Abstract
Animal personality is characterized by consistent among-individual differences in behavior across time and contexts and can influence a wide range of ecological and evolutionary processes. Although consistent behavioral differences have been linked to variation in behaviors that create or modify extended phenotypes, their role in shaping the deployment of chemical effectors that induce phenotypic changes in other organisms remains largely unexplored. Venom provides an ideal system for addressing this question because venomous animals can regulate how much venom they deploy, and the quantity delivered can strongly influence the resulting phenotype expressed by the recipient. In this study, we subjected 48 subadult and adult southern Pacific rattlesnakes (Crotalus oreganus helleri) to standardized venom extractions to examine the repeatability of venomous bite characteristics across three trials. All measured bite attributes exhibited significant repeatability, including number of strikes during tubing for restraint, bite occurrence during the extraction, number of venom pulses from the fangs, volume of venom expended, and venom volume per pulse. Larger snakes expended more venom and venom expenditure increased with the number of venom pulses delivered, but consistent among-individual differences persisted in all traits after accounting for these factors. These results indicate that, similar to other defensive traits, venom deployment comprises a repeatable component of the defensive behavior of rattlesnakes. Although repeatability of venom expenditure needs to be tested in additional contexts, our study demonstrates consistent individual differences in the deployment of a chemical effector used to generate an extended phenotype, thus highlighting the importance of consistent behavioral variation in determining how organisms deploy chemical weapons and other bioactive compounds that alter the behavior, physiology, or fitness of other organisms.
Introduction
In recent decades, the study of animal personality has shifted from treating individual behavioral variation as unexplained noise to recognizing it as both a source of phenotypic diversity (King and Figueredo, 1997; Pennisi, 2016) and a product of natural selection (; Wilson et al., 2019). Animal personality is generally defined as consistent among-individual differences in behavior that are repeatable across time and/or contexts, independent of factors such as sex, age, body size, dominance rank, or other sources of behavioral variation (Gosling, 2001; Réale et al., 2007; ; Wolf and Weissing, 2012; Kaiser and Müller, 2021). Because consistency across time and contexts is a defining feature of animal personality, repeatability provides a quantitative means of assessing consistent individual differences in behavior. These consistent behavioral differences have become a subject of intense scrutiny in behavioral ecology, and have now been documented across a wide range of taxa (Gosling, 2001; Réale et al., 2007; Vonk et al., 2017; ; ), including cephalopods (Mazza et al., 2024), arthropods (Rádai et al., 2022), fishes (Luchiari and Maximino, 2023), amphibians (Kelleher et al., 2018), reptiles (Waters et al., 2017), birds (van Oers and Naguib, 2013; ), and mammals (Finkemeier et al., 2018; Savazza et al., 2026).
Personality research has focused on several commonly recognized axes, including shyness–boldness, exploration–avoidance, activity, aggressiveness, and sociability (Réale et al., 2007; ; ), and on correlations among these traits that form behavioral syndromes (Sih et al., 2004; Réale et al., 2010). Interest in animal personality has grown rapidly because consistent behavioral differences can influence cognition, stress responses, development, fitness, life-history strategies, sociality, and wellbeing, while also providing insight into the evolutionary, developmental, mechanistic, and ecological processes responsible for generating and maintaining behavioral variation within populations (Stamps and Groothuis, 2010a; Sih et al., 2015; Dougherty and Guillette, 2018; Wilson et al., 2019; ; ; Gartland et al., 2022; Laskowski et al., 2022; MacKinlay and Shaw, 2023).
An extended phenotype is a genetically influenced trait expressed outside an organism’s physical body that directly impacts its survival and reproductive success (). Examples include environmental modification (e.g., spider web, bird nest, beaver dam, and other created or modified physical structures), manipulation of another organism through direct interaction (e.g., altered host behavior by a parasite invading its body to facilitate transmission), and indirect manipulation at a distance (e.g., intensive feeding of a cuckoo chick that manipulates its foster parents). An organism’s unique personality traits, like bold exploration or meticulous caution, can dictate how it builds, modifies, or interacts with its extended phenotype. A more aggressive spider, for instance, might construct a web with a greater number of sticky silk lines to more effectively capture prey (DiRienzo et al., 2020), making the external structure a tangible, physical manifestation of its behavioral personality. Although personalities may serve as the behavioral engine that shapes extended phenotypes, studies that explore the role of repeatability (the key attribute of personality) in creating or modifying the extended phenotypes of animals remain scarce.
Venom deployment can also generate an extended phenotypic trait (e.g., Martinson et al., 2014; Hughes and Libersat, 2018; Pecina et al., 2020; Jenner et al., 2025b). Venom delivery systems introduce toxins through a wound, thereby causing pathophysiological injury to the targeted organism (Nelsen et al., 2014; Jenner et al., 2025a, b). Through envenomation, the venom producer manipulates a living victim by inducing a specific state, such as altered behavior, pain, immobility, and/or death, which can be viewed as an extended phenotype of the venomous organism. The resulting phenotype is jointly determined by the venom producer’s biochemical (venom composition) and behavioral (dose and delivery site) traits and the target organism’s physiological and behavioral responses. Venom delivery systems are now recognized to be present among diverse organisms, including animals, plants, fungi, protists, bacteria, and viruses (Hayes et al., 2025).
Although venom can serve a remarkable variety of roles, it most often functions within a predatory and/or antipredator capacity (Jenner and Undheim, 2017). Despite its well-documented benefits, reliance on venom entails non-trivial costs, including venom production and storage, usually within glands (Li et al., 2005; McCue, 2006; Enzor et al., 2011; Nisani et al., 2012; Morgenstern and King, 2013; Smith and Wang, 2014); mechanisms that prevent self-intoxication (Munekiyo and Mackessy, 2005; Mackessy, 2022; Ukken et al., 2022; ); production and maintenance of venom delivery structures (e.g., stingers, fangs, spines, ducts); and ecological risks associated with reduced venom availability during periods of replenishment (Hayes et al., 2008). Accordingly, natural selection should favor mechanisms, including decision-making processes, that promote the judicious use of venom. Indeed, the quantity of venom deployed often varies depending on target identity and context, a behavioral phenomenon known as venom metering and sometimes venom optimization (Hayes et al., 2002; Hayes et al., 2008; Morgenstern and King, 2013; ; Schendel et al., 2019).
Given the phylogenetic ubiquity of venom, its adaptive utility for inducing extended phenotypes, and the capacity of organisms to meter venom delivery, we can anticipate both among-individual variation and within-individual consistency in venom use, providing an opportunity to examine consistent individual differences in a chemical effector (Hernández Duran et al., 2021). However, studies examining repeatability in use of venom or other chemical effectors for predation, defense, and competition remain remarkably scarce. Among the few examples for predation, personality influences whether the spider Drassodes lapidosus relies on venom or silk when subduing prey (); whether individuals of the social spider Stegodyphus sarasinorum participate in prey capture using silk (); and the number of prey envenomated by the spider Zodarion rubidum during a single encounter (). The few examples for defense include proclivities of the honeybee Apis mellifera to sting and inject venom into an attacker (Kannan et al., 2025) and the millipede Narceus americanus to secrete benzoquinone as a “poison” on its body surface when attacked (Duchesne and Careau, 2022). Personality also modulates toxungen application (Nelsen et al., 2014) to the body surface and/or venom spraying into the bite-generated wounds of heterospecific competitors by the ants Lasius niger (Menges et al., 2022) and Linepithema humile (Neumann and Pinter-Wollman, 2019). To our knowledge, no study has examined repeatable individual differences in the quantity of chemical effectors deployed to induce extended phenotypes during predatory, defensive, or competitive interactions.
