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A novel technique to measure chronic levels of corticosterone in turtles living around a major roadway
Abstract and Figures
Conservation biology integrates multiple disciplines to expand the ability to identify threats to populations and develop
mitigation for these threats. Road ecology is a branch of conservation biology that examines interactions between wildlife
and roadways. Although the direct threats of road mortality and habitat fragmentation posed by roads have received much attention,
a clear understanding of the indirect physiological effects of roads on wildlife is lacking. Chronic physiological stress
can lower immune function, affect reproductive rates and reduce life expectancy; thus, it has the potential to induce long-lasting
effects on populations. Reptiles are globally in decline, and roads are known to have negative effects on reptile populations;
however, it is unknown whether individual responses to roads and traffic result in chronic stress that creates an additional
threat to population viability. We successfully extracted reliable measures of corticosterone (CORT), a known, commonly used
biomarker for physiological stress, from claw trimmings from painted turtles (Chrysemys picta) captured at three study sites (road-impacted site, control site and validation site). Corticosterone levels in claws were
evaluated as a measure of chronic stress in turtles because CORT is deposited during growth of the claw and could provide
an opportunity to examine past long-term stress levels. While male turtles had higher CORT levels on average than females,
there was no difference in the level of CORT between the road-impacted and control site, nor was there a relationship between
CORT and turtle body condition. In validating a novel approach for non-invasive measurement of long-term CORT levels in a
keratinized tissue in wild reptiles, our study provides a new avenue for research in the field of stress physiology.
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... Studies of corticosterone in keratinized tissues of reptiles are limited in number compared to hair and feather glucocorticoid studies in avian and mammalian species, but warrant continued research, as they can be obtained in a minimally invasive manner and may reflect more chronic glucocorticoid levels than can be measured in plasma or other tissues (Berkvens et al., 2013;Baxter-Gilbert et al., 2014;Matas et al., 2016;Carbajal et al., 2018;Hamilton et al., 2018;Gormally and Romero, 2020;Dillon et al., 2021). The chronic presence of a stressor can lead to a persistent physiologic stress response and continuous activation of the hypothalamic-pituitary-adrenal axis due to reduced efficacy of negative feedback mechanisms (Sapolsky, 1992;Sheriff et al., 2009). ...
... In reptiles, corticosterone has been quantified from turtle and chameleon claws (Baxter-Gilbert et al., 2014;Matas et al., 2016), as well as the shed skin of lizards (Carbajal et al., 2018;Zena et al., 2022) and snakes (Berkvens et al., 2013). No effect of a chronic stressor was reflected in shedskin corticosterone in snakes in the study by Berkvens et al. (2013). ...
... Shed skins were stored individually in sealed plastic freezer bags (Ziploc; SC Johnson, Racine, WI, USA) at 4 C (39.2 F) at the animal care facility for up to 2 wk until they could be transferred to laboratory storage. After transfer, they were maintained at room temperature until extraction (Baxter-Gilbert et al., 2014;Carbajal et al., 2018). ...
Renal disease is often identified as a cause of morbidity and mortality in avian patients. However, currently, early antemortem detection of renal disease in avian patients is difficult. Anatomical and physiological differences between mammals and birds mean the use of commonly employed diagnostic testing (ie, measurement of blood urea nitrogen [BUN] and serum creatinine, urinalysis, and ultrasonography) are either nondiagnostic or difficult to achieve. Symmetric dimethylarginine (SDMA) is considered a more sensitive marker for renal disease in humans, dogs, and cats. However, SDMA has not yet been assessed for diagnostic use in any psittacine species. In this study, we establish reference ranges for SDMA in both Hispaniolan Amazon parrots (Amazona ventralis, HAP) and Quaker parrots (Myiopsitta monachus, QP). Blood was collected from 23 Amazon parrots and 32 Quaker parrots maintained in research facilities. Measurement of SDMA through a commercially available immunoassay (IA-SDMA) as well as creatinine, BUN, uric acid, phosphorus, calcium, sodium, potassium, and chloride were determined through IDEXX Laboratories. Plasma SDMA concentrations ranged from 6 to 15 µg/dL and 3 to 15 µg/dL for the HAP and QP, respectively. Sex was a confounding factor for the QP population, but sex did not have a significant effect on SDMA for the HAP population. No significant correlations were identified between SDMA concentrations and other parameters in either psittacine species. Our results show proof of concept for the IA-SDMA and provide reference intervals for SDMA in HAP and QP. Further investigation is required to determine the validity of this assay and the predictive power of SDMA in the detection of renal impairment for parrots and other common companion birds.
