The genetic regulatory architecture and epigenomic basis for age-related changes in rattlesnake venom.

Developmental phenotypic changes can evolve under selection imposed by age- and size-related ecological differences. Many of these changes occur through programmed alterations to gene expression patterns, but the molecular mechanisms and gene-regulatory networks underlying these adaptive changes remain poorly understood. Many venomous snakes, including the eastern diamondback rattlesnake (Crotalus adamanteus), undergo correlated changes in diet and venom expression as snakes grow larger with age, providing models for identifying mechanisms of timed expression changes that underlie adaptive life history traits. By combining a highly contiguous, chromosome-level genome assembly with measures of expression, chromatin accessibility, and histone modifications, we identified cis-regulatory elements and trans-regulatory factors controlling venom ontogeny in the venom glands of C. adamanteus. Ontogenetic expression changes were significantly correlated with epigenomic changes within genes, immediately adjacent to genes (e.g., promoters), and more distant from genes (e.g., enhancers). We identified 37 candidate transcription factors (TFs), with the vast majority being up-regulated in adults. The ontogenetic change is largely driven by an increase in the expression of TFs associated with growth signaling, transcriptional activation, and circadian rhythm/biological timing systems in adults with corresponding epigenomic changes near the differentially expressed venom genes. However, both expression activation and repression contributed to the composition of both adult and juvenile venoms, demonstrating the complexity and potential evolvability of gene regulation for this trait. Overall, given that age-based trait variation is common across the tree of life, we provide a framework for understanding gene-regulatory-network-driven life-history evolution more broadly.

Assessing Target Specificity of the Small Molecule Inhibitor MARIMASTAT to Snake Venom Toxins: A Novel Application of Thermal Proteome Profiling.

New treatments that circumvent the pitfalls of traditional antivenom therapies are critical to address the problem of snakebite globally. Numerous snake venom toxin inhibitors have shown promising cross-species neutralization of medically significant venom toxins in vivo and in vitro. The development of high-throughput approaches for the screening of such inhibitors could accelerate their identification, testing, and implementation and thus holds exciting potential for improving the treatments and outcomes of snakebite envenomation worldwide. Energetics-based proteomic approaches, including thermal proteome profiling and proteome integral solubility alteration (PISA) assays, represent "deep proteomics" methods for high throughput, proteome-wide identification of drug targets and ligands. In the following study, we apply thermal proteome profiling and PISA methods to characterize the interactions between venom toxin proteoforms in Crotalus atrox (Western Diamondback Rattlesnake) and the snake venom metalloprotease (SVMP) inhibitor marimastat. We investigate its venom proteome-wide effects and characterize its interactions with specific SVMP proteoforms, as well as its potential targeting of non-SVMP venom toxin families. We also compare the performance of PISA thermal window and soluble supernatant with insoluble precipitate using two inhibitor concentrations, providing the first demonstration of the utility of a sensitive high-throughput PISA-based approach to assess the direct targets of small molecule inhibitors for snake venom.

Functional genomic diversity is correlated with neutral genomic diversity in populations of an endangered rattlesnake.

Theory predicts that genetic erosion in small, isolated populations of endangered species can be assessed using estimates of neutral genetic variation, yet this widely used approach has recently been questioned in the genomics era. Here, we leverage a chromosome-level genome assembly of an endangered rattlesnake (Sistrurus catenatus) combined with whole genome resequencing data (N = 110 individuals) to evaluate the relationship between levels of genome-wide neutral and functional diversity over historical and future timescales. As predicted, we found positive correlations between genome-wide estimates of neutral genetic diversity (π) and inferred levels of adaptive variation and an estimate of inbreeding mutation load, and a negative relationship between neutral diversity and an estimate of drift mutation load. However, these correlations were half as strong for projected future levels of neutral diversity based on contemporary effective population sizes. Broadly, our results confirm that estimates of neutral genetic diversity provide an accurate measure of genetic erosion in populations of a threatened vertebrate. They also provide nuance to the neutral-functional diversity controversy by suggesting that while these correlations exist, anthropogenetic impacts may have weakened these associations in the recent past and into the future.

