Contributions of water-borne corticosterone as one non-invasive biomarker in assessing nitrate pollution stress in tadpoles of Rana temporaria.

Among a multitude of stressors to which wildlife is exposed, environmental pollution is a pervasive one that poses a serious threat. The permeable skin of amphibians is likely to increase direct contact of the body with pollutants, making them a group worth studying to access environmental quality. Consequently, finding reliable and complementary biomarkers that will present detectable and predictable changes in response to pollutants is essential to identify pollution sublethal effects on amphibians and to investigate whether these are in part responsible for population declines. The glucocorticoid hormone corticosterone (CORT), involved in many metabolic functions, is often used to measure the physiological stress response to environmental stressors in amphibians. In this study, we evaluated whether water-borne CORT can serve as a non-invasive biomarker for nitrate pollution stress in the European common frog (Rana temporaria) by comparing the effect of nitrate exposure on hormone release rates and on other physiological downstream biomarkers, i.e., ultimate physiological effects of the stressor. Specifically, we investigated the effect of different nitrate concentrations (0, 10, 50, and 100 mg/L) on water-borne CORT release rates, age, size, and body condition. Exposure to nitrate pollution significantly increased age at metamorphosis and water-borne CORT release rates, and led to reduced mass and body condition, but only at higher nitrate concentrations (i.e., 50 and 100 mg/L). Considering this similar sensitivity to other acknowledged biomarkers, water-borne CORT was a reliable biomarker of physiological stress in R. temporaria exposed to nitrate pollution stress in a controlled single-stressor laboratory approach. Thus, water-borne CORT is a promising method to be included in more holistic approaches. We recommend that such approaches keep testing multiple biomarker combinations, as species are exposed to several stressors likely to interact and produce varied outcomes in different biomarkers in their natural habitats.

Short-term responses of Rana arvalis tadpoles to pH and predator stress: adaptive divergence in behavioural and physiological plasticity?

Environmental stress is a major driver of ecological and evolutionary processes in nature. To cope with stress, organisms can adjust through phenotypic plasticity and/or adapt through genetic change. Here, we compared short-term behavioural (activity) and physiological (corticosterone levels, CORT) responses of Rana arvalis tadpoles from two divergent populations (acid origin, AOP, versus neutral origin, NOP) to acid and predator stress. Tadpoles were initially reared in benign conditions at pH 7 and then exposed to a combination of two pH (acid versus neutral) and two predator cue (predator cue versus no predator cue) treatments. We assessed behavioural activity within the first 15 min, and tissue CORT within 8 and 24 h of stress exposure. Both AOP and NOP tadpoles reduced their activity in acidic pH, but the response to the predator cue differed between the populations: AOP tadpoles increased whereas NOP tadpoles decreased their activity. The AOP and NOP tadpoles differed also in their CORT responses, with AOP being more responsive (CORT levels of NOP tadpoles did not differ statistically across treatments). After 8 h exposure, AOP tadpoles had elevated CORT levels in the acid-predator cue treatment and after 24 h exposure they had elevated CORT levels in all three stress treatments (relative to the benign neutral-no-cue treatment). These results suggest that adaptation to environmental acidification in R. arvalis is mediated, in part, via behavioural and hormonal plasticity.

Context dependent variation in corticosterone and phenotypic divergence of Rana arvalis populations along an acidification gradient.

BACKGROUND: Physiological processes, as immediate responses to the environment, are important mechanisms of phenotypic plasticity and can influence evolution at ecological time scales. In stressful environments, physiological stress responses of individuals are initiated and integrated via the release of hormones, such as corticosterone (CORT). In vertebrates, CORT influences energy metabolism and resource allocation to multiple fitness traits (e.g. growth and morphology) and can be an important mediator of rapid adaptation to environmental stress, such as acidification. The moor frog, Rana arvalis, shows adaptive divergence in larval life-histories and predator defense traits along an acidification gradient in Sweden. Here we take a first step to understanding the role of CORT in this adaptive divergence. We conducted a fully factorial laboratory experiment and reared tadpoles from three populations (one acidic, one neutral and one intermediate pH origin) in two pH treatments (Acid versus Neutral pH) from hatching to metamorphosis. We tested how the populations differ in tadpole CORT profiles and how CORT is associated with tadpole life-history and morphological traits. RESULTS: We found clear differences among the populations in CORT profiles across different developmental stages, but only weak effects of pH treatment on CORT. Tadpoles from the acid origin population had, on average, lower CORT levels than tadpoles from the neutral origin population, and the intermediate pH origin population had intermediate CORT levels. Overall, tadpoles with higher CORT levels developed faster and had shorter and shallower tails, as well as shallower tail muscles. CONCLUSIONS: Our common garden results indicate among population divergence in CORT levels, likely reflecting acidification mediated divergent selection on tadpole physiology, concomitant to selection on larval life-histories and morphology. However, CORT levels were highly environmental context dependent. Jointly these results indicate a potential role for CORT as a mediator of multi-trait divergence along environmental stress gradients in natural populations. At the same time, the population level differences and high context dependency in CORT levels suggest that snapshot assessment of CORT in nature may not be reliable bioindicators of stress.

