Water-borne Hormone Research Articles

PREVALENCE OF RANAVIRUS IN SPOTTED SALAMANDER (AMBYSTOMA MACULATUM) LARVAE FROM CREATED VERNAL POOLS IN WEST VIRGINIA, USA.

Ranavirosis is a disease of high concern for amphibians due to widespread documentation of its lethal and sublethal impacts and its high transmission potential across populations and species. We investigated whether spotted salamander (Ambystoma maculatum) ranavirus prevalence and viral load were associated with habitat characteristics, genetic diversity, corticosterone levels, and body size. In 2015 and 2016, we sampled 34 recently created vernal pools in the Monongahela National Forest, West Virginia, USA. We collected tail clippings from 1,128 spotted salamander larvae and waterborne hormone samples from 436 of those larvae, along with eight environmental characteristics of the pools. Over the 2-yr period, we detected ranavirus in 62% of pools, with prevalence ranging from 0% to 63% (mean, 7.68%). Spotted salamander size was positively correlated with ranavirus presence and viral load; however, we did not find associations between ranavirus prevalence or viral load and habitat characteristics, spotted salamander genetic diversity, relatedness, effective number of breeders, or corticosterone levels. The widespread occurrence of ranavirus in the vernal pools illustrates the potential for rapid natural introduction of the pathogen to created wetlands. Managers could consider monitoring local distributions of ranavirus before creation of new vernal pools to guide strategic placement of the wetlands to minimize occurrence and prevalence of this pathogen.

Baseline corticosterone levels in spadefoot toads reflect alternate larval diets one year later

Early-life environmental variation can influence later-life physiology, such as the regulation of glucocorticoids. However, characterizing the effects of environmental factors on hormone regulation can be hampered when assessing animals that are small and require destructive sampling to collect blood. Using spadefoot toads (genus Spea), we evaluated whether waterborne corticosterone (CORT) measures could be used as a proxy for plasma CORT measures, detect stress-induced levels of CORT, and detect larval diet-induced changes in CORT regulation after metamorphosed individuals were maintained for 1 year under common garden conditions. We found that waterborne CORT measures were correlated with plasma CORT measures and could be used to detect stress-induced levels of CORT. Further, larval diet type significantly influenced baseline plasma CORT levels 1-year post-metamorphosis: adults that had consumed live prey as larvae had higher plasma CORT levels than adults that had consumed detritus as larvae. However, waterborne measures failed to reflect these differences, possibly due to low sample size. Our study demonstrates the utility of the waterborne hormone assay in assessing variation in baseline and stress-induced CORT levels in adult spadefoots. However, resolving more subtle differences that arise through developmental plasticity will require larger samples sizes when using the waterborne assay.

Differences in Corticosterone Release Rates of Larval Spring Salamanders (Gyrinophilus porphyriticus) in Response to Native Fish Presence.

Simple SummaryIn amphibians, glucocorticoid hormones play a key role in the response to predation stress. Predators can directly affect prey via injury and death, but they can also have indirect effects due to the activity of glucocorticoids. The regulation of glucocorticoids can differ between populations that have co-evolved with predators and those that have not. We measured glucocorticoids at baseline and in response to a novel stressor in free-living larval salamanders that either live with or without fish predators naturally. We found that salamanders living with fish predators had lower measures of glucocorticoids than those without fish predators. Our study indicates that predator presence alters glucocorticoid regulation, which may allow species to better cope with native and introduced predators.Invasive fish predators are an important factor causing amphibian declines and may have direct and indirect effects on amphibian survival. For example, early non-lethal exposure to these stressors may reduce survival in later life stages, especially in biphasic species. In amphibians, the glucocorticoid hormone corticosterone is released by the hypothalamo–pituitary–interrenal axis (HPI), as an adaptive physiological response to environmental stressors. The corticosterone response (baseline and response to acute stressors) is highly flexible and context dependent, and this variation can allow individuals to alter their phenotype and behavior with environmental changes, ultimately increasing survival. We sampled larvae of the spring salamander (Gyrinophilus porphyriticus) from two streams that each contained predatory brook trout (Slavelinus fontinalis) in the lower reaches and no predatory brook trout in the upper reaches. We measured baseline and stress-induced corticosterone release rates of larvae from the lower and upper reaches using a non-invasive water-borne hormone assay. We hypothesized that corticosterone release rates would differ between larvae from fish-present reaches and larvae from fish-free reaches. We found that baseline and stressor-induced corticosterone release rates were downregulated in larvae from reaches with fish predators. These results indicate that individuals from reaches with predatory trout are responding to fish predators by downregulating corticosterone while maintaining an active HPI axis. This may allow larvae more time to grow before metamorphosing, while also allowing them to physiologically respond to novel stressors. However, prolonged downregulation of corticosterone release rates can impact growth in post-metamorphic individuals.