Snake venom gene expression is coordinated by novel regulatory architecture and the integration of multiple co-opted vertebrate pathways.

Understanding how regulatory mechanisms evolve is critical for understanding the processes that give rise to novel phenotypes. Snake venom systems represent a valuable and tractable model for testing hypotheses related to the evolution of novel regulatory networks, yet the regulatory mechanisms underlying venom production remain poorly understood. Here, we use functional genomics approaches to investigate venom regulatory architecture in the prairie rattlesnake and identify cis-regulatory sequences (enhancers and promoters), trans-regulatory transcription factors, and integrated signaling cascades involved in the regulation of snake venom genes. We find evidence that two conserved vertebrate pathways, the extracellular signal-regulated kinase and unfolded protein response pathways, were co-opted to regulate snake venom. In one large venom gene family (snake venom serine proteases), this co-option was likely facilitated by the activity of transposable elements. Patterns of snake venom gene enhancer conservation, in some cases spanning 50 million yr of lineage divergence, highlight early origins and subsequent lineage-specific adaptations that have accompanied the evolution of venom regulatory architecture. We also identify features of chromatin structure involved in venom regulation, including topologically associated domains and CTCF loops that underscore the potential importance of novel chromatin structure to coevolve when duplicated genes evolve new regulatory control. Our findings provide a model for understanding how novel regulatory systems may evolve through a combination of genomic processes, including tandem duplication of genes and regulatory sequences, cis-regulatory sequence seeding by transposable elements, and diverse transcriptional regulatory proteins controlled by a co-opted regulatory cascade.

A novel broad spectrum venom metalloproteinase autoinhibitor in the rattlesnake Crotalus atrox evolved via a shift in paralog function.

The complexity of snake venom composition reflects adaptation to the diversity of prey and may be driven at times by a coevolutionary arms race between snakes and venom-resistant prey. However, many snakes are also resistant to their own venom due to serum-borne inhibitors of venom toxins, which raises the question of how snake autoinhibitors maintain their efficacy as venom proteins evolve. To investigate this potential three-way arms race among venom, prey, and autoinhibitors, we have identified and traced the evolutionary origin of serum inhibitors of snake venom metalloproteinases (SVMPs) in the Western Diamondback rattlesnake Crotalus atrox which possesses the largest known battery of SVMP genes among crotalids examined. We found that C. atrox expresses five members of a Fetuin A-related metalloproteinase inhibitor family but that one family member, FETUA-3, is the major SVMP inhibitor that binds to approximately 20 different C. atrox SVMPs and inhibits activities of all three SVMP classes. We show that the fetua-3 gene arose deep within crotalid evolution before the origin of New World species but, surprisingly, fetua-3 belongs to a different paralog group than previously identified SVMP inhibitors in Asian and South American crotalids. Conversely, the C. atrox FETUA-2 ortholog of previously characterized crotalid SVMP inhibitors shows limited activity against C. atrox SVMPs. These results reveal that there has been a functional evolutionary shift in the major SVMP inhibitor in the C. atrox lineage as the SVMP family expanded and diversified in the Crotalus lineage. This broad-spectrum inhibitor may be of potential therapeutic interest.

Distinct regulatory networks control toxin gene expression in elapid and viperid snakes.