... Enzyme immunoassays (EIAs) were quantified using methods described in Baxter- Gilbert et al. (2014) and Stewart et al. (2020). Species specific differences in fecal hormone metabolite profiles are typically assessed using biochemical, physiological and biological validation techniques that ensure appropriate selection of EIAs (Palme 2019). ...
Male and female rodents experience different selective pressures associated with reproductive costs. Thus, we may expect the expression of different Pace-of-life (POL) strategies between sexes. Further, the pace-of-life syndrome (POLS) hypothesis and anisogamy predict differences in the costs of gamete production, where variation in life history trait expression should follow a fast-slow continuum such that males and females might exist on opposite ends of the spectrum. However, males and females could express a similar POL strategy in systems where the reproductive costs are consistent between sexes or where selective pressures force a similar directionality of traits. We used standardized behavioural tests and fecal glucocorticoid metabolite (FGM) concentrations to measure potential differences in POL strategies among three rodent species in Algonquin Provincial Park. We hypothesized that differences in reproductive costs between males and females would result in differences in POL traits along the fast-slow continuum. We predicted that males would express more explorative behaviours and have a higher level FGMs than female counterparts. We found little support for the POLS hypothesis between sexes. Instead, we suggest that the species observed may express a similar directionality of selection for the observed behavioural traits, where both sexes express similar relationships in POL. Thus, some male rodents may be more explorative to accommodate the increase of energetic stress associated with mate acquisition, while females may share similar trait expressions to accommodate the increased metabolic demand for the care and development of young.
... To quantify corticosterone in dried extracts, we used a corticosterone enzyme immunoassay (CJM006, provided by C. Munro, University of California, Davis, CA) described by Baxter-Gilbert et al. (2014). Here, extracts were first reconstituted in a 150-mL enzyme immunoassay buffer solution, resulting in a fourfold concentration, and then immediately analyzed in duplicate. ...
... Some turtles inhabiting the vicinity of roads with intensive traffic, develop a tolerance towards stressors related to the passing motor vehicles. The levels of stress hormones in individuals of a population of Painted turtles (Chrysemys picta), living around an important motorway in Canada were comparable to those of the control group of animals living far away from any roads [53]. The vicinity of road E 87, connecting the Industrial zone of Burgas and Kraymorie was constructed within a site which is optimally suited for E. orbicularis (see [54,55]). ...
We report on the impact of vehicle traffic on a local population of European pond turtles ( Emys orbicularis ) inhabiting the city of Burgas (Bulgaria). We performed a six-year monitoring program to collect data on the presence of turtles on road E 87 in the south region of the city. The road section splits a Natura 2000 site and crosses through an artificial water basin with fresh to brackish water. We visited the road section in irregular intervals from 3 to over 20 days in the period March to October from 2016 to 2021. For the period of the monitoring, we were able to detect a total of over 300 specimens from all ontogenetic stages (33 juveniles, 74 subadults and 224 adults). During our field surveys in 2016, we detected a total of 47 live and 23 dead adults. In the next years, we found more dead adult turtles than live ones. Similar results were recorded for subadults. In the juveniles, the dead specimens were almost equal to those which were detected alive. Our results indicate an impact of the traffic on the local population of E. orbicularis . We discuss the design and position of constructions, which can mitigate the conflict and reduce the potential for Wildlife-vehicle collisions (WVC).
