot and reversed phase HPLC (RP-HPLC) were used to characterize the venom of the C. o. helleri male, the C. s. scutulatus female and their two hybrid offspring as they aged. In general, Crotalus o. helleri × C. s. scutulatus hybrid venoms appeared to exhibit overlapping parental venom profiles, and several different enzyme activity patterns. Both hybrids expressed C. o. helleri father-specific myotoxins as well as C. s. scutulatus mother-specific Mojave toxin. Snake venom metalloprotease activity displayed apparent sex-influenced expression patterns, while hybrid serine protease activities were intermediate to parental activities. The C. s. scutulatus × C. o. helleri hybrid male's venom profile provided the strongest evidence that type I and type II venom characteristics are expressed simultaneously in hybrid venoms, as this snake contained distinctive characteristics of both parental species. However, the possibility of sex-influenced development of metalloprotease activity, as seen in the ontogenetic shifts of the hybrid female, may influence the levels of expression of both type I and type II characteristics in hybrid venoms. Ultimately, the chronological analysis of this known hybrid system reveals the most distinct characteristics that can be used in determining successful hybridization between snakes that follow the type I-type II trend in rattlesnake venom composition, namely the presence of metalloprotease activity and Mojave toxin.

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

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

Snake Venomics of Crotalus tigris: The Minimalist Toxin Arsenal of the Deadliest Neartic Rattlesnake Venom. Evolutionary Clues for Generating a Pan-Specific Antivenom against Crotalid Type II Venoms

We report the proteomic and antivenomic characterization of Crotalus tigris venom. This venom exhibits the highest lethality for mice among rattlesnakes and the simplest toxin proteome reported to date. The venom proteome of C. tigris comprises 7-8 gene products from 6 toxin families; the presynaptic β-neurotoxic heterodimeric PLA(2), Mojave toxin, and two serine proteinases comprise, respectively, 66 and 27% of the C. tigris toxin arsenal, whereas a VEGF-like protein, a CRISP molecule, a medium-sized disintegrin, and 1-2 PIII-SVMPs each represent 0.1-5% of the total venom proteome. This toxin profile really explains the systemic neuro- and myotoxic effects observed in envenomated animals. In addition, we found that venom lethality of C. tigris and other North American rattlesnake type II venoms correlates with the concentration of Mojave toxin A-subunit, supporting the view that the neurotoxic venom phenotype of crotalid type II venoms may be described as a single-allele adaptation. Our data suggest that the evolutionary trend toward neurotoxicity, which has been also reported for the South American rattlesnakes, may have resulted by pedomorphism. The ability of an experimental antivenom to effectively immunodeplete proteins from the type II venoms of C. tigris, Crotalus horridus , Crotalus oreganus helleri, Crotalus scutulatus scutulatus, and Sistrurus catenatus catenatus indicated the feasibility of generating a pan-American anti-Crotalus type II antivenom, suggested by the identification of shared evolutionary trends among South and North American Crotalus species.

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.

Quantitative Analysis of Snake Venoms Using Soluble Polymer-based Isotope Labeling

We present the design and synthesis of a new quantitative strategy termed soluble polymer-based isotope labeling (SoPIL) and its application as a novel and inclusive method for the identification and relative quantification of individual proteins in complex snake venoms. The SoPIL reagent selectively captures and isolates cysteine-containing peptides, and the subsequent tagged peptides are released and analyzed using nanoflow liquid chromatography-tandem mass spectrometry. The SoPIL strategy was used to quantify venom proteins from two pairs of venomous snakes: Crotalus scutulatus scutulatus type A, C. scutulatus scutulatus type B, Crotalus oreganus helleri, and Bothrops colombiensis. The hemorrhagic, hemolytic, clotting ability, and fibrinogenolytic activities of crude venoms were measured and correlated with difference in protein abundance determined by the SoPIL analysis. The SoPIL approach could provide an efficient and widely applicable tool for quantitative proteomics.

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.

The Link between Mating Season and Male Reproductive Anatomy in the Rattlesnakes Crotalus viridis oreganus and Crotalus viridis helleri

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The Link between Mating Season and Male Reproductive Anatomy in the Rattlesnakes Crotalus viridis oreganus and Crotalus viridis helleri

The Link between Mating Season and Male Reproductive Anatomy in the Rattlesnakes Crotalus viridis oreganus and Crotalus viridis helleri