BACKGROUND: Venom systems are ideal models to study genetic regulatory mechanisms that underpin evolutionary novelty. Snake venom glands are thought to share a common origin, but there are major distinctions between venom toxins from the medically significant snake families Elapidae and Viperidae, and toxin gene regulatory investigations in elapid snakes have been limited. Here, we used high-throughput RNA-sequencing to profile gene expression and microRNAs between active (milked) and resting (unmilked) venom glands in an elapid (Eastern Brown Snake, Pseudonaja textilis), in addition to comparative genomics, to identify cis- and trans-acting regulation of venom production in an elapid in comparison to viperids (Crotalus viridis and C. tigris). RESULTS: Although there is conservation in high-level mechanistic pathways regulating venom production (unfolded protein response, Notch signaling and cholesterol homeostasis), there are differences in the regulation of histone methylation enzymes, transcription factors, and microRNAs in venom glands from these two snake families. Histone methyltransferases and transcription factor (TF) specificity protein 1 (Sp1) were highly upregulated in the milked elapid venom gland in comparison to the viperids, whereas nuclear factor I (NFI) TFs were upregulated after viperid venom milking. Sp1 and NFI cis-regulatory elements were common to toxin gene promoter regions, but many unique elements were also present between elapid and viperid toxins. The presence of Sp1 binding sites across multiple elapid toxin gene promoter regions that have been experimentally determined to regulate expression, in addition to upregulation of Sp1 after venom milking, suggests this transcription factor is involved in elapid toxin expression. microRNA profiles were distinctive between milked and unmilked venom glands for both snake families, and microRNAs were predicted to target a diversity of toxin transcripts in the elapid P. textilis venom gland, but only snake venom metalloproteinase transcripts in the viperid C. viridis venom gland. These results suggest differences in toxin gene posttranscriptional regulation between the elapid P. textilis and viperid C. viridis. CONCLUSIONS: Our comparative transcriptomic and genomic analyses between toxin genes and isoforms in elapid and viperid snakes suggests independent toxin regulation between these two snake families, demonstrating multiple different regulatory mechanisms underpin a venomous phenotype.

Genetic mechanisms and biological processes underlying host response to ophidiomycosis (snake fungal disease) inferred from tissue-specific transcriptome analyses.

Emerging infectious diseases in wildlife species caused by pathogenic fungi are of growing concern, yet crucial knowledge gaps remain for diseases with potentially large impacts. For example, there is detailed knowledge about host pathology and mechanisms underlying response for chytridiomycosis in amphibians and white-nose syndrome in bats, but such information is lacking for other more recently described fungal infections. One such disease is ophidiomycosis, caused by the fungus Ophidiomyces ophidiicola, which has been identified in many species of snakes, yet the biological mechanisms and molecular changes occurring during infection are unknown. To gain this information, we performed a controlled experimental infection in captive Prairie rattlesnakes (Crotalus viridis) with O. ophidiicola at two different temperatures: 20 and 26°C. We then compared liver, kidney, and skin transcriptomes to assess tissue-specific genetic responses to O. ophidiicola infection. Given previous histopathological studies and the fact that snakes are ectotherms, we expected highest fungal activity on skin and a significant impact of temperature on host response. Although we found fungal activity to be localized on skin, most of the differential gene expression occurred in internal tissues. Infected snakes at the lower temperature had the highest host mortality whereas two-thirds of the infected snakes at the higher temperature survived. Our results suggest that ophidiomycosis is likely a systemic disease with long-term effects on host response. Our analysis also identified candidate protein coding genes that are potentially involved in host response, providing genetic tools for studies of host response to ophidiomycosis in natural populations.

Crotamine derived from Crotalus durissus terrificus venom combined with drugs increases in vitro antibacterial and antifungal activities.

This study investigated crotamine (CTA), a peptide derived from the venom of the South American rattlesnake Crotalus durissus terrificus, known for its exceptional cell penetration potential. The objective was to explore the antibacterial and antifungal activity of CTA, its ability to inhibit efflux pumps and evaluate the effectiveness of its pharmacological combination with antibiotics and antifungals. In microbiological assays, CTA in combination with antibiotics was tested against strains of S. aureus and the inhibition of NorA, Tet(K) and MepA efflux pumps was also evaluated. CTA alone did not present clinically relevant direct antibacterial action, presenting MIC > 209.7 µM against strains S. aureus 1199B, IS-58, K2068. The standard efflux pump inhibitor CCCP showed significant effects in all negative relationships to assay reproducibility. Against the S. aureus 1199B strain, CTA (20.5 µM) associated with norfloxacin diluted 10 × (320.67 µM) showed a potentiating effect, in relation to the control. Against the S. aureus IS-58 strain, the CTA associated with tetracycline did not show a significant combinatorial effect, either with 2304 or 230.4 µM tetracycline. CTA at a concentration of 2.05 µM associated with ciprofloxacin at a concentration of 309.4 µM showed a significant potentiating effect. In association with EtBr, CTA at concentrations of 2.05 and 20.5 µM potentiated the effect in all strains tested, reducing the prevention of NorA, Tet(K) and MepA efflux pumps. In the C. albicans strain, a potentiating effect of fluconazole (334.3 µM) was observed when combined with CTA (2.05 µM). Against the C. tropicalis strain, a significant effect was also observed in the association of fluconazole 334.3 µM, where CTA 2.05 µM considerably reduced fungal growth and decreased the potentiation of fluconazole. Against the C. krusei strain, no significant potentiating effect of fluconazole was obtained by CTA. Our results indicate that CTA in pharmacological combination potentiates the effects of antibiotics and antifungal. This represents a new and promising antimicrobial strategy for treating a wide variety of infections.