... Some turtles inhabiting the vicinity of roads with intensive tra c, develop a tolerance towards stressors related to the passing motor vehicles. The levels of stress hormones in individuals of a population of Painted turtles (Chrysemys picta), living around an important motorway in Canada were comparable to those of the control group of animals living far away from any roads [53]. The section of road E 87, connecting the Industrial zone of Burgas and Kraymorie was constructed within a site which is optimally suited for E. orbicularis (see [54,55]). ...
We report on the impact of vehicle traffic on a local population of European pond turtles (Emys orbicularis) inhabiting the city of Burgas (Bulgaria). We performed a six-year monitoring program to collect data on the presence of turtles on road E 87 in the south region of the city. The road section splits a Natura 2000 site and crosses through an artificial water basin with fresh to brackish water. We visited the road section in irregular intervals from 3 to over 20 days in the period March to October from 2016 to 2021. For the period of the monitoring, we were able to detect a total of over 300 specimens from all ontogenetic stages (33 juveniles, 74 subadults and 224 adults). During our field surveys in 2016, we detected a total of 47 live and 23 dead adults. In the next years, we found more dead adult turtles than live ones. Similar results were recorded for subadults. In the juveniles, the dead specimens were almost equal to those which were detected alive. Our results indicate an impact of the traffic on the local population of E. orbicularis. We discuss the design and position of constructions, which can mitigate the conflict and reduce the potential for WVC.
... This can be further complicated by time spent in traps, if turtles find being in traps to be stressful (Cash et al., 1997). Without being able to account for handling time, further research is needed to assess if PFAS exposure is influencing stress in turtles; additionally, measuring CORT in claws/keratin may be a better indicator of long-term chronic stress in turtles (Gormally and Romero, 2020;Baxter-Gilbert et al., 2014)). Although some site and sex hormone trends are apparent, blood PFAS only showed potential relationships with progesterone in gravid females and blood CORT in males. ...
Ecological and conservation research has focused on documenting wildlife's behavioural and demographic responses to anthropogenic disturbance. Yet, a significant gap remains in our understanding of the physiological mechanisms that underlie behavioural or demographic adaptations to human activities.
Glucocorticoid concentrations (GCCs) are widely considered as relevant endpoints to assess the physiological response of wildlife to natural or anthropogenic stressors, yet recent studies show mixed outcomes concerning GCC shifts in disturbed populations. Consequently, uncertainties remain around the role of GCCs as reliable indicators of the effect of anthropogenic disturbance on organisms' physiology, likely due to the masking effect of several confounding factors.
To unveil generalizable patterns of physiological effects of human disturbance on wildlife, we collected GCCs measured in conspecific populations living in undisturbed and disturbed areas from 121 studies and 114 species of terrestrial vertebrates, and assessed the roles of disturbance type and species traits in shaping GCC shifts using a phylogenetic meta‐analysis of log response ratios (lnRRΔ).
We found a 36% increase in GCCs due to human disturbance in mammals (lnRRΔ = 0.304), but no evidence of a generalizable effect for birds, non‐avian reptiles and amphibians. Mammals showed similar responses to all forms of anthropogenic disturbance, while birds showed 24.6% increase in GCCs for habitat conversion only (lnRRΔ = 0.221). Furthermore, change in GCCs tended to be higher in large‐sized than in small‐sized mammals, but comparatively lower in herbivorous mammals. Finally, we found non‐migratory birds to elicit stronger responses compared with migratory birds (lnRRΔ = 0.234).
Our study provides novel evidence supporting the influential roles of diet and body mass in mammals, and migratory behaviours in birds, in shaping glucocorticoid responses to human disturbance, establishing a link between magnitude of physiological responses and pace‐of‐life‐related traits. Furthermore, we found GCC trends to be coherent with known demographic and behavioural responses, suggesting that GCC shifts might be costly to sustain, thus serving as potential early‐warning signals of population decline. Finally, we found consistency in the magnitude of GCC shifts in mammals facing all types of human disturbance, highlighting that even seemingly innocuous interactions with humans may still induce substantial physiological changes.
