PHYSIOLOGY: REPTILES

CROCODILES

Ganswindt S. B., Myburgh J. G., Cameron E. Z., Ganswindt A. (2014): Non-invasive assessment of adrenocortical function in captive Nile crocodiles (Crocodylus niloticus). Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 177: 11-17.
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The occurrence of stress-inducing factors in captive crocodilians is a concern, since chronic stress can negatively affect animal health and reproduction, and hence production. Monitoring stress in wild crocodiles could also be beneficial for assessing the state of health in populations which are potentially threatened by environmental pollution. In both cases, a non-invasive approach to assess adrenocortical function as a measure of stress would be preferable, as animals are not disturbed during sample collection, and therefore sampling is feedback-free. So far, however, such a non-invasive method has not been established for any crocodilian species. As an initial step, we therefore examined the suitability of two enzyme-immunoassays, detecting faecal glucocorticoid metabolites (FGMs) with a 11β,21-diol-20-one and 5β-3α-ol-11-one structure, respectively, for monitoring stress-related physiological responses in captive Nile crocodiles (Crocodylus niloticus). An adrenocorticotropic hormone (ACTH) challenge was performed on 10 sub-adult crocodiles, resulting in an overall increase in serum corticosterone levels of 272% above the pre-injection levels 5 h post-injection. Saline-treated control animals (n = 8) showed an overall increase of 156% in serum corticosterone levels 5 h post-administration. Faecal samples pre- and post-injection could be obtained from three of the six individually housed crocodiles, resulting in FGM concentrations 136–380% above pre-injection levels, always detected in the first sample collected post-treatment (7–15 days post-injection). FGM concentrations seem comparatively stable at ambient temperatures for up to 72 h post-defaecation. In conclusion, non-invasive hormone monitoring can be used for assessing adrenocortical function in captive Nile crocodiles based on FGM analysis.

Hamilton M. T., Finger Jr J. W., Elsey R. M., Mastromonaco G. F., Tuberville T. D. (2018): Corticosterone in American alligator (Alligator mississippiensis) tail scutes: evaluating the feasibility of using unconventional samples for investigating environmental stressors. General and Comparative Endocrinology 268: 7-13.
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Baseline plasma corticosterone (CORT) concentrations have been widely used to investigate the effects of stressors in wild and captive crocodilians. However, collecting baseline plasma CORT samples from wild crocodilians may be particularly difficult due to the capture and handling protocols used for large individuals. Thus, it may prove beneficial to use recently modified techniques for extracting CORT deposited in keratinized and non-keratinized tissues to better quantify the effects of long-term stress in crocodilians. In this study, we investigated the feasibility of using American alligator (Alligator mississippiensis) tail scute tissues to quantify CORT by collecting blood and tail scutes from 40 alligators before and after a short-term handling stressor. The objective of the current study was to better understand CORT deposition in crocodilian scutes and whether short-term increases in CORT could be detected. We found that CORT can be reliably extracted from alligator scute tissue and quantified using a commercially available enzyme immunoassay. However, there was a significant increase in scute CORT concentrations following an alligator being exposed to a short-term stressor (p = 0.017), although the magnitude of change was less than observed in plasma samples from the same individuals (p = 0.002). Furthermore, our results indicate that there was a significant effect of body condition on an alligator’s post-stressor CORT concentration (p = 0.02). While our study is among the first to experimentally examine the usefulness of tissue CORT in crocodilians, a combination of field and laboratory experiments are needed to better understand deposition rates of CORT in scute tissues and to further validate the usefulness of tissue glucocorticoids for evaluating the effects of stress.

LIZARDS

Kummrow M. S., Gilman C., Mackie P., Smith D. A., Mastromonaco G. F. (2011): Noninvasive analysis of fecal reproductive hormone metabolites in female veiled chameleons (Chamaeleo calyptratus) by enzyme immunoassay. Zoo Biology 30: 95-115.
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The noninvasive technique of gonadal steroid metabolite measurement in feces for evaluation of reproductive activity has proven an effective and important tool for population management in various captive species, but has not yet been validated and used in reptile species. In this study, enzyme immunoassays (EIAs) were validated for the analysis of fecal samples from female veiled chameleons (Chamaeleo calyptratus) for estrogen (E2), testosterone (T), and progesterone (P) and their metabolites. High performance liquid chromatography and physiological methods (GnRH stimulation) were used for the validation of the assays. Biological events, such as skin color changes indicative of ovarian activity and oviposition, correlated with the cyclical pattern of E2, T and P metabolites in feces over a period of two reproductive cycles. This is the first study to report frequent longitudinal measurements of fecal hormone levels by EIA in a reptile species.