The relevance of vascular adjustments to hemodynamic control in the face of temperature change in Crotalus durissus.

The presence of cardiac shunts in ectothermic tetrapods is thought to be consistent with active vascular modulations for proper hemodynamic support. Local control of blood flow modulates tissue perfusion and thus systemic conductance (Gsys) is assumed to increase with body temperature (Tb) to accommodate higher aerobic demand. However, the general increase of Gsys presses for a higher right-to-left (R-L) shunt, which reduces arterial oxygen concentration. In contrast, Tb reduction leads to a Gsys decrease and a left-to-right shunt, which purportedly increases pulmonary perfusion and plasma filtration in the respiratory area. This investigation addressed the role of compensatory vascular adjustments in the face of the metabolic alterations caused by Tb change in the South American rattlesnake (Crotalus durissus). Cardiovascular recordings were performed in decerebrated rattlesnake preparations at 10, 20 and 30°C. The rise in Tb increased metabolic demand, and correlated with an augmentation in heart rate. Although cardiac output increased, systemic stroke volume reduced while pulmonary stroke volume remained stable. Although that resulted in a proportionally higher increase in pulmonary blood flow, the R-L shunt was maintained. While the systemic compliance of large arteries was the most relevant factor in regulating arterial systemic blood pressure, peripheral conductance of pulmonary circulation was the major factor influencing the final cardiac shunt. Such dynamic adjustment of systemic compliance and pulmonary resistance for shunt modulation has not been demonstrated before and contrasts with previous knowledge on shunt control.

SEQUENCE SLIDER: integration of structural and genetic data to characterize isoforms from natural sources.

Proteins isolated from natural sources can be composed of a mixture of isoforms with similar physicochemical properties that coexist in the final steps of purification. Yet, even where unverified, the assumed sequence is enforced throughout the structural studies. Herein, we propose a novel perspective to address the usually neglected sequence heterogeneity of natural products by integrating biophysical, genetic and structural data in our program SEQUENCE SLIDER. The aim is to assess the evidence supporting chemical composition in structure determination. Locally, we interrogate the experimental map to establish which side chains are supported by the structural data, and the genetic information relating sequence conservation is integrated into this statistic. Hence, we build a constrained peptide database, containing most probable sequences to interpret mass spectrometry data (MS). In parallel, we perform MS de novo sequencing with genomic-based algorithms to detect point mutations. We calibrated SLIDER with Gallus gallus lysozyme, whose sequence is unequivocally established and numerous natural isoforms are reported. We used SLIDER to characterize a metalloproteinase and a phospholipase A2-like protein from the venom of Bothrops moojeni and a crotoxin from Crotalus durissus collilineatus. This integrated approach offers a more realistic structural descriptor to characterize macromolecules isolated from natural sources.

The Tiger Rattlesnake genome reveals a complex genotype underlying a simple venom phenotype.

Variation in gene regulation is ubiquitous, yet identifying the mechanisms producing such variation, especially for complex traits, is challenging. Snake venoms provide a model system for studying the phenotypic impacts of regulatory variation in complex traits because of their genetic tractability. Here, we sequence the genome of the Tiger Rattlesnake, which possesses the simplest and most toxic venom of any rattlesnake species, to determine whether the simple venom phenotype is the result of a simple genotype through gene loss or a complex genotype mediated through regulatory mechanisms. We generate the most contiguous snake-genome assembly to date and use this genome to show that gene loss, chromatin accessibility, and methylation levels all contribute to the production of the simplest, most toxic rattlesnake venom. We provide the most complete characterization of the venom gene-regulatory network to date and identify key mechanisms mediating phenotypic variation across a polygenic regulatory network.