Rattlesnakes (genera Crotalus and Sistrurus) are ideally suited for assessing the potential influence of personality on chemical effector deployment. While research on snake behavior has historically been limited compared to other animal groups, interest has steadily increased over the past two decades (Doody et al., 2013; Schuett et al., 2016; Doody et al., 2021), with the study of consistent individual differences remaining largely underexplored until recently (Waters et al., 2017). Rattlesnakes exhibit distinctive defensive displays and, when biting, meter variable quantities of venom depending on context (predatory vs. defensive strikes), level of hunger, size and species of prey, perceived level of threat, and population-level exposure to predators and antagonists (reviewed in Hayes et al., 2002; Hayes et al., 2008; Herbert and Hayes, 2008; Hayes et al., 2024). Distinct personalities in proclivity to bite defensively have been identified in two species of rattlesnakes (Crotalus atrox, ; C. oreganus, Gibert et al., 2022) and inferred in two others (C. scutulatus and C. viridis, Maag et al., 2025). Personality in defensive bites has also been demonstrated in another viperid species, Vipera ammodytes (van Zele et al., 2026).
In this study, we measured individual repeatability of defensive venom expenditure in subadult and adult southern Pacific rattlesnakes (C. oreganus helleri) during venom extractions. We tested whether individuals differed consistently across trials in five behaviors: tendency to strike defensively during capture, proclivity to bite during the ensuing venom extraction, number of venom pulses ejected from the fangs, quantity of venom expended, and venom expended per pulse. By quantifying repeatability, we assessed the consistency of individual differences in these defensive behaviors across the trials. These behaviors are related to the aggressive axis measured in other animal studies, which can encompass predatory, defensive, or competitive contexts (Pruitt and Riechert, 2012). We hypothesized that individual snakes would exhibit consistent behavioral tendencies across trials and predicted that there would be repeatability in and correlations among these five behaviors.
Materials and methods
Experimental subjects and housing
We tested 35 adult C. o. helleri (70–113 cm; 22 males and 13 females) that were wild caught from across southern California and had been maintained in captivity for 2–10+ years. We also tested 13 subadult (30.5–66 cm; 7 males and 6 females) C. o. helleri that had been born and maintained in captivity up to 4 years and were from four different clutches. We individually housed adult snakes in clear plastic tubs (50L × 40W × 18H cm) or Vision cages (56L × 74W × 30H cm; Vision Racks and Cages, Neenah, WI, USA) with a water dish, plastic shelter, and butcher paper substrate. Subadults were housed in smaller plastic tubs (39L × 25W × 24H cm). Snakes were maintained at 29 °C with a 12:12 light:dark cycle and were offered thawed rodent prey every two weeks. Snakes were given at least one week to digest their meal prior to trials.
Testing procedure
We tested each snake three times with two weeks (12–15 days) separating the trials. Trials began when one investigator (always CEM) removed a snake from its home cage by restraining it in a clear plastic tube with the snake’s head extended out the end. We recorded the number of strikes that occurred while restraining the snake in the plastic tube. Another investigator (always WKH) then grasped the head of the snake with 45-cm metal tongs and encouraged the snake to bite a parafilm-covered beaker secured in place by a ring stand (Hayes et al., 2020; Figure 1). The snakes were given 30 sec to bite the beaker and the trial concluded once a bite occurred. If the snake did not bite the beaker after 30 sec, we discontinued the trial. We recorded the trials at 240 fps on an iPhone 11 Pro (Apple, Inc., Cupertino, California, USA). We then collected the venom from the beaker with a micro-pipettor and transferred it to a 2-mL microfuge tube, centrifuged the sample to ensure all fluid was at the bottom of the tube, and froze it at -20 C. Venom volume was later measured using graduated 0.5–2 mL pipets. Videos were analyzed via JWatcher (v 1.0) by one investigator (CEM) who was blinded to individual and trial order. Behaviors recorded from the videos included: bite occurrence (yes/no) and number of venom pulses. Snakes were observed to bite and expulse venom unilaterally (a venom pulse from a single fang during retraction from an extended position) and/or bilaterally (a venom pulse from each of both fangs, often but not always simultaneously; Hayes et al., 2008; Hayes, 2008). The number of venom pulses was summed for the two fangs (c.f. Hayes et al., 2024). Lastly, we wanted to know whether consistent among-individual differences influence the quantity of venom expulsed per pulse, so we derived this measure by dividing venom volume by number of pulses.
Figure 1
To ensure consistency across trials, we recorded both temperature and handling time. Because temperature can influence defensive behavior in pitvipers (Whitford et al., 2020), we measured each snake’s external body temperature prior to trials using a thermal gun (26.3–31.40 C; mean ± 1 SD: 29.0 ± 0.8 C; Helect Infrared Laser Thermometer, Shenzhen JEWY Tech Co., Shenzhen, China). Preliminary analyses indicated that body temperature did not significantly influence bite characteristics and was therefore excluded from further analyses to help reduce overparameterization of the models. We also recorded handling time, which was the time required to tube each snake, as handling can induce stress. Snakes housed in Vision cages took longer to tube than those in plastic tubs (mean ± 1 SD: 46 ± 31 and 33 ± 23 sec, respectively; ANOVA: p = 0.015). However, preliminary analyses indicated that handling time only influenced the number of strikes, and was therefore excluded from all other analyses.
Statistical analyses
We conducted all analyses in RStudio (v.4.5.0; R Core Team, 2025). Alpha was set to 0.05 and all p-values were calculated with the ANOVA function using the car package, with data distributions assessed visually and using Shapiro-Wilk tests. Because of missing data (failed video capture or unreliable venom retrieval), sample sizes varied somewhat among statistical models.
Repeatability of behavior across time is an essential criterion used to characterize consistent individual differences in animal personality (Réale et al., 2007; ; ). Thus, for each of the five behaviors (strikes during tubing, bite occurrence, venom pulses, venom volume, and venom per pulse), we measured repeatability (R) among trials using generalized linear mixed-effects models (GLMM) fitted by restricted maximum likelihood with the rptR package (Stoffel et al., 2017). We log-transformed number of strikes and venom volume (after adding a constant of 0.001 to each to avoid zeros) before fitting them to a Gaussian distribution. We also fitted bite occurrence to a binary distribution and venom pulses to a Gaussian distribution. For all models, we treated SVL and trial number as fixed factors and included snake ID as a random effect. We set bootstraps to 1000 permutations.
To investigate the effect of sex, SVL, and trial number on the four behaviors suitable for analysis (we omitted venom per pulse, as it was derived from and explained by venom pulses and venom volume), we relied on linear mixed models (LMMs) and GLMMs using the packages lme4 () and glmmTMB (), respectively. We treated sex, SVL, and trial number as fixed factors and snake ID as a random effect. We also included handling time as a fixed factor in the mixed model for number of strikes, number of strikes as a fixed factor in the models for venom pulses and venom volume (inclusion created instability in the model for bite occurrence), and venom pulses as a fixed factor in the model for venom volume. Preliminary analyses indicated that time in captivity was not a significant factor prior to the venom extraction, so it was omitted from analyses to reduce parameterization. We analyzed number of strikes using a GLMM with a Poisson distribution and bite occurrence using a GLMM with a binomial distribution. We examined venom pulses and log-transformed venom volume using LMMs. For significant main effects, we conducted post-hoc comparisons of estimated marginal means using the emmeans package (Lenth, 2025), with p-values adjusted for false discovery rate (FDR; ).
Results
Repeatability
Results of the repeatability analyses are presented in Table 1. All five behaviors (strikes during tubing, bite occurrence, venom pulses, venom volume, and venom per pulse) were significantly repeatable across trials. Repeatability coefficients varied between 0.27 and 0.47, indicating moderate repeatability. Repeatability was highest for number of venom pulses (R = 0.47) and lowest for bite occurrence (R = 0.27), with the latter having low confidence.
Table 1
| Variable | Trials | Type of model | R | 95% CI | p-value |
|---|---|---|---|---|---|
| Strikes | 144 | Gaussian with log-transformed data | 0.36 | 0.15–0.52 | < 0.001 |
| Bite occurrence | 142 | Binary | 0.26 | 0.00–0.90 | 0.009 |
| Venom pulses | 142 | Gaussian | 0.47 | 0.28–0.63 | < 0.001 |
| Venom volume | 139 | Gaussian with log-transformed data | 0.41 | 0.21–0.56 | < 0.001 |
| Venom per pulse | 137 | Gaussian | 0.43 | 0.24–0.58 | <0.001 |
Summary of repeatability (R) estimates with 95% confidence intervals (CI) for four behaviors measured during multiple venom extractions (three across a 6-wk period) of 13 subadult and 35 adult southern Pacific rattlesnakes (Crotalus oreganus helleri)
Sample size (number of trials) is given for each estimate. Bold values indicate statistical significance (p < 0.05).
