Motor control: Snake neurons speed up.

How are motor neurons tuned for very different jobs? Classic work has focused on variations in motor neuron size and their premotor networks. New results in rattlesnakes show that shifting a motor neuron's temporal precision can be as simple as changing its potassium channel conductance.

Pit viper thermography: the pit organ used by crotaline snakes to detect thermal contrast has poor spatial resolution.

Pit vipers detect infrared radiation by means of temperature contrasts created on their pit organ membranes. Signals from pit organs integrate with visual signals in the optic tectum, leading to the conjecture that the facial pits operate as an extension of the visual system. Because similar mechanisms underlie thermal imaging technology, imagery from thermal cameras is often used to infer how pit vipers perceive their environment. However, pit organs lack a focusing mechanism, and biophysical models predict that pit organs should have poor spatial resolution compared with thermal imaging cameras. Nevertheless, behavioral studies occasionally suggest pits may have better resolution than predicted by biophysical models, indicating that processing in the central nervous system may improve imaging. To estimate the spatial resolution of the neural image informing behavior, we recorded snake responses evoked by targets moving across backgrounds composed of two contrasting temperatures with an average temperature equal to the target temperature. An unresolved background would appear uniform; thus, the target would be detectable only if the background pattern were resolved. Western rattlesnakes (Crotalus oreganus) displayed no statistically significant responses to targets presented in front of patterned backgrounds, regardless of the temperature contrasts or spatial frequencies within the background, but responded strongly to targets presented in front of homogeneous backgrounds. We found no evidence that the pit organ system can resolve spatial details subtending an angle of 9 deg or less. We discuss the implications of these results for understanding pit organ function in ecologically relevant habitats with thermal heterogeneity.

Baroreflex responses of decerebrate rattlesnakes (Crotalus durissus) are comparable to awake animals.

A decerebrate rattlesnake, Crotalus durissus, has previously been used as a model Squamate for cardiovascular studies. It enabled instrumentation for concomitant recordings of diverse variables that showed autonomic responses. However, to validate the preparation and its scope for use, it is necessary to assess how close its cardiovascular variables are to non-decerebrate snakes and the effectiveness of its autonomic responses. Similarly, it is important to analyze its recovery profile after instrumentation and observe if it maintains stability throughout the duration of experimental protocol. Here we have objectively assessed these points by comparing decerebrate preparations and non-decerebrate snakes, after the occlusive cannulation of the vertebral artery. We have assessed cardiovascular variables and the baroreflex to analyze the presence, magnitude and stability of complex autonomic-controlled parameters as indicators of autonomic nervous system (ANS) functionality. After instrumentation, mean heart rates were high but recovered to stable values within 24 h. Mean arterial pressure stabilized within 24 h in control snakes and 48 h in decerebrate preparations. After that, both parameters remained stable. The operational gain and effectiveness index of the baroreflex recovered within the first 6 h after instrumentation in both experimental groups. In addition, the baroreflex capacities and its limits were also equivalent between the groups. These experiments demonstrated that decerebrate preparations and inactive, non-decerebrate snakes showed comparable recovery profiles following anesthesia and cannulation, maintained similar values of cardiovascular variables during experimental manipulation and exhibited functional, ANS modulated reflexes. Accordingly, the present results attest the relevance of this decerebrate preparation for studies on cardiovascular modulation.

Origins, genomic structure and copy number variation of snake venom myotoxins.

Crotamine, myotoxin a and homologs are short peptides that often comprise major fractions of rattlesnake venoms and have been extensively studied for their bioactive properties. These toxins are thought to be important for rapidly immobilizing mammalian prey and are implicated in serious, and sometimes fatal, responses to envenomation in humans. While high quality reference genomes for multiple venomous snakes are available, the loci that encode myotoxins have not been successfully assembled in any existing genome assembly. Here, we integrate new and existing genomic and transcriptomic data from the Prairie Rattlesnake (Crotalus viridis viridis) to reconstruct, characterize, and infer the chromosomal locations of myotoxin-encoding loci. We integrate long-read transcriptomics (Pacific Bioscience's Iso-Seq) and short-read RNA-seq to infer gene sequence diversity and characterize patterns of myotoxin and paralogous ß-defensin expression across multiple tissues. We also identify two long non-coding RNA sequences which both encode functional myotoxins, demonstrating a newly discovered source of venom coding sequence diversity. We also integrate long-range mate-pair chromatin contact data and linked-read sequencing to infer the structure and chromosomal locations of the three myotoxin-like loci. Further, we conclude that the venom-associated myotoxin is located on chromosome 1 and is adjacent to non-venom paralogs. Consistent with this locus contributing to venom composition, we find evidence that the promoter of this gene is selectively open in venom gland tissue and contains transcription factor binding sites implicated in broad trans-regulatory pathways that regulate snake venoms. This study provides the best genomic reconstruction of myotoxin loci to date and raises questions about the physiological roles and interplay between myotoxin and related genes, as well as the genomic origins of snake venom variation.