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Dredging is commonly used to maintain navigational channels in freshwater lakes such as Lake Erie, and the dredged material may be discarded in confined disposal facilities (CDFs). The effects of these CDFs on wildlife health are largely unknown. We compared health assessments of painted turtles (Chrysemys picta) at two sites in southwestern Lake Erie, Ohio, USA in May 2022: CDF for dredged material (n=27), and a protected coastal marsh (PCM; n=24). Each turtle underwent a physical examination, blood collection for hematology and plasma biochemistry profiles, and testing for Chlamydia spp., herpesviruses, ranavirus (frog virus 3), and Mycoplasmopsis spp. via oral and cloacal swabs. Six turtles were positive for Chlamydia spp. (11.8%; 95% confidence interval [CI]: 4.4%–23.9%), two from the CDF and four from the PCM. One Chlamydia-positive turtle was co-infected with herpesvirus (2.0%; 95% CI: 0%–10.5%) with 96.3% homology with emydid herpesvirus. No ranavirus or Mycoplasmopsis species were detected (0%, 95% CI: 0%–6.7%). Turtles captured from the CDF had significantly higher heterophil percentages, heterophil/lymphocyte ratios, and glucose levels, but significantly lower total protein and calcium/phosphorus ratios than turtles from PCM. Turtles living in the CDF were hyperglycemic, potentially indicating increased stress levels compared to the normoglycemic turtles from PCM. As humans continue to alter wetland habitats, the impact of anthropogenic sites such as CDFs on freshwater chelonian health and welfare should be monitored.
Hormone monitoring of at-risk species can be valuable for evaluation of individual physiological status. Traditional non-invasive endocrine monitoring from urine and faeces typically captures only a short window in time, poorly reflecting long-term hormone fluctuations. We examined toenail trimmings collected from African (Loxodonta africana) and Asian (Elephas maximus) elephants during routine foot care, to determine if long-term hormone patterns are preserved in these slow-growing keratinized tissues. We first measured the growth rate of elephant toenails biweekly for one year, to establish the temporal delay between deposition of hormones into nail tissue (at the proximal nail bed) and collection of toenail trimmings months later (at the distal tip of the nail). In African elephants, toenails grew ~0.18 ± 0.015 mm/day (mean ± SEM) and in Asian elephants, toenails grew ~0.24 ± 0.034 mm/day. This slow growth rate, combined with the large toenail size of elephants, may mean that toenails could contain a ‘hormone timeline’ of over a year between the nail bed and nail tip. Progesterone, testosterone and cortisol were readily detectable using commercial enzyme immunoassays, and all assays passed validations, indicating that these hormones can be accurately quantified in elephant toenail extract. In most cases, variations in hormone concentrations reflected expected physiological patterns for adult females and males (e.g. ovarian cycling and musth) and matched individual health records from participating zoos. Progesterone patterns aligned with our calculations of temporal delay, aligning with female ovarian cycling from over six months prior. Unexpectedly, male testosterone patterns aligned with current musth status at the time of sample collection (i.e. rather than prior musth status). Though this sample type will require further study, these results indicate that preserved hormone patterns in elephant toenails could give conservationists a new tool to aid management of elephant populations.
Effective and targeted conservation action requires detailed information about species, their distribution, systematics and ecology as well as the distribution of threat processes which affect them. Knowledge of reptilian diversity remains surprisingly disparate, and innovative means of gaining rapid insight into the status of reptiles are needed in order to highlight urgent conservation cases and inform environmental policy with appropriate biodiversity information in a timely manner. We present the first ever global analysis of extinction risk in reptiles, based on a random representative sample of 1500 species (16% of all currently known species). To our knowledge, our results provide the first analysis of the global conservation status and distribution patterns of reptiles and the threats affecting them, highlighting conservation priorities and knowledge gaps which need to be addressed urgently to ensure the continued survival of the world’s reptiles. Nearly one in five reptilian species are threatened with extinction, with another one in five species classed as Data Deficient. The proportion of threatened reptile species is highest in freshwater environments, tropical regions and on oceanic islands, while data deficiency was highest in tropical areas, such as Central Africa and Southeast Asia, and among fossorial reptiles. Our results emphasise the need for research attention to be focussed on tropical areas which are experiencing the most dramatic rates of habitat loss, on fossorial reptiles for which there is a chronic lack of data, and on certain taxa such as snakes for which extinction risk may currently be underestimated due to lack of population information. Conservation actions specifically need to mitigate the effects of human-induced habitat loss and harvesting, which are the predominant threats to reptiles.