Snakes on a plain: biotic and abiotic factors determine venom compositional variation in a wide-ranging generalist rattlesnake.

BACKGROUND: Snake venoms are trophic adaptations that represent an ideal model to examine the evolutionary factors that shape polymorphic traits under strong natural selection. Venom compositional variation is substantial within and among venomous snake species. However, the forces shaping this phenotypic complexity, as well as the potential integrated roles of biotic and abiotic factors, have received little attention. Here, we investigate geographic variation in venom composition in a wide-ranging rattlesnake (Crotalus viridis viridis) and contextualize this variation by investigating dietary, phylogenetic, and environmental variables that covary with venom. RESULTS: Using shotgun proteomics, venom biochemical profiling, and lethality assays, we identify 2 distinct divergent phenotypes that characterize major axes of venom variation in this species: a myotoxin-rich phenotype and a snake venom metalloprotease (SVMP)-rich phenotype. We find that dietary availability and temperature-related abiotic factors are correlated with geographic trends in venom composition. CONCLUSIONS: Our findings highlight the potential for snake venoms to vary extensively within species, for this variation to be driven by biotic and abiotic factors, and for the importance of integrating biotic and abiotic variation for understanding complex trait evolution. Links between venom variation and variation in biotic and abiotic factors indicate that venom variation likely results from substantial geographic variation in selection regimes that determine the efficacy of venom phenotypes across populations and snake species. Our results highlight the cascading influence of abiotic factors on biotic factors that ultimately shape venom phenotype, providing evidence for a central role of local selection as a key driver of venom variation.

High thermal quality rookeries facilitate high thermoregulatory accuracy in pregnant female rattlesnakes.

Temperature is a primary factor influencing organismal development, and the fluctuating daily and seasonal thermal regimes of temperate climates may challenge the ability of viviparous reptiles to optimize body temperatures during gestation. Testing how viviparous reptiles navigate highly variable thermal conditions (e.g., relatively cold nights and/or highly fluctuating temperatures) is a powerful way to understand how they use microhabitats for thermoregulatory benefits. We assessed the thermal ecology of pregnant and non-pregnant female Prairie Rattlesnakes (Crotalus viridis) inhabiting a high-elevation, montane shrubland in northwest Colorado throughout their short summer active season, addressing the thermal consequences of microhabitat selection with a focus on thermoregulation of pregnant females at communal rookery sites. We deployed operative temperature models to collect data on the thermal quality of microhabitats used by the snakes, and calculated thermoregulatory accuracy of the snakes by comparing their field-active body temperatures with preferred body temperatures of snakes placed in a thermal gradient. Pregnant females inhabited rocky, hilltop rookeries that had higher thermal quality due to higher and less variable nighttime temperatures compared to microhabitats in the surrounding prairie. Pregnant females therefore thermoregulated more accurately than non-pregnant females. The difference was most pronounced during the night, when pregnant females at rookeries maintained higher body temperatures than non-pregnant snakes in the prairie. Our results support the hypothesis that one major reason female rattlesnakes at high latitudes and/or high elevations forgo migration and gestate at communal, rocky, hilltop rookeries is that, relative to prairie microhabitats, they provide better conditions for thermoregulation during pregnancy.

Ruthenium Antivenom Inhibits the Defibrinogenating Activity of Crotalus adamanteus Venom in Rabbits.

Eastern Diamondback Rattlesnake (Crotalus adamanteus) envenomation is a medical emergency encountered in the Southeastern United States. The venom contains a snake venom thrombin-like enzyme (SVTLE) that is defibrinogenating, causing coagulopathy without effects on platelets in humans. This investigation utilized thrombelastographic methods to document this coagulopathy kinetically on the molecular level in a rabbit model of envenomation via the analyses of whole blood samples without and with platelet inhibition. Subsequently, the administration of a novel ruthenium compound containing site-directed antivenom abrogated the coagulopathic effects of envenomation in whole blood without platelet inhibition and significantly diminished loss of coagulation in platelet-inhibited samples. This investigation provides coagulation kinetic insights into the molecular interactions and results of SVTLE on fibrinogen-dependent coagulation and confirmation of the efficacy of a ruthenium antivenom. These results serve as a rationale to investigate the coagulopathic effects of other venoms with this model and assess the efficacy of this site-directed antivenom.