Kalliokoski O., Timm J. A., Ibsen I. B., Hau J., Frederiksen A. M. B., Bertelsen M. F. (2012): Fecal glucocorticoid response to environmental stressors in green iguanas (Iguana iguana). General and Comparative Endocrinology 177: 93-97.
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Quantification of glucocorticoid metabolites in feces has been shown to be a powerful tool in evaluating well-being in vertebrates. Little is known however about the hypothalamic–pituitary–adrenal axis response to stressors, and consequent glucocorticoid excretion, in reptiles. In a longitudinal study, fecal corticosterone metabolite (FCM) levels in green iguanas (Iguana iguana) were quantified during periods of rest and exposure to hypothesized stressors. FCM quantification was combined with behavioral analysis to further contextualize the measured increases. It was shown that both daily 5-minute handling/restraint, as well as housing devoid of climbing opportunity, resulted in increased FCM excretion. Behavioral analysis suggested that the iguanas were chronically stressed by the lack of climbing opportunity, whereas handling may have induced only a transient stress response. The experimental design, using repeated periods of stressor-exposure, also revealed a facilitating effect, where the two stressors potentiated one another. Furthermore, the order of the two stressors was found to be important. The study provides insight into the functioning of the hormonal stress response in green iguanas, and to the refining of their housing and handling.

Halliday W. D., Paterson J. E., Patterson L. D., Cooke S. J., Blouin-Demers G. (2014): Testosterone, body size, and sexual signals predict parasite load in Yarrow’s Spiny Lizards (Sceloporus jarrovii). Canadian Journal of Zoology 92: 1075-1082.
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Parasite load significantly impacts host health and fitness and may vary substantially among individuals within a population. The immunocompetence handicap hypothesis posits that sexual signals are honest indicators of male quality because they are maintained by testosterone, an immunosuppressant that yields higher parasite loads. Additionally, testosterone may influence parasite load by increasing activity levels. We examined these two hypotheses in a wild population of Yarrow’s Spiny Lizards (Sceloporus jarrovii Cope, 1875) in Arizona. We (i) compared fecal testosterone levels to ectoparasite and haemoparasite loads, (ii) tested if sexual signals (total coloured area, aggression, and head size), locomotor activity, and body size correlated with testosterone levels, and (iii) compared sexual signals, locomotor activity, and body size to parasite load. Ectoparasite loads increased with total coloured area and tended to increase with testosterone, but this latter relationship was only nearly significant. Parasite loads increased with body size. Thus, we found some support for the immunocompetence handicap hypothesis and none for the activity hypothesis. Our results are consistent with an alternative hypothesis that larger individuals have more parasites because they have more surface area and (or) have had longer to accumulate parasites. Future studies should examine the relative contributions of testosterone and glucocorticoids in driving variation in parasite loads.

Matas D., Keren-Rotem T., Koren L. (2016): A method to determine integrated steroid levels in wildlife claws. General and Comparative Endocrinology 230: 26-28.
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Glucocorticoids act throughout life to regulate numerous physiological and behavioral processes. Their levels are therefore highly labile, reacting to varying conditions and stressors. Hence, measuring glucocorticoids (and other steroids) in wildlife is challenging, and devising methods that are unaffected by the stress of capture and handling should be explored. Here we use the tip of free-ranging chameleons’ claws that were cut to allow individual identification, and report a steroids extraction and quantification method. Claw steroids present an integrated level representing the period of claw growth. We found that we could measure corticosterone in small amounts of chameleon claw matrix using commercial EIA kits. Using this method, we learned that in wild male chameleons, claw corticosterone levels were associated with body size. We suggest that claw-testing can potentially provide an ideal matrix for wildlife biomonitoring.

