Matthew L. Holding

Coevolution of venom function and venom resistance in a rattlesnake predator and its squirrel prey

Measuring local adaptation can provide insights into how coevolution occurs between predators and prey. Specifically, theory predicts that local adaptation in functionally matched traits of predators and prey will not be detected when coevolution is governed by escalating arms races, whereas it will be present when coevolution occurs through an alternate mechanism of phenotype matching. Here, we analyse local adaptation in venom activity and prey resistance across 12 populations of Northern Pacific rattlesnakes and California ground squirrels, an interaction that has often been described as an arms race. Assays of venom function and squirrel resistance show substantial geographical variation (influenced by site elevation) in both venom metalloproteinase activity and resistance factor effectiveness. We demonstrate local adaptation in the effectiveness of rattlesnake venom to overcoming present squirrel resistance, suggesting that phenotype matching plays a role in the coevolution of these molecular traits. Further, the predator was the locally adapted antagonist in this interaction, arguing that rattlesnakes are evolutionarily ahead of their squirrel prey. Phenotype matching needs to be considered as an important mechanism influencing coevolution between venomous animals and resistant prey.

Physiological and Behavioral Effects of Repeated Handling and Short-Distance Translocations on Free-Ranging Northern Pacific Rattlesnakes (Crotalus oreganus oreganus)

Abstract Translocation, a management and conservation strategy used commonly in which animals are moved from their sites of origin to other localities, has proven controversial. We examined the physiological and behavioral impacts of repeated handling and short-distance translocation on rattlesnakes, which are often translocated from areas of human use because of a perceived threat to people. Northern Pacific Rattlesnakes (Crotalus oreganus oreganus) were radiotracked for 2 months, during which time one of three treatments was imposed weekly: translocation, walk and release at that days capture site (handling control), and undisturbed control. At both the beginning (spring) and the end (summer) of the study, blood samples were obtained before and after an acute handling stressor, and plasma concentrations of corticosterone (CORT) and testosterone (T) were determined. All rattlesnakes showed a CORT stress response, but baseline and stressed concentrations of neither hormone were affected by either translocation or handling. However, the response of both hormones to stress differed between spring and summer, with a greater increase in CORT and a detectable decrease in T occurring in summer. Activity range size was affected by translocation, whereas no effects on snake behaviors recorded during observer approach were detected. Rattlesnakes appear quite resilient to the potential impacts of frequent handling or short-distance translocation.

Roads are associated with a blunted stress response in a North American pit viper

Whereas numerous studies have examined roads as anthropogenic stressors in birds and mammals, comparatively few studies have been undertaken on reptiles. We investigated plasma corticosterone (CORT) levels at baseline and following 30min of restraint stress in free-ranging copperhead snakes (Agkistrodon contortrix) captured within the forest interior or while in contact with public roads. There was no difference in baseline CORT levels between snakes in the forest and on roads. Copperheads responded to restraint stress by increasing plasma levels of CORT; however snakes on roads exhibited a lower CORT stress response compared to forest snakes. Additionally, among snakes captured on roads there was a negative association between road traffic and baseline CORT, stressed CORT, and the magnitude of the CORT response. Our results suggest that roads are associated with a blunted stress response in copperheads. Reduced stress responses may be indicative of acclimation, the inhibited ability to mount a stress response in the face of prolonged chronic stress, or that road environments select for individuals with lower CORT responsiveness. Either scenario could result in increased road mortality if snakes do not perceive roads as a potential threat.

Wet- and Dry-Season Steroid Hormone Profiles and Stress Reactivity of an Insular Dwarf Snake, the Hog Island Boa (Boa constrictor imperator)

Field endocrine studies providing new comparisons for inference into the evolutionary and ecological factors shaping organismal physiology are important, often yielding novel physiological insights. Here, we explored factors associated with the sex steroid hormone concentrations and adrenocortical response to capture stress in Hog Island boas (Boa constrictor imperator) in the Cayos Cochinos archipelago of Honduras to generate comparative field hormone data from a tropical reptile and test the island tameness hypothesis. Baseline concentrations of testosterone, corticosterone, estradiol, and progesterone were measured during the wet and dry seasons, and an acute stressor of 1 h in a cloth bag was used to assess the stress response. Plasma steroid concentrations in these snakes were generally low in comparison to other taxa. Higher testosterone concentrations in males and higher estradiol and corticosterone concentrations in females were observed during the wet season compared to the dry season, which may be indicative of mating activities and vitellogenesis during this period. Snakes displayed a 15-fold increase in corticosterone concentrations in response to capture stress, a rise that was not impacted by whether a snake had been captured during previous years. The adrenocortical stress response was greater in males and positively related to body temperature. We suggest that this system merits future inquiries into the physiology and behavior of B. c. imperator, particularly as a model for studying insular impacts on diverse life history characters.