Scaling and relations of morphology with locomotor kinematics in the sidewinder rattlesnake Crotalus cerastes.

The movement of limbless terrestrial animals differs fundamentally from that of limbed animals, yet few scaling studies of their locomotor kinematics and morphology are available. We examined scaling and relations of morphology and locomotion in sidewinder rattlesnakes (Crotalus cerastes). During sidewinding locomotion, a snake lifts sections of its body up and forward while other sections maintain static ground contact. We used high-speed video to quantify whole-animal speed and acceleration; the height to which body sections are lifted; and the frequency, wavelength, amplitude and skew angle (degree of tilting) of the body wave. Kinematic variables were not sexually dimorphic, and most did not deviate from isometry, except wave amplitude. Larger sidewinders were not faster, contrary to many results from limbed terrestrial animals. Free from the need to maintain dynamic similarity (because their locomotion is dominated by friction rather than inertia), limbless species may have greater freedom to modulate speed independently of body size. Path analysis supported: (1) a hypothesized relationship between body width and wavelength, indicating that stouter sidewinders form looser curves; (2) a strong relationship between cycle frequency and whole-animal speed; and (3) weaker effects of wavelength (positive) and amplitude (negative) on speed. We suggest that sidewinding snakes may face a limit on stride length (to which amplitude and wavelength both contribute), beyond which they sacrifice stability. Thus, increasing frequency may be the best way to increase speed. Finally, frequency and skew angle were correlated, a result that deserves future study from the standpoint of both kinematics and physiology.

Crotalus atrox venom-induced cellular toxicity: Early wound progression involves reactive oxygen species.

Understanding the mechanisms that produce cellular cytotoxicity is fundamental in the field of toxicology. Cytotoxic stimuli can include organic toxins such as hemorrhagic snake venom, which can lead to secondary complications such as the development of necrotic tissue and profuse scarring. These clinical manifestations mimic cytotoxic responses induce by other organic compounds such as organic acids. We used hemorrhagic snake venom and human embryonic kidney cells (HEK 293T) as a model system to better understand the cellular responses involved in venom induced cytotoxicity. Cells stimulated with Crotalus atrox (CA) (western diamondback) venom for 4 or 10 h demonstrated significant cytotoxicity. Results from 2',7'-Dichlorodihydrofluorescein diacetate (H2 DCF-DA) assays determine CA venom stimulation induces a robust production of reactive oxygen species (ROS) over a 3-h time course. In contrast, pretreatment with polyethylene glycol (PEG)-catalase or N-acetyl cysteine (NAC) prior to CA venom stimulation significantly blunted H2 DCFDA fluorescence fold changes and showed greater cytoprotective effects than cells stimulated with CA venom alone. Pre- incubating HEK293T cells with the NADPH oxidase (NOX) pan-inhibitor VAS2870 prior venom stimulation significantly minimized the venom-induced oxidative burst at early timepoints (≤2 h). Collectively, our experiments show that pre-application of antioxidants reduces CA venom induce cellular toxicity. This result highlights the importance of ROS in the early stages of cytotoxicity and suggests muting ROS production in noxious injuries may increase positive clinical outcomes.

Crotalus Durissus Ruruima: Current Knowledge on Natural History, Medical Importance, and Clinical Toxinology.

Crotalus durissus ruruima is a rattlesnake subspecies mainly found in Roraima, the northernmost state of Brazil. Envenomings caused by this subspecies lead to severe clinical manifestations (e.g. respiratory muscle paralysis, rhabdomyolysis, and acute renal failure) that can lead to the victim's death. In this review, we comprehensively describe C. d. ruruima biology and the challenges this subspecies poses for human health, including morphology, distribution, epidemiology, venom cocktail, clinical envenoming, and the current and future specific treatment of envenomings by this snake. Moreover, this review presents maps of the distribution of the snake subspecies and evidence that this species is responsible for some of the most severe envenomings in the country and causes the highest lethality rates. Finally, we also discuss the efficacy of the Brazilian horse-derived antivenoms to treat C. d. ruruima envenomings in Roraima state.