Boyer J. F., Swierk L. (2017): Rapid body color brightening is associated with exposure to a stressor in an Anolis lizard. Canadian Journal of Zoology 95: 213-219.
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Many species use color change to optimize body coloration to changing environmental conditions, and drivers of rapid color change in natural populations are numerous and poorly understood. We examined factors influencing body coloration in the Water Anole (Anolis aquaticus Taylor, 1956), a lizard possessing color-changing stripes along the length of its body. We quantified the color of three body regions (the eye stripe, lateral stripe, and dorsum) before and after exposure to a mild stressor (handling and restraint). Based on current understanding of the genus Anolis Daudin, 1802, we hypothesized that exposure to a stressor would generate genus-typical skin darkening (i.e., increased melanism). Contrary to expectations, stress consistently brightened body coloration: eye and lateral stripes transitioned from brown to pale blue and green and the dorsum became lighter brown. Sex, size, and body temperature did not correlate with any aspect of body coloration, and a laboratory experiment confirmed that light exposure did not drive brightening. We propose that color change may serve to reduce conspicuousness through disruptive camouflage; lizards tended to display brighter stripes on mottled green–brown substrates. Together, these results improve our understanding of Anolis color change diversity and emphasize the need for a broader interpretation of the mechanism and functions of color change across taxa.

Carbajal A., Tallo-Parra O., Monclús L., Aresté M., Fernández-Bellon H., Almagro V., Lopez-Bejar M. (2018): Corticosterone measurement in Komodo dragon shed skin. Herpetological Journal 28: 110-116.
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The analysis of corticosterone (CORT), the main glucocorticoid in reptiles, via blood or faeces provides an index of hormone concentrations over a relatively short time period. Unlike these conventional matrices, snake shed skin is supposed to incorporate circulating CORT over the period of skin growth, thus reflecting long-term retrospective levels of the hormone. The present study aimed to assess the feasibility to extract CORT from shed skin of Komodo dragon and biochemically validate the quantification of the hormone by enzyme immunoassay (EIA). Additionally, possible sources of variation in shed skin CORT that could reflect biological variation were examined (sex, age, body region and season of ecdysis). Results of the biochemical validation showed that CORT can be reliably measured in shed skin of Komodo dragon by EIA through the presented methodology. Males presented statistically higher levels of CORT than females, and when accounting for males’ seasonal differences, concentrations decreased significantly from spring to summer. Juveniles showed higher CORT values than adults, however, results should be interpreted with caution since the model revealed that date of ecdysis was significantly influencing CORT levels. Besides that, concentrations of CORT were not influenced by body region. Overall, the present study demonstrates a potential biological source of variation in shed skin CORT concentrations due to sex, age and season of skin ecdysis. Combined with other indicators, detection of CORT concentrations in shed skin could allow a systematic control of Komodo dragon’s physiology, offering a useful tool for zoo management and providing key data for the species conservation.

Scheun J., Greeff D., Ganswindt A. (2018): Non-invasive monitoring of glucocorticoid metabolite concentrations in urine and faeces of the Sungazer (Smaug giganteus). PeerJ 6: e6132.
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Developing non-invasive techniques for monitoring physiological stress responses has been conducted in a number of mammal and bird species, revolutionizing field-based endocrinology and conservation practices. However, studies validating and monitoring glucocorticoid concentrations in reptiles are still limited. The aim of the study was to validate a method for monitoring glucocorticoid metabolite concentrations in urine (uGCM) and faeces (fGCM) of the cordylid lizard, the Sungazer (Smaug giganteus). An adrenocorticotropic hormone (ACTH) challenge was conducted on one male and two females with both urine and faecal material being collected during baseline and post-injection periods. Steroid extracts were analysed with four enzyme immunoassays (EIAs)namely: 11-oxoaetiocholanolone, 5α-pregnane-3β-11β-21-triol-20-one, tetrahydrocorticosterone, and corticosterone. A considerable response in fGCM and uGCM concentrations following ACTH administration was observed in all subjects, with the 5α-pregnane-3β-11β-21-triol-20-one and tetrahydrocorticosterone EIAs appearing to be the most suited for monitoring alterations in glucocorticoid metabolite concentrations in S. giganteus using faeces or urine as hormone matrix. Both EIAs showed a significantly higher concentration of glucocorticoid metabolites in faeces compared to urine for both sexes. Collectively, the findings of this study confirmed that both urine and faeces can be used to non-invasively assess adrenocortical function in S. giganteus.