Coping With Urban Habitats Via Glucocorticoid Regulation: Physiology, Behavior, and Life History in Stream Fishes.

As environments become urbanized, tolerant species become more prevalent. The physiological, behavioral and life-history mechanisms associated with the success of such species in urbanized habitats are not well understood, especially in freshwater ecosystems. Here, we examined the glucocorticoid (GC) profiles, life-history traits, and behavior of two species of fish across a gradient of urbanization to understand coping capacity and associated trade-offs. We studied the tolerant live-bearing Western Mosquitofish (Gambusia affinis) for two years and the slightly less tolerant, egg-laying, Blacktail Shiner (Cyprinella venusta) for one year. We used a water-borne hormone method to examine baseline, stress-induced, and recovery cortisol release rates across six streams with differing degrees of urbanization. We also measured life-history traits related to reproduction, and for G. affinis, we measured shoaling behavior and individual activity in a novel arena. Both species showed a trend for reduced stress responsiveness in more urbanized streams, accompanied by higher reproductive output. Although not all populations fit this trend, these results suggest that GC suppression may be adaptive for coping with urban habitats. In G. affinis, GC recovery increased with urbanization, and individuals with the lowest stress response and highest recovery had the greatest reproductive allotment, suggesting that rapid return to baseline GC levels is also an important coping mechanism. In G. affinis, urban populations showed altered life-history trade-offs whereas behavioral traits did not vary systematically with urbanization. Thus, these tolerant species of fish may cope with anthropogenically modified streams by altering their GC profiles and life-history trade-offs. These results contribute to understanding the mechanisms driving species-specific adaptations and thereby community structure in freshwater systems associated with land-use converted areas.

Effects of salinity and a glucocorticoid antagonist, RU486, on waterborne aldosterone and corticosterone of northern leopard frog larvae

Increased salinity is an emerging contaminant of concern for aquatic taxa. For amphibians exposed to salinity, there is scarce information about the physiological effects and changes in osmoregulatory hormones such as corticosterone (CORT) and aldosterone (ALDO). Recent studies have quantified effects of salinity on CORT physiology of amphibians based on waterborne hormone collection methods, but much less is known about ALDO in iono- and osmoregulation of amphibians. We re-assayed waterborne hormone samples from a previous study to investigate effects of salinity (sodium chloride, NaCl) and a glucocorticoid receptor antagonist (RU486) on ALDO of northern leopard frog (Rana pipiens) larvae. We also investigated relationships between ALDO and CORT. Waterborne ALDO marginally decreased with increasing salinity and was, unexpectedly, positively correlated with baseline and stress-induced waterborne CORT. Importantly, ALDO increased when larvae were exposed to RU486, suggesting that RU486 may also suppress mineralocorticoid receptors or that negative feedback of ALDO is mediated through glucocorticoid receptors. Alternatively, CORT increases with RU486 treatment and might be a substrate for ALDO synthesis, which could account for increases in ALDO with RU486 treatment and the correlation between CORT and ALDO. ALDO was negatively correlated with percent water, such that larvae secreting more ALDO retained less water. Although sample sizes were limited and further validation and studies are warranted, our findings expand our understanding of adrenal steroid responses to salinization in amphibians and proposes new hypotheses regarding the co-regulation of ALDO and CORT.

Artificial light at night (ALAN) affects the stress physiology but not the behavior or growth of Rana berlandieri and Bufo valliceps.