We generated plots of these behaviors (excluding venom per pulse, which was mathematically derived from other measures) to visualize repeatability across all of the 48 individuals. Nearly half of the snakes (23; 47.9%) never struck while being tubed, whereas 12 (25.0%) struck during a single trial, eight (16.7%) struck during two trials, and five (10.4%) struck during all three trials (Figure 2A). Once secured in the tube and pressed against the membrane-covered beaker’s edge, all snakes bit the membrane during at least one of the trials (Figure 2B). Seven snakes (14.6%) bit only once during the trials, refusing at other times to engage the beaker membrane with their fangs. Nine snakes (18.8%) bit the membrane in two trials and 32 (66.7%) did so in all of their trials. All snakes delivered at least one wet bite (Figure 2C). We did not quantify unilateral and bilateral venom pulses, although they were frequent, with snakes delivering an unequal number of venom pulses from the two fangs in 64 (45.1%) of the 142 venom extractions (two videos were lost). Twelve snakes (25.0%) refused to give more than two pulses of venom during any of their trials, whereas 36 (75.0%) delivered multiple pulses of venom at least once from one or both of their fangs. Fourteen (29.2%) gave three pulses of venom from at least one fang during a trial. Snakes generally delivered similar quantities of venom across multiple trials, with the amount injected remaining relatively consistent for each individual despite differences associated with body size (Figure 2D).
Figure 2
Effects of sex, body size, and trial number on behaviors
Table 2 summarizes the results of the LMM and GLMM tests for strikes during tubing, bite occurrence, number of venom pulses, and venom volume. For number of strikes during tubing, we detected a significant effect of handling time (p < 0.001), with snakes that struck more often taking longer to tube (Figure 3A). Trial number was not a significant factor (p = 0.056), although strike frequency showed a trend toward more strikes in the third trial compared to the first (FDR-adjusted p = 0.0497, 95% CI 0.10–0.24; Figure 3B). For bite occurrence and number of venom pulses, no factors significantly explained their variance. For venom volume, body size (SVL) exerted a significant effect (p < 0.001), with larger snakes expending more venom in an exponential relationship (Figure 4A). The number of venom pulses (p < 0.001) was also significant, with snakes delivering more venom pulses also expending more venom (Figure 4B). The trial number (p = 0.32) and number of strikes during tubing (p = 0.23) failed to influence venom yield when biting the membrane.
Table 2
| Variable | Type of model | df | F or χ2 | 95% CI | p-value | |
|---|---|---|---|---|---|---|
| Strikes | GLMM with Poisson distribution (N = 144 trials) | |||||
| Sex | 1 | 2.66 | -1.82–0.17 | 0.10 | ||
| SVL | 1 | 0.21 | -0.03–0.019 | 0.64 | ||
| Trial number | 2 | 5.71 | 0.056 | |||
| Handling time | 1 | 31.92 | 0.015–0.03 | < 0.001 | ||
| Bite occurrence | GLMM with binomial distribution (N = 142 trials) | |||||
| Sex | 1 | 0.81 | -0.88–4.18 | 0.37 | ||
| SVL | 1 | 0.32 | -0.03–0.05 | 0.56 | ||
| Trial number | 2 | 0.44 | 0.80 | |||
| Venom pulses | LMM (N = 142 trials) | |||||
| Sex | 1, 44.6 | 1.10 | -0.32–1.09 | 0.30 | ||
| SVL | 1, 43.3 | 2.74 | -0.002–0.03 | 0.11 | ||
| Trial number | 2, 91.1 | 1.23 | 0.30 | |||
| Strikes | 1, 134.7 | 3.39 | -0.006–0.27 | 0.068 | ||
| Venom volume | LMM with log-transformed data (N = 138 trials) | |||||
| Sex | 1, 46.5 | 1.5 | -0.18–0.83 | 0.22 | ||
| SVL | 1, 46.4 | 27.79 | 0.02–0.45 | < 0.001 | ||
| Trial number | 2, 91.0 | 1.16 | 0.32 | |||
| Strikes | 1, 114.2 | 1.40 | -0.04–0.20 | 0.23 | ||
| Venom pulses | 1, 128.2 | 112.5 | 0.65–0.94 | < 0.001 | ||
Summary of results from linear mixed models (LMMs with F-values) and generalized linear mixed models (GLMMs with χ2-values) that evaluated the effects of sex, body size (snout-vent length, SVL), trial number, and handling time on the four measured behaviors of rattlesnakes.
Bold values indicate statistical significance (p < 0.05). 95% confidence intervals are reported for individual model coefficients; however, a single 95% confidence interval is not applicable to trial number in the omnibus tests.
Figure 3
Figure 4
Discussion
In this study, we examined whether southern Pacific rattlesnakes exhibit consistent among-individual differences in five defensive behaviors associated with venom extractions. As hypothesized, we observed repeatability in the number of strikes elicited during tubing, as well as bite occurrence, number of venom pulses from the two fangs, and volume of venom expended during venom extractions. Moreover, repeatability of venom expended resulted from not just number of venom pulses, but also from the amount of venom ejected per pulse. However, we were surprised by the weak association between defensive strikes prior to tubing and the behaviors that ensued when given an opportunity to bite the membrane-covered beaker. Nevertheless, our findings indicate that consistent among-individual differences contribute to variation in venom expenditure during defensive bites by males and females alike and by snakes of varying age. At a broader level, we provide the first evidence to our knowledge that consistent among-individual differences are associated with variation in the quantity of a chemical effector released to elicit an extended phenotype in another organism.
Our findings add to previous studies of venomous snakes that have similarly reported repeatability in defensive rattling and striking (Gibert et al., 2022; ; ; Maag et al., 2025; van Zele et al., 2026). Although rattlesnakes can issue bluff strikes and dry bites (Rowe and Owings, 1990; Young and Zahn, 2001; Hayes et al., 2002; Hayes et al., 2008), which can effectively deter more timid predators and/or antagonists, venom use comprises an integral component of the typical defensive behavior of rattlesnakes. Variation in venom expenditure may have important fitness consequences. For example, insufficient venom expended during a defensive bite may fail to deter a predator, potentially increasing the risk of death, whereas excessive use could lead subsequently to a missed meal or chance to defend itself (Hayes, 1995; Hayes et al., 2002; Hayes et al., 2008; Herbert and Hayes, 2008). Because personality traits are subject to natural selection through their effects on survival and reproduction (McDougall et al., 2006; Réale et al., 2007), consistent among-individual differences in venom expenditure may have fitness consequences and could represent an adaptive component of defensive behavior.
We identified several factors, both morphological and behavioral, that can influence venom expenditure. Larger rattlesnakes expended more venom during the extractions than smaller individuals, which was consistent not only with prior venom extraction studies (Glenn and Straight, 1982; Mackessy et al., 2003) but also measurements from actual predatory (Hayes, 1991) and defensive (Herbert, 1998; Rehling, 2002; Herbert and Hayes, 2009) bites. We expected this relationship because as snakes grow in length, a single dimension, storage capacity in their venom gland expands in three dimensions. In addition to storage capacity, the volume of venom expulsed is a product of the number of venom pulses and the quantity of venom expulsed per pulse. Both of these latter measures were repeatable, supporting the view that rattlesnakes are capable of metering venom via several mechanisms under their control (Hayes et al., 2008), and that these mechanisms may vary consistently among individuals.