Hudson S. B., Robertson M. W., Wilcoxen T. E. (2019): Fecal glucocorticoid response to periodic social stress in male Green Anoles, Anolis carolinensis. Copeia 107: 653-660.
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Agonistic encounters necessary for territory establishment and maintenance can be stressful for those involved. Stress responsiveness associated with territorial behavior can occur on both acute and chronic temporal scales contingent upon social status. Social interactions that recur for territory maintenance pose periodic stressors that incur variable physiological costs across social ranks. Adult males of the Green Anole, Anolis carolinensis, experience stressful social encounters during territorial disputes as individuals contest status within a dominance hierarchy. Dominant males in stable territories are known to exhibit greener body coloration and lower levels of stress hormone, corticosterone, relative to their subordinate counterparts. Periodic interactions with novel competitors, however, may induce comparable levels of cumulative glucocorticoid secretion regardless of social status. Glucocorticoid metabolites excreted in feces can be quantified to assess the chronic hypothalamic–pituitary–adrenal (HPA) axis response to periodic social stressors. Fecal glucocorticoid metabolite (FGM) levels in male A. carolinensis were hypothesized to increase in response to novel social encounters that simulated territory establishment and maintenance. Adrenocortical response to recurring episodes of territoriality was predicted to generate similar longitudinal FGM levels across social ranks. FGM analysis was combined with behavioral assessment of body coloration to further contextualize measured stress levels of dominant and subordinate anoles. Prolonged social interaction led to similarly increased levels of fecal glucocorticoid metabolites in both dominant and subordinate anoles relative to those that were solitary. This study provides an alternative perspective on the activity of the HPA axis in dominant–subordinate relationships of A. carolinensis over prolonged periods of territoriality.

Megía-Palma R., Arregui L., Pozo I., Žagar A., Serén N., Carretero M. A., Merino S. (2020): Geographic patterns of stress in insular lizards reveal anthropogenic and climatic signatures. Science of the Total Environment 749: 141655.
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Anthropization of insular ecosystems may have negative impacts on native populations of lizards, which provide core ecosystem services on islands. We aimed to identify environmental factors to explain the interlocal variation in faecal glucocorticoids, parasite intensity, and body condition in populations of insular lizards. A cross-sectional design during the summer of 2017 and 2018 was used to sample 611 adult lizards, Gallotia galloti. Interlocal variation of three stress indicators was analysed in response to environmental variables across a wide environmental gradient in Tenerife (Canary Islands): (i) concentration of faecal glucocorticoids, (ii) intensities of infection by hematic parasites, and (iii) body condition. The data, with low spatial autocorrelation, were analysed using multimodel inference and model cross-validation. Bioclimatic variables associated with the extreme hot and dry climate of summer were the most informative predictors. Interlocal variation in faecal corticosterone in males was best fitted to a model that included the maximum temperature of the warmest month, although the best predictor was habitat anthropization. The thermal annual range, associated with extreme thermal events, was positively related to faecal corticosterone in females. Extreme hot temperatures were positively related to the median parasite intensities in both sexes, while the highest mean intensities of infection were found in females from the most xeric coastal localities. None of the predictors tested, including faecal glucocorticoids, explained individual or interlocal variation in body condition. Effects of human pressure and climate change on insular populations of lizards can be additive. However, the uncoupled relationship found between body condition and the faecal glucocorticoid content suggests that current negative effects may be aggravated during drought periods in summer. Given the impact of climate change on islands, our results may be of application to other archipelagos, where lizards also play key ecological roles.

Kummrow M. S., Pimm R. H., Mackie P. M., Tabh J., Mastromonaco G. F. (2021): Fecal adrenal hormone patterns during ovulatory and non-ovulatory reproductive cycles in female veiled chameleons (Chamaeleo calyptratus). General and Comparative Endocrinology 310: 113822.
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The relationship between the reproductive (hypothalamic-pituitary-gonadal; HPG) and adrenal (hypothalamic-pituitary-adrenal; HPA) hormone axes is complex and can vary depending on the species and environmental factors affecting an individual. In an effort to understand this relationship in female veiled chameleons (Chamaeleo calyptratus), the patterns of fecal metabolites of corticosterone (C), estradiol (E), testosterone (T), and progesterone (P) were analyzed by enzyme immunoassay (EIA) during ovulatory (OC; eggs laid) and non-ovulatory cycles (NOC; no eggs laid). Glucocorticoid (GC) metabolites in the fecal extracts were characterized by HPLC and corticosterone EIA performance was assessed by parallelism, accuracy and precision tests. The results indicated that the assay chosen reliably measured the hormone metabolites present in the fecal extracts. Regular, cyclical hormone metabolite patterns consisting of an E peak followed by peaks of T, P and C in close succession were observed during both ovulatory and non-ovulatory cycles; relative levels of P and C, however, were higher during ovulatory cycles. Corticosterone metabolite levels, in particular, increased throughout vitellogenesis and peaked in late vitellogenesis (in non-ovulatory cycles) or around the time of ovulation, and remained elevated throughout the gravid period, falling just prior to oviposition. The results provide evidence of variation in glucocorticoid production throughout different stages of the reproductive cycle, including a role in the ovulatory process; the physiology, however, remains unclear.