Side-impact collision: mechanics of obstacle negotiation in sidewinding snakes.

Snakes excel at moving through cluttered environments, and heterogeneities can be used as propulsive contacts for snakes performing lateral undulation. However, sidewinding, which is often associated with sandy deserts, cuts a broad path through its environment that may increase its vulnerability to obstacles. Our prior work demonstrated that sidewinding can be represented as a pair of orthogonal body waves (vertical and horizontal) that can be independently modulated to achieve high maneuverability and incline ascent, suggesting that sidewinders may also use template modulations to negotiate obstacles. To test this hypothesis, we recorded overhead video of four sidewinder rattlesnakes (Crotalus cerastes) crossing a line of vertical pegs placed in the substrate. Snakes used three methods to traverse the obstacles: a Propagate Through behavior in which the lifted moving portion of the snake was deformed around the peg and dragged through as the snake continued sidewinding (115/160 runs), Reversal turns that reorient the snake entirely (35/160), or switching to Concertina locomotion (10/160). The Propagate Through response was only used if the anterior-most region of static contact would propagate along a path anterior to the peg, or if a new region of static contact could be formed near the head to satisfy this condition; otherwise, snakes could only use Reversal turns or switch to Concertina locomotion. Reversal turns allowed the snake to re-orient and either escape without further peg contact or re-orient into a posture amenable to using the Propagate Through response. We developed an algorithm to reproduce the Propagate Through behavior in a robophysical model using a modulation of the two-wave template. This range of behavioral strategies provides sidewinders with a versatile range of options for effectively negotiating obstacles in their natural habitat, as well as provide insights into the design and control of robotic systems dealing with heterogeneous habitats.

Physiological demands and signaling associated with snake venom production and storage illustrated by transcriptional analyses of venom glands.

Despite the extensive body of research on snake venom, many facets of snake venom systems, such as the physiology and regulation of the venom gland itself, remain virtually unstudied. Here, we use time series gene expression analyses of the rattlesnake venom gland in comparison with several non-venom tissues to characterize physiological and cellular processes associated with venom production and to highlight key distinctions of venom gland cellular and physiological function. We find consistent evidence for activation of stress response pathways in the venom gland, suggesting that mitigation of cellular stress is a crucial component of venom production. Additionally, we demonstrate evidence for an unappreciated degree of cellular and secretory activity in the steady state venom gland relative to other secretory tissues and identify vacuolar ATPases as the likely mechanisms driving acidification of the venom gland lumen during venom production and storage.

Comparative thermal ecology parameters of the mexican dusky rattlesnake (Crotalus triseriatus).

Montane habitats exhibit a high degree of thermal heterogeneity, and thus provide considerable thermoregulatory challenges for ectotherms. Comparative analyses provide an opportunity to understand how variation in abiotic factors (e.g., operative temperatures, thermal quality) can affect life history traits within species. We studied the thermal ecology of three populations of the rattlesnake Crotalus triseriatus inhabiting different volcanoes in the central region of Mexico using the Hertz et al. (1993) protocol. The average body temperature of dusky rattlesnakes from the three study sites was 22.4 °C; mean active body temperature was higher in site 2 than in sites 1 and 3, but no differences between females, males and juveniles nor an interaction among site and sex was found. The thermal quality was low in the three sites, particularly in sites 1 and 3. Thermoregulation accuracy statistically differed among populations: individuals from site 2 were more accurate thermoregulating, while individuals from site 1 were the least accurate. Compared to other snakes, dusky rattlesnakes can be considered as a eurythermic species, which can often be active at relatively low body temperatures.

Exposure to fluctuating temperatures leads to reduced immunity and to stress response in rattlesnakes.