Artificial light at night (ALAN) alters the natural light dark patterns in ecosystems. ALAN can have a suite of effects on community structure and is a driver of evolutionary processes that influences a range of behavioral and physiological traits. Our understanding of possible effects of ALAN across species amphibians is lacking and research is warranted as ALAN could contribute to stress and declines of amphibian populations, particularly in urban areas. We tested the hypothesis that exposure to constant light or pulsed ALAN would physiologically stress Rio Grande leopard frog (Rana berlandieri) and Gulf Coast toad (Bufo valliceps) tadpoles. We reared tadpoles under constant or pulsed (on and off again) ALAN for 14 days and measured corticosterone release rates over time using a non-invasive water-borne hormone protocol. ALAN treatments did not affect behavior or growth. Tadpoles of both species had higher corticosterone (cort) release rates after 14 days of constant light exposure. Leopard frog tadpoles had lower cort release rates after exposure to pulsed ALAN while toad tadpoles had higher cort release rates. These results suggest that short-term exposure to constant or pulsed light at night may contribute to stress in tadpoles but that each species differentially modulated their cort response to ALAN exposure and a subsequent stressor. This flexibility in the upregulation and downregulation of hypothalamic-pituitary-interrenal axis response may indicate an alternative mechanism for diminishing the deleterious effects of chronic stress. Nonetheless, ALAN should be considered in management and conservation plans for amphibians.

Behavioral and Endocrine Alterations to Partner Interactions and Offspring Care during Periods of Conflict

Biparental care has evolved to ensure successful rearing of offspring. However, separation during periods of care can lead to conflicts that might negatively impact pair bonds and offspring care. In this study, pair-bonded convict cichlids (Amatitlania nigrofasciata) were observed for changes in behavior toward their partners and offspring before and after a period of separation. Males and females were designated either as a Resident (remain with offspring) or Removed (separated from partner and offspring for 5 days) individual. Behaviors between partners and toward offspring were measured before and after separation, and compared to the levels of behavior of control pairs (never separated), as well as individuals introduced to a novel partner instead. Cortisol levels of Resident male and female A. nigrofasciata were assayed using water-borne hormone collection before and after separation. Aggression between pair bond members did increase following reintroduction, but did not lead to the termination of pair bonds. Resident females showed more aggression to novel partners than Resident males. Offspring care decreased in both Resident and Removed females. Experimental pairs decreased the amount of time spent interacting with intruders. Cortisol levels were significantly higher among experimental pairs compared with control pairs that did not experience a separation. Females (both control and experimental) showed small, yet significant increases in cortisol levels, while both control and experimental males did not. These results suggest that while pair bonds appear resilient, prolonged separations influence pair bond and parental care dynamics, both behaviorally and hormonally, and require pairs to re-establish roles, resulting in less time caring for offspring.

Collective action reduces androgen responsiveness with implications for shoaling dynamics in stickleback fish

Androgens, traditionally viewed as hormones that regulate secondary sexual characteristics and reproduction in male vertebrates, are often modulated by social stimuli. High levels of the ‘social hormone’ testosterone (T) are linked to aggression, dominance, and competition. Low T levels, in contrast, promote sociopositive behaviours such as affiliation, social tolerance, and cooperation, which can be crucial for group-level, collective behaviours. Here, we test the hypothesis that - in a collective context - low T levels should be favourable, using non-reproductive male and female stickleback fish (Gasterosteus aculeatus) and non-invasive waterborne hormone analysis. In line with our predictions, we show that the fishes' T levels were significantly lower during shoaling compared to when alone, with high-T individuals showing the largest decrease. Ruling out stress-induced T suppression and increased T conversion into oestradiol, we find evidence that shoaling directly affects androgen responsiveness. We also show that groups characterized by lower mean T exhibit less hierarchical leader-follower dynamics, suggesting that low T promotes egalitarianism. Overall, we show that collective action results in lower T levels, which may serve to promote coordination and group performance. Our study, together with recent complementary findings in humans, emphasizes the importance of low T for the expression of sociopositive behaviour across vertebrates, suggesting similarities in endocrine mechanisms.

Non-invasive Methods for Measuring and Monitoring Stress Physiology in Imperiled Amphibians

Global climate change is negatively impacting global biodiversity and ectothermic vertebrates, with amphibians being the most imperiled vertebrate taxa. Increased mean global atmospheric temperatures, high rates of habitat degradation, and exposure to infectious diseases, such as chytridiomycosis, have contributed to population declines and extinctions of rare and endangered amphibian species. Field-based monitoring of physiological endocrine traits can help determine the sub-lethal effects of environmental stressors and provide early alerts when populations are chronically stressed. Recent advances in amphibian stress endocrinology include the development and use of non-invasive methods to quantify the glucocorticoid, or stress biomarker, corticosterone. Non-invasive methods, such as urinary, skin and buccal swabs, and water-borne hormone monitoring methods (suited for terrestrial and aquatic dwelling species), are available to quantify baseline and short-term physiological stress responses of amphibians under field settings. In this review, we illustrate, by using two case studies of aquatic and terrestrial amphibian species, the applications of non-invasive corticosterone monitoring methods to advance the ecological knowledge and conservation of imperiled amphibian species, discuss the limitations of these methods, and provide future directions for the use of non-invasive hormone monitoring methods. We highlight the use of non-invasive field endocrinology methods to monitor the impacts of environmental stressors on the physiology of amphibians, which can be applied to advance ecological research, conservation, and management of imperiled species.