Scheun J., Campbell R., Ganswindt A., McIntyre T. (2021): Hot and bothered: alterations in faecal glucocorticoid metabolite concentrations of the sungazer lizard, in response to an increase in environmental temperature. African Zoology 56: 222-230.
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Despite the commonly held belief that reptiles are immune to extreme temperatures, global warming is predicted to result in the loss of 40% of all reptile species by 2080. In order to understand the effects of elevated temperature on African reptile physiology and health, additional research is required. We studied the physiological stress response of sungazer lizards (Smaug giganteus) facing elevated temperatures within captivity. Control animals (1M, 2F) were kept at 30 °C throughout the 13-week study period. Test animals (1M, 2F) were exposed to 30 °C for four weeks (stage 1), 39 °C for six weeks (stage 2) and 30 °C for three weeks (stage 3). Faecal samples were collected from both control and test animals to monitor faecal glucocorticoid metabolite (fGCM) concentrations as a proxy of physiological stress. Although the fGCM levels of control females remained constant, test females showed an acute fGCM increase following the increase and subsequent decrease of temperatures. The test male had significantly higher fGCM levels during stage 3, compared with stage 1 and 2. The control male, although displaying constant fGCM levels throughout the study, had higher fGCM levels than the test male. The results indicate that a considerable temporal increase in environmental temperature did not lead to chronically elevated adrenocortical activity in sungazer lizards. It is likely that inherent traits within the species allow the species to adapt to such changes. Furthermore, the endangered status of the species and limited availability of study animals restricted the sample size in this study to a few, captive-based lizards. Therefore, although our results are an important addition to reptile conservation, they should be extrapolated to free-ranging populations with caution. Future research should consider the effects of chronically elevated temperature in relation to water and food shortages, as well as interindividual variation in physiological responses.

Amdekar M. S., Thaker M. (2022): Colours of stress in male Indian rock agamas predict testosterone levels but not performance. Hormones and Behavior 144: 105214.
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Rapid physiological colour change offers dynamic signalling opportunities that can reveal distinct information to receivers in different contexts. Information content in dynamic colours, however, is largely unexplored. In males of the Indian rock agama (Psammophilus dorsalis), stressful events, including male-male agonistic interactions, induce a colour change, wherein the dorsal band turns yellow and the lateral bands turn orange. We aimed to determine whether these pigment-based dynamic colours convey information about individual quality. Using an agamid-specific visual model, we first quantified the chromatic and achromatic contrasts of each colour component displayed by males during handling stress, which induces the maximal response of aggression-typical colours. We then measured baseline testosterone levels, morphology (body mass and size), and performance measures (bite force and sprint speed) of these lizards. Chromatic contrasts of the dorsal yellow and lateral orange bands, individually and relative to each other (internal pair), were negatively correlated with testosterone levels, while the chromatic contrast of the internal pair was positively correlated with body condition. The lack of an association between colour contrasts and both bite force and sprint speed indicate that the conspicuousness of colours expressed during stressful events, such as agonistic interactions, do not reveal male performance ability. Despite our expectations of a positive relationship with testosterone, morphology (body condition), and performance (bite force, sprint speed), we find that for P. dorsalis, the conspicuousness of stress-induced colours provide only some information about individual quality. We speculate that the dynamicity of physiological colours may influence their function as content-containing signals in social interactions.