The rattlesnakes also showed a trend of striking more during the third trial than the first. Change might be expected since animals can alter their defensive behavior through learned experiences with predators (Fanselow, 2018). Adult pitvipers, for example, can become habituated to repeated disturbance over a time period of several days (Glaudas, 2004; Glaudas et al., 2006; Place and Abramson, 2008), which may reduce the intensity of defensive responses. However, strike frequency in our study trended towards increasing across successive trials (Figure 3B) across a longer time period of four weeks, suggesting that the snakes had not become habituated to the disturbance. Instead, snakes may have shifted toward a behaviorally defensive strategy, relying more on striking during latter trials based on negative experiences from previous trials. Thus, repeated exposure to a stressor may cause snakes to alter components of their defensive strategy in different directions depending on the interval duration.
Currently, relatively few studies have examined consistent individual differences in species that pose significant human-wildlife conflict. As urbanization expands and rattlesnake habitats become fragmented and destroyed, rattlesnakes increasingly interact with humans (Woodroffe et al., 2005; Frank et al., 2019; Phillips et al., 2019), leading to frequent human-wildlife conflict (; ; Dunn et al., 2024) including human envenomations (Ruha et al., 2017; Greene et al., 2021). The southern Pacific rattlesnake (C. o. helleri) is responsible for the majority of medically significant snakebites in southern California (; Wasserberger et al., 2006), highlighting the importance of understanding the factors that influence defensive behavior in this species. Assessing individual variation in behavior has been suggested to be important for understanding animal responses to mitigation strategies (), and may therefore improve management practices. Because defensive biting and venom expenditure are linked to individual behavioral tendencies, some snakes may be less inclined to bite when encountered, whereas others may be more likely to bite and inject large amounts of venom. The clinical severity of a venomous snake bite has been shown to increase with snake size, as larger individuals typically inject greater volumes of venom (Janes Jr et al., 2010; ). This suggests that risk to humans may vary not only with size, but on an individual basis. Consequently, some individual snakes may pose a greater threat to the public than others. Additionally, because consistent behavioral differences are shaped by natural selection and can enhance adaptation to local conditions (McDougall et al., 2006; Réale et al., 2007), features of environmental heterogeneity, such as habitat type (Dubuc-Messier et al., 2017), forest structure (Mortelliti and Brehm, 2020; ), altitude (Qu et al., 2019), human disturbance (), and predator/antagonist level (Hayes et al., 2024), can influence behavior at the population level. Understanding how environmental factors shape consistent individual differences in the defensive responses of rattlesnakes, and how these traits vary across populations and species, could improve management strategies, inform more effective public safety messaging, and contribute to conservation efforts by helping mitigate human–wildlife conflict (; Malhotra et al., 2021; MacKinlay and Shaw, 2023).
At a broader level, our results have implications for understanding the evolution of extended phenotypes. Extended phenotypes are often viewed through the lens of architectural design, niche construction, and host manipulation (Schaedelin and Taborsky, 2009; ; Hughes and Libersat, 2018; Perez et al., 2020), yet the behavioral processes that govern their expression remain comparatively understudied. By demonstrating that consistent among-individual differences are associated with variation in the quantity of venom deployed, we show that consistent among-individual behavioral variation can shape not only whether an extended phenotype is expressed, but also its magnitude. Because venom-induced phenotypes in a target organism depend in part on venom dose, individual differences in venom expenditure may generate corresponding differences in the intensity, duration, or effectiveness of the extended phenotype itself. These findings highlight consistent among-individual differences as a potentially important source of variation linking genotype, behavior, and extended phenotypic expression. Similar approaches may prove fruitful for understanding how other chemical effectors modify the behavior, physiology, or fitness of recipients, including toxungens, pheromones, and other manipulative compounds. Integrating personality research with the study of chemical ecology and extended phenotypes may therefore provide new insights into how selection acts on both the production and strategic deployment of biochemical traits that influence other organisms.
Studies of animal personality are strengthened when behavioral assays demonstrate ecological relevance, repeatability, and convergent and discriminant validity (). Although our results demonstrated repeatability in venom deployment, the ecological relevance of the experiment may be limited and additional evidence across contexts is needed to determine whether these differences represent broader behavioral tendencies. Snakes were given the opportunity to bite the beaker voluntarily rather than forcibly, and venom glands were not manually compressed, which is typical of many venom extractions (Glenn and Straight, 1982; Hayes et al., 2020). However, venom was expended under heightened stress, in an artificial environment, and when fully constrained with no alternative options for defense other than biting (e.g., fleeing, displaying, bluff strikes, brief strikes). The context of venom deployment during venom extraction may be comparable to that of a snake largely immobilized in the clutches of a predator or antagonist, but the kinematics of biting and venom expulsion differ from those of bites delivered during defensive strikes from an unrestrained snake. Venom extractions often induce multiple venom pulses, whereas defensive strikes are typically brief and presumably allow just a single venom pulse (Herbert, 1998; 2007; Hayes et al., 2008). Previous studies on repeatable behavior in pitvipers have found that behaviors measured in the laboratory do not necessarily correlate with those observed in free-ranging individuals (Maag et al., 2025), likely due to contextual differences between field and laboratory assays. Holding wild snakes in captivity may elevate stress and increase defensive behavior. Alternatively, adult pitvipers can habituate to repeated disturbance in captivity (Glaudas, 2004; Glaudas et al., 2006), potentially reducing defensive behavior compared to those observed in the wild. In addition, natural environments are more biotically and abiotically complex, which may contribute to greater behavioral variability in free ranging animals. Our study animals had been maintained in captivity for an extended time period (1–10 years) and their behavior may not be indicative of free-ranging snake behavior, although we did not detect an effect of captivity duration in either this study or in a prior study (Hayes et al., 2024). Nevertheless, even under artificial conditions, consistent differences among individuals may still reflect meaningful variation in behavior, particularly if similar relative differences are maintained across different contexts. Future studies should aim to examine the repeatability of bite characteristics under more natural conditions, such as in free-ranging snakes, to gain a better understanding of how captivity can alter behavior.
Personality is also characterized by consistency across time and contexts, which few studies explore adequately. Ideally, studies should explore individuality across ontogeny () and through multiple rather than single behavioral assays (Niemelä and Dingemanse, 2018; ), as selection may act more so on an individual’s behavioral plasticity (Stamps and Groothuis, 2010b; Wuerz and Krüger, 2015) to better accommodate the influences of state (e.g., body temperature, proximity to cover, hunger, recent ingestion, gestation) and experience. Our study was limited to a relatively narrow timeframe (four weeks) for the individual snakes, although we found that consistent individual differences exist in young and old alike, which suggests the possibility that consistent individual differences may persist through life. For contexts, it would be helpful to measure whether the relative level of aggressiveness in venom extractions is consistent with that exhibited during induced strikes when the snake is not physically restrained and predatory strikes at one or more prey species or prey of varying size.
Conclusion
This study is the first to report repeatability in bite characteristics and venom expenditure in pitvipers and is among the few studies to examine repeatable behavior in rattlesnakes. Consistent with previous research, we found evidence of stable individual differences in behavior across time, indicating that venom deployment is a repeatable component of defensive behavior. These findings expand our understanding of behavioral variation in rattlesnakes and suggest that individual differences influence how animals deploy chemical weapons and other bioactive compounds that alter the behavior, physiology, or fitness of other organisms. More broadly, our results suggest that consistent individual differences may influence not only whether a chemical effector is deployed, but also the magnitude of the resulting extended phenotype. Because the effects of venom on a recipient depend in part on the amount of venom deployed, consistent differences in venom expenditure could translate into meaningful differences in the intensity or effectiveness of envenomation. In rattlesnakes, this variation may also have implications for human-wildlife conflict, because individuals that differ consistently in defensive behavior and venom expenditure may present different levels of risk when encounters occur. Improving our understanding of the factors influencing venom deployment may therefore help to reduce conflict and better inform mitigation practices. Future studies should examine bite characteristics in more natural contexts, such as with free-ranging snakes or through in situ observations and include both defensive and predatory contexts. Investigating consistent individual differences in the deployment of venom and other chemical effectors across contexts and taxa may help clarify the broader role of behavioral variation in shaping defensive, predatory, and competitive behaviors in animals.