We examined the reproductive ecology of black-legged kittiwakes Rissa tridactyla in several breeding colonies in the North Pacific to test if inter-annual changes in the Pacific Decadal Oscillation (PDO), Winter Ice Cover (ICI), or local sea-surface temperature (SST) predict changes in productivity (fledglings per nest) or nutritional stress (corticosterone). We explored the implications of the observed variation in productivity and stress for projected population dynamics based on a previously demonstrated corticosterone-survival relationship. Although productivity was highly variable (0 to 0.9 fledglings nest(-1)), the relationships between productivity and environmental indices were weak, with local SST providing slightly more explanatory power than PDO or ICI, suggesting that local factors rather than large-scale climate variability may determine variation in productivity. The relationships between stress and environmental indices were stronger than the relationship between productivity and environment. The measured response of stress to environment showed opposite signs between the southern and northern colonies, and typically implied annual mortality rates varying from 11 to 17%. The observed relationships between climate and stress indicate that anticipated warming might bring at least short-term demographic benefits for kittiwakes in the Bering shelf region, while having negative impacts on birds breeding in the Gulf of Alaska and western Aleutians. We predict decline (without immigration) for colonies with the lowest productivity and conclude that climate variability is likely to affect survival of North Pacific kittiwakes on a region-specific basis. Longevity of these birds may not always be sufficient to buffer their populations from low reproductive performance.
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 day's 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.
Walter Cannon and his students and coworkers conducted an initial series of experiments from 1910 to 1911 that examined the link between emotional experiences and the secretion of epinephrine from the adrenal medulla. Employing an intestinal strip bioassay for the semiquantitative study of epinephrine secretion, he and his students determined that epinephrine secretion increased dramatically when a quiescent animal was exposed to a stressful or threatening stimulus. In addition, Cannon's laboratory used increases in glucose in the urine (glycosuria) as an indirect measure of epinephrine secretion with similar results. The connection between epinephrine secretion and increases in blood glucose led Cannon to write the following entry in his journal on January 20, 1911: "Got idea that adrenals in excitement serve to affect muscular power and mobilize sugar for muscular use - thus in wild state readiness for fight or run!" In time, "fight or run" was transformed into the more familiar fight or flight.
Effective and targeted conservation action requires detailed information about species, their distribution, systematics and ecology as well as the distribution of threat processes which affect them. Knowledge of reptilian diversity remains surprisingly disparate, and innovative means of gaining rapid insight into the status of reptiles are needed in order to highlight urgent conservation cases and inform environmental policy with appropriate biodiversity information in a timely manner. We present the first ever global analysis of extinction risk in reptiles, based on a random representative sample of 1500 species (16% of all currently known species). To our knowledge, our results provide the first analysis of the global conservation status and distribution patterns of reptiles and the threats affecting them, highlighting conservation priorities and knowledge gaps which need to be addressed urgently to ensure the continued survival of the world’s reptiles. Nearly one in five reptilian species are threatened with extinction, with another one in five species classed as Data Deficient. The proportion of threatened reptile species is highest in freshwater environments, tropical regions and on oceanic islands, while data deficiency was highest in tropical areas, such as Central Africa and Southeast Asia, and among fossorial reptiles. Our results emphasise the need for research attention to be focussed on tropical areas which are experiencing the most dramatic rates of habitat loss, on fossorial reptiles for which there is a chronic lack of data, and on certain taxa such as snakes for which extinction risk may currently be underestimated due to lack of population information. Conservation actions specifically need to mitigate the effects of human-induced habitat loss and harvesting, which are the predominant threats to reptiles.