Martín J., Barja I., Rodríguez-Ruiz G., Recio P., Cuervo J. J. (2022): Hidden but potentially stressed: A non-invasive technique to quantify fecal glucocorticoid levels in a fossorial amphisbaenian reptile. Animals 13: 109.
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To understand wildlife responses to the changing environment, it is useful to examine their physiological responses and particularly their endocrine status. Here, we validated an enzyme immunoassay (EIA) to non-invasively quantify fecal corticosterone metabolites (FCM) in the fossorial amphisbaenian reptile Trogonophis wiegmanni from North Africa. We supplemented animals assigned to the treatment group with corticosterone dissolved in oil applied non-invasively on the skin for several days, while control groups received the oil-alone solution. Fresh feces were collected at the end of the supplementation period, and FCM levels were quantified by an EIA. Basal FCM levels were similar for both treatments and increased at the end of the test, but FCM increased significantly more in corticosterone-treated animals. A further examination of FCM levels in a wild population of this amphisbaenian did not find overall sexual, size or seasonal differences but showed a high range of variation among individuals. This suggests that different uncontrolled intrinsic or local environmental variables might increase the circulating glucocorticoid levels of different individuals. Our results confirmed the suitability of EIA for analyzing physiological changes in FCM in this amphisbaenian species. This technique may be useful for understanding and remediating the little-explored potential stressors of the soil environment that may negatively affect the health state of fossorial reptiles.

Megía-Palma R., Barja I., Barrientos R. (2022): Fecal glucocorticoid metabolites and ectoparasites as biomarkers of heat stress close to roads in a Mediterranean lizard. Science of The Total Environment 802: 149919.
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Differences between air and ground temperatures are expected to narrow with the advance of the season in temperate regions (aka seasonal restriction in the availability of thermal microhabitats), which may activate behavioral and physiological responses of ectotherm species adapted to temperate climates. However, according to cost-benefit models of ectotherm thermoregulation, we hypothesize that these responses may also carry some costs. We quantified seasonal shifts in thermoregulatory precision, concentration of fecal glucocorticoid metabolites, and load of ectoparasites in a Mediterranean lizard, Psammodromus algirus. We also tested whether the proximity to a road, a putative source of chronic stress, can facilitate the glucocorticoid-mediated response of lizards to heat stress. As expected, differences between body and environmental temperatures narrowed during the reproductive season and lizards responded by increasing their thermoregulatory precision and the secretion of glucocorticoids, as indicated by metabolites in feces. Interestingly, lizards tended to have higher glucocorticoid concentration when captured far from the road. This might reflect either a putative impairment of the glucocorticoid-mediated response of the lizards to heat stress close to the road or the plastic capability of P. algirus to acclimate to sources of moderate chronic stress. In the latter direction, the increase of both glucocorticoid metabolites and thermoregulatory precision supported that this Mediterranean species responds to environmental thermal restrictions with adaptive behavioral and physiological mechanisms. However, this was also associated with an increase in its susceptibility to ectoparasites, which represents an added cost to the current cost-benefit models of ectotherm thermoregulation.

Zena L. A., Dillon D., Hunt K. E., Navas C. A., Bícego K. C., Buck C. L. (2022): Seasonal changes in steroid and thyroid hormone content in shed skins of the tegu lizard Salvator merianae. Journal of Comparative Physiology B 192: 127-139.
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Sampling blood for endocrine analysis from some species may not be practical or ethical. Quantification of hormones extracted from nontypical sample types, such as keratinized tissues, offers a less invasive alternative to the traditional collection and analysis of blood. Here, we aimed to validate assays by using parallelism and accuracy tests for quantification of testosterone, corticosterone, progesterone, and triiodothyronine (T3) in shed skins of tegu lizards. We assessed whether hormone content of sheds varied across one year similar to what was previously detected in plasma samples. In addition, we aimed to identify the phase relationship between hormone levels of shed skin and plasma levels obtained from the same animals. High frequency of shedding occurred during the active season for tegus (spring/summer), while shedding ceased during hibernation (winter). All hormones measured in shed skins exhibited seasonal changes in concentration. Levels of testosterone in shed skins of male tegus correlated positively with plasma testosterone levels, while corticosterone in both males and females exhibited an inverse relationship between sample types for the same month of collection. An inverse relationship was found when accounting for a lag time of 3 and 4 months between sheds and plasma testosterone. These results indicate that endocrine content of sheds may be confounded by factors (i.e., seasons, environmental temperature, thermoregulatory behavior, among others) that affect frequency of molting, skin blood perfusion, and therefore hormone transfer from the bloodstream and deposition in sheds of squamates.