Meerkat close calling patterns are linked to sex, social category, season and wind, but not fecal glucocorticoid metabolite concentrations.

It is well established that animal vocalizations can encode information regarding a sender's identity, sex, age, body size, social rank and group membership. However, the association between physiological parameters, particularly stress hormone levels, and vocal behavior is still not well understood. The cooperatively breeding African meerkats (Suricata suricatta) live in family groups with despotic social hierarchies. During foraging, individuals emit close calls that help maintain group cohesion. These contact calls are acoustically distinctive and variable in rate across individuals, yet, information on which factors influence close calling behavior is missing. The aim of this study was to identify proximate factors that influence variation in call rate and acoustic structure of meerkat close calls. Specifically, we investigated whether close calling behavior is associated with sex, age and rank, or stress hormone output (i.e., measured as fecal glucocorticoid metabolite (fGCM) concentrations) as individual traits of the caller, as well as with environmental conditions (weather) and reproductive seasonality. To disentangle the effects of these factors on vocal behavior, we analyzed sound recordings and assessed fGCM concentrations in 64 wild but habituated meerkats from 9 groups during the reproductive and non-reproductive seasons. Dominant females and one-year old males called at significantly higher rates compared to other social categories during the reproductive season. Additionally, dominant females produced close calls with the lowest mean fundamental frequencies (F0) and the longest mean pulse durations. Windy conditions were associated with significantly higher call rates during the non-reproductive season. Fecal GCM concentrations were unrelated to close calling behavior. Our findings suggest that meerkat close calling behavior conveys information regarding the sex and social category of the caller, but shows no association with fGCM concentrations. The change in call rate in response to variation in the social and ecological environments individuals experience indicates some degree of flexibility in vocal production.

Validation of a Fecal Glucocorticoid Assay to Assess Adrenocortical Activity in Meerkats Using Physiological and Biological Stimuli.

In mammals, glucocorticoid (i.e. GC) levels have been associated with specific life-history stages and transitions, reproductive strategies, and a plethora of behaviors. Assessment of adrenocortical activity via measurement of glucocorticoid metabolites in feces (FGCM) has greatly facilitated data collection from wild animals, due to its non-invasive nature, and thus has become an established tool in behavioral ecology and conservation biology. The aim of our study was to validate a fecal glucocorticoid assay for assessing adrenocortical activity in meerkats (Suricata suricatta), by comparing the suitability of three GC enzyme immunoassays (corticosterone, 11ß-hydroxyetiocholanolone and 11oxo-etiocholanolone) in detecting FGCM increases in adult males and females following a pharmacological challenge with adrenocorticotropic hormone (ACTH) and biological stimuli. In addition, we investigated the time course characterizing FGCM excretion, the effect of age, sex and time of day on FGCM levels and assessed the potential effects of soil contamination (sand) on FGCM patterns. Our results show that the group specific 11ß-hydroxyetiocholanolone assay was most sensitive to FGCM alterations, detecting significant and most distinctive elevations in FGCM levels around 25 h after ACTH administration. We found no age and sex differences in basal FGCM or on peak response levels to ACTH, but a marked diurnal pattern, with FGCM levels being substantially higher in the morning than later during the day. Soil contamination did not significantly affect FGCM patterns. Our results emphasize the importance of conducting assay validations to characterize species-specific endocrine excretion patterns, a crucial step to all animal endocrinology studies using a non-invasive approach.