Water-borne and plasma corticosterone are not correlated in spotted salamanders.

Water‐borne hormone measurement is a noninvasive method suitable for amphibians of all sizes that are otherwise difficult to sample. For this method, containment‐water is assayed for hormones released by the animal. Originally developed in fish, the method has expanded to amphibians, but requires additional species‐specific validations. We wanted to determine physiological relevance of water‐borne corticosterone in spotted salamanders (Ambystoma maculatum) by comparing concentrations to those taken using established corticosterone sampling methods, such as plasma. Using a mixture of field and laboratory studies, we compared water‐borne corticosterone levels to other traditional methods of sampling corticosterone for spotted salamander larvae, metamorphs, and adults. Despite multiple attempts, and detecting differences between age groups, we found no correlations between water‐borne and plasma corticosterone levels in any age group. Water‐borne sampling measures a rate of release; whereas plasma is the concentration circulating in the blood. The unique units of measurement may inherently prevent correlations between the two. These two methods may also require different interpretations of the data and the physiological meaning. We also note caveats with the method, including how to account for differences in body size and life history stages. Collectively, our results illustrate the importance of careful validation of water‐borne hormone levels in each species in order to understand its physiological significance.

Validation of water-borne cortisol and corticosterone in tadpoles: Recovery rate from an acute stressor, repeatability, and evaluating rearing methods

Amphibian populations are declining globally, so understanding how individuals respond to anthropogenic and environmental stressors may aid conservation efforts. Using a non-invasive water-borne hormone assay, we measured the release rates of two glucocorticoid hormones, corticosterone and cortisol, in Rio Grande Leopard frog, Rana berlandieri, tadpoles. We validated this method pharmacologically and biologically using an adrenocorticotropic hormone (ACTH) challenge, exposure to exogenous corticosterone, and an agitation test. We calculated the repeatability of hormone release rates, the recovery time from an acute stressor, and explored rearing methods for tadpoles. Tadpole corticosterone release rates increased following an ACTH challenge, exposure to exogenous corticosterone, and agitation, validating the use of water-borne hormone methods in this species. After exposure to an acute stressor via agitation, corticosterone release rates began to decline after 2 h and were lowest after 6 h, suggesting a relatively rapid recovery from an acute stressor. Tadpoles reared in groups had higher corticosterone release rates than tadpoles reared individually, and lost mass by Day 7, while tadpoles reared individually did not show a stress response, therefore either rearing method is viable, but have differing physiological costs for tadpoles. Repeatability of corticosterone release rates was moderate to high in R. berlandieri tadpoles, indicating that this species can show a response to selection and potentially respond to rapid environmental change. Our results show that the water-borne hormone assay is a viable way to measure glucocorticoids in this species and is useful in the field of conservation physiology for rare and endangered species.

Habituation and individual variation in the endocrine stress response in the Trinidadian guppy (Poecilia reticulata)

The vertebrate stress response enables individuals to react to and cope with environmental challenges. A crucial aspect of the stress response is the elevation of circulating glucocorticoids. However, continued activation of the stress response under repeated exposure to stressors can be damaging to fitness. Under certain circumstances it may therefore be adaptive to habituate to repeated exposures to a particular stressor by reducing the magnitude of any associated release of glucocorticoids. Here, we investigate whether Trinidadian guppies (Poecilia reticulata) habituate to repeated exposure to a mild stressor, using a waterborne hormone sampling approach that has previously been shown to elicit a stress response in small fish. We also test for individual variation in the extent of habituation to this stressor. Concentrating on freely circulating cortisol, we found that the first exposure to the assay induced high cortisol release rates but that guppies tended to habituate quickly to subsequent exposures. There were consistent differences among individuals in their average cortisol release rate (after accounting for effects of variables such as body size) over repeated exposures. Our analyses did not find evidence of individual differences in habituation rate, although limitations in statistical power could account for this finding. We repeated the analysis for free 11-ketotestosterone, which can also respond to stressors, but found no obvious habituation pattern and no among-individual variation. We also present data on conjugated forms of both hormones, which were repeatable but did not show the expected time-lagged habituation effect. We discuss consistent individual differences around the general pattern of habituation in the flexible stress response, and highlight the potential for individual variation in habituation to facilitate selection against the deleterious effects of chronic stress.