Statements
Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Ethics statement
The animal study was approved by Loma Linda University Institutional Animal Care and Use Committee (protocol 24-025). The study was conducted in accordance with the local legislation and institutional requirements.
Author contributions
CM: Conceptualization, Data curation, Formal analysis, Investigation, Writing – original draft, Writing – review & editing. WH: Conceptualization, Investigation, Supervision, Writing – review & editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. Funding was provided by the Loma Linda University Department of Earth and Biological Sciences. The journal provided a fee waiver for publication.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that generative AI was used in the creation of this manuscript. During the preparation of this manuscript, ChatGPT, Jenni, and Perplexity were used to assist with locating relevant literature and citations. ChatGPT was also used during the later stages of drafting to improve language and readability, provide coding assistance for data visualization and statistical analyses, and generate suggestions for the manuscript title. All AI-generated outputs, suggestions, and code were reviewed, verified, and extensively revised by the authors. The authors take full responsibility for the accuracy, integrity, and content of the final published work.
Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
References
1
AtkinsM. C.HowarthC. R.RusselloM. A.TomalJ. H.LarsenK. W. (2022). Evidence of intrapopulation differences in rattlesnake defensive behavior across neighboring habitats. Behav. Ecol. Sociobiol.76, 3. doi: 10.1007/s00265-021-03100-6
2
BaileyN. W. (2012). Evolutionary models of extended phenotypes. Trends Ecol. Evol.27, 561–569. doi: 10.1016/j.tree.2012.05.011
3
BatemanH. L.BrownJ. A.LarsonK. L.AndradeR.HughesB. (2021). Unwanted residential wildlife: Evaluating social-ecological patterns for snake removals. Global Ecol. Conserv.27, e01601. doi: 10.1016/j.gecco.2021.e01601
4
BatesD.MächlerM.BolkerB.WalkerS. (2015). Fitting linear mixed-effects models using lme4. J. Stat. Software67, 1–48. doi: 10.18637/jss.v067.i01
5
BeleyurT.BellurD. U.SomanathanH. (2015). Long-term behavioural consistency in prey capture but not in web maintenance in a social spider. Behav. Ecol. Sociobiol.69, 1019–1028. doi: 10.1007/s00265-015-1915-z
6
BellA. M.HankisonS. J.LaskowskiK. L. (2009). The repeatability of behaviour: a meta-analysis. Anim. Behav.77, 771–783. doi: 10.1016/j.anbehav.2008.12.022
7
BenjaminiY.HochbergY. (1995). Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Society: Ser. B. (Methodological)57, 289–300. doi: 10.1111/j.2517-6161.1995.tb02031.x
8
BeydizadaN.PekárS. (2023). Personality predicts mode of attack in a generalist ground spider predator. Behav. Ecol.34, 42–49. doi: 10.1093/beheco/arac095
9
BeydizadaN. I.PekárS. (2026). Personality is associated with the hunting strategy in a specialized ant-eating spider. Curr. Zool., zoag025. doi: 10.1093/cz/zoag025
10
BiroP. A.StampsJ. A. (2008). Are animal personality traits linked to life-history productivity? Trends Ecol. Evol.23, 361–368. doi: 10.1016/j.tree.2008.04.003
11
BlackwellB. F.DeVaultT. L.Fernández-JuricicE.GeseE. M.Gilbert-NortonL.BreckS. W. (2016). No single solution: application of behavioural principles in mitigating human–wildlife conflict. Anim. Behav.120, 245–254. doi: 10.1016/j.anbehav.2016.07.013
12
BrehmA. M.MortellitiA. (2024). Environmental heterogeneity modifies the link between personality and survival in fluctuating small mammal populations. J. Anim. Ecol.93, 196–207. doi: 10.1111/1365-2656.14037
13
BrooksM. E.KristensenK.Van BenthemK. J.MagnussonA.BergC. W.NielsenA.et al. (2017). glmmTMB balances speed and flexibility among packages for zero-inflated generalized linear mixed modeling. R J.9, 378–400. doi: 10.32614/RJ-2017-066
14
BushS. P.GreenS. M.MoynihanJ. A.HayesW. K.CardwellM. D. (2002). Crotalidae polyvalent immune Fab (ovine) antivenom is efficacious for envenomations by Southern Pacific rattlesnakes (Crotalus helleri). Ann. Emergency Med.40, 619–624. doi: 10.1067/mem.2002.129939
15
CabreraD.NilssonJ.GriffenB. (2021a). The development of animal personality across ontogeny: A cross-species review. Anim. Behav.173, 137–144. doi: 10.1016/j.anbehav.2021.01.003
16
CabreraD.NilssonJ. R.GriffenB. D. (2021b). The development of animal personality across ontogeny: a cross-species review. Anim. Behav.173, 137–144. doi: 10.1016/j.anbehav.2021.01.003
17
CarterA. J.FeeneyW. E.MarshallH. H.CowlishawG.HeinsohnR. (2013). Animal personality: what are behavioural ecologists measuring? Biol. Rev.88, 465–475. doi: 10.1111/brv.12007
18
ČernýJ.HejníkováM.PodlahováŠ.Čapková FrydrychováR.SábováM.ŠtěrbováH.et al. (2026). Self-toxicity and tolerance mechanisms of honeybee venom in honeybees. J. Exp. Biol.229, jeb251886. doi: 10.1242/jeb.251886
19
ChengJ.HeH.ZhangM.GanB.NiuH.ZhangH. (2023). A review to the methods of personality measurements in wild animals. Wildl. Lett.1, 131–142. doi: 10.1002/wll2.12022
20
CockremJ. F. (2022). Individual variation, personality, and the ability of animals to cope with climate change. Front. Ecol. Evol.10, 897314. doi: 10.3389/fevo.2022.897314
21
CooperA.NelsenD.HayesW. (2017). “ The strategic use of venom by spiders,” in Evolution of Venomous Animals and Their Toxins. Eds. GopalakrishnakoneP.MalhotraA. ( Springer Netherlands, Dordrecht, Netherlands), 145–166.
22
CorbitA. G.HayesW. K. (2016). Factors that influence the clinical severity of venomous snakebites in Southern California. Toxicon117, 106. doi: 10.1016/j.toxicon.2016.04.014
23
CorbitA. G.HayesW. K. (2022). Human–wildlife conflict at a suburban–wildlands interface: Effects of short-and long-distance translocations on red diamond rattlesnake (Crotalus ruber) activity and survival. Diversity14, 130. doi: 10.3390/d14020130
24
Da CunhaO.HorneL. M.JohnsonJ. D. (2023). Personally rattled: A unique protocol to support the presence of personality and behavioral syndromes in rattlesnakes. Behav. Ecol. Sociobiol.77, 115. doi: 10.1007/s00265-023-03391-x
25
Da CunhaO.MeadJ. J.SanchezB. A.PollardK. J.JohnsonJ. D.SeymoureB. M. (2025). The relationship between movement and personality is dependent upon the seasons in the western diamond-backed rattlesnake. Anim. Behav.223, 123181. doi: 10.1016/j.anbehav.2025.123181
26
DawkinsR. (1982). The Extended Phenotype: The Long Reach of the Gene (Oxford, UK: Oxford University Press).
27
de AlmeidaG. N.RamosG.de JesusL. G.BrancoM. E. C.de AzevedoC. S.Sant’AnnaA. C. (2024). Temperament of Psittaciformes: A systematic review. Appl. Anim. Behav. Sci.277, 106348. doi: 10.1016/j.applanim.2024.106348
28
DingemanseN. J.KazemA. J.RéaleD.WrightJ. (2010). Behavioural reaction norms: animal personality meets individual plasticity. Trends Ecol. Evol.25, 81–89. doi: 10.1016/j.tree.2009.07.013
29
DingemanseN. J.RéaleD. (2005). Natural selection and animal personality. Behaviour142, 1159–1184. doi: 10.1163/156853905774539445
30
DingemanseN. J.WrightJ. (2020). Criteria for acceptable studies of animal personality and behavioural syndromes. Ethology126, 865–869. doi: 10.1111/eth.13082
31
DiRienzoN.SchraftH.MontiglioP.BradleyC.DornhausA. (2020). Foraging behavior and extended phenotype independently affect foraging success in spiders. Behav. Ecol.31, 1242–1249. doi: 10.1093/beheco/araa080
32
DoodyJ. S.BurghardtG. M.DinetsV. (2013). Breaking the social–non-social dichotomy: a role for reptiles in rertebrate social behavior research? Ethology119, 95–103. doi: 10.1111/eth.12047
33
DoodyJ. S.DinetsV.BurghardtG. M. (2021). The Secret Social Lives of Reptiles (Baltimore, MD: John Hopkins University Press).