Evaluating the Performance of De Novo Assembly Methods for Venom-Gland Transcriptomics

Venom-gland transcriptomics is a key tool in the study of the evolution, ecology, function, and pharmacology of animal venoms. In particular, gene-expression variation and coding sequences gained through transcriptomics provide key information for explaining functional venom variation over both ecological and evolutionary timescales. The accuracy and usefulness of inferences made through transcriptomics, however, is limited by the accuracy of the transcriptome assembly, which is a bioinformatic problem with several possible solutions. Several methods have been employed to assemble venom-gland transcriptomes, with the Trinity assembler being the most commonly applied among them. Although previous evidence of variation in performance among assembly software exists, particularly regarding recovery of difficult-to-assemble multigene families such as snake venom metalloproteinases, much work to date still employs a single assembly method. We evaluated the performance of several commonly used de novo assembly methods for the recovery of both nontoxin transcripts and complete, high-quality venom-gene transcripts across eleven snake and four scorpion transcriptomes. We varied k-mer sizes used by some assemblers to evaluate the impact of k-mer length on transcript recovery. We showed that the recovery of nontoxin transcripts and toxin transcripts is best accomplished through different assembly software, with SDT at smaller k-mer lengths and Trinity being best for nontoxin recovery and a combination of SeqMan NGen and a seed-and-extend approach implemented in Extender as the best means of recovering a complete set of toxin transcripts. In particular, Extender was the only means tested capable of assembling multiple isoforms of the diverse snake venom metalloproteinase family, while traditional approaches such as Trinity recovered at most one metalloproteinase transcript. Our work demonstrated that traditional metrics of assembly performance are not predictive of performance in the recovery of complete and high quality toxin genes. Instead, effective venom-gland transcriptomic studies should combine and quality-filter the results of several assemblers with varying algorithmic strategies.

No safety in the trees: Local and species-level adaptation of an arboreal squirrel to the venom of sympatric rattlesnakes.

Within some species, squirrels respond to variable selection from venomous snake predators by showing population-level variation in resistance, while between species, some rattlesnakes possess venom that is more effective at overcoming venom resistance in different species of squirrels. A functional evaluation of resistance variation to venom within and between species of squirrels and snakes can link resistance variation to its evolutionary causes across these different evolutionary scales. To do this, we compared the effectiveness of squirrel sera in inhibiting rattlesnake (Crotalus spp.) venom metalloproteinase activity between populations and between species to test for a response to local variation in selection from a single rattlesnake predator and for specialization of two resistant squirrel species to each of their distinct sympatric snake predators. We found that Timber Rattlesnake (Crotalus horridus) venom inhibition by Eastern gray squirrels (Sciurus carolinensis) is higher at a site where the rattlesnakes are present, which suggests selection may maintain venom resistance in populations separated by short distances. Next, we performed a reciprocal cross of venoms and sera from two rattlesnake and two squirrel species. This showed that squirrel resistance is lower when tested against venom from allopatric compared to sympatric rattlesnake species, demonstrating that squirrel inhibitors are specialized to sympatric venom and suggesting a tradeoff in terms of specialization to the venom of a specific species of rattlesnake predator. This pattern can be explained if inhibitors must recognize venom proteins and resistance evolution tracks venom evolution.

Evaluating the thermal effects of translocation in a large‐bodied pitviper

Acute stressors can be costly, often requiring alteration of normal physiological processes to mitigate their effects. Animal translocation may be a very stressful event and result in a reduced ability to maintain homeostasis. The impacts of translocation on the thermal ecology of ectothermic vertebrates, which may rely on preferred habitats for thermoregulation, are currently unknown. In this study, 22 adult male Northern Pacific rattlesnakes (Crotalus oreganus oreganus) were implanted with automated temperature loggers and radio-tracked. Snakes were assigned to one of three treatments: translocation, handling control, and undisturbed control. Short-distance translocation (SDT) and handling treatments were applied weekly for 6 weeks. Hourly body temperature (Tb ) was recorded during the course of the study. Mean Tb was impacted in a time-dependent fashion, where translocated snakes had lower mean Tb than handled controls during the first week of the study only, especially the first 24 hr after translocation. Separating the dataset into day and night revealed that this effect was localized to Tb variation during the day only. Variance in temperature was not impacted by translocation or handling. Snake body mass and time of year were the major factors influencing the thermal profiles of these rattlesnakes. Thermal ecology in male rattlesnakes is resilient to SDT, suggesting that they quickly resume normal behaviors following repeated bouts of acute capture stress and disturbance of their spatial ecology. This study provides support for SDT as a safe measure for mitigating human-snake interactions and facilitating conservation practices regarding male snakes, which are the most frequently encountered sex.