The Waiting and Mating Game: Condition Dependent Mate Sampling in Female Gray Treefrogs (Hyla versicolor)

Strong sexual selection by receivers can lead to the evolution of elaborate courtship behaviors in signalers. However the process by which receivers sample signalers and execute mate choice under complex signaling conditions—and thus the realized strength of sexual selection—is poorly understood. Moreover, receivers can vary in condition, which can further influence mate sampling strategies. Using wild female frogs we tested two hypotheses at the intersection of these important problems: that some of the individual variation in mate sampling is explained by (1) the reproductive urgency hypothesis, which predicts that receivers in a more urgent reproductive state will sample mates less and/or (2) the reproductive investment hypothesis, which predicts that receivers that have invested less in the current reproductive effort will sample mates less. Eastern gray treefrogs, Hyla versicolor, were collected in amplexus and repeatedly tested for phonotaxis behavior using a dynamic playback assay. To evaluate if hormonal mechanisms explained variation in the mate sampling, three steroid hormones, estradiol, progesterone, and corticosterone, were collected using a noninvasive water-borne hormone assay, validated for this species in the present study. Finally, we measured clutch size (investment) and the duration of time required for each female to oviposit after being reunited with their male mate (urgency). We found repeatability in many of the behaviors, including mate sampling. We found that females with higher concentrations estradiol and corticosterone made quicker choices, and that females with higher progesterone sampled mates more. We also found that female frogs in a more urgent reproductive state had lower concentrations of progesterone and estradiol, thereby providing the first evidence of a relationship between gonadal hormones and reproductive urgency. Collectively we found some support for the reproductive urgency but not the investment hypothesis. Thus, even though a female frog’s reproductive readiness is a highly transient life history stage, fine scale variation in her reproductive timeline could mitigate the strength of directional selection.

Evolution of steroid hormones in reproductive females of the threespine stickleback fish

Hormones play a prominent role in animal development, mediating the expression of traits and coordinating phenotypic responses to the environment. Their role as physiological integrators has implications for how populations respond to natural selection and can impact the speed and direction of evolutionary change. However, many emerging and established fish models with the potential to be ecologically or evolutionarily informative are small-bodied, making hormone sampling through traditional methods (whole-body or plasma) lethal or highly disruptive. Sampling methodology has thus restricted study design, often limiting sample sizes, and has prevented the study of at-risk/endangered populations. We utilize water-borne hormone sampling, a minimally invasive method of measuring the rate of steroid hormone release across the gills and further validate this method in a novel, evolutionary context. First, we compare water-borne hormone measures of cortisol with those quantified from plasma and whole-body samples collected from the same individuals to establish the relationship between concentrations quantified via the three methods. We then compare the release of steroid hormones in three populations of threespine stickleback to establish the sensitivity of this tool in measuring within-individual and between-individual variation in biologically relevant contexts (reproductive stages), and in assessing differences among populations with distinct evolutionary histories. We demonstrate a strong positive relationship between cortisol concentrations measured with water-borne, plasma, and whole-body collection techniques. Tracking estradiol and testosterone throughout clutch production in females produced anticipated patterns associated with growing and maturing eggs, with divergence in estradiol production in one population. Additionally, differences among populations in cortisol levels at ovulation paralleled the relative presence of a social stressor, and thus expected energetic needs within each population. We confirm that water-borne hormone sampling is sufficiently sensitive to capture biologically relevant fluctuations in steroid hormones between environmental contexts and demonstrate that among-population differences are detectable. This technique can be applied broadly to small fish to answer important ecological and evolutionary questions. By linking population variation in hormones and the multivariate phenotype, this technique will help elucidate both proximate mechanisms underlying phenotypic development and variation, and the way hormone networks alter evolutionary responses to selection.

Physiological responses to elevated temperature across the geographic range of a terrestrial salamander.