Martín J., Barja I., Rodríguez-Ruiz G., Recio P., Cuervo J. J. (2023): Hidden but potentially stressed: A non-invasive technique to quantify fecal glucocorticoid levels in a fossorial amphisbaenian reptile. Animals 13: 109.
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SNAKES

Berkvens C. N., Hyatt C., Gilman C., Pearl D. L., Barker I. K., Mastromonaco G. F. (2013): Validation of a shed skin corticosterone enzyme immunoassay in the African House Snake (Lamprophis fuliginosus) and its evaluation in the Eastern Massasauga Rattlesnake (Sistrurus catenatus catenatus). General and Comparative Endocrinology 194: 1-9.
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This study investigates the use of an enzyme immunoassay to measure keratin glucocorticoid concentrations in reptilian shed skins. Keratin glucocorticoid concentrations were compared to fecal glucocorticoid concentrations during the period of keratin growth in the African House Snake (Lamprophis fuliginosus) and the Eastern Massasauga Rattlesnake (Sistrurus catenatus catenatus). Biochemical validation was performed for the shed skin and fecal corticosterone enzyme immunoassays in the African House Snake. Biological and physiological validations were attempted in the African House Snake. A statistically significant positive association was detected between shed skin corticosterone and the mean fecal corticosterone metabolites from 3 weeks before to 1 week after the previous ecdysis in the African House Snake. A statistically significant difference was not detected between the shed skin corticosterone concentrations of the minimally handled control and the weekly handled (or experimentally stressed) African House Snakes. Adrenocorticotropic hormone stimulation did not result in the physiological validation anticipated for shed skin corticosterone concentrations in the African House Snake.

Augustine L., Baskir E., Kozlowski C. P., Hammack S., Elden J., Wanner M. D., Franklin A. D., Powell D. M. (2022): Investigating welfare metrics for snakes at the Saint Louis Zoo. Animals 12: 373.
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Modern herpetoculture has seen a rise in welfare-related habitat modifications, although ethologically-informed enclosure design and evidence-based husbandry are lacking. The diversity that exists within snakes complicates standardizing snake welfare assessment tools and evaluation techniques. Utilizing behavioral indicators in conjunction with physiological measures, such as fecal glucocorticoid metabolite concentrations, could aid in the validation of evidence-based metrics for evaluating snake welfare. We increased habitat cleaning, to identify behavioral or physiological indicators that might indicate heightened arousal in snakes as a response to the disturbance. While glucocorticoid metabolite concentrations increased significantly during a period of increased disturbance, this increase was not associated with a significant increase in tongue-flicking, a behavior previously associated with arousal in snakes. Locomotion behavior and the proportion of time spent exposed were also not affected by more frequent habitat cleaning. These results demonstrate the need to further investigate the behavioral and physiological responses of snakes to different aspects of animal care at a species and individual level. They also highlight the need to collect baseline behavioral and physiological data for animals, in order to make meaningful comparisons when evaluating changes in animal care.

Racine H., Guthrie K. S., Hill T., Loughman Z. (2022): Impact of indigestible materials on the efficiency of fecal corticosterone immunoassay testing in Pituophis species. Animals 12: 1410.
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Measuring fecal glucocorticoid metabolites (FGM) has recently become a sought-after method for assessing stress in animals. While there are many benefits to this methodology, there are also recognized limitations, including the apprehensive interpretation of results. While many factors can influence FGM levels, we aimed to standardize and improve these methods in snakes. Fecal samples were collected from Pituophis species and FGMs were extracted by two different sample collection methods: (1) fecal sample containing undigested materials and (2) fecal samples with undigested materials removed. These extracts were then used to quantify FGM concentrations using a corticosterone EIA kit. The results indicated that the samples with the undigestible materials removed had a 95% increase in overall yield (p < 0.01). Since the collected fecal samples contain 75% undigestible materials by weight, these results support our hypothesis that removing these materials will improve extraction methods for a more reliable measurement of corticosterone. This is the first step towards standardizing the methods for assessing stress by measuring fecal glucocorticoid metabolites in snakes.