Phosphate-Buffered Saline and Dimethyl Sulfoxide Enhance the Antivenom Action of Ruthenium Chloride against Crotalus atrox Venom in Human Plasma-A Preliminary Report.

Ruthenium chloride (RuCl3) is widely utilized for synthesis and catalysis of numerous compounds in academia and industry and is utilized as a key molecule in a variety of compounds with medical applications. Interestingly, RuCl3 has been demonstrated to modulate human plasmatic coagulation and serves as a constituent of a compounded inorganic antivenom that neutralizes the coagulopathic effects of snake venom in vitro and in vivo. Using thrombelastography, this investigation sought to determine if RuCl3 inhibition of the fibrinogenolytic effects of Crotalus atrox venom could be modulated by vehicle composition in human plasma. Venom was exposed to RuCl3 in 0.9% NaCl, phosphate-buffered saline (PBS), or 0.9% NaCl containing 1% dimethyl sulfoxide (DMSO). RuCl3 inhibited venom-mediated delay in the onset of thrombus formation, decreased clot growth velocity, and decreased clot strength. PBS and DMSO enhanced the effects of RuCl3. It is concluded that while a Ru-based cation is responsible for significant inhibition of venom activity, a combination of Ru-based ions containing phosphate and DMSO enhances RuCl3-mediated venom inhibition. Additional investigation is indicated to determine what specific Ru-containing molecules cause venom inhibition and what other combinations of inorganic/organic compounds may enhance the antivenom effects of RuCl3.

Fab Antivenom Reversal of Neurotoxicity Caused by a Juvenile Crotalus horridus Lacking Canebrake Toxin.

We present a case of neurotoxic effects in a pediatric patient after envenomation by a timber rattlesnake (Crotalus horridus) in the Appalachian upstate of South Carolina. Though some members of this species are capable of primarily neurotoxic envenomation, there is heterogeneity in venom composition, and neurotoxic timber rattlesnakes are not endemic to the Appalachian region. However, neurotoxic effects caused by C horridus species lacking typical neurotoxins have been suspected, though not previously confirmed in the medical literature. This case presents a patient who was envenomated by a genotypically confirmed non-neurotoxic C horridus but who nevertheless presented with symptoms consistent with primary neurotoxicity. Neurotoxic effects can be variable in their response to traditional antivenom, though this patient demonstrated rapid response to treatment, representing a novel case in the literature of neurotoxic effects from a snake lacking typical neurotoxins with documented improvement with traditional antivenom.

SCHIZANGIELLA INFECTIONS IN AN EASTERN RATSNAKE (PANTHEROPHIS ALLEGHANIENSIS) AND A TIMBER RATTLESNAKE (CROTALUS HORRIDUS).

This report describes Schizangiella infections in colubrid and viperid snakes. A captive eastern ratsnake (Pantherophis alleghaniensis) was presented for a large intraoral mass associated with the mandible. The mass was debulked and histologic examination revealed severe, granulomatous stomatitis with intralesional fungi exhibiting morphologic features consistent with Schizangiella serpentis. PCR and sequencing of affected tissues confirmed S. serpentis. Because of declining health, the ratsnake was euthanized and postmortem examination identified a disseminated S. serpentis infection involving the skeletal musculature, lung, kidney, mesentery, and mandible. A wild-caught timber rattlesnake (Crotalus horridus) was presented for cutaneous lesions, weakness, and lethargy and later died. Postmortem examination revealed a mass-like structure in the esophagus characterized by high numbers of Schizangiella-like fungi associated with extensive granulomatous inflammation; the snake also had cutaneous mycosis suggestive of ophidiomycosis. This is the first report to document the unique morphologic features of S. serpentis in tissues and the presentation of schizangiellosis in snakes. Schizangiellosis should be considered as a differential diagnosis for nodular lesions involving the oral cavity and/or the gastrointestinal tract of snakes.