34
DoughertyL. R.GuilletteL. M. (2018). Linking personality and cognition: A meta-analysis. Philos. Trans. R. Soc. B. Biol. Sci.373, 20170282. doi: 10.1098/rstb.2017.0282
35
Dubuc-MessierG.RéaleD.PerretP.CharmantierA. (2017). Environmental heterogeneity and population differences in blue tits personality traits. Behav. Ecol.28, 448–459. doi: 10.1093/beheco/arw148
36
DuchesneA. G.CareauV. (2022). Toxic personalities: are chemical defences and boldness correlated? Behav. Ecol. Sociobiol.76, 108. doi: 10.1007/s00265-022-03217-2
37
DunnA. R.MorzilloA. T.LarsonL.-E.ChristoffelR. A. (2024). Spatial analysis of attitudes toward timber rattlesnakes (Crotalus horridus) where encounters occur within an exurban landscape. Soc Nat. Resour.37, 251–269. doi: 10.1080/08941920.2023.2269554
38
EnzorL. A.WilbornR. E.BennettW. A. (2011). Toxicity and metabolic costs of the Atlantic stingray (Dasyatis sabina) venom delivery system in relation to its role in life history. J. Exp. Mar. Biol. Ecol.409, 235–239. doi: 10.1016/j.jembe.2011.08.026
39
FanselowM. S. (2018). The role of learning in threat imminence and defensive behaviors. Curr. Opin. Behav. Sci.24, 44–49. doi: 10.1016/j.cobeha.2018.03.003
40
FinkemeierM.-A.LangbeinJ.PuppeB. (2018). Personality research in mammalian farm animals: concepts, measures, and relationship to welfare. Front. Vet. Sci.5, 131. doi: 10.3389/fvets.2018.00131
41
FrankB.GlikmanJ. A.MarchiniS. (2019). Human–Wildlife Interactions: Turning Conflict Into Coexistence (Cambridge, United Kingdom: Cambridge University Press).
42
GartlandL. A.FirthJ. A.LaskowskiK. L.JeansonR.IoannouC. C. (2022). Sociability as a personality trait in animals: methods, causes and consequences. Biol. Rev.97, 802–816. doi: 10.1111/brv.12823
43
GibertR. G.MaagD. W.SandersL. N.ClarkR. W. (2022). Investigating personality in vipers: individual rattlesnakes exhibit consistent behavioral responses in defensive and exploratory contexts. Behav. Ecol. Sociobiol.76, 132. doi: 10.1007/s00265-022-03239-w
44
GlaudasX. (2004). Do cottonmouths (Agkistrodon piscivorus) habituate to human confrontations? Southeast. Nat.3, 129–138. doi: 10.1656/1528-7092(2004)003[0129:DCAPHT]2.0.CO;2
45
GlaudasX.WinneC. T.FedewaL. A. (2006). Ontogeny of anti‐predator behavioral habituation in cottonmouths (Agkistrodon piscivorus). Ethology112, 608–615. doi: 10.1111/j.1439-0310.2005.01183.x
46
GlennJ. L. S.StraightR.C. (1982). “ The rattlesnakes and their venom yield and lethal toxicity,” in Rattlesnake Venoms: Their Actions and Treatment. Ed. TuA. T. (New York, NY: Marcel Dekker), 3–119.
47
GoslingS. D. (2001). From mice to men: what can we learn about personality from animal research? Psychol. Bull.127, 45. doi: 10.1037/0033-2909.127.1.45
48
GreeneS. C.FoltJ.WyattK.BrandehoffN. P. (2021). Epidemiology of fatal snakebites in the United States 1989–2018. Am. J. Emergency Med.45, 309–316. doi: 10.1016/j.ajem.2020.08.083
49
HayesW. K. (1991). Ontogeny of striking, prey-handling and envenomation behavior of prairie rattlesnakes (Crotalus v. viridis). Toxicon, 29 (7), 867–875.
50
HayesW. K. (1995). Venom metering by juvenile prairie rattlesnakes, Crotalus v. viridis: effects of prey size and experience. Anim. Behav.50, 33–40. doi: 10.1006/anbe.1995.0218
51
HayesW. K.HerbertS. S.HarrisonJ. R.WileyK. L. (2008). Spitting versus biting: differential venom gland contraction regulates venom expenditure in the black-necked spitting cobra, Naja nigricollis nigricollis. Journal of Herpetology, 42 (3), 453–460.
52
HayesW. K.FoxG. A.NelsenD. R. (2020). “ Venom collection from spiders and snakes: voluntary and involuntary extractions (“milking”) and venom gland extractions,” in Snake and Spider Toxins: Methods and Protocols. Ed. PrielA. ( Springer US, New York, NY), 53–71.
53
HayesW. K.GrenE. C. K.NelsenD. R.CorbitA. G.CooperA. M.FoxG. A.et al. (2025). It’s a small world after all: The remarkable but overlooked diversity of venomous organisms, with candidates among plants, fungi, protists, bacteria, and viruses. Toxins17, 99. doi: 10.3390/toxins17030099
54
HayesW. K.HerbertS. S.RehlingG. C.GennaroJ. F. (2002). “ Factors that influence venom expenditure in viperids and other snake species during predatory and defensive contexts,” in Biology of the Vipers. Eds. SchuettG. W.HoggrenH.DouglasM. E.GreeneH. W. ( Eagle Mountain Publishing, Eagle Mountain, Utah, USA), 207–233.
55
HayesW. K.PersonC. E.FoxG. A.KingJ. L.BriggsE.GrenE. C. (2024). Paradoxical exception to island tameness: increased defensiveness in an Insular population of rattlesnakes. Toxins16, 157. doi: 10.3390/toxins16030157
56
HerbertS. S. (1998). Factors Influencing Venom Expenditure During Defensive Bites by Cottonmouths (Agkistrodon Piscivorus) and Rattlesnakes (Crotalus Viridis, Crotalus Atrox) (Loma Linda, CA: Loma Linda University). Biology M.S. Thesis, Loma Linda University.
57
HerbertS. S. (2007). Venom Expenditure by Viperid and Elapid Snakes: Mechanisms, Adaptation, and Application (Loma Linda, CA: Loma Linda University). Biology Ph.D. Dissertation, Loma Linda University.
58
HerbertS.HayesW. (2008). “ Venom expenditure by rattlesnakes and killing effectiveness in rodent prey: do rattlesnakes expend optimal amounts of venom,” in The Biology of Rattlesnakes. Eds. HayesW. K.BushS. P.BeamanK. R.CardwellM. D. (Loma Linda, CA: Loma Linda University Press), 221–228.
59
HerbertS. S.HayesW. K. (2009). Denim clothing reduces venom expenditure by rattlesnakes striking defensively at model human limbs. Ann. Emergency Med.54, 830–836. doi: 10.1016/j.annemergmed.2009.09.022
60
Hernández DuranL.WilsonD. T.BriffaM.RymerT. L. (2021). Beyond spider personality: The relationships between behavioral, physiological, and environmental factors. Ecol. Evol.11, 2974–2989. doi: 10.1002/ece3.7243
61
HughesD. P.LibersatF. (2018). Neuroparasitology of parasite–insect associations. Annu. Rev. Entomol.63, 471–487. doi: 10.1146/annurev-ento-020117-043234
62
JanesD. N.BushS. P.KolluruG. R. (2010). Large snake size suggests increased snakebite severity in patients bitten by rattlesnakes in Southern California. Wilderness Environ. Med.21, 120–126. doi: 10.1016/j.wem.2010.01.010
63
JennerR. A.CasewellN. R.UndheimE. A. B. (2025a). Venom versus poison: delivery mode matters. Trends Ecol. Evol.40, 1046-1047. doi: 10.1016/j.tree.2025.09.019
64
JennerR. A.CasewellN. R.UndheimE. A. B. (2025b). What is animal venom? Rethinking a manipulative weapon. Trends Ecol. Evol.40, 852–861. doi: 10.1016/j.tree.2025.05.009
65
JennerR. A.UndheimE. A. B. (2017). Venom: The Secrets of Natures's Deadliest Weapon (Washington, DC: Smithsonian Books).