Good vibrations: Assessing the stability of snake venom composition after researcher-induced disturbance in the laboratory

Abstract Phenotypic plasticity contributes to intraspecific variation in traits of many animal species. Venom is an integral trait to the success and survival of many snake species, and potential plasticity in venom composition is important to account for in the context of basic research as well as in human medicine for treating the various symptoms of snakebite and producing effective anti‐venoms. Researchers may unknowingly induce changes in venom variation by subjecting snakes to novel disturbances and potential stressors. We explored phenotypic plasticity in snake venom composition over time in captive Pacific rattlesnakes (Crotalus oreganus) exposed to vibration treatment, compared to an undisturbed control group. Venom composition did not change significantly in response to vibration, nor was there a detectable effect of overall time in captivity, even though snakes re‐synthesized venom stores while subjected to novel disturbance in the laboratory. This result indicates that venom composition is a highly repeatable phenotype over short time spans and that the composition of venom within adult individuals may be resistant to or unaffected by researcher‐induced disturbance. On the other hand, the change in venom composition, measured as movement along the first principle component of venom phenotype space, was associated with baseline corticosterone (CORT) levels in the snakes. While differential forms of researcher‐induced disturbance may not affect venom composition, significant changes in baseline CORT, or chronic stress, may affect the venom phenotype, and further investigations will be necessary to assess the nature of the relationship between CORT and venom protein expression. HighlightsIndividual snake venoms were highly repeatable in snakes that were exposed to a novel stressor.Initial and final individual snake venoms sampled with a 2 week interval were not significantly different in snakes acclimated to laboratory for at least 10 days.Corticosterone was correlated with slight changes in venom composition after re‐synthesis.

Local prey community composition and genetic distance predict venom divergence among populations of the northern Pacific rattlesnake (Crotalus oreganus)

Identifying the environmental correlates of divergence in functional traits between populations can provide insights into the evolutionary mechanisms that generate local adaptation. Here, we assess patterns of population differentiation in expressed venom proteins in Northern Pacific rattlesnakes (Crotalus oreganus) from 13 locations across California. We evaluate the relative importance of major biotic (prey species community composition), abiotic (temperature, precipitation and elevation) and genetic factors (genetic distance based on RAD‐seq loci) as correlates of population divergence in venom phenotypes. We found that over half of the variation in venom composition is associated with among‐population differentiation for genetic and environmental variables and that this variation occurred along axes defining previously observed functional trade‐offs between venom proteins that have neurotoxic, myotoxic and hemorrhagic effects. Surprisingly, genetic differentiation among populations was the best predictor of venom divergence, accounting for 46% of overall variation, whereas differences in prey community composition and abiotic factors explained smaller amounts of variation (23% and 19%, respectively). The association between genetic differentiation and venom composition could be due to an isolation‐by‐distance effect or, more likely, an isolation‐by‐environment effect where selection against recent migrants is strong, producing a correlation between neutral genetic differentiation and venom differentiation. Our findings suggest that even coarse estimates of prey community composition can be useful in understanding the selection pressures acting on patterns of venom protein expression. Additionally, our results suggest that factors other than adaptation to spatial variation in prey need to be considered when explaining population divergence in venom.

Confronting Scientific Misconceptions by Fostering a Classroom of Scientists in the Introductory Biology Lab

Abstract A fundamental component of science curricula is the understanding of scientific inquiry. Although recent trends favor using student inquiry to learn concepts through hands-on activities, it is often unclear to students where the line is drawn between the content and the process of science. This activity explicitly introduces students to the processes of science and allows the classroom to become a scientific community where independent studies are performed, shared, and revised. We designed this activity to be relatively independent of the chosen content, allowing instructors to utilize the presented framework for classes of various disciplines and education levels.