COMPARING THE EFFECTS OF LITHIUM HEPARIN AND DIPOTASSIUM ETHYLENEDIAMINETETRAACETIC ACID ON HEMATOLOGIC VALUES IN PRAIRIE RATTLESNAKES (CROTALUS VIRIDIS) AND LAKE ERIE WATER SNAKES (NERODIA SIPEDON INSULARUM).

Anticoagulants prevent clotting of blood samples and preserve cellular morphology for hematologic evaluations, but studies comparing anticoagulants are limited in snakes. The objective of this study was to evaluate the effects of lithium heparin (LH) and dipotassium ethylenediaminetetraacetic acid (EDTA) on hematologic values in prairie rattlesnakes (PR; Crotalus viridis, n = 16) and Lake Erie water snakes (LEWS; Nerodia sipedon insularum, n = 21). Venipuncture was performed and blood samples were immediately aliquoted into LH and EDTA microtainers. Packed cell volume (PCV), total solids (TS), 100-cell differential counts, and Avian Leukopet white blood cell counts (WBC) were determined for each anticoagulant. Passing-Bablok regression and Bland-Altman plots revealed that anticoagulant choice did not constantly or proportionally bias the values of any WBC parameter. Mixed models demonstrated that blood anticoagulated with EDTA had higher PCV in PR (P = 0.04) and TS in both species (P < 0.05). However, the magnitude of the differences attributable to anticoagulant choice was relatively small and likely not clinically important. Hemolysis was not appreciated in any samples. Our findings demonstrate that LH and EDTA are equally appropriate for use in PR and LEWS, but may require separate reference values.

Inferring population connectivity in eastern massasauga rattlesnakes (Sistrurus catenatus) using landscape genetics.

Assessing the environmental factors that influence the ability of a threatened species to move through a landscape can be used to identify conservation actions that connect isolated populations. However, direct observations of species' movement are often limited, making the development of alternate approaches necessary. Here we use landscape genetic analyses to assess the impact of landscape features on the movement of individuals between local populations of a threatened snake, the eastern massasauga rattlesnake (Sistrurus catenatus). We linked connectivity data with habitat information from two landscapes of similar size: a large region of unfragmented habitat and a previously studied fragmented landscape consisting of isolated patches of habitat. We used this analysis to identify features of the landscape where modification or acquisition would enhance population connectivity in the fragmented region. We found evidence that current connectivity was impacted by both contemporary land-cover features, especially roads, and inherent landscape features such as elevation. Next, we derived estimates of expected movement ability using a recently developed pedigree-based approach and least-cost paths through the unfragmented landscape. We then used our pedigree and resistance map to estimate resistance polygons of the potential extent for S. catenatus movement in the fragmented landscape. These polygons identify possible sites for future corridors connecting currently isolated populations in this landscape by linking the impact of future habitat modification or land acquisition to dispersal ability in this species. Overall, our study shows how modeling landscape resistance across differently fragmented landscapes can identify habitat features that affect contemporary movement in threatened species in fragmented landscapes and how this information can be used to guide mitigation actions whose goal is to connect isolated populations.

Neurotoxicity Associated With the Southern Pacific Rattlesnake (Crotalushelleri).

The Southern Pacific rattlesnake (Crotalus helleri) is commonly encountered throughout Southern California. Typical toxicity includes tissue injury and hematologic toxicity. However, neurotoxicity is not commonly reported with rattlesnake envenomations, other than infrequently with select species, including the Mojave rattlesnake (Crotalus scutulatus scutulatus). Importantly, clinical neurotoxicity has not been well described with the Southern Pacific rattlesnake, the only rattlesnake in the city of Los Angeles, along with the Southern and coastal regions of Los Angeles County. In this case series, 7 patients envenomated by the Southern Pacific rattlesnake with significant neurotoxicity, including dysarthria, ataxia, and myokymia, are presented. Clinicians practicing in this region should be aware of evolving patterns of toxicity associated with the Southern Pacific rattlesnake.