Widespread species often possess physiological mechanisms for coping with thermal heterogeneity, and uncovering these mechanisms provides insight into species' responses to climate change. The emergence of non-invasive corticosterone (CORT) assays allows us to rapidly assess physiological responses to environmental change on a large scale. We lack, however, a basic understanding of how temperature affects CORT, and whether temperature and CORT interactively affect performance. Here, we examined the effects of elevated temperature on CORT and whole-organism performance in a terrestrial salamander, Plethodon cinereus, across a latitudinal gradient. Using water-borne hormone assays, we found that raising ambient temperature from 15 to 25°C increased CORT release at a similar rate for salamanders from all sites. However, CORT release rates were higher overall in the warmest, southernmost site. Elevated temperatures also affected physiological performance, but the effects differed among sites. Ingestion rate increased in salamanders from the warmer sites but remained the same for those from cooler sites. Mass gain was reduced for most individuals, although this reduction was more dramatic in salamanders from the cooler sites. We also found a temperature-dependent relationship between CORT and food conversion efficiency (i.e. the amount of mass gained per unit food ingested). CORT was negatively related to food conversion efficiency at 25°C but was unrelated at 15°C. Thus, the energetic gains of elevated ingestion rates may be counteracted by elevated CORT release rates experienced by salamanders in warmer environments. By integrating multiple physiological metrics, we highlight the complex relationships between temperature and individual responses to warming climates.

The effects of ozonation on select waterborne steroid hormones in recirculation aquaculture systems containing sexually mature Atlantic salmon Salmo salar

Steroid hormones have been shown to accumulate in recirculation aquaculture system (RAS) water over time; however, their influence on the reproductive physiology of fish within RAS remains unknown. Whether ozonation impacts waterborne hormone levels in RAS has likewise not been fully evaluated. To this end, a controlled 3-month study was conducted in 6 replicated RAS containing a mixture of sexually mature and immature Atlantic salmon Salmo salar to determine whether ozone, as typically applied in RAS to improve water quality, is associated with a reduction in waterborne hormones. Post-smolt Atlantic salmon (1253±15g) were stocked into each RAS; 109 of 264 fish placed in each system were sexually mature males, and 5 were mature females. Water ozonation, controlled using an ORP set-point of 290–300mV, was applied with the pure oxygen feed gas within the low-head oxygenators of 3 randomly selected RAS, while the remaining 3 RAS did not receive ozone. The RAS hydraulic retention time was 6.9±0.3 days. Study fish were raised under these conditions for 12 weeks; during weeks 10 and 12, triplicate water samples were collected from the following locations in each RAS: i) culture tank, ii) makeup water, iii) pre-biofilter, iv) post-biofilter, and v) post-gas conditioning. Concentrations of 3 waterborne hormones – testosterone, 11-ketotestosterone (11-KT), and estradiol (17β-estradiol) – were quantified using enzyme immunoassays (EIA). Estradiol was significantly reduced by ozonation; testosterone and 11-KT were also reduced by ozonation, although these reductions were not observed across all sampling locations and events. Testosterone and 11-KT concentrations, however, were significantly reduced following water passage through the biofilters of both ozonated and non-ozonated RAS. The results of this study demonstrate the potential for ozone to be used in RAS as a means of preventing the accumulation of steroid hormones. Further research is required to assess whether reducing hormones in this manner impacts precocious sexual maturation in RAS-produced Atlantic salmon.

Cortisol coregulation in fish.

Cortisol coregulation, which is the up- or down-regulation of partners’ physiological stress responses, has been described for individuals with strong attachment bonds, e.g. parents and their children, and romantic relationship partners. Research into moderating effects on cortisol coregulation suggests stronger covariation among distressed partners. Whether cortisol coregulation is unique to humans or can also be found in other species that share universal features of the vertebrate stress response remains unexplored. Using a repeated measures approach and non-invasive waterborne hormone analysis, we test the hypothesis that dyads of three-spined stickleback fish (Gasterosteus aculeatus) coregulate their cortisol levels in shared environments. Dyadic cortisol levels were unrelated when cohabiting (home tank), but significantly covaried when sharing a more stressful (as indicated by higher cortisol levels) environment (open field). Time-lag analysis further revealed that open field cortisol levels were predicted by partner’s cortisol levels prior to the shared experience. To our knowledge, this study provides the first evidence for coregulatory processes on cortisol responses in a non-human animal that lacks strong bonds and social attachment relationships, suggesting a shared evolutionary origin of cortisol coregulation in vertebrates. From an adaptive perspective, cortisol coregulation may serve to reduce risk in challenging, potentially threatening situations.