Ectothermic organisms often experience considerable variation in their body temperature throughout the circadian cycle. However, studies focusing on the measurement of physiological traits are usually performed under constant temperature regimes. This mismatch between thermal exposure in the field and experimental conditions could act as a stressor agent, as physiological functions are strongly influenced by temperature. Herein, we asked the question whether constant thermal regimes would cause a stress response and impact the immunity of the South American rattlesnake (Crotalus durissus) when compared with a fluctuating thermal regime. We addressed this question by determining heterophil:lymphocyte (H:L) ratio, plasma bacteria-killing ability (BKA) and corticosterone (CORT) levels in snakes kept under a constant temperature regime at 30°C, and under a fluctuating regime that oscillated between 25°C at night and 35°C during the day. The experiments had a mirrored design, in which half of the snakes were subjected to a fluctuating-to-constant treatment, while the other half was exposed to a constant-to-fluctuating treatment. The shift from constant to fluctuating thermal regime was accompanied by an increase in plasma CORT levels, indicating the activation of a stress response. Exposure to a fluctuating thermal regime at the onset of the experiments induced a decrease in the BKA of rattlesnakes. H:L ratio was not affected by treatments and, therefore, the shift between thermal regimes seems to have acted as a low-intensity stressor. Our results suggest that removal from temperatures close to the snake's preferred body temperature triggers a stress response in rattlesnakes.

Thermal regime effects on the resting metabolic rate of rattlesnakes depend on temperature range.

While ectothermic organisms often experience considerable circadian variation in body temperature under natural conditions, the study of the effects of temperature on metabolic rates are traditionally based on subjecting animals to constant temperature regimes. Whether data resulting from constant-temperature experiments accurately predicts temperature effects under more natural fluctuating temperature regimes remains uncertain. To address such possibility, we measured the resting metabolic rates of the South American rattlesnakes (Crotalus durissus) under constant and circadian fluctuating thermal regimes in a range of temperatures. Metabolic rates measured at constant 20 °C and 25 °C did not differ from the rates measured at fluctuating regimes with corresponding mean temperatures. However, the difference between thermal regimes increased with temperature, with the metabolic rate measured at constant 30 °C being greater than that measured at the fluctuating thermal regime with corresponding mean temperature. Therefore, our results indicate that thermal regime effects on rattlesnakes' metabolism is dependent on temperature range. Broadly, our results highlight the importance of considering multi-factorial attributes of temperature variation in the exam of its effects over functional traits. Such approach provides a more solid support for inferences about temperature effects on the life history, ecology and conservation of ectothermic organisms.

Evaluation of the sequence method as a tool to assess spontaneous baroreflex in reptiles.

The sequence method is an alternative to the traditional pharmacological approach (i.e., the Oxford technique) used to calculate baroreflex gain (G) in mammals. Although the sequence method assesses baroreflex by measuring spontaneous events of blood pressure regulation, the pharmacological method relies on the injection of vasoactive drugs that impact the baroreflex mechanism itself. The sequence method might be relevant for dynamic measurement of baroreflex modulation but it was never validated for vertebrates with low heart rate. Hence, we tested the sequence method in three species of reptiles and compared the results with those provided by the classic pharmacological method. G was similar between both methods and values correlated when parameters for the sequence method were set at delay 0 or 1 (i.e., the baroreflex system responds immediately to blood pressure changes or after 1 heartbeat). Calculation of the baroreflex effectiveness index was adequate at a minimum of 300 cycles and a delay of 1 for the three species. Therefore, the sequence method has been validated to investigate baroreflex regulation in reptiles, enabling studies during dynamic alterations in homeostasis.

Rattlesnakes Must Drink: Meal Consumption Does Not Improve Hydration State.

Water is critical to survival, yet free-standing water is often rare in deserts and seasonally dry environments. Thus, many dry-adapted species utilize either metabolic (that produced from metabolism) or dietary (that found in food) water to meet their hydric needs. It is suspected that desert reptiles can fulfill their hydric needs solely through dietary water intake. However, food consumption does not improve the hydration state of Gila monsters (Heloderma suspectum), a binge-feeding desert lizard, and meal consumption by snakes leads to increased voluntary drinking. Therefore, we predicted that meal consumption would not improve hydration state in dehydrated western diamondbacked rattlesnakes (Crotalus atrox). We found no significant difference in the change in plasma osmolality between moderately dehydrated snakes that consumed a meal and those that did not. In fact, snakes that received a meal reached severe dehydration more than a week sooner than snakes that did not receive a meal. Additionally, consuming a meal when severely dehydrated did not reduce plasma osmolality, whereas severely dehydrated snakes provided with water ab lib. returned to a normosmotic state. These results provide the most convincing evidence that carnivorous desert reptiles likely rely on free-standing water to fulfill their hydric needs. These species have thus had to adapt to sporadic water availability through minimizing water loss and tolerating considerable variation in plasma osmolality.

