Commonly collected thermal performance data can inform species distributions in a data-limited invader.

Predicting potential distributions of species in new areas is challenging. Physiological data can improve interpretation of predicted distributions and can be used in directed distribution models. Nonnative species provide useful case studies. Panther chameleons (Furcifer pardalis) are native to Madagascar and have established populations in Florida, USA, but standard correlative distribution modeling predicts no suitable habitat for F. pardalis there. We evaluated commonly collected thermal traits- thermal performance, tolerance, and preference-of F. pardalis and the acclimatization potential of these traits during exposure to naturally-occurring environmental conditions in North Central Florida. Though we observed temperature-dependent thermal performance, chameleons maintained similar thermal limits, performance, and preferences across seasons, despite long-term exposure to cool temperatures. Using the physiological data collected, we developed distribution models that varied in restriction: time-dependent exposure near and below critical thermal minima, predicted activity windows, and predicted performance thresholds. Our application of commonly collected physiological data improved interpretations on potential distributions of F. pardalis, compared with correlative distribution modeling approaches that predicted no suitable area in Florida. These straightforward approaches can be applied to other species with existing physiological data or after brief experiments on a limited number of individuals, as demonstrated here.

Dermatomycosis Caused by Paranannizziopsis australasiensis in Nonnative Panther Chameleons (Furcifer pardalis) Captured in Central Florida, USA.

Emergent fungal pathogens in herpetofauna are a concern in both wild and captive populations. We diagnosed dermatomycosis by Paranannizziopsis australasiensis in two panther chameleons (Furcifer pardalis) and suspected it in eight others captured from an established free-living nonnative population in Florida, USA. Chameleons developed skin lesions following recent exposure to cold weather conditions while housed in captivity, approximately 10 mo after capture and 12 wk after being placed in outdoor enclosures. Affected animals were treated with oral voriconazole and terbinafine until most cases resolved; however, medications were ultimately discontinued. Paranannizziopsis australasiensis has not previously been described in chameleons, nor in animals originating from a free-ranging population in the USA. Although the source of P. australasiensis infection is uncertain, we discuss several scenarios related to the pet trade and unique situation of chameleon "ranching" present in the USA.

Divergent Serpentoviruses in Free-Ranging Invasive Pythons and Native Colubrids in Southern Florida, United States.

Burmese python (Python bivittatus) is an invasive snake that has significantly affected ecosystems in southern Florida, United States. Aside from direct predation and competition, invasive species can also introduce nonnative pathogens that can adversely affect native species. The subfamily Serpentovirinae (order Nidovirales) is composed of positive-sense RNA viruses primarily found in reptiles. Some serpentoviruses, such as shingleback nidovirus, are associated with mortalities in wild populations, while others, including ball python nidovirus and green tree python nidovirus can be a major cause of disease and mortality in captive animals. To determine if serpentoviruses were present in invasive Burmese pythons in southern Florida, oral swabs were collected from both free-ranging and long-term captive snakes. Swabs were screened for the presence of serpentovirus by reverse transcription PCR and sequenced. A total serpentovirus prevalence of 27.8% was detected in 318 python samples. Of the initial swabs from 172 free-ranging pythons, 42 (24.4%) were positive for multiple divergent viral sequences comprising four clades across the sampling range. Both sex and snout-vent length were statistically significant factors in virus prevalence, with larger male snakes having the highest prevalence. Sampling location was statistically significant in circulating virus sequence. Mild clinical signs and lesions consistent with serpentovirus infection were observed in a subset of sampled pythons. Testing of native snakes (n = 219, 18 species) in part of the python range found no evidence of python virus spillover; however, five individual native snakes (2.3%) representing three species were PCR positive for unique, divergent serpentoviruses. Calculated pairwise uncorrected distance analysis indicated the newly discovered virus sequences likely represent three novel genera in the subfamily Serpentovirinae. This study is the first to characterize serpentovirus in wild free-ranging pythons or in any free-ranging North America reptile. Though the risk these viruses pose to the invasive and native species is unknown, the potential for spillover to native herpetofauna warrants further investigation.

Snap-freezing in the Field: Effect of Sample Holding Time on Performance of Bactericidal Assays.