66
KaiserM. I.MüllerC. (2021). What is an animal personality? Biol. Philos.36, 15. doi: 10.1007/s10539-020-09776-w
67
KannanK.GaliziaC. G.NouvianM. (2025). Consistency and individuality of honeybee stinging behaviour across time and social contexts. R. Soc Open Sci.12, 241295. doi: 10.1098/rsos.241295
68
KelleherS. R.SillaA. J.ByrneP. G. (2018). Animal personality and behavioral syndromes in amphibians: a review of the evidence, experimental approaches, and implications for conservation. Behav. Ecol. Sociobiol.72, 79. doi: 10.1007/s00265-018-2493-7
69
KingJ. E.FigueredoA. J. (1997). The five-factor model plus dominance in chimpanzee personality. J. Res. Pers.31, 257–271. doi: 10.1006/jrpe.1997.2179
70
LaskowskiK. L.ChangC.-C.SheehyK.AguiñagaJ. (2022). Consistent individual behavioral variation: what do we know and where are we going? Annu. Rev. Ecol. Evol. Syst.53, 161–182. doi: 10.1146/annurev-ecolsys-102220-011451
71
LenthR. V. (2025). emmeans: Estimated Marginal Means, aka Least-Squares Means. R package version 1.11.1. Available online at: https://CRAN.R-project.org/package=emmeans.
72
LiM.FryB. G.KiniR. M. (2005). Eggs-only diet: Its implications for the toxin profile changes and ecology of the marbled sea snake (Aipysurus eydouxii). J. Mol. Evol.60, 81–89. doi: 10.1007/s00239-004-0138-0
73
LuchiariA. C.MaximinoC. (2023). Fish personality: Meta-theoretical issues, personality dimensions, and applications to neuroscience and psychopathology. Pers. Neurosci.6, e9. doi: 10.1017/pen.2023.3
74
MaagD. W.FrancioliY. Z.GoetzM. T.SandersL. N.LopezX.CastoeT. A.et al. (2025). Variation in defensive and exploratory behaviors across a rattlesnake (Crotalus scutulatus × viridis) hybrid zone in southwestern New Mexico. Sci. Rep.15, 11989. doi: 10.1038/s41598-025-96155-8
75
MackessyS. P. (2022). Venom production and secretion in reptiles. J. Exp. Biol.225, jeb227348. doi: 10.1242/jeb.227348
76
MackessyS. P.WilliamsK.AshtonK. G. (2003). Ontogenetic variation in venom composition and diet of Crotalus oreganus concolor: A case of venom paedomorphosis? Copeia2003, 769–782. doi: 10.1643/ha03-037.1
77
MacKinlayR. D.ShawR. C. (2023). A systematic review of animal personality in conservation science. Conserv. Biol.37, e13935. doi: 10.1111/cobi.13935
78
MalhotraA.WüsterW.OwensJ. B.HodgesC. W.JesudasanA.ChG.et al. (2021). Promoting co-existence between humans and venomous snakes through increasing the herpetological knowledge base. Toxicon: X12, 100081. doi: 10.1016/j.toxcx.2021.100081
79
MartinsonE. O.WheelerD.WrightJ.MrinaliniSiebertA. L.WerrenJ. H. (2014). Nasonia vitripennis venom causes targeted gene expression changes in its fly host. Mol. Ecol.23, 5918–5933. doi: 10.1111/mec.12967
80
MazzaV.PacchiarottiG.StrinatiC.CarliniA.MatherJ. A.CarereC. (2024). Personality dimensions in the common octopus (Octopus vulgaris). Behaviour161, 913–948. doi: 10.1163/1568539X-bja10290
81
McCueM. D. (2006). Cost of producing venom in three North American pitviper species. Copeia2006, 818–825. doi: 10.1643/0045-8511(2006)6[818:COPVIT]2.0.CO;2
82
McDougallP. T.RéaleD.SolD.ReaderS. M. (2006). Wildlife conservation and animal temperament: causes and consequences of evolutionary change for captive, reintroduced, and wild populations. Anim. Conserv.9, 39–48. doi: 10.1111/j.1469-1795.2005.00004.x
83
MengesV.SpäthS.MenzelF. (2022). Temporally consistent behavioural variation between wild ant colonies is robust to strong seasonal and thermal variation. Anim. Behav.196, 151-163. doi: 10.1016/j.anbehav.2022.10.014
84
MorgensternD.KingG. F. (2013). The venom optimization hypothesis revisited. Toxicon63, 120–128. doi: 10.1016/j.toxicon.2012.11.022
85
MortellitiA.BrehmA. M. (2020). Environmental heterogeneity and population density affect the functional diversity of personality traits in small mammal populations. Proc. R. Soc. B. Biol. Sci.287, 20201713. doi: 10.1098/rspb.2020.1713
86
MunekiyoS. M.MackessyS. P. (2005). Presence of peptide inhibitors in rattlesnake venoms and their effects on endogenous metalloproteases. Toxicon45, 255–263. doi: 10.1016/j.toxicon.2004.10.009
87
NelsenD. R.NisaniZ.CooperA. M.FoxG. A.GrenE. C. K.CorbitA. G.et al. (2014). Poisons, toxungens, and venoms: redefining and classifying toxic biological secretions and the organisms that employ them. Biol. Rev.89, 450–465. doi: 10.1111/brv.12062
88
NeumannK. M.Pinter-WollmanN. (2019). Collective responses to heterospecifics emerge from individual differences in aggression. Behav. Ecol.30, 801–808. doi: 10.1093/beheco/arz017
89
NiemeläP.DingemanseN. (2018). On the usage of single measurements in behavioural ecology research on individual differences. Anim. Behav.145, 99–105. doi: 10.1016/j.anbehav.2018.09.012
90
NisaniZ.BoskovicD. S.DunbarS. G.KellnW.HayesW. K. (2012). Investigating the chemical profile of regenerated scorpion (Parabuthus transvaalicus) venom in relation to metabolic cost and toxicity. Toxicon60, 315–323. doi: 10.1016/j.toxicon.2012.04.343
91
PecinaP.VidličkaĽ.ProkopP. (2020). Testing adaptive significance of host manipulation with a parasitoid wasp. Ethology126, 735–740. doi: 10.1111/eth.13023
92
PennisiE. (2016). The power of personality. Science352, 644–647. doi: 10.1126/science.352.6286.644
93
PerezD. M.GardnerJ. L.MedinaI. (2020). Climate as an evolutionary driver of nest morphology in birds: A review. Front. Ecol. Evol.8. doi: 10.3389/fevo.2020.566018
94
PhillipsC.LipmanG. S.GugelmannH.DoeringK.LungD. (2019). Snakebites and climate change in California 1997–2017. Clin. Toxicol.57, 168–174. doi: 10.1080/15563650.2018.1508690
95
PlaceA. J.AbramsonC. I. (2008). Habituation of the rattle response in western diamondback rattlesnakes, Crotalus atrox. Copeia2008, 835–843. doi: 10.1643/ce-06-246
96
PruittJ. N.RiechertS. E. (2012). The ecological consequences of temperament in spiders. Curr. Zool.58, 589–596. doi: 10.1093/czoolo/58.4.589
97
QuJ.RéaleD.FletcherQ. E.ZhangY. (2019). Among-population divergence in personality is linked to altitude in plateau pikas (Ochotona curzoniae). Front. Zool.16, 26. doi: 10.1186/s12983-019-0329-6
98
RádaiZ.KissJ.NagyN. A.SomogyiA.Á.FülöpA.TóthZ.et al. (2022). State and physiology behind personality in arthropods: a review. Behav. Ecol. Sociobiol.76, 150. doi: 10.1007/s00265-022-03259-6
99
R Core Team (2025). R: A Language and Environment for Statistical Computing (Vienna, Austria: R Foundation for Statistical Computing).