Neuronal Substrates for Infrared Contrast Enhancement and Motion Detection in Rattlesnakes.

Pit vipers detect infrared (IR) radiation with loreal pit organs [1] that are connected to the hindbrain by trigeminal nerve fibers [2-4]. The pattern of central afferent termination forms a topographical representation of the sensory periphery within the nucleus of the lateral descending trigeminal tract (LTTD) [4-7]. All LTTD neurons project to another specialized, ipsilateral hindbrain area, the nucleus reticularis caloris (RC) [8-11], before IR signals are integrated with visual signals in the optic tectum [12, 13]. Pit-organ-innervating afferent fibers provoke in individual LTTD neurons a direct, robust spike activity upon peripheral activation [7, 14]. This discharge is truncated by an indirect, delayed synaptic inhibition from afferent fibers of adjacent sensory areas through parallel microcircuitry that converges with afferent fibers onto the same target neurons [7]. Here, we determined the impact of this interaction on IR contrast enhancement and/or motion detection in LTTD and RC neurons using isolated whole-brain preparations of rattlesnakes with intact pit organs. Simulated and real IR source motion provoked weak directional tuning of the discharge in LTTD neurons and RC neurons expressed a strong, motion-direction-differentiating activity. The hierarchically increasing motion sensitivity potentially derives from a direction-specific inhibition or spike frequency adaptation of LTTD neuronal discharge that becomes further pronounced by convergent projections onto individual RC neurons. The emerging signaling pattern complies with contrast enhancement (LTTD) and extraction of movement-related signals (RC), thereby forming a motion detection mechanism that encodes moving IR sources relative to the ambient temperature [14].

Respiratory sinus arrhythmia is a major component of heart rate variability in undisturbed, remotely monitored rattlesnakes, Crotalus durissus.

ECG recordings were obtained using an implanted telemetry device from the South American rattlesnake, Crotalus durissus, held under stable conditions without restraining cables or interaction with researchers. Mean heart rate (fH) recovered rapidly (<24 h) from anaesthesia and operative procedures. This preceded a more gradual development of heart rate variability (HRV), with instantaneous fH increasing during each lung ventilation cycle. Atropine injection increased mean fH and abolished HRV. Complete autonomic blockade revealed a cholinergic tonus on the heart of 55% and an adrenergic tonus of 37%. Power spectral analysis of HRV identified a peak at the same frequency as ventilation. This correlation was sustained after temperature changes and it was more evident, marked by a more prominent power spectrum peak, when ventilation is less episodic. This HRV component is homologous to that observed in mammals, termed respiratory sinus arrhythmia (RSA). Evidence for instantaneous control of fH indicated rapid conduction of activity in the cardiac efferent nervous supply, as supported by the description of myelinated fibres in the cardiac vagus. Establishment of HRV 10 days after surgical intervention seems a reliable indicator of the re-establishment of control of integrative functions by the autonomic nervous system. We suggest that this criterion could be applied to other animals exposed to natural or imposed trauma, thus improving protocols involving animal handling, including veterinarian procedures.

Venom composition of adult Western Diamondback Rattlesnakes (Crotalus atrox) maintained under controlled diet and environmental conditions shows only minor changes.

Many species of snakes produce venom as a chemical means of procuring potentially fractious prey. Studies have increasingly focused on venom compositional variation between and within individual snakes of the same species/subspecies, with significant differences often being observed. This variation in composition has been attributed to differences in age, season, diet, and environment, suggesting that these factors could help explain the inter- and intra-specific variation found in some snake venoms, perhaps via some type of feedback mechanism(s). To address several of these possible sources of variation, this study utilized wild-caught Western Diamondback Rattlesnakes (Crotalus atrox) from Cochise Co., AZ. Sixteen adult C. atrox were maintained in the lab on a diet of NSA mice for eight months to determine whether venom composition changed in captivity under a static diet in a stable environment. Reducing 1-D SDS-PAGE, fibrinogen degradation assays, reversed-phase HPLC, and MALDI-TOF mass spectrometry revealed only minor differences over time within individuals. Venom L-amino acid oxidase (LAAO) and phosphodiesterase activities significantly increased over the course of captivity, with no changes occurring in azocasein metalloproteinase, kallikrein-like serine proteinase (KLSP), or thrombin-like serine proteinase (TLSP) activities. Snake total length was positively correlated with TLSP activity and negatively correlated with LAAO and KLSP activity. There was typically a much higher degree of variation between individuals than within individuals for all analyses performed and measurements collected. Because the overall "fingerprint" of each snake's venom remained more/less constant, it is concluded that biologically significant changes in venom composition did not occur within individual C. atrox as a function of captivity/diet. However, this study does indicate that differences in activity levels do occur in minor venom enzyme components, but the differences observed are likely to be of minimal significance to the production of antivenom or to subsequent treatment of human envenomations.