Comparative analyses in biology rely on the quality of available data. Methodological differences among studies may introduce variation in results that obscure patterns. In the field of eco-immunology, functional immune assays such as antimicrobial capacity assays are widely used for among-species applications. Sample storage time and animal handling time can influence assay results in some species, but how sample holding time prior to freezing influences assay results is unknown. Sample holding time can vary widely in field studies on wild animals, prompting the need to understand the implications of such variation on assay results. We investigated the hypothesis that sample holding time prior to freezing influences assay results in six species (Leiocephalus carinatus, Iguana iguana, Loxodonta africana, Ceratotherium simum, Columba livia, and Buteo swainsoni) by comparing antibacterial capacity of serum with varying processing times prior to snap-freezing. Blood was collected once from each individual and aliquots were placed on ice and assigned different holding times (0, 30, 60, 180, and 240 min), after which each sample was centrifuged, then serum was separated and snap-frozen on dry ice and stored at -80ºC for 60 days prior to assaying. For each aliquot, we conducted antibacterial capacity assays with serial dilutions of serum inoculated with E. coli and extracted the dilution at 50% antibacterial capacity for analysis. We found a decrease in antibacterial capacity with increased holding time in one of the six species tested (B. swainsoni), driven in part by complete loss of antibacterial capacity in some individuals at the 240-min time point. While the majority of species' antibacterial capacity were not affected, our results demonstrate the need to conduct pilot assays spanning the anticipated variation in sample holding times to develop appropriate field protocols.

Physiological effects of capture and short-term captivity in an invasive snake species, the Burmese python (Python bivittatus) in Florida.

It is important to evaluate the role of captivity as a potential stressor. An understanding of stress responses to capture and transition to captivity may inform the limitations of laboratory studies on wild animals, aid in understanding the consequences of introducing animals into captive environments, and help predict which species may be successful invasives. We investigated physiological effects of captivity by comparing at-capture blood variables in wild Burmese pythons (Python bivittatus) in Florida to pythons recently brought into captivity (1-109 days). We conducted an acute restraint test by collecting samples at baseline (immediately at handling) and one hour post-restraint across wild field-sampled (n = 19) and recently-captive (n = 33) pythons to evaluate fluctuations in plasma corticosterone, bacterial killing ability, antibody response, leukogram, and serpentovirus infection. We observed higher baseline plasma corticosterone and monocytes in recently captive compared to wild snakes, which both subsided in snakes held for a longer time in captivity, and a mild decrease in lymphocytes in the middle of the captivity period. Functional immunity and viral infection were not affected by captivity, and pythons maintained restraint-induced responses in corticosterone, heterophil to lymphocyte ratio, and monocyte counts throughout captivity. Prevalence for serpentovirus was 50%, though infection status was related to sampling date rather than captivity, indicating that viral infection may be seasonal. The history of Burmese python as a common captive animal for research and pet trade, as well as its general resilience to effects of capture and short-term captivity, may contribute to its invasion success in Florida.

Experimental Manipulation of Corticosterone Does Not Affect Venom Composition or Functional Activity in Free-Ranging Rattlesnakes.

AbstractVenom is an integral feeding trait in many animal species. Although venom often varies ontogenetically, little is known about the proximate physiological mediators of venom variation within individuals. The glucocorticoid hormone corticosterone (CORT) can alter the transcription and activation of proteins, including homologues of snake venom components such as snake venom metalloproteinases (SVMPs) and phospholipase A2 (PLA2). CORT is endogenously produced by snakes, varies seasonally and also in response to stress, and is a candidate endogenous mediator of changes in venom composition and functional activity. Here, we tested the hypothesis that CORT induces changes in snake venom by sampling the venom of wild adult rattlesnakes before and after they were treated with either empty (control) or CORT-filled (treatment) Silastic implants. We measured longitudinal changes in whole-venom composition, whole-venom total protein content, and enzymatic activity of SVMP and PLA2 components of venom. We also assessed the within-individual repeatability of venom components. Despite successfully elevating plasma CORT in the treatment group, we found no effect of CORT treatment or average plasma CORT level on any venom variables measured. Except for total protein content, venom components were highly repeatable within individuals ([Formula: see text]). Our results indicate that the effects of CORT, a hormone commonly associated with stress and metabolic functions, in adult rattlesnake venom are negligible. Our findings bode well for venom researchers and biomedical applications that rely on the consistency of venoms produced from potentially stressed individuals and provide an experimental framework for future studies of proximate mediators of venom variation across an individual's life span.