TURTLES

Rittenhouse C. D., Millspaugh J. J., Washburn B. E., Hubbard M. W. (2005): Effects of radiotransmitters on fecal glucocorticoid metabolite levels of three‐toed box turtles in captivity. Wildlife Society Bulletin 33: 706-713.
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The increased use of radiotelemetry for studying movement, resource selection, and population demographics in reptiles necessitates closer examination of the assumption that radiotransmitter attachment does not bias study results. We determined the effects of radiotransmitter attachment on fecal glucocorticoid metabolite levels of wild three-toed box turtles (Terrapene carolina triunguis) in captivity. During May 2002 we captured 11 adult three-toed box turtles in central Missouri. We housed turtles in individual pens in a semi-natural outdoor setting. We radiotagged 6 turtles, and the remaining 5 turtles served as controls. We captured and handled all turtles similarly during treatments. We collected feces daily prior to attachment (14 June–05 July 2002), while transmitters were attached (06 July–02 August 2002), and after transmitters were removed (03 August–24 August 2002). We conducted a standard assay validation and found that the assay accurately and precisely quantified fecal glucocorticoid metabolites of box turtles. We did not find a significant effect of radiotransmitter attachment on fecal glucocorticoid metabolite levels of three-toed box turtles (F_1,9=0.404, P=0.541). Fecal glucocorticoid metabolite levels of control and treatment turtles increased significantly during the study (F_2,166=7.874, P=0.001), but there was no treatment:period interaction (F_2,166=0.856, P=0.427). Additionally, we did not find a significant relationship between glucocorticoid metabolite levels and time in captivity (r²=0.01, F_1,179=2.89, P=0.091) or maximum daily temperature (r² < 0.01, F1,179=0.301, P=0.584). Our results suggested that radiotransmitter attachment did not significantly increase fecal glucocorticoid metabolite levels in adult three-toed box turtles; however, we conducted our study in captivity and sample sizes were small. Thus, more research is needed to assess potential effects of radiotransmitters on turtles in the wild. We believe this study is the first to validate the use of fecal glucocorticoid metabolite measures for reptiles, which might prove useful in other research studies.

Baxter-Gilbert J. H., Riley J. L., Mastromonaco G. F., Litzgus J. D., Lesbarrères D. (2014): A novel technique to measure chronic levels of corticosterone in turtles living around a major roadway. Conservation Physiology 2: cou036.
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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.

Umapathy G., Deepak V., Kumar V., Chandrasekhar M., Vasudevan K. (2015): Endocrine profiling of endangered tropical chelonians using noninvasive fecal steroid analyses. Chelonian Conservation and Biology 14: 108-115.
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The present study demonstrates the feasibility of in situ endocrine profiling of the Cochin cane turtle (Vijayachelys silvatica) and the Travancore tortoise (Indotestudo travancorica) using noninvasive fecal-steroid analyses. Reproductive (testosterone, progesterone, estradiol) and stress hormones (Glucocorticoids) and their metabolites were detected in fecal samples with unambiguous signatures during their breeding seasons. We validate immunoassays for monitoring reproduction and stress level using fecal steroid analysis.

Weigand N. M., Wagner R. B., Tonra C. M., Popescu V. D. (2019): Proximity to highways has limited influence on space use and physiology of terrestrial testudines. European Journal of Wildlife Research 65: 80.
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Transportation infrastructure is one of the mainstays of human modification of terrestrial landscapes. Turtle populations are highly affected by roads through direct mortality, contributing to population declines. However, sub-lethal effects, such as increased physiological stress, may indirectly affect turtle demographic rates, particularly in populations recently exposed to roads. We took advantage of a unique study system in southeast Ohio, where an intact forest was bisected by a four-lane highway in 2013, exposing eastern box turtles (Terrapene carolina carolina) to a new threat. The goal of this study was to evaluate ecological, physiological, and behavioral effects of exposure to a new road by comparing a roadside turtle population to a control population in a nearby roadless area, and guide mitigation on new and existing roadways. We used a unique combination of radio telemetry to assess space use, behavior, and habitat selection of turtle, and bioassay techniques to analyze chronic stress using corticosterone stored in nail keratin. We found no differences in home range sizes and habitat selection between the two sites, but roadside turtles showed strong highway avoidance, despite spending a significant amount of time in its immediate vicinity. All turtles selected for higher woody debris and understory vegetation cover, and males at both sites selected for higher canopy cover. Corticosterone concentrations from nails collected upon initial capture (2017) did not differ between the two sites, but males showed a wider range of variation. Corticosterone concentrations were significantly higher in 2018, with roadside animals showing the highest levels, but they were not correlated with home range size or proximity to highway. As such, further work is needed to evaluate indirect effects of multiple stressors on turtle endocrinology and their demographic implications, as well as the level of demographic compensation resulting from road avoidance.