A Non-Invasive Water-Borne Assay of Stress Hormones in Aquatic Salamanders

Declines in amphibian populations are due to an array of anthropogenic and natural factors requiring a need to detect and monitor populations that are either in decline or at risk of decline. Physiological biomarkers, such as glucocorticoid stress hormones, present a viable option to monitor stress levels and, thus, the physiological health of vertebrate populations. We performed preliminary experiments necessary for the use of water-borne hormone assays to measure corticosterone (CORT) as an assessment of aspects of chronic stress for the management and conservation of three species of aquatic salamanders in central Texas (Eurycea, Plethodontidae). We examined the time for CORT levels to peak in response to capture and handling in populations of E. nana, E. sosorum, and E. tonkawae. Peak CORT release rates differed among species, with peak releases generally occurring in under 3 h. This highlights the need to obtain a timeline for CORT to mount so that peak rates are captured during the leaching phase. We also examined the responsiveness of the hypothalamic-pituitary-interrenal (HPI) axis using an adrenocorticotropic hormone (ACTH) challenge or exposure to an external stressor (agitation). Eurycea sosorum (one population) and E. tonkawae showed HPI responsiveness. To examine the relationship between field and laboratory CORT release rates, we compared baseline CORT release rates between captive and wild-caught populations of all three species. Once the preliminary studies have been performed, water-borne CORT release rates can be an effective biomarker to monitor whether a population is experiencing chronic stress. Water-borne hormone collection also allows for repeated sampling of individuals over time. Our experiments provide a foundation for future studies relating stress to HPI responsiveness in aquatic Eurycea, which, in turn, will help inform management of both wild and captive populations.

Hormonal Responses to Noncontact Aggression in Convict Cichlid Fish.

This study explored whether convict cichlid fish mount a hormonal response to aggressive encounters where dominance status remains unresolved. Hormone samples were collected at two time points before an aggressive interaction to obtain confinement-induced and baseline measures, and at one time point following a contest across a clear partition (experimental) or exposure to an opaque partition with an opponent on the opposite side (control). There was no overall significant effect of treatment (control vs. experimental) on hormone release rates but there were trends for cortisol and testosterone (T). A priori linear contrasts showed that individuals that engaged in aggressive interactions had lower postfight cortisol and T release rates than controls, suggesting that aggression, in this context, might attenuate the synthesis of both hormones. Cortisol decreased significantly between initial confinement and baseline, indicating that individuals habituate to the water-borne hormone collection procedure. Contrary to expectation, individuals with higher baseline T and 11-ketotestosterone (KT) release rates took longer to initiate conflict. None of the other measures of behavior were predicted by baseline hormone release rates, and contest behavior did not predict postfight hormone release rates. There was a significant positive relationship between KT and T at all time points. As with studies that employ mirror image stimulation, we found no hormonal response to unresolved contests despite high levels of aggressive behavior. Our study is unique because we demonstrate that animals engaged in conflict with live opponents also do not mount a significant hormonal response when clear dominance relationships are not established.

The relationship between handling time and cortisol release rates changes as a function of brain parasite densities in California killifish Fundulus parvipinnis.

This study validated a technique for non-invasive hormone measurements in California killifish Fundulus parvipinnis, and looked for associations between cortisol (a stress hormone) and 11-ketotestosterone (KT, an androgen) release rates and the density or intensity of the trematode parasites Euhaplorchis californiensis (EUHA) and Renicola buchanani (RENB) in wild-caught, naturally infected F. parvipinnis. In experiment 1, F. parvipinnis were exposed to an acute stressor by lowering water levels to dorsal-fin height and repeatedly handling the fish over the course of an hour. Neither parasite was found to influence cortisol release rates in response to this acute stressor. In experiment 2, different F. parvipinnis were exposed on four consecutive days to the procedure for collecting water-borne hormone levels and release rates of 11-KT and cortisol were quantified. This design examined whether F. parvipinnis perceived the water-borne collection procedure to be a stressor, while also exploring how parasites influenced hormone release rates under conditions less stressful than those in experiment 1. No association was found between RENB and hormone release rates, or between EUHA and 11-KT release rates. The interaction between EUHA density and handling time, however, was an important predictor of cortisol release rates. The relationship between handling time and cortisol release rates was negative for F. parvipinnis harbouring low or intermediate density infections, and became positive for fish harbouring high densities of EUHA.