Body size impacts critical thermal maximum measurements in lizards.

Understanding the mechanisms behind critical thermal maxima (CTmax; the high body temperature at which neuromuscular coordination is lost) of organisms is central to understanding ectotherm thermal tolerance. Body size is an often overlooked variable that may affect interpretation of CTmax, and consequently, how CTmax is used to evaluate mechanistic hypotheses of thermal tolerance. We tested the hypothesis that body size affects CTmax and its interpretation in two experimental contexts. First, in four Sceloporus species, we examined how inter- and intraspecific variation in body size affected CTmax at normoxic and experimentally induced hypoxic conditions, and cloacal heating rate under normoxic conditions. Negative relationships between body size and CTmax were exaggerated in larger species, and hypoxia-related reductions in CTmax were unaffected by body size. Smaller individuals had faster cloacal heating rates and higher CTmax, and variation in cloacal heating rate affected CTmax in the largest species. Second, we examined how body size interacted with the location of body temperature measurements (i.e., cloaca vs. brain) in Sceloporus occidentalis, then compared this in living and deceased lizards. Brain temperatures were consistently lower than cloacal temperatures. Smaller lizards had larger brain-cloacal temperature differences than larger lizards, due to a slower cloacal heating rate in large lizards. Both live and dead lizards had lower brain than cloacal temperatures, suggesting living lizards do not actively maintain lower brain temperatures when they cannot pant. Thermal inertia influences CTmax data in complex ways, and body size should therefore be considered in studies involving CTmax data on species with variable sizes.

Gradual and Discrete Ontogenetic Shifts in Rattlesnake Venom Composition and Assessment of Hormonal and Ecological Correlates.

Ontogenetic shifts in venom occur in many snakes but establishing their nature as gradual or discrete processes required additional study. We profiled shifts in venom expression from the neonate to adult sizes of two rattlesnake species, the eastern diamondback and the timber rattlesnake. We used serial sampling and venom chromatographic profiling to test if ontogenetic change occurs gradually or discretely. We found evidence for gradual shifts in overall venom composition in six of eight snakes, which sometimes spanned more than two years. Most chromatographic peaks shift gradually, but one quarter shift in a discrete fashion. Analysis of published diet data showed gradual shifts in overall diet composition across the range of body sizes attained by our eight study animals, while the shifts in abundance of different prey classes varied in form from gradual to discrete. Testosterone concentrations were correlated with the change in venom protein composition, but the relationship is not strong enough to suggest causation. Venom research employing simple juvenile versus adult size thresholds may be failing to account for continuous variation in venom composition lifespan. Our results imply that venom shifts represent adaptive matches to dietary shifts and highlight venom for studies of alternative gene regulatory mechanisms.

A tale of two islands: evidence for impaired stress response and altered immune functions in an insular pit viper following ecological disturbance.

The frequency and intensity of ecological perturbations affecting wild animal populations is expected to increase in the future with animals facing numerous global threats. Seahorse Key is a continental island off mainland Florida that has historically been a major rookery for several species of waterbirds. As a result of an unknown disturbance, the entire rookery abandoned Seahorse Key in April 2015 and shifted nesting activities to nearby Snake Key, resulting in an influx of food resources in the form of fish carrion to resident Florida cottonmouth snakes (Agkistrodon conanti), while snakes on Seahorse Key experienced a drastic reduction in food resources. Our objective was to assess plasma corticosterone concentrations, corticosterone negative feedback using dexamethasone, blood glucose, body condition, packed cell volume, natural antibody agglutination, white blood cell counts and ratios and erythrocyte sedimentation rate to characterize the long-term effects of differential resource availability in these two snake populations 3 years after this major ecological disturbance. We collected blood samples at three time points from cottonmouths on Seahorse Key (n = 6 individuals) and Snake Key (n = 13 individuals) in fall 2018. In due consideration of the small sample size, our study shows evidence that 3 years after the shift in waterbird nesting Seahorse Key cottonmouths exhibit a dampened acute stress response and presumptive impaired innate immune functions relative to cottonmouths on Snake Key. These results highlight the context-dependent nature of biomarkers and implicate the significant decrease in food resources on Seahorse Key in altering hormonal stress responses and innate immune functions, possibly leading to unknown long-term downstream effects. This study assessed the response of a wild population of pit viper to ecological disturbance in situ with the aim to improve our understanding of how animals cope with such perturbations and improve our capacity to make informed decisions for conservation.