Photoreceptors morphology and genetics of the visual pigments of Bothrops jararaca and Crotalus durissus terrificus (Serpentes, Viperidae).

Snakes inhabit a great variety of habitats, whose spectral quality of light may vary a lot and influence specific adaptations of their visual system. In this study, we investigated the genetics of the visual opsins and the morphology of retinal photoreceptors, of two nocturnal snakes from the Viperidae family, Bothrops jararaca and Crotalus durissus terrificus, which inhabit preferentially the Atlantic Rain Forest and the Brazilian Savannah, respectively. Total RNA was extracted from homogenized retinas and converted to cDNA. The opsin genes expressed in snake retinas, LWS, RH1, and SWS1, were amplified by polymerase chain reactions (PCRs) and sequenced. The absorption peak (λmax) of the opsins were estimated based on amino acids located at specific spectral tuning sites. Photoreceptor cell populations were analyzed using immunohistochemistry with anti-opsin antibodies. Results showed the same morphological cell populations and same opsins absorption peaks, in both viperid species: double and single cones with LWS photopigment and λmax at ∼555 nm; single cones with SWS1 photopigment and λmax at ∼360 nm; and rods with the rhodopsin RH1 photopigment and λmax at ∼500 nm. The results indicate adaptations to nocturnal habit in both species despite the differences in habitat, and the possibility of a dichromatic color vision at photopic conditions.

Infrared-sensing snakes select ambush orientation based on thermal backgrounds.

Sensory information drives the ecology and behaviour of animals, and some animals are able to detect environmental cues unavailable to us. For example, rattlesnakes use infrared (IR) radiation to detect warm prey at night when visual cues are reduced. Until recently these sensory worlds have been inaccessible to human observers; now technology can allow us to "eavesdrop" on these species and understand how sensory perception drives ecology and behaviour. We used thermography and computer simulations to examine how prey-background temperature contrast and areas of temperature transitions influence the angular orientation of free-ranging rattlesnakes once they have selected an ambush site. We tracked free-ranging sidewinder rattlesnakes Crotalus cerastes to their selected ambush sites and recorded 360° near-ground thermographic panoramas from the centre of the ambush site. A computer simulation then moved a simulated prey item across the panorama and computed a contrast index for all directions. Rattlesnakes did not face ambush directions that offered stronger contrast than average, but they demonstrated a striking tendency to face directions with strong thermal transitions. Background transitions likely create a readily detected, rapidly changing stimulus when a prey animal passes. Quantifications of sensory environments like this one can boost our comprehension of how sensory function impacts the ecology, behaviour, and evolution of animals.

When one phenotype is not enough: divergent evolutionary trajectories govern venom variation in a widespread rattlesnake species.

Understanding the origin and maintenance of phenotypic variation, particularly across a continuous spatial distribution, represents a key challenge in evolutionary biology. For this, animal venoms represent ideal study systems: they are complex, variable, yet easily quantifiable molecular phenotypes with a clear function. Rattlesnakes display tremendous variation in their venom composition, mostly through strongly dichotomous venom strategies, which may even coexist within a single species. Here, through dense, widespread population-level sampling of the Mojave rattlesnake, Crotalus scutulatus, we show that genomic structural variation at multiple loci underlies extreme geographical variation in venom composition, which is maintained despite extensive gene flow. Unexpectedly, neither diet composition nor neutral population structure explain venom variation. Instead, venom divergence is strongly correlated with environmental conditions. Individual toxin genes correlate with distinct environmental factors, suggesting that different selective pressures can act on individual loci independently of their co-expression patterns or genomic proximity. Our results challenge common assumptions about diet composition as the key selective driver of snake venom evolution and emphasize how the interplay between genomic architecture and local-scale spatial heterogeneity in selective pressures may facilitate the retention of adaptive functional polymorphisms across a continuous space.