100
RéaleD.DingemanseN. J.KazemA. J.WrightJ. (2010). Evolutionary and ecological approaches to the study of personality. Philos. Trans. R. Soc. B. Biol. Sci.365, 3937–3946. doi: 10.1098/rstb.2010.0222
101
RéaleD.ReaderS. M.SolD.McDougallP. T.DingemanseN. J. (2007). Integrating animal temperament within ecology and evolution. Biol. Rev.82, 291–318. doi: 10.1111/j.1469-185X.2007.00010.x
102
RehlingG. C. (2002). Venom Expenditure in Multiple Bites by Rattlesnakes and Cottonmouths (Loma Linda, CA: Loma Linda University). Biology M.S. Thesis, Loma Linda University.
103
RoweM. P.OwingsD. H. (1990). Probing, assessment, and management during interactions between ground squirrels and rattlesnakes. Ethology86, 237–249. doi: 10.1111/j.1439-0310.1990.tb00432.x
104
RuhaA.-M.KleinschmidtK. C.GreeneS.SpyresM. B.BrentJ.WaxP.et al. (2017). The epidemiology, clinical course, and management of snakebites in the north american snakebite registry. J. Med. Toxicol.13, 309–320. doi: 10.1007/s13181-017-0633-5
105
SavazzaS.MerzM. R.MortellitiA. (2026). Measuring personality in wild small mammals: a review of methods and proposal for a standardised approach. Mammal Rev.56, e70010. doi: 10.1111/mam.70010
106
SchaedelinF. C.TaborskyM. (2009). Extended phenotypes as signals. Biol. Rev.84, 293–313. doi: 10.1111/j.1469-185X.2008.00075.x
107
SchendelV.RashL. D.JennerR. A.UndheimE. A. B. (2019). The diversity of venom: The importance of behavior and venom system morphology in understanding its ecology and evolution. Toxins11, 666. doi: 10.3390/toxins11110666
108
SchuettG. W.ClarkR. W.ReppR. A.AmarelloM.SmithC. F.GreeneH. W. (2016). “ Social behavior of rattlesnakes: a shifting paradigm,” in Rattlesnakes of Arizona. Eds. SchuettG. W.FeldnerM. J.ReisererR. S.SmithC. F. ( Eco Publishers, Rodeo, New Mexico, USA), 161–244.
109
SihA.BellA.JohnsonJ. C. (2004). Behavioral syndromes: an ecological and evolutionary overview. Trends Ecol. Evol.19, 372–378. doi: 10.1016/j.tree.2004.04.009
110
SihA.MathotK. J.MoirónM.MontiglioP.-O.WolfM.DingemanseN. J. (2015). Animal personality and state–behaviour feedbacks: a review and guide for empiricists. Trends Ecol. Evol.30, 50–60. doi: 10.1016/j.tree.2014.11.004
111
SmithA. S.WangZ. (2014). Hypothalamic oxytocin mediates social buffering of the stress response. Biol. Psychiatry76, 281–288. doi: 10.1016/j.biopsych.2013.09.017
112
StampsJ.GroothuisT. G. (2010a). The development of animal personality: relevance, concepts and perspectives. Biol. Rev.85, 301–325. doi: 10.1111/j.1469-185X.2009.00103.x
113
StampsJ. A.GroothuisT. G. G. (2010b). Developmental perspectives on personality: Implications for ecological and evolutionary studies of individual differences. Philos. Trans. R. Soc. B. Biol. Sci.365, 4029–4041. doi: 10.1098/rstb.2010.0218
114
StoffelM. A.NakagawaS.SchielzethH. (2017). rptR: repeatability estimation and variance decomposition by generalized linear mixed‐effects models. Methods Ecol. Evol.8, 1639–1644. doi: 10.1111/2041-210X.12797
115
UkkenF. P.DowellN. L.HajraM.CarrollS. B. (2022). A novel broad spectrum venom metalloproteinase autoinhibitor in the rattlesnake Crotalus atrox evolved via a shift in paralog function. Proc. Natl. Acad. Sci.119, e2214880119. doi: 10.1073/pnas.2214880119
116
van OersK.NaguibM. (2013). “ Avian personality,” in Animal Personalities: Behavior, Physiology, and Evolution. Eds. C.C.M.D. ( University of Chicago Press, Chicago, IL), 66–95.
117
van ZeleN.NicotT.KsasR.LengagneT.LourdaisO. (2026). Quantitative honesty in snakes: acoustic warning is influenced by sex, personality and correlates with bite risk in the nose-horned viper. Proc. R. Soc. B. Biol. Sci.293, 20252698. doi: 10.1098/rspb.2025.2698
118
VonkJ.WeissA.KuczajS. (2017). Personality in Nonhuman Animals (Cham, Switzerland: Springer International Publishing).
119
WasserbergerJ.OrdogG.MerkinT. E. (2006). Southern Pacific rattlesnake bite: a unique clinical challenge. J. Emergency Med.31, 263–266. doi: 10.1016/j.jemermed.2005.09.018
120
WatersR. M.BowersB. B.BurghardtG. M. (2017). “ Personality and individuality in reptile behavior,” in Personality in Nonhuman Animals. Eds. VonkJ.WeissA.KuczajS. ( Springer International Publishing, Cham, Switzerland), 153–184.
121
WhitfordM. D.FreymillerG. A.HighamT. E.ClarkR. W. (2020). The effects of temperature on the defensive strikes of rattlesnakes. J. Exp. Biol.223, jeb223859. doi: 10.1242/jeb.223859
122
WilsonV.GuentherA.ØverliØ.SeltmannM. W.AltschulD. (2019). Future directions for personality research: Contributing new insights to the understanding of animal behavior. Animals9, 240. doi: 10.3390/ani9050240
123
WolfM.WeissingF. J. (2012). Animal personalities: consequences for ecology and evolution. Trends Ecol. Evol.27, 452–461. doi: 10.1016/j.tree.2012.05.001
124
WoodroffeR.ThirgoodS.RabinowitzA. (2005). “ The impact of human-wildlife conflict on natural systems,” in People and Wildlife, Conflict or Co-Existence? Eds. WoodroffeR.ThirgoodS.RabinowitzA. ( Cambridge University Press, Cambridge, UK), 1–12.
125
WuerzY.KrügerO. (2015). Personality over ontogeny in zebra finches: long-term repeatable traits but unstable behavioural syndromes. Front. Zool.12 Suppl 1, S9. doi: 10.1186/1742-9994-12-s1-s9
126
YoungB. A.ZahnK. (2001). Venom flow in rattlesnakes: mechanics and metering. J. Exp. Biol.204, 4345–4351. doi: 10.1242/jeb.204.24.4345
Summary
Keywords
antipredatory behavior, chemical effector, chemical weaponry, pitviper, reptile, southern pacific rattlesnake, temperament, venom delivery
Citation
Martin CE and Hayes WK (2026) Repeatability in venom expenditure during defensive bites in rattlesnakes: use of a chemical weapon deployed to generate an extended phenotype. Front. Ethol. 5:1918065. doi: 10.3389/fetho.2026.1918065
Received
24 June 2026
Revised
03 September 2026
Accepted
08 September 2026
Published
08 October 2026
Volume
5 - 2026
Edited by
Stephanie Ann Poindexter, University at Buffalo, United States
Reviewed by
Ahmed Karmaoui, Moulay Ismail University, Morocco
Narmin Beydizada, Hubei University, China
Updates
Copyright
© 2026 Martin and Hayes.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Chelsea E. Martin, chemartin94@gmail.com
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.