Effects of oxygen on responses to heating in two lizard species sampled along an elevational gradient.

Thermal tolerance is an important variable in predictive models about the effects of global climate change on species distributions, yet the physiological mechanisms responsible for reduced performance at high temperatures in air-breathing vertebrates are not clear. We conducted an experiment to examine how oxygen affects three variables exhibited by ectotherms as they heat-gaping threshold, panting threshold, and loss of righting response (the latter indicating the critical thermal maximum)-in two lizard species along an elevational (and therefore environmental oxygen partial pressure) gradient. Oxygen partial pressure did not impact these variables in either species. We also exposed lizards at each elevation to severely hypoxic gas to evaluate their responses to hypoxia. Severely low oxygen partial pressure treatments significantly reduced the gaping threshold, panting threshold, and critical thermal maximum. Further, under these extreme hypoxic conditions, these variables were strongly and positively related to partial pressure of oxygen. In an elevation where both species overlapped, the thermal tolerance of the high elevation species was less affected by hypoxia than that of the low elevation species, suggesting the high elevation species may be adapted to lower oxygen partial pressures. In the high elevation species, female lizards had higher thermal tolerance than males. Our data suggest that oxygen impacts the thermal tolerance of lizards, but only under severely hypoxic conditions, possibly as a result of hypoxia-induced anapyrexia.

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

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.

Physiological and behavioral effects of exogenous corticosterone in a free-ranging ectotherm.

In the face of global change, free-ranging organisms are expected to experience more unpredictable stressors. An understanding of how organisms with different life history strategies will respond to such changes is an integral part of biodiversity conservation. Corticosterone (CORT) levels are often used as metrics to assess the population health of wild vertebrates, despite the fact that the stress response and its effects on organismal function are highly variable. Our understanding of the stress response is primarily derived from studies on endotherms, leading to some contention on the effects of chronic stress across and within taxa. We assessed the behavioral and hormonal responses to experimentally elevated stress hormone levels in a free-ranging, arid-adapted ectotherm, the Southern Pacific rattlesnake (Crotalus helleri). Plasma CORT was significantly elevated in CORT-implanted snakes 15days after implantation. Implantation with CORT did not affect testosterone (T) levels or defensive behavior. Interestingly, we observed increased defensive behavior in snakes with more stable daily body temperatures and in snakes with higher plasma T during handling (tubing). Regardless of treatment group, those individuals with lower baseline CORT levels and higher body temperatures tended to exhibit greater increases in CORT levels following a standardized stressor. These results suggest that CORT may not mediate physiological and behavioral trait expression in arid-adapted ectotherms such as rattlesnakes.

Oxygen concentration affects upper thermal tolerance in a terrestrial vertebrate.

We tested the oxygen limitation hypothesis, which states that animals decline in performance and reach the upper limits of their thermal tolerance when the metabolic demand for oxygen at high temperatures exceeds the circulatory system's ability to supply adequate oxygen, in air-breathing lizards exposed to air with different oxygen concentrations. Lizards exposed to hypoxic air (6% O2) gaped, panted, and lost their righting response at significantly lower temperatures than lizards exposed to normoxic (21% O2) or hyperoxic (35% O2) air. A greater proportion of lizards in the hyperoxic treatment were able to withstand body temperatures above 44°C than in the normoxic treatment. We also found that female lizards had a higher panting threshold than male lizards, while sex had no effect on gaping threshold and loss of righting response. Body size affected the temperature at which lizards lost the righting response, with larger lizards losing the response at lower temperatures than smaller lizards when exposed to hypoxic conditions. These data suggest that oxygen limitation plays a mechanistic role in the thermal